From 3e0793fdd494b97cdf0591e88bb06a7ba6080b4e Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Tue, 30 Jul 2024 16:03:02 +0300 Subject: [PATCH 01/36] Install kraken2 and bracken --- modules.json | 10 ++ .../nf-core/bracken/bracken/environment.yml | 7 + modules/nf-core/bracken/bracken/main.nf | 52 ++++++ modules/nf-core/bracken/bracken/meta.yml | 51 ++++++ .../bracken/bracken/tests/main.nf.test | 121 ++++++++++++++ .../bracken/bracken/tests/main.nf.test.snap | 157 ++++++++++++++++++ .../bracken/bracken/tests/nextflow.config | 5 + .../nf-core/bracken/bracken/tests/tags.yml | 2 + .../nf-core/kraken2/kraken2/environment.yml | 9 + modules/nf-core/kraken2/kraken2/main.nf | 85 ++++++++++ modules/nf-core/kraken2/kraken2/meta.yml | 78 +++++++++ .../kraken2/kraken2/tests/main.nf.test | 143 ++++++++++++++++ .../kraken2/kraken2/tests/main.nf.test.snap | 74 +++++++++ .../nf-core/kraken2/kraken2/tests/tags.yml | 3 + 14 files changed, 797 insertions(+) create mode 100644 modules/nf-core/bracken/bracken/environment.yml create mode 100644 modules/nf-core/bracken/bracken/main.nf create mode 100644 modules/nf-core/bracken/bracken/meta.yml create mode 100644 modules/nf-core/bracken/bracken/tests/main.nf.test create mode 100644 modules/nf-core/bracken/bracken/tests/main.nf.test.snap create mode 100644 modules/nf-core/bracken/bracken/tests/nextflow.config create mode 100644 modules/nf-core/bracken/bracken/tests/tags.yml create mode 100644 modules/nf-core/kraken2/kraken2/environment.yml create mode 100644 modules/nf-core/kraken2/kraken2/main.nf create mode 100644 modules/nf-core/kraken2/kraken2/meta.yml create mode 100644 modules/nf-core/kraken2/kraken2/tests/main.nf.test create mode 100644 modules/nf-core/kraken2/kraken2/tests/main.nf.test.snap create mode 100644 modules/nf-core/kraken2/kraken2/tests/tags.yml diff --git a/modules.json b/modules.json index f197e3f65..ac86bcd98 100644 --- a/modules.json +++ b/modules.json @@ -15,6 +15,11 @@ "git_sha": "81b90194ce9911dbd55bba2c65c6919f6677abc4", "installed_by": ["modules"] }, + "bracken/bracken": { + "branch": "master", + "git_sha": "f362bca32735a848feb314d601db1cee584ee0d1", + "installed_by": ["modules"] + }, "cat/fastq": { "branch": "master", "git_sha": "4fc983ad0b30e6e32696fa7d980c76c7bfe1c03e", @@ -90,6 +95,11 @@ "git_sha": "de5811dd9ca15af1e131806001bcaae909e42021", "installed_by": ["modules", "quantify_pseudo_alignment"] }, + "kraken2/kraken2": { + "branch": "master", + "git_sha": "ca87ad032a62f025f0c373facacef2df0c5411b2", + "installed_by": ["modules"] + }, "multiqc": { "branch": "master", "git_sha": "b80f5fd12ff7c43938f424dd76392a2704fa2396", diff --git a/modules/nf-core/bracken/bracken/environment.yml b/modules/nf-core/bracken/bracken/environment.yml new file mode 100644 index 000000000..6eb5b1b9a --- /dev/null +++ b/modules/nf-core/bracken/bracken/environment.yml @@ -0,0 +1,7 @@ +name: bracken_bracken +channels: + - conda-forge + - bioconda + - defaults +dependencies: + - "bioconda::bracken=2.9" diff --git a/modules/nf-core/bracken/bracken/main.nf b/modules/nf-core/bracken/bracken/main.nf new file mode 100644 index 000000000..caded9edd --- /dev/null +++ b/modules/nf-core/bracken/bracken/main.nf @@ -0,0 +1,52 @@ +process BRACKEN_BRACKEN { + tag "$meta.id" + label 'process_low' + + conda "${moduleDir}/environment.yml" + container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ? + 'https://depot.galaxyproject.org/singularity/bracken:2.9--py38h2494328_0': + 'biocontainers/bracken:2.9--py38h2494328_0' }" + + input: + tuple val(meta), path(kraken_report) + path database + + output: + tuple val(meta), path(bracken_report) , emit: reports + tuple val(meta), path("*bracken_species.txt"), emit: txt + path "versions.yml" , emit: versions + + when: + task.ext.when == null || task.ext.when + + script: + def args = task.ext.args ?: "" + def prefix = task.ext.prefix ?: "${meta.id}" + bracken_report = "${prefix}.tsv" + """ + bracken \\ + ${args} \\ + -d '${database}' \\ + -i '${kraken_report}' \\ + -o '${bracken_report}' + + cat <<-END_VERSIONS > versions.yml + "${task.process}": + bracken: \$(echo \$(bracken -v) | cut -f2 -d'v') + END_VERSIONS + """ + + stub: + def args = task.ext.args ?: '' + def prefix = task.ext.prefix ?: "${meta.id}" + bracken_report = "${prefix}.tsv" + """ + touch ${prefix}.tsv + touch ${prefix}_bracken_species.txt + + cat <<-END_VERSIONS > versions.yml + "${task.process}": + bracken: \$(echo \$(bracken -v) | cut -f2 -d'v') + END_VERSIONS + """ +} diff --git a/modules/nf-core/bracken/bracken/meta.yml b/modules/nf-core/bracken/bracken/meta.yml new file mode 100644 index 000000000..b7ff4489f --- /dev/null +++ b/modules/nf-core/bracken/bracken/meta.yml @@ -0,0 +1,51 @@ +name: bracken_bracken +description: Re-estimate taxonomic abundance of metagenomic samples analyzed by kraken. +keywords: + - bracken + - metagenomics + - abundance + - kraken2 +tools: + - bracken: + description: Bracken (Bayesian Reestimation of Abundance with KrakEN) is a highly accurate statistical method that computes the abundance of species in DNA sequences from a metagenomics sample. + homepage: https://ccb.jhu.edu/software/bracken/ + documentation: https://ccb.jhu.edu/software/bracken/index.shtml?t=manual + tool_dev_url: https://github.com/jenniferlu717/Bracken + doi: "10.7717/peerj-cs.104" + licence: ["GPL v3"] +input: + - meta: + type: map + description: | + Groovy Map containing sample information + e.g. [ id:'test', single_end:false ] + - kraken_report: + type: file + description: TSV file with six columns coming from kraken2 output + pattern: "*.{tsv}" + - database: + type: file + description: Directory containing the kraken2/Bracken files for analysis + pattern: "*" +output: + - meta: + type: map + description: | + Groovy Map containing sample information + e.g. [ id:'test', single_end:false ] + - versions: + type: file + description: File containing software versions + pattern: "versions.yml" + - reports: + type: file + description: TSV output report of the re-estimated abundances + pattern: "*.{tsv}" + - txt: + type: file + description: TXT file of bracken corrected results of Kraken2 report output + pattern: "*.txt" +authors: + - "@Midnighter" +maintainers: + - "@Midnighter" diff --git a/modules/nf-core/bracken/bracken/tests/main.nf.test b/modules/nf-core/bracken/bracken/tests/main.nf.test new file mode 100644 index 000000000..59c1398e5 --- /dev/null +++ b/modules/nf-core/bracken/bracken/tests/main.nf.test @@ -0,0 +1,121 @@ +nextflow_process { + + name "Test Process BRACKEN_BRACKEN" + script "../main.nf" + process "BRACKEN_BRACKEN" + + tag "modules" + tag "modules_nfcore" + tag "bracken" + tag "bracken/bracken" + tag "kraken2/kraken2" + tag "untar" + + setup { + run ("UNTAR") { + script "../../../untar/main.nf" + process { + """ + input[0] = [[],file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/db/kraken2_bracken.tar.gz', checkIfExists: true)] + """ + } + } + } + + test("sarscov2 - single-end - fastq") { + + setup { + run("KRAKEN2_KRAKEN2") { + script "../../../kraken2/kraken2/main.nf" + process { + """ + input[0] = [[id: 'test', single_end: true], file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_1.fastq.gz', checkIfExists: true)] + input[1] = UNTAR.out.untar.map{it[1]} + input[2] = false + input[3] = false + """ + } + } + } + + when { + process { + """ + input[0] = KRAKEN2_KRAKEN2.out.report + input[1] = UNTAR.out.untar.map{it[1]} + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot(process.out).match() } + ) + } + + } + +test("sarscov2 - paired-end - fastq") { + + setup { + run("KRAKEN2_KRAKEN2") { + script "../../../kraken2/kraken2/main.nf" + process { + """ + input[0] = [ + [id: 'test', single_end: false], + [ + file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_1.fastq.gz', checkIfExists: true), + file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_2.fastq.gz', checkIfExists: true) + ] + ] + input[1] = UNTAR.out.untar.map{it[1]} + input[2] = false + input[3] = false + """ + } + } + } + + when { + process { + """ + input[0] = KRAKEN2_KRAKEN2.out.report + input[1] = UNTAR.out.untar.map{it[1]} + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot(process.out).match() } + ) + } + + } + + test("sarscov2 - stub - fastq") { + + options "-stub" + + when { + process { + """ + input[0] = [[id: 'test'],file(params.modules_testdata_base_path + 'generic/txt/hello.txt', checkIfExists: true)] + input[1] = UNTAR.out.untar.map{it[1]} + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot(process.out).match() } + ) + } + + } + +} diff --git a/modules/nf-core/bracken/bracken/tests/main.nf.test.snap b/modules/nf-core/bracken/bracken/tests/main.nf.test.snap new file mode 100644 index 000000000..5aadb7b70 --- /dev/null +++ b/modules/nf-core/bracken/bracken/tests/main.nf.test.snap @@ -0,0 +1,157 @@ +{ + "sarscov2 - single-end - fastq": { + "content": [ + { + "0": [ + [ + { + "id": "test", + "single_end": true + }, + "test.tsv:md5,4a21ae14ff8a0311d55f139af5247838" + ] + ], + "1": [ + [ + { + "id": "test", + "single_end": true + }, + "test.kraken2.report_bracken_species.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + ] + ], + "2": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ], + "reports": [ + [ + { + "id": "test", + "single_end": true + }, + "test.tsv:md5,4a21ae14ff8a0311d55f139af5247838" + ] + ], + "txt": [ + [ + { + "id": "test", + "single_end": true + }, + "test.kraken2.report_bracken_species.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + ] + ], + "versions": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ] + } + ], + "meta": { + "nf-test": "0.8.4", + "nextflow": "24.03.0" + }, + "timestamp": "2024-04-16T14:14:27.122403998" + }, + "sarscov2 - paired-end - fastq": { + "content": [ + { + "0": [ + [ + { + "id": "test", + "single_end": false + }, + "test.tsv:md5,4a21ae14ff8a0311d55f139af5247838" + ] + ], + "1": [ + [ + { + "id": "test", + "single_end": false + }, + "test.kraken2.report_bracken_species.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + ] + ], + "2": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ], + "reports": [ + [ + { + "id": "test", + "single_end": false + }, + "test.tsv:md5,4a21ae14ff8a0311d55f139af5247838" + ] + ], + "txt": [ + [ + { + "id": "test", + "single_end": false + }, + "test.kraken2.report_bracken_species.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + ] + ], + "versions": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ] + } + ], + "meta": { + "nf-test": "0.8.4", + "nextflow": "24.03.0" + }, + "timestamp": "2024-04-16T14:19:15.627050252" + }, + "sarscov2 - stub - fastq": { + "content": [ + { + "0": [ + [ + { + "id": "test" + }, + "test.tsv:md5,d41d8cd98f00b204e9800998ecf8427e" + ] + ], + "1": [ + [ + { + "id": "test" + }, + "test_bracken_species.txt:md5,d41d8cd98f00b204e9800998ecf8427e" + ] + ], + "2": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ], + "reports": [ + [ + { + "id": "test" + }, + "test.tsv:md5,d41d8cd98f00b204e9800998ecf8427e" + ] + ], + "txt": [ + [ + { + "id": "test" + }, + "test_bracken_species.txt:md5,d41d8cd98f00b204e9800998ecf8427e" + ] + ], + "versions": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ] + } + ], + "meta": { + "nf-test": "0.8.4", + "nextflow": "24.03.0" + }, + "timestamp": "2024-04-16T14:14:36.5929911" + } +} \ No newline at end of file diff --git a/modules/nf-core/bracken/bracken/tests/nextflow.config b/modules/nf-core/bracken/bracken/tests/nextflow.config new file mode 100644 index 000000000..b550b7489 --- /dev/null +++ b/modules/nf-core/bracken/bracken/tests/nextflow.config @@ -0,0 +1,5 @@ +process { + withName: BRACKEN_BRACKEN { + ext.args = "-l S -t 10 -r 150" + } +} diff --git a/modules/nf-core/bracken/bracken/tests/tags.yml b/modules/nf-core/bracken/bracken/tests/tags.yml new file mode 100644 index 000000000..6a2cbd384 --- /dev/null +++ b/modules/nf-core/bracken/bracken/tests/tags.yml @@ -0,0 +1,2 @@ +bracken/bracken: + - "modules/nf-core/bracken/bracken/**" diff --git a/modules/nf-core/kraken2/kraken2/environment.yml b/modules/nf-core/kraken2/kraken2/environment.yml new file mode 100644 index 000000000..0c067feeb --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/environment.yml @@ -0,0 +1,9 @@ +name: kraken2_kraken2 +channels: + - conda-forge + - bioconda + - defaults +dependencies: + - "bioconda::kraken2=2.1.3" + - "coreutils=9.4" + - "pigz=2.8" diff --git a/modules/nf-core/kraken2/kraken2/main.nf b/modules/nf-core/kraken2/kraken2/main.nf new file mode 100644 index 000000000..364a6fe24 --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/main.nf @@ -0,0 +1,85 @@ +process KRAKEN2_KRAKEN2 { + tag "$meta.id" + label 'process_high' + + conda "${moduleDir}/environment.yml" + container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ? + 'https://depot.galaxyproject.org/singularity/mulled-v2-8706a1dd73c6cc426e12dd4dd33a5e917b3989ae:c8cbdc8ff4101e6745f8ede6eb5261ef98bdaff4-0' : + 'biocontainers/mulled-v2-8706a1dd73c6cc426e12dd4dd33a5e917b3989ae:c8cbdc8ff4101e6745f8ede6eb5261ef98bdaff4-0' }" + + input: + tuple val(meta), path(reads) + path db + val save_output_fastqs + val save_reads_assignment + + output: + tuple val(meta), path('*.classified{.,_}*') , optional:true, emit: classified_reads_fastq + tuple val(meta), path('*.unclassified{.,_}*') , optional:true, emit: unclassified_reads_fastq + tuple val(meta), path('*classifiedreads.txt') , optional:true, emit: classified_reads_assignment + tuple val(meta), path('*report.txt') , emit: report + path "versions.yml" , emit: versions + + when: + task.ext.when == null || task.ext.when + + script: + def args = task.ext.args ?: '' + def prefix = task.ext.prefix ?: "${meta.id}" + def paired = meta.single_end ? "" : "--paired" + def classified = meta.single_end ? "${prefix}.classified.fastq" : "${prefix}.classified#.fastq" + def unclassified = meta.single_end ? "${prefix}.unclassified.fastq" : "${prefix}.unclassified#.fastq" + def classified_option = save_output_fastqs ? "--classified-out ${classified}" : "" + def unclassified_option = save_output_fastqs ? "--unclassified-out ${unclassified}" : "" + def readclassification_option = save_reads_assignment ? "--output ${prefix}.kraken2.classifiedreads.txt" : "--output /dev/null" + def compress_reads_command = save_output_fastqs ? "pigz -p $task.cpus *.fastq" : "" + + """ + kraken2 \\ + --db $db \\ + --threads $task.cpus \\ + --report ${prefix}.kraken2.report.txt \\ + --gzip-compressed \\ + $unclassified_option \\ + $classified_option \\ + $readclassification_option \\ + $paired \\ + $args \\ + $reads + + $compress_reads_command + + cat <<-END_VERSIONS > versions.yml + "${task.process}": + kraken2: \$(echo \$(kraken2 --version 2>&1) | sed 's/^.*Kraken version //; s/ .*\$//') + pigz: \$( pigz --version 2>&1 | sed 's/pigz //g' ) + END_VERSIONS + """ + + stub: + def args = task.ext.args ?: '' + def prefix = task.ext.prefix ?: "${meta.id}" + def paired = meta.single_end ? "" : "--paired" + def classified = meta.single_end ? "${prefix}.classified.fastq.gz" : "${prefix}.classified_1.fastq.gz ${prefix}.classified_2.fastq.gz" + def unclassified = meta.single_end ? "${prefix}.unclassified.fastq.gz" : "${prefix}.unclassified_1.fastq.gz ${prefix}.unclassified_2.fastq.gz" + def readclassification_option = save_reads_assignment ? "--output ${prefix}.kraken2.classifiedreads.txt" : "--output /dev/null" + def compress_reads_command = save_output_fastqs ? "pigz -p $task.cpus *.fastq" : "" + + """ + touch ${prefix}.kraken2.report.txt + if [ "$save_output_fastqs" == "true" ]; then + touch $classified + touch $unclassified + fi + if [ "$save_reads_assignment" == "true" ]; then + touch ${prefix}.kraken2.classifiedreads.txt + fi + + cat <<-END_VERSIONS > versions.yml + "${task.process}": + kraken2: \$(echo \$(kraken2 --version 2>&1) | sed 's/^.*Kraken version //; s/ .*\$//') + pigz: \$( pigz --version 2>&1 | sed 's/pigz //g' ) + END_VERSIONS + """ + +} diff --git a/modules/nf-core/kraken2/kraken2/meta.yml b/modules/nf-core/kraken2/kraken2/meta.yml new file mode 100644 index 000000000..7909ffe7e --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/meta.yml @@ -0,0 +1,78 @@ +name: kraken2_kraken2 +description: Classifies metagenomic sequence data +keywords: + - classify + - metagenomics + - fastq + - db +tools: + - kraken2: + description: | + Kraken2 is a taxonomic sequence classifier that assigns taxonomic labels to sequence reads + homepage: https://ccb.jhu.edu/software/kraken2/ + documentation: https://github.com/DerrickWood/kraken2/wiki/Manual + doi: 10.1186/s13059-019-1891-0 + licence: ["MIT"] +input: + - meta: + type: map + description: | + Groovy Map containing sample information + e.g. [ id:'test', single_end:false ] + - reads: + type: file + description: | + List of input FastQ files of size 1 and 2 for single-end and paired-end data, + respectively. + - db: + type: directory + description: Kraken2 database + - save_output_fastqs: + type: string + description: | + If true, optional commands are added to save classified and unclassified reads + as fastq files + - save_reads_assignment: + type: string + description: | + If true, an optional command is added to save a file reporting the taxonomic + classification of each input read +output: + - meta: + type: map + description: | + Groovy Map containing sample information + e.g. [ id:'test', single_end:false ] + - classified_reads_fastq: + type: file + description: | + Reads classified as belonging to any of the taxa + on the Kraken2 database. + pattern: "*{fastq.gz}" + - unclassified_reads_fastq: + type: file + description: | + Reads not classified to any of the taxa + on the Kraken2 database. + pattern: "*{fastq.gz}" + - classified_reads_assignment: + type: file + description: | + Kraken2 output file indicating the taxonomic assignment of + each input read + - report: + type: file + description: | + Kraken2 report containing stats about classified + and not classifed reads. + pattern: "*.{report.txt}" + - versions: + type: file + description: File containing software versions + pattern: "versions.yml" +authors: + - "@joseespinosa" + - "@drpatelh" +maintainers: + - "@joseespinosa" + - "@drpatelh" diff --git a/modules/nf-core/kraken2/kraken2/tests/main.nf.test b/modules/nf-core/kraken2/kraken2/tests/main.nf.test new file mode 100644 index 000000000..4c5130217 --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/tests/main.nf.test @@ -0,0 +1,143 @@ +nextflow_process { + name "Test Process KRAKEN2_KRAKEN2" + script "../main.nf" + process "KRAKEN2_KRAKEN2" + tag "modules" + tag "modules_nfcore" + tag "untar" + tag "kraken2" + tag "kraken2/kraken2" + + setup { + run("UNTAR") { + script "modules/nf-core/untar/main.nf" + process { + """ + input[0] = Channel.of([ + [], + file( + params.test_data['sarscov2']['genome']['kraken2_tar_gz'], + checkIfExists: true + ) + ]) + """ + } + } + } + + test("sarscov2 illumina single end [fastq]") { + when { + process { + """ + input[0] = [ + [ id:'test', single_end:true ], // meta map + [ file( + params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], + checkIfExists: true + )] + ] + input[1] = UNTAR.out.untar.map{ it[1] } + input[2] = true + input[3] = false + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot( + process.out.report, + process.out.versions, + ).match() + }, + { assert process.out.classified_reads_fastq.get(0).get(1) ==~ ".*/test.classified.fastq.gz" }, + { assert process.out.unclassified_reads_fastq.get(0).get(1) ==~ ".*/test.unclassified.fastq.gz" }, + ) + } + } + + test("sarscov2 illumina paired end [fastq]") { + when { + params { + outdir = "$outputDir" + } + + process { + """ + input[0] = [ + [ id:'test', single_end:false ], // meta map + [ + file( + params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], + checkIfExists: true + ), + file( + params.test_data['sarscov2']['illumina']['test_2_fastq_gz'], + checkIfExists: true + ) + + ] + ] + input[1] = UNTAR.out.untar.map{ it[1] } + input[2] = true + input[3] = false + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot( + process.out.report, + process.out.versions, + ).match() + }, + { assert process.out.classified_reads_fastq.get(0).get(1).get(0) + ==~ ".*/test.classified_1.fastq.gz" }, + { assert process.out.classified_reads_fastq.get(0).get(1).get(1) + ==~ ".*/test.classified_2.fastq.gz" }, + { assert process.out.unclassified_reads_fastq.get(0).get(1).get(0) + ==~ ".*/test.unclassified_1.fastq.gz" }, + { assert process.out.unclassified_reads_fastq.get(0).get(1).get(1) + ==~ ".*/test.unclassified_2.fastq.gz" }, + ) + } + } + + test("sarscov2 illumina single end [fastq] + save_reads_assignment") { + when { + params { + outdir = "$outputDir" + } + + process { + """ + input[0] = [ + [ id:'test', single_end:true ], // meta map + [ file( + params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], + checkIfExists: true + )] + ] + input[1] = UNTAR.out.untar.map{ it[1] } + input[2] = false + input[3] = true + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot( + process.out.report, + process.out.classified_reads_assignment, + process.out.versions, + ).match() + }, + ) + } + } +} diff --git a/modules/nf-core/kraken2/kraken2/tests/main.nf.test.snap b/modules/nf-core/kraken2/kraken2/tests/main.nf.test.snap new file mode 100644 index 000000000..b432f878f --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/tests/main.nf.test.snap @@ -0,0 +1,74 @@ +{ + "sarscov2 illumina single end [fastq]": { + "content": [ + [ + [ + { + "id": "test", + "single_end": true + }, + "test.kraken2.report.txt:md5,4227755fe40478b8d7dc8634b489761e" + ] + ], + [ + "versions.yml:md5,79adf2ca1cfc625cb77e391b27142c43" + ] + ], + "meta": { + "nf-test": "0.8.4", + "nextflow": "23.10.1" + }, + "timestamp": "2024-04-04T18:47:03.745692" + }, + "sarscov2 illumina paired end [fastq]": { + "content": [ + [ + [ + { + "id": "test", + "single_end": false + }, + "test.kraken2.report.txt:md5,4227755fe40478b8d7dc8634b489761e" + ] + ], + [ + "versions.yml:md5,79adf2ca1cfc625cb77e391b27142c43" + ] + ], + "meta": { + "nf-test": "0.8.4", + "nextflow": "23.10.1" + }, + "timestamp": "2024-04-04T18:47:13.75649" + }, + "sarscov2 illumina single end [fastq] + save_reads_assignment": { + "content": [ + [ + [ + { + "id": "test", + "single_end": true + }, + "test.kraken2.report.txt:md5,4227755fe40478b8d7dc8634b489761e" + ] + ], + [ + [ + { + "id": "test", + "single_end": true + }, + "test.kraken2.classifiedreads.txt:md5,e7a90531f0d8d777316515c36fe4cae0" + ] + ], + [ + "versions.yml:md5,79adf2ca1cfc625cb77e391b27142c43" + ] + ], + "meta": { + "nf-test": "0.8.4", + "nextflow": "23.10.1" + }, + "timestamp": "2024-04-04T18:47:22.459465" + } +} \ No newline at end of file diff --git a/modules/nf-core/kraken2/kraken2/tests/tags.yml b/modules/nf-core/kraken2/kraken2/tests/tags.yml new file mode 100644 index 000000000..9ebfd7ab6 --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/tests/tags.yml @@ -0,0 +1,3 @@ +kraken2/kraken2: + - modules/nf-core/kraken2/kraken2/** + - modules/nf-core/untar/** From a7281e5ed53903d44bb6c5ad272f68834e067eb0 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Tue, 30 Jul 2024 16:12:13 +0300 Subject: [PATCH 02/36] Change nextflow configurations --- modules/nf-core/bracken/bracken/nextflow.config | 11 +++++++++++ modules/nf-core/kraken2/kraken2/nextflow.config | 13 +++++++++++++ nextflow.config | 5 ++++- 3 files changed, 28 insertions(+), 1 deletion(-) create mode 100644 modules/nf-core/bracken/bracken/nextflow.config create mode 100644 modules/nf-core/kraken2/kraken2/nextflow.config diff --git a/modules/nf-core/bracken/bracken/nextflow.config b/modules/nf-core/bracken/bracken/nextflow.config new file mode 100644 index 000000000..8089e551f --- /dev/null +++ b/modules/nf-core/bracken/bracken/nextflow.config @@ -0,0 +1,11 @@ +if (!params.skip_alignment && !params.skip_qc) { + if (!params.skip_kraken2) { + withName: 'BRACKEN' { + ext.args = "-l ${params.bracken_precision}" + publishDir = [ + path: { "${params.outdir}/bracken" }, + mode: params.publish_dir_mode + ] + } + } +} \ No newline at end of file diff --git a/modules/nf-core/kraken2/kraken2/nextflow.config b/modules/nf-core/kraken2/kraken2/nextflow.config new file mode 100644 index 000000000..a81d68de3 --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/nextflow.config @@ -0,0 +1,13 @@ +if (!params.skip_alignment && !params.skip_qc) { + if (!params.skip_kraken2) { + process { + withName: 'KRAKEN2' { + ext.args = "--confidence 0.05 --minimum-hit-groups 3" + publishDir = [ + path: { "${params.outdir}/kraken2/kraken_reports" }, + mode: params.publish_dir_mode + ] + } + } + } +} \ No newline at end of file diff --git a/nextflow.config b/nextflow.config index 1c46d7c2e..47833626c 100644 --- a/nextflow.config +++ b/nextflow.config @@ -73,7 +73,7 @@ params { kallisto_quant_fraglen = 200 kallisto_quant_fraglen_sd = 200 save_merged_fastq = false - save_unaligned = false + save_unaligned = true save_align_intermeds = false skip_markduplicates = false skip_alignment = false @@ -89,6 +89,9 @@ params { skip_preseq = true skip_dupradar = false skip_qualimap = false + skip_kraken2 = false + kraken_db = null + bracken_precision = "G" skip_rseqc = false skip_biotype_qc = false skip_deseq2_qc = false From 734919e309047f46c46fae6848fb128bd4323d4b Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Tue, 30 Jul 2024 16:31:40 +0300 Subject: [PATCH 03/36] Initial addition of kraken and bracken to pipeline --- workflows/rnaseq/main.nf | 41 +++++++++++++++++++++++++++++++--------- 1 file changed, 32 insertions(+), 9 deletions(-) diff --git a/workflows/rnaseq/main.nf b/workflows/rnaseq/main.nf index 7bf5d36e0..b86079c1b 100755 --- a/workflows/rnaseq/main.nf +++ b/workflows/rnaseq/main.nf @@ -38,6 +38,8 @@ include { PRESEQ_LCEXTRAP } from '../../modules/nf-core/preseq/lcextrap' include { QUALIMAP_RNASEQ } from '../../modules/nf-core/qualimap/rnaseq' include { STRINGTIE_STRINGTIE } from '../../modules/nf-core/stringtie/stringtie' include { SUBREAD_FEATURECOUNTS } from '../../modules/nf-core/subread/featurecounts' +include { KRAKEN2_BUILD } from '../modules/nf-core/kraken2/build/main' +include { BRACKEN_BUILD } from '../modules/nf-core/bracken/build/main' include { MULTIQC } from '../../modules/nf-core/multiqc' include { UMITOOLS_PREPAREFORRSEM as UMITOOLS_PREPAREFORSALMON } from '../../modules/nf-core/umitools/prepareforrsem' include { BEDTOOLS_GENOMECOV as BEDTOOLS_GENOMECOV_FW } from '../../modules/nf-core/bedtools/genomecov' @@ -158,9 +160,10 @@ workflow RNASEQ { // // SUBWORKFLOW: Alignment with STAR and gene/transcript quantification with Salmon // - ch_genome_bam = Channel.empty() - ch_genome_bam_index = Channel.empty() - ch_star_log = Channel.empty() + ch_genome_bam = Channel.empty() + ch_genome_bam_index = Channel.empty() + ch_star_log = Channel.empty() + ch_unaligned_sequences = Channel.empty() if (!params.skip_alignment && params.aligner == 'star_salmon') { // Check if an AWS iGenome has been provided to use the appropriate version of STAR def is_aws_igenome = false @@ -180,10 +183,11 @@ workflow RNASEQ { is_aws_igenome, ch_fasta.map { [ [:], it ] } ) - ch_genome_bam = ALIGN_STAR.out.bam - ch_genome_bam_index = ALIGN_STAR.out.bai - ch_transcriptome_bam = ALIGN_STAR.out.bam_transcript - ch_star_log = ALIGN_STAR.out.log_final + ch_genome_bam = ALIGN_STAR.out.bam + ch_genome_bam_index = ALIGN_STAR.out.bai + ch_transcriptome_bam = ALIGN_STAR.out.bam_transcript + ch_star_log = ALIGN_STAR.out.log_final + ch_unaligned_sequences = ALIGN_STAR.out.fastq ch_multiqc_files = ch_multiqc_files.mix(ALIGN_STAR.out.stats.collect{it[1]}) ch_multiqc_files = ch_multiqc_files.mix(ALIGN_STAR.out.flagstat.collect{it[1]}) ch_multiqc_files = ch_multiqc_files.mix(ALIGN_STAR.out.idxstats.collect{it[1]}) @@ -337,8 +341,9 @@ workflow RNASEQ { ch_splicesites.map { [ [:], it ] }, ch_fasta.map { [ [:], it ] } ) - ch_genome_bam = FASTQ_ALIGN_HISAT2.out.bam - ch_genome_bam_index = FASTQ_ALIGN_HISAT2.out.bai + ch_genome_bam = FASTQ_ALIGN_HISAT2.out.bam + ch_genome_bam_index = FASTQ_ALIGN_HISAT2.out.bai + ch_unaligned_sequences = FASTQ_ALIGN_HISAT2.out.fastq ch_multiqc_files = ch_multiqc_files.mix(FASTQ_ALIGN_HISAT2.out.stats.collect{it[1]}) ch_multiqc_files = ch_multiqc_files.mix(FASTQ_ALIGN_HISAT2.out.flagstat.collect{it[1]}) ch_multiqc_files = ch_multiqc_files.mix(FASTQ_ALIGN_HISAT2.out.idxstats.collect{it[1]}) @@ -634,6 +639,24 @@ workflow RNASEQ { ch_multiqc_files = ch_multiqc_files.mix(ch_fail_strand_multiqc.collectFile(name: 'fail_strand_check_mqc.tsv')) } + + if (!params.skip_kraken2) { + KRAKEN2 ( + ch_unaligned_sequences, + params.kraken_db, + false, //TODO Not saving read alignments. Should this be modifiable? + false + ) + ch_kraken_reports = KRAKEN2.out.report + ch_versions = ch_versions.mix(KRAKEN2.out.versions) + + BRACKEN ( + ch_kraken_reports, + kraken_db + ) + ch_versions = ch_versions.mix(BRACKEN.out.versions) + ch_multiqc_files = ch_multiqc_files.mix(BRACKEN.out.reports.collect().ifEmpty([])) + } } // From 8695f5093b7e2a02ed3f7dd6b53ff90607d0e2a5 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Wed, 31 Jul 2024 10:38:30 +0300 Subject: [PATCH 04/36] Fix include statements --- workflows/rnaseq/main.nf | 18 +++++++++--------- 1 file changed, 9 insertions(+), 9 deletions(-) diff --git a/workflows/rnaseq/main.nf b/workflows/rnaseq/main.nf index b86079c1b..a514c05b0 100755 --- a/workflows/rnaseq/main.nf +++ b/workflows/rnaseq/main.nf @@ -32,15 +32,15 @@ include { getInferexperimentStrandedness } from '../../subworkflows/local/utils_ // // MODULE: Installed directly from nf-core/modules // -include { DUPRADAR } from '../../modules/nf-core/dupradar' -include { SAMTOOLS_SORT } from '../../modules/nf-core/samtools/sort' -include { PRESEQ_LCEXTRAP } from '../../modules/nf-core/preseq/lcextrap' -include { QUALIMAP_RNASEQ } from '../../modules/nf-core/qualimap/rnaseq' -include { STRINGTIE_STRINGTIE } from '../../modules/nf-core/stringtie/stringtie' -include { SUBREAD_FEATURECOUNTS } from '../../modules/nf-core/subread/featurecounts' -include { KRAKEN2_BUILD } from '../modules/nf-core/kraken2/build/main' -include { BRACKEN_BUILD } from '../modules/nf-core/bracken/build/main' -include { MULTIQC } from '../../modules/nf-core/multiqc' +include { DUPRADAR } from '../../modules/nf-core/dupradar' +include { SAMTOOLS_SORT } from '../../modules/nf-core/samtools/sort' +include { PRESEQ_LCEXTRAP } from '../../modules/nf-core/preseq/lcextrap' +include { QUALIMAP_RNASEQ } from '../../modules/nf-core/qualimap/rnaseq' +include { STRINGTIE_STRINGTIE } from '../../modules/nf-core/stringtie/stringtie' +include { SUBREAD_FEATURECOUNTS } from '../../modules/nf-core/subread/featurecounts' +include { KRAKEN2_KRAKEN2 as KRAKEN2 } from '../modules/nf-core/kraken2/kraken2/main' +include { BRACKEN_BRACKEN as BRACKEN } from '../modules/nf-core/bracken/bracken/main' +include { MULTIQC } from '../../modules/nf-core/multiqc' include { UMITOOLS_PREPAREFORRSEM as UMITOOLS_PREPAREFORSALMON } from '../../modules/nf-core/umitools/prepareforrsem' include { BEDTOOLS_GENOMECOV as BEDTOOLS_GENOMECOV_FW } from '../../modules/nf-core/bedtools/genomecov' include { BEDTOOLS_GENOMECOV as BEDTOOLS_GENOMECOV_REV } from '../../modules/nf-core/bedtools/genomecov' From 44ba28282edc17586b8395c1018f05e3f6067daa Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Wed, 31 Jul 2024 16:10:43 +0300 Subject: [PATCH 05/36] Fix include statements --- workflows/rnaseq/main.nf | 6 +++--- 1 file changed, 3 insertions(+), 3 deletions(-) diff --git a/workflows/rnaseq/main.nf b/workflows/rnaseq/main.nf index a514c05b0..d7602e2a6 100755 --- a/workflows/rnaseq/main.nf +++ b/workflows/rnaseq/main.nf @@ -38,8 +38,8 @@ include { PRESEQ_LCEXTRAP } from '../../modules/nf-core/preseq/lcextr include { QUALIMAP_RNASEQ } from '../../modules/nf-core/qualimap/rnaseq' include { STRINGTIE_STRINGTIE } from '../../modules/nf-core/stringtie/stringtie' include { SUBREAD_FEATURECOUNTS } from '../../modules/nf-core/subread/featurecounts' -include { KRAKEN2_KRAKEN2 as KRAKEN2 } from '../modules/nf-core/kraken2/kraken2/main' -include { BRACKEN_BRACKEN as BRACKEN } from '../modules/nf-core/bracken/bracken/main' +include { KRAKEN2_KRAKEN2 as KRAKEN2 } from '../../modules/nf-core/kraken2/kraken2/main' +include { BRACKEN_BRACKEN as BRACKEN } from '../../modules/nf-core/bracken/bracken/main' include { MULTIQC } from '../../modules/nf-core/multiqc' include { UMITOOLS_PREPAREFORRSEM as UMITOOLS_PREPAREFORSALMON } from '../../modules/nf-core/umitools/prepareforrsem' include { BEDTOOLS_GENOMECOV as BEDTOOLS_GENOMECOV_FW } from '../../modules/nf-core/bedtools/genomecov' @@ -652,7 +652,7 @@ workflow RNASEQ { BRACKEN ( ch_kraken_reports, - kraken_db + params.kraken_db ) ch_versions = ch_versions.mix(BRACKEN.out.versions) ch_multiqc_files = ch_multiqc_files.mix(BRACKEN.out.reports.collect().ifEmpty([])) From 3657e3e7eca46588c108ecb1daad8f511db82072 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Mon, 5 Aug 2024 10:23:21 +0300 Subject: [PATCH 06/36] Include module config files --- workflows/rnaseq/nextflow.config | 2 ++ 1 file changed, 2 insertions(+) diff --git a/workflows/rnaseq/nextflow.config b/workflows/rnaseq/nextflow.config index c7eeac733..9cbf0cd30 100644 --- a/workflows/rnaseq/nextflow.config +++ b/workflows/rnaseq/nextflow.config @@ -8,6 +8,8 @@ includeConfig "../../modules/nf-core/qualimap/rnaseq/nextflow.config" includeConfig "../../modules/nf-core/sortmerna/nextflow.config" includeConfig "../../modules/nf-core/stringtie/stringtie/nextflow.config" includeConfig "../../modules/nf-core/subread/featurecounts/nextflow.config" +includeConfig "../../modules/nf-core/kraken2/kraken2/nextflow.config" +includeConfig "../../modules/nf-core/bracken/bracken/nextflow.config" includeConfig "../../subworkflows/local/align_star/nextflow.config" includeConfig "../../subworkflows/local/quantify_rsem/nextflow.config" includeConfig "../../subworkflows/nf-core/quantify_pseudo_alignment/nextflow.config" From 7ab90f670bf6ee7ef580eb16ac6b0a1740ed49c1 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Mon, 5 Aug 2024 10:23:51 +0300 Subject: [PATCH 07/36] Use presence of kraken_db to determine bracken run, add save params --- .../nf-core/bracken/bracken/nextflow.config | 2 +- .../nf-core/kraken2/kraken2/nextflow.config | 2 +- nextflow.config | 4 ++- workflows/rnaseq/main.nf | 25 ++++++++++++------- 4 files changed, 21 insertions(+), 12 deletions(-) diff --git a/modules/nf-core/bracken/bracken/nextflow.config b/modules/nf-core/bracken/bracken/nextflow.config index 8089e551f..cf7b1cad6 100644 --- a/modules/nf-core/bracken/bracken/nextflow.config +++ b/modules/nf-core/bracken/bracken/nextflow.config @@ -1,5 +1,5 @@ if (!params.skip_alignment && !params.skip_qc) { - if (!params.skip_kraken2) { + if (params.kraken_db && !params.skip_bracken) { withName: 'BRACKEN' { ext.args = "-l ${params.bracken_precision}" publishDir = [ diff --git a/modules/nf-core/kraken2/kraken2/nextflow.config b/modules/nf-core/kraken2/kraken2/nextflow.config index a81d68de3..0070ad9fd 100644 --- a/modules/nf-core/kraken2/kraken2/nextflow.config +++ b/modules/nf-core/kraken2/kraken2/nextflow.config @@ -1,5 +1,5 @@ if (!params.skip_alignment && !params.skip_qc) { - if (!params.skip_kraken2) { + if (params.kraken_db) { process { withName: 'KRAKEN2' { ext.args = "--confidence 0.05 --minimum-hit-groups 3" diff --git a/nextflow.config b/nextflow.config index 47833626c..5e2d8a9f2 100644 --- a/nextflow.config +++ b/nextflow.config @@ -89,8 +89,10 @@ params { skip_preseq = true skip_dupradar = false skip_qualimap = false - skip_kraken2 = false kraken_db = null + skip_bracken = false + save_kraken_assignments = false + save_kraken_unassigned = false bracken_precision = "G" skip_rseqc = false skip_biotype_qc = false diff --git a/workflows/rnaseq/main.nf b/workflows/rnaseq/main.nf index d7602e2a6..e5aeff0a1 100755 --- a/workflows/rnaseq/main.nf +++ b/workflows/rnaseq/main.nf @@ -640,22 +640,29 @@ workflow RNASEQ { ch_multiqc_files = ch_multiqc_files.mix(ch_fail_strand_multiqc.collectFile(name: 'fail_strand_check_mqc.tsv')) } - if (!params.skip_kraken2) { + if (params.kraken_db) { KRAKEN2 ( ch_unaligned_sequences, params.kraken_db, - false, //TODO Not saving read alignments. Should this be modifiable? - false + params.save_kraken_assignments, + params.save_kraken_unassigned ) ch_kraken_reports = KRAKEN2.out.report ch_versions = ch_versions.mix(KRAKEN2.out.versions) - BRACKEN ( - ch_kraken_reports, - params.kraken_db - ) - ch_versions = ch_versions.mix(BRACKEN.out.versions) - ch_multiqc_files = ch_multiqc_files.mix(BRACKEN.out.reports.collect().ifEmpty([])) + // Only put Kraken2 output in MultiQC if Bracken is not run + if (params.skip_bracken) { + ch_multiqc_files = ch_multiqc_files.mix(KRAKEN2.out.reports.collect{it[1]}.ifEmpty([])) + } + + if (!params.skip_bracken) { + BRACKEN ( + ch_kraken_reports, + params.kraken_db + ) + ch_versions = ch_versions.mix(BRACKEN.out.versions) + ch_multiqc_files = ch_multiqc_files.mix(BRACKEN.out.reports.collect{it[1]}.ifEmpty([])) + } } } From 39cfdef38a9b3ba44c40b652cd0680c5ff6f146d Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Mon, 5 Aug 2024 16:24:38 +0300 Subject: [PATCH 08/36] Fix multiqc for Bracken/Kraken --- workflows/rnaseq/assets/multiqc/multiqc_config.yml | 8 ++++++++ workflows/rnaseq/main.nf | 4 ++-- 2 files changed, 10 insertions(+), 2 deletions(-) diff --git a/workflows/rnaseq/assets/multiqc/multiqc_config.yml b/workflows/rnaseq/assets/multiqc/multiqc_config.yml index 2bf39b16c..5d144cbdf 100644 --- a/workflows/rnaseq/assets/multiqc/multiqc_config.yml +++ b/workflows/rnaseq/assets/multiqc/multiqc_config.yml @@ -30,6 +30,8 @@ run_modules: - preseq - rseqc - qualimap + - kraken + - bracken # Order of modules top_modules: @@ -105,6 +107,12 @@ sp: samtools/idxstats: fn: "*.idxstats*" + kraken: + fn: "*report.txt" + + bracken: + fn: "*bracken_species.txt" + rseqc/bam_stat: fn: "*.bam_stat.txt" rseqc/gene_body_coverage: diff --git a/workflows/rnaseq/main.nf b/workflows/rnaseq/main.nf index e5aeff0a1..323db5d02 100755 --- a/workflows/rnaseq/main.nf +++ b/workflows/rnaseq/main.nf @@ -652,7 +652,7 @@ workflow RNASEQ { // Only put Kraken2 output in MultiQC if Bracken is not run if (params.skip_bracken) { - ch_multiqc_files = ch_multiqc_files.mix(KRAKEN2.out.reports.collect{it[1]}.ifEmpty([])) + ch_multiqc_files = ch_multiqc_files.mix(KRAKEN2.out.report.collect{it[1]}) } if (!params.skip_bracken) { @@ -661,7 +661,7 @@ workflow RNASEQ { params.kraken_db ) ch_versions = ch_versions.mix(BRACKEN.out.versions) - ch_multiqc_files = ch_multiqc_files.mix(BRACKEN.out.reports.collect{it[1]}.ifEmpty([])) + ch_multiqc_files = ch_multiqc_files.mix(BRACKEN.out.txt.collect{it[1]}) } } } From 2e9c24398f2fbc76920dc389284b2b42ee41a952 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Mon, 5 Aug 2024 16:31:44 +0300 Subject: [PATCH 09/36] Add Bracken/Kraken citations --- CITATIONS.md | 8 ++++++++ 1 file changed, 8 insertions(+) diff --git a/CITATIONS.md b/CITATIONS.md index 48a60b8e4..5eaeea7f8 100644 --- a/CITATIONS.md +++ b/CITATIONS.md @@ -16,6 +16,10 @@ > Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010 Mar 15;26(6):841-2. doi: 10.1093/bioinformatics/btq033. Epub 2010 Jan 28. PubMed PMID: 20110278; PubMed Central PMCID: PMC2832824. +- [Bracken](https://doi.org/10.7717/peerj-cs.104) + + > Lu, J., Breitwieser, F. P., Thielen, P., & Salzberg, S. L. (2017). Bracken: estimating species abundance in metagenomics data. PeerJ. Computer Science, 3(e104), e104. https://doi.org/10.7717/peerj-cs.104 + - [fastp](https://www.ncbi.nlm.nih.gov/pubmed/30423086/) > Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018 Sep 1;34(17):i884-i890. doi: 10.1093/bioinformatics/bty560. PubMed PMID: 30423086; PubMed Central PMCID: PMC6129281. @@ -38,6 +42,10 @@ > Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019 Aug;37(8):907-915. doi: 10.1038/s41587-019-0201-4. Epub 2019 Aug 2. PubMed PMID: 31375807. +- [Kraken2](https://doi.org/10.1186/s13059-019-1891-0) + + > Wood, D. E., Lu, J., & Langmead, B. (2019). Improved metagenomic analysis with Kraken 2. Genome Biology, 20(1), 257. https://doi.org/10.1186/s13059-019-1891-0 + - [MultiQC](https://pubmed.ncbi.nlm.nih.gov/27312411/) > Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016 Oct 1;32(19):3047-8. doi: 10.1093/bioinformatics/btw354. Epub 2016 Jun 16. PubMed PMID: 27312411; PubMed Central PMCID: PMC5039924. From 03314c9b280b6af42d4658f94a4736d079945a5b Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Tue, 6 Aug 2024 12:49:33 +0300 Subject: [PATCH 10/36] Fix bracken config --- modules/nf-core/bracken/bracken/nextflow.config | 14 ++++++++------ 1 file changed, 8 insertions(+), 6 deletions(-) diff --git a/modules/nf-core/bracken/bracken/nextflow.config b/modules/nf-core/bracken/bracken/nextflow.config index cf7b1cad6..e6d396652 100644 --- a/modules/nf-core/bracken/bracken/nextflow.config +++ b/modules/nf-core/bracken/bracken/nextflow.config @@ -1,11 +1,13 @@ if (!params.skip_alignment && !params.skip_qc) { if (params.kraken_db && !params.skip_bracken) { - withName: 'BRACKEN' { - ext.args = "-l ${params.bracken_precision}" - publishDir = [ - path: { "${params.outdir}/bracken" }, - mode: params.publish_dir_mode - ] + process { + withName: 'BRACKEN' { + ext.args = "-l ${params.bracken_precision}" + publishDir = [ + path: { "${params.outdir}/bracken" }, + mode: params.publish_dir_mode + ] + } } } } \ No newline at end of file From 7913cbca1cf060fdc37b000f249dec57a089cb28 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Wed, 7 Aug 2024 07:37:08 +0000 Subject: [PATCH 11/36] Update bracken --- modules.json | 2 +- modules/nf-core/bracken/bracken/main.nf | 9 +- .../bracken/bracken/tests/genus_test.config | 5 ++ .../bracken/bracken/tests/main.nf.test | 48 ++++++++++- .../bracken/bracken/tests/main.nf.test.snap | 83 +++++++++++++++---- 5 files changed, 127 insertions(+), 20 deletions(-) create mode 100644 modules/nf-core/bracken/bracken/tests/genus_test.config diff --git a/modules.json b/modules.json index ac86bcd98..20b52c3a1 100644 --- a/modules.json +++ b/modules.json @@ -17,7 +17,7 @@ }, "bracken/bracken": { "branch": "master", - "git_sha": "f362bca32735a848feb314d601db1cee584ee0d1", + "git_sha": "c214fad97b328eb6d6233f779be9ba44814a9136", "installed_by": ["modules"] }, "cat/fastq": { diff --git a/modules/nf-core/bracken/bracken/main.nf b/modules/nf-core/bracken/bracken/main.nf index caded9edd..e3d32fb21 100644 --- a/modules/nf-core/bracken/bracken/main.nf +++ b/modules/nf-core/bracken/bracken/main.nf @@ -13,7 +13,7 @@ process BRACKEN_BRACKEN { output: tuple val(meta), path(bracken_report) , emit: reports - tuple val(meta), path("*bracken_species.txt"), emit: txt + tuple val(meta), path(bracken_kraken_style_report), emit: txt path "versions.yml" , emit: versions when: @@ -23,12 +23,14 @@ process BRACKEN_BRACKEN { def args = task.ext.args ?: "" def prefix = task.ext.prefix ?: "${meta.id}" bracken_report = "${prefix}.tsv" + bracken_kraken_style_report = "${prefix}.kraken2.report_bracken.txt" """ bracken \\ ${args} \\ -d '${database}' \\ -i '${kraken_report}' \\ - -o '${bracken_report}' + -o '${bracken_report}' \\ + -w '${bracken_kraken_style_report}' cat <<-END_VERSIONS > versions.yml "${task.process}": @@ -40,9 +42,10 @@ process BRACKEN_BRACKEN { def args = task.ext.args ?: '' def prefix = task.ext.prefix ?: "${meta.id}" bracken_report = "${prefix}.tsv" + bracken_kraken_style_report = "${prefix}.kraken2.report_bracken.txt" """ touch ${prefix}.tsv - touch ${prefix}_bracken_species.txt + touch ${bracken_kraken_style_report} cat <<-END_VERSIONS > versions.yml "${task.process}": diff --git a/modules/nf-core/bracken/bracken/tests/genus_test.config b/modules/nf-core/bracken/bracken/tests/genus_test.config new file mode 100644 index 000000000..bc5f63ae0 --- /dev/null +++ b/modules/nf-core/bracken/bracken/tests/genus_test.config @@ -0,0 +1,5 @@ +process { + withName: BRACKEN_BRACKEN { + ext.args = "-l G -t 10 -r 150" + } +} diff --git a/modules/nf-core/bracken/bracken/tests/main.nf.test b/modules/nf-core/bracken/bracken/tests/main.nf.test index 59c1398e5..9d2105ded 100644 --- a/modules/nf-core/bracken/bracken/tests/main.nf.test +++ b/modules/nf-core/bracken/bracken/tests/main.nf.test @@ -24,6 +24,8 @@ nextflow_process { test("sarscov2 - single-end - fastq") { + config "./nextflow.config" + setup { run("KRAKEN2_KRAKEN2") { script "../../../kraken2/kraken2/main.nf" @@ -56,7 +58,51 @@ nextflow_process { } -test("sarscov2 - paired-end - fastq") { + test("sarscov2 - paired-end - fastq - genus config") { + + config "./genus_test.config" + + setup { + run("KRAKEN2_KRAKEN2") { + script "../../../kraken2/kraken2/main.nf" + process { + """ + input[0] = [ + [id: 'test', single_end: false], + [ + file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_1.fastq.gz', checkIfExists: true), + file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_2.fastq.gz', checkIfExists: true) + ] + ] + input[1] = UNTAR.out.untar.map{it[1]} + input[2] = false + input[3] = false + """ + } + } + } + + when { + process { + """ + input[0] = KRAKEN2_KRAKEN2.out.report + input[1] = UNTAR.out.untar.map{it[1]} + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot(process.out).match() } + ) + } + + } + + test("sarscov2 - paired-end - fastq") { + + config "./nextflow.config" setup { run("KRAKEN2_KRAKEN2") { diff --git a/modules/nf-core/bracken/bracken/tests/main.nf.test.snap b/modules/nf-core/bracken/bracken/tests/main.nf.test.snap index 5aadb7b70..c97a4c71b 100644 --- a/modules/nf-core/bracken/bracken/tests/main.nf.test.snap +++ b/modules/nf-core/bracken/bracken/tests/main.nf.test.snap @@ -17,7 +17,7 @@ "id": "test", "single_end": true }, - "test.kraken2.report_bracken_species.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + "test.kraken2.report_bracken.txt:md5,ca0fbeedc4353b5fdd081688823a33df" ] ], "2": [ @@ -38,7 +38,7 @@ "id": "test", "single_end": true }, - "test.kraken2.report_bracken_species.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + "test.kraken2.report_bracken.txt:md5,ca0fbeedc4353b5fdd081688823a33df" ] ], "versions": [ @@ -47,10 +47,63 @@ } ], "meta": { - "nf-test": "0.8.4", - "nextflow": "24.03.0" + "nf-test": "0.9.0", + "nextflow": "24.04.4" }, - "timestamp": "2024-04-16T14:14:27.122403998" + "timestamp": "2024-08-06T11:35:03.995620397" + }, + "sarscov2 - paired-end - fastq - genus config": { + "content": [ + { + "0": [ + [ + { + "id": "test", + "single_end": false + }, + "test.tsv:md5,f609b09d6edb5ebc1ea1435d1dd46cde" + ] + ], + "1": [ + [ + { + "id": "test", + "single_end": false + }, + "test.kraken2.report_bracken.txt:md5,2ce58814420a3690da1f08e10e8d3a30" + ] + ], + "2": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ], + "reports": [ + [ + { + "id": "test", + "single_end": false + }, + "test.tsv:md5,f609b09d6edb5ebc1ea1435d1dd46cde" + ] + ], + "txt": [ + [ + { + "id": "test", + "single_end": false + }, + "test.kraken2.report_bracken.txt:md5,2ce58814420a3690da1f08e10e8d3a30" + ] + ], + "versions": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ] + } + ], + "meta": { + "nf-test": "0.9.0", + "nextflow": "24.04.4" + }, + "timestamp": "2024-08-06T12:13:33.399680181" }, "sarscov2 - paired-end - fastq": { "content": [ @@ -70,7 +123,7 @@ "id": "test", "single_end": false }, - "test.kraken2.report_bracken_species.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + "test.kraken2.report_bracken.txt:md5,ca0fbeedc4353b5fdd081688823a33df" ] ], "2": [ @@ -91,7 +144,7 @@ "id": "test", "single_end": false }, - "test.kraken2.report_bracken_species.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + "test.kraken2.report_bracken.txt:md5,ca0fbeedc4353b5fdd081688823a33df" ] ], "versions": [ @@ -100,10 +153,10 @@ } ], "meta": { - "nf-test": "0.8.4", - "nextflow": "24.03.0" + "nf-test": "0.9.0", + "nextflow": "24.04.4" }, - "timestamp": "2024-04-16T14:19:15.627050252" + "timestamp": "2024-08-06T12:09:15.465609745" }, "sarscov2 - stub - fastq": { "content": [ @@ -121,7 +174,7 @@ { "id": "test" }, - "test_bracken_species.txt:md5,d41d8cd98f00b204e9800998ecf8427e" + "test.kraken2.report_bracken.txt:md5,d41d8cd98f00b204e9800998ecf8427e" ] ], "2": [ @@ -140,7 +193,7 @@ { "id": "test" }, - "test_bracken_species.txt:md5,d41d8cd98f00b204e9800998ecf8427e" + "test.kraken2.report_bracken.txt:md5,d41d8cd98f00b204e9800998ecf8427e" ] ], "versions": [ @@ -149,9 +202,9 @@ } ], "meta": { - "nf-test": "0.8.4", - "nextflow": "24.03.0" + "nf-test": "0.9.0", + "nextflow": "24.04.4" }, - "timestamp": "2024-04-16T14:14:36.5929911" + "timestamp": "2024-08-06T11:35:42.965471207" } } \ No newline at end of file From 063b4ac86ca3690991dc6832b2270ea9e37cba34 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Wed, 7 Aug 2024 07:45:20 +0000 Subject: [PATCH 12/36] Adjust multiqc configs for updated bracken --- workflows/rnaseq/assets/multiqc/multiqc_config.yml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/workflows/rnaseq/assets/multiqc/multiqc_config.yml b/workflows/rnaseq/assets/multiqc/multiqc_config.yml index 5d144cbdf..a2d33206a 100644 --- a/workflows/rnaseq/assets/multiqc/multiqc_config.yml +++ b/workflows/rnaseq/assets/multiqc/multiqc_config.yml @@ -111,7 +111,7 @@ sp: fn: "*report.txt" bracken: - fn: "*bracken_species.txt" + fn: "*.kraken2.report_bracken.txt" rseqc/bam_stat: fn: "*.bam_stat.txt" From 075bca8b9fff53c017fc60d21d0e072a8cc9648c Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Wed, 7 Aug 2024 09:38:12 +0000 Subject: [PATCH 13/36] Add input validation and update scheme --- nextflow.config | 2 +- nextflow_schema.json | 39 ++++++++++++++++-- .../utils_nfcore_rnaseq_pipeline/main.nf | 41 +++++++++++++++++++ 3 files changed, 77 insertions(+), 5 deletions(-) diff --git a/nextflow.config b/nextflow.config index 5e2d8a9f2..9de4b2009 100644 --- a/nextflow.config +++ b/nextflow.config @@ -73,7 +73,7 @@ params { kallisto_quant_fraglen = 200 kallisto_quant_fraglen_sd = 200 save_merged_fastq = false - save_unaligned = true + save_unaligned = false save_align_intermeds = false skip_markduplicates = false skip_alignment = false diff --git a/nextflow_schema.json b/nextflow_schema.json index d89b6398e..fe58cca84 100644 --- a/nextflow_schema.json +++ b/nextflow_schema.json @@ -411,7 +411,7 @@ }, "min_mapped_reads": { "type": "number", - "default": 5.0, + "default": 5, "fa_icon": "fas fa-percentage", "description": "Minimum percentage of uniquely mapped reads below which samples are removed from further processing.", "help_text": "Some downstream steps in the pipeline will fail if this threshold is too low." @@ -456,14 +456,14 @@ "stranded_threshold": { "type": "number", "minimum": 0.5, - "maximum": 1.0, + "maximum": 1, "default": 0.8, "description": "The fraction of stranded reads that must be assigned to a strandedness for confident assignment. Must be at least 0.5." }, "unstranded_threshold": { "type": "number", - "minimum": 0.0, - "maximum": 1.0, + "minimum": 0, + "maximum": 1, "default": 0.1, "description": "The difference in fraction of stranded reads assigned to 'forward' and 'reverse' below which a sample is classified as 'unstranded'. By default the forward and reverse fractions must differ by less than 0.1 for the sample to be called as unstranded." } @@ -518,6 +518,18 @@ "fa_icon": "fas fa-save", "description": "Where possible, save unaligned reads from either STAR, HISAT2 or Salmon to the results directory.", "help_text": "This may either be in the form of FastQ or BAM files depending on the options available for that particular tool." + }, + "save_kraken_assignments": { + "type": "boolean", + "fa_icon": "fas fa-save", + "description": "Save read-by-read assignments from Kraken2.", + "help_text": "`--kraken_db` parameter must be provided." + }, + "save_kraken_unassigned": { + "type": "boolean", + "fa_icon": "fas fa-save", + "description": "Save reads that were not given assignment from Kraken2.", + "help_text": "`--kraken_db` parameter must be provided." } } }, @@ -539,6 +551,19 @@ "default": "bam_stat,inner_distance,infer_experiment,junction_annotation,junction_saturation,read_distribution,read_duplication", "fa_icon": "fas fa-chart-pie", "description": "Specify the RSeQC modules to run." + }, + "kraken_db": { + "type": "string", + "description": "Run Kraken2 and Bracken with provided database on unaligned reads.", + "help_text": "See [pre-built Kraken/Bracken databases](https://benlangmead.github.io/aws-indexes/k2). This option must be ran with `--save_unaligned`", + "fa_icon": "fas fa-bacteria" + }, + "bracken_precision": { + "type": "string", + "default": "G", + "fa_icon": "fas fa-tree", + "description": "Taxonomic level for Bracken abundance estimations.", + "help_text": "Options are D, P, C, O, F, G, S." } } }, @@ -637,6 +662,12 @@ "fa_icon": "fas fa-fast-forward", "description": "Skip DESeq2 PCA and heatmap plotting." }, + "skip_bracken": { + "type": "boolean", + "description": "Skip Bracken.", + "fa_icon": "fas fa-fast-forward", + "help_text": "If running Kraken2, skips Bracken post-processing." + }, "skip_multiqc": { "type": "boolean", "description": "Skip MultiQC.", diff --git a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf index 8022a5c1b..989bc8f5c 100644 --- a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf +++ b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf @@ -256,6 +256,27 @@ def validateInputParameters() { } } + // Check that unaligned reads are saved when using Kraken2 + if (params.kraken_db) { + if (!params.save_unaligned) { + error("Please provide --save_unaligned when using Kraken2 to save unaligned reads for analysis.") + } + + if (params.aligner = 'star_rsem') { + error("Kraken2 cannot be used with --aligner star_rsem since unaligned reads are not saved. Please use --aligner star_salmon or --aligner hisat2.") + } + } + + // Check that Bracken skipped option is not given when not running kraken + if (!params.kraken_db && params.skip_bracken) { + skipBrackenWithoutKrackenWarn() + } + + // Check that no kraken options are given when not running kraken + if (!params.kraken_db && (params.save_kraken_assignments || params.save_kraken_unassigned)) { + krakenArgumentsWithoutKrakenDBWarn() + } + // Check which RSeQC modules we are running def valid_rseqc_modules = ['bam_stat', 'inner_distance', 'infer_experiment', 'junction_annotation', 'junction_saturation', 'read_distribution', 'read_duplication', 'tin'] def rseqc_modules = params.rseqc_modules ? params.rseqc_modules.split(',').collect{ it.trim().toLowerCase() } : [] @@ -471,6 +492,26 @@ def additionaFastaIndexWarn(index) { "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" } +// +// Print a warning if using '--skip_bracken' without '--kraken_db' +// +def skipBrackenWithoutKrackenWarn() { + log.warn "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n" + + " '--skip_bracken' parameter has been provided without '--kraken_db'.\n" + + " Neither bracken or kraken will run.\n" + + "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" +} + +// +// Print a warning if save_kraken_assignments or save_kraken_unassigned is provided without kraken_db +// +def krakenArgumentsWithoutKrakenDBWarn() { + log.warn "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n" + + " '--save_kraken_assignments' or '--save_kraken_unassigned' parameters have been provided without '--kraken_db'.\n" + + " Kraken2 is not being run so neither will be saved.\n" + + "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" +} + // // Function to generate an error if contigs in genome fasta file > 512 Mbp // From a0a225c565fb490bb92ee04c1dca5de45ba6b606 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Wed, 7 Aug 2024 12:27:46 +0000 Subject: [PATCH 14/36] Change default Bracken precision to species --- nextflow.config | 2 +- nextflow_schema.json | 2 +- 2 files changed, 2 insertions(+), 2 deletions(-) diff --git a/nextflow.config b/nextflow.config index 9de4b2009..33cb94f49 100644 --- a/nextflow.config +++ b/nextflow.config @@ -93,7 +93,7 @@ params { skip_bracken = false save_kraken_assignments = false save_kraken_unassigned = false - bracken_precision = "G" + bracken_precision = "S" skip_rseqc = false skip_biotype_qc = false skip_deseq2_qc = false diff --git a/nextflow_schema.json b/nextflow_schema.json index fe58cca84..436f4c4ad 100644 --- a/nextflow_schema.json +++ b/nextflow_schema.json @@ -560,7 +560,7 @@ }, "bracken_precision": { "type": "string", - "default": "G", + "default": "S", "fa_icon": "fas fa-tree", "description": "Taxonomic level for Bracken abundance estimations.", "help_text": "Options are D, P, C, O, F, G, S." From bd4ea1caa77ce9e24f96c62092920392093a90f4 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Wed, 7 Aug 2024 12:28:04 +0000 Subject: [PATCH 15/36] Debug input validation --- .../utils_nfcore_rnaseq_pipeline/main.nf | 36 +++++++++++++------ 1 file changed, 26 insertions(+), 10 deletions(-) diff --git a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf index 989bc8f5c..3f5765272 100644 --- a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf +++ b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf @@ -262,19 +262,25 @@ def validateInputParameters() { error("Please provide --save_unaligned when using Kraken2 to save unaligned reads for analysis.") } - if (params.aligner = 'star_rsem') { + if (params.aligner == 'star_rsem') { error("Kraken2 cannot be used with --aligner star_rsem since unaligned reads are not saved. Please use --aligner star_salmon or --aligner hisat2.") } } - // Check that Bracken skipped option is not given when not running kraken - if (!params.kraken_db && params.skip_bracken) { - skipBrackenWithoutKrackenWarn() - } + //Check that Kraken/Bracken parameters are not provided without the --kraken_db + if (!params.kraken_db) { + + if (!params.bracken_precision.equals('S')) { + brackenPrecisionWithoutKrakenDBWarn() + } + + if (params.skip_bracken) { + skipBrackenWithoutKrackenWarn() + } - // Check that no kraken options are given when not running kraken - if (!params.kraken_db && (params.save_kraken_assignments || params.save_kraken_unassigned)) { - krakenArgumentsWithoutKrakenDBWarn() + if (params.save_kraken_assignments || params.save_kraken_unassigned) { + krakenArgumentsWithoutKrakenDBWarn() + } } // Check which RSeQC modules we are running @@ -498,12 +504,12 @@ def additionaFastaIndexWarn(index) { def skipBrackenWithoutKrackenWarn() { log.warn "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n" + " '--skip_bracken' parameter has been provided without '--kraken_db'.\n" + - " Neither bracken or kraken will run.\n" + + " Neither Bracken nor Kraken2 will run.\n" + "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" } // -// Print a warning if save_kraken_assignments or save_kraken_unassigned is provided without kraken_db +// Print a warning if --save_kraken_assignments or --save_kraken_unassigned is provided without --kraken_db // def krakenArgumentsWithoutKrakenDBWarn() { log.warn "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n" + @@ -512,6 +518,16 @@ def krakenArgumentsWithoutKrakenDBWarn() { "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" } +/// +/// Print a warning if --bracken-precision is provided without --kraken_db +/// +def brackenPrecisionWithoutKrakenDBWarn() { + log.warn "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n" + + " '--bracken-precision' parameter has been provided without '--kraken_db'.\n" + + " Bracken will not run so precision will not be set.\n" + + "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" +} + // // Function to generate an error if contigs in genome fasta file > 512 Mbp // From 77898275bed7e0a8ae48084e0aed763a77da4ed8 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Wed, 7 Aug 2024 12:28:44 +0000 Subject: [PATCH 16/36] Documentation/output image --- README.md | 1 + docs/images/bracken-top-n-plot.png | Bin 0 -> 55992 bytes docs/output.md | 23 ++++++++++++++++++++++- 3 files changed, 23 insertions(+), 1 deletion(-) create mode 100644 docs/images/bracken-top-n-plot.png diff --git a/README.md b/README.md index 411dfd3f4..15fe29d97 100644 --- a/README.md +++ b/README.md @@ -44,6 +44,7 @@ 3. [`dupRadar`](https://bioconductor.org/packages/release/bioc/html/dupRadar.html) 4. [`Preseq`](http://smithlabresearch.org/software/preseq/) 5. [`DESeq2`](https://bioconductor.org/packages/release/bioc/html/DESeq2.html) + 6. [`Kraken2`](https://ccb.jhu.edu/software/kraken2/) -> [`Bracken`](https://ccb.jhu.edu/software/bracken/) on unaligned sequences; _optional_ 15. Pseudoalignment and quantification ([`Salmon`](https://combine-lab.github.io/salmon/) or ['Kallisto'](https://pachterlab.github.io/kallisto/); _optional_) 16. Present QC for raw read, alignment, gene biotype, sample similarity, and strand-specificity checks ([`MultiQC`](http://multiqc.info/), [`R`](https://www.r-project.org/)) diff --git a/docs/images/bracken-top-n-plot.png b/docs/images/bracken-top-n-plot.png new file mode 100644 index 0000000000000000000000000000000000000000..ac2d485f67e90c389be6f169b0ff79dbae36f67e GIT binary patch literal 55992 zcmeFacT|*HvnCD*h$u!76=+aIkRT#CBOD}25@|9tlCwyX3?@v_0+N%6B*{4kQ9+a@ zH%L~AO;ndCEj1e6|J(l~2E3(voL;d66=?9Q}BCKjKA_EkVMb?$3${%IoSHY9# zzq4(*gcBCNOG$I9#Bf@+o-5FQYm|AyU}PgwM^G!94=cT0uIJuofvXSP^Yz!YU$~Nz zWp^r_JPfb;Q2omj-r9@E#F>60Zmbl2ReRVp)*Ozz!r;&y-LGerU=gW%Kh^lnoR9%+4JK@NynNYZ z0`ML^hWp_0sL)?Nuwn()M*RIht@D(Bxnr4`8hOdUu_AT-qfYJ7dwWRHOG1vNeU=kl z>e4)eW;vEsKN=bKW8ac&Q~7MKPfkSDZKZlHcPQWnUD_OSs~10_-rKpgDX}M*Oa1(E zH!{4_cWGc`yXPsRXlK$R4?V(tdSmOx5GFh65_q<_aCNDQPe)#h8xhw(nQY(K^k_V3 z6}>(Ymsk0O?-DW>GOjSs9VPMmSYgJ;_q$T(cj^q@d`p81+>6rnJK`&Gh7|4cokr+$ 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zUE?(P7u4}vIKOm)Eg@;2AWIs81`(2}>NXhq=x_#|xq!1AKyit&|DX zLM&H%PP8k!CEG`yVSn@Ry3bymD_C90S3mY=^wp+-2y~vx{@4UAkFvkqZ22dxaTinK zlHfh2H2;^dvzaVuibW)vrcl}q${`egiHZGedSG|nPM5R) E3$MultiQC - DESeq2 sample similarity plot

+### Kraken2/Bracken + +
+Output files + +- `kraken2/kraken_reports` + - `*.kraken2.report.txt`: Classification of unaligned reads in the Kraken report format. See the [kraken2 manual](https://github.com/DerrickWood/kraken2/wiki/Manual#output-formats) for more details + - `*.classified*.fastq.gz` If `--save_kraken_alignments`, outputs fastq file for each sample with each classified read annotated with taxonomic identification from Kraken2. + - `*.unclassified*.fastq.gz` If `save_kraken_unassigned`, outputs fastq file with all reads that were not classified by Kraken2. +- `bracken/` + - `*.kraken2.report_bracken.txt`: Kraken-style reports of the Bracken abundance estimate results. See the [kraken2 manual](https://github.com/DerrickWood/kraken2/wiki/Manual#output-formats) for more details. + - `*.tsv` Summary of estimated reads for each taxon member at the given classification level and what corrections were made from Kraken2. + +
+ +[Kraken2](https://ccb.jhu.edu/software/kraken2/) is a taxonomic classification tool that uses k-mer matches paired with a lowest common ancestory (LCA) algorithm to classify species reads. [Bracken](https://ccb.jhu.edu/software/bracken/) is a statistical method to generate abundance estimates based off of the Kraken2 output. These algorithms are run on unaligned sequences to detect potential contamination of samples. MultiQC reports the top 5 taxon members detected at the level of classification used for Bracken, with toggles available for higher taxonomic levels. If Bracken is skipped, MultiQC will report the top 5 species detected by Kraken2. + +![MultiQC - Bracken top species plot](images/bracken-top-n-plot.png) + + ### MultiQC
@@ -673,7 +694,7 @@ Results generated by MultiQC collate pipeline QC from supported tools i.e. FastQ ### Pseudoalignment -The principal output files are the same between Salmon and Kallsto: +The principal output files are the same between Salmon and Kallisto:
Output files From 336a7b02c8f75d0347f946901c85a1fd6e459ac5 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Wed, 7 Aug 2024 16:16:38 +0300 Subject: [PATCH 17/36] Update changelog --- CHANGELOG.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/CHANGELOG.md b/CHANGELOG.md index e62628fc6..801b577e8 100644 --- a/CHANGELOG.md +++ b/CHANGELOG.md @@ -22,7 +22,7 @@ Special thanks to the following for their contributions to the release: Thank you to everyone else that has contributed by reporting bugs, enhancements or in any other way, shape or form. ### Enhancements & fixes - +- [PR #1351](https://github.com/nf-core/rnaseq/pull/1351) - Adding Kraken2/Bracken on unaligned reads as an additional quality control step to detect sample contamination - [PR #1186](https://github.com/nf-core/rnaseq/pull/1186) - Properly update qualimap/rnaseq module (ie not patch) - [PR #1197](https://github.com/nf-core/rnaseq/pull/1197) - Delete lib directory and replace with utils\_\* subworkflows - [PR #1199](https://github.com/nf-core/rnaseq/pull/1199) - Replace modules.config with more modular config files per module/subworkflow/workflow From a1e1d99d185f6fdd408b23a36a777dcb51ecb3e7 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Fri, 9 Aug 2024 10:40:22 +0300 Subject: [PATCH 18/36] Update Kraken2 module --- modules.json | 2 +- modules/nf-core/kraken2/kraken2/tests/main.nf.test | 12 ++++++------ 2 files changed, 7 insertions(+), 7 deletions(-) diff --git a/modules.json b/modules.json index 20b52c3a1..089f9f21e 100644 --- a/modules.json +++ b/modules.json @@ -97,7 +97,7 @@ }, "kraken2/kraken2": { "branch": "master", - "git_sha": "ca87ad032a62f025f0c373facacef2df0c5411b2", + "git_sha": "a13d5d945742a60bbef6e5c177e81cda540f75dc", "installed_by": ["modules"] }, "multiqc": { diff --git a/modules/nf-core/kraken2/kraken2/tests/main.nf.test b/modules/nf-core/kraken2/kraken2/tests/main.nf.test index 4c5130217..c0843df29 100644 --- a/modules/nf-core/kraken2/kraken2/tests/main.nf.test +++ b/modules/nf-core/kraken2/kraken2/tests/main.nf.test @@ -16,7 +16,7 @@ nextflow_process { input[0] = Channel.of([ [], file( - params.test_data['sarscov2']['genome']['kraken2_tar_gz'], + params.modules_testdata_base_path + "genomics/sarscov2/genome/db/kraken2.tar.gz", checkIfExists: true ) ]) @@ -32,7 +32,7 @@ nextflow_process { input[0] = [ [ id:'test', single_end:true ], // meta map [ file( - params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], + params.modules_testdata_base_path + "genomics/sarscov2/illumina/fastq/test_1.fastq.gz", checkIfExists: true )] ] @@ -69,16 +69,16 @@ nextflow_process { [ id:'test', single_end:false ], // meta map [ file( - params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], + params.modules_testdata_base_path + "genomics/sarscov2/illumina/fastq/test_1.fastq.gz", checkIfExists: true ), file( - params.test_data['sarscov2']['illumina']['test_2_fastq_gz'], + params.modules_testdata_base_path + "genomics/sarscov2/illumina/fastq/test_2.fastq.gz", checkIfExists: true ) + ] ] - ] input[1] = UNTAR.out.untar.map{ it[1] } input[2] = true input[3] = false @@ -117,7 +117,7 @@ nextflow_process { input[0] = [ [ id:'test', single_end:true ], // meta map [ file( - params.test_data['sarscov2']['illumina']['test_1_fastq_gz'], + params.modules_testdata_base_path + "genomics/sarscov2/illumina/fastq/test_1.fastq.gz", checkIfExists: true )] ] From 33c94836fba117a612b569ab1c5e2f64ebe5846c Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Fri, 9 Aug 2024 12:59:23 +0300 Subject: [PATCH 19/36] Linting --- CHANGELOG.md | 1 + docs/output.md | 1 - 2 files changed, 1 insertion(+), 1 deletion(-) diff --git a/CHANGELOG.md b/CHANGELOG.md index 801b577e8..f571b434a 100644 --- a/CHANGELOG.md +++ b/CHANGELOG.md @@ -22,6 +22,7 @@ Special thanks to the following for their contributions to the release: Thank you to everyone else that has contributed by reporting bugs, enhancements or in any other way, shape or form. ### Enhancements & fixes + - [PR #1351](https://github.com/nf-core/rnaseq/pull/1351) - Adding Kraken2/Bracken on unaligned reads as an additional quality control step to detect sample contamination - [PR #1186](https://github.com/nf-core/rnaseq/pull/1186) - Properly update qualimap/rnaseq module (ie not patch) - [PR #1197](https://github.com/nf-core/rnaseq/pull/1197) - Delete lib directory and replace with utils\_\* subworkflows diff --git a/docs/output.md b/docs/output.md index 9a6b73ef8..144caabd5 100644 --- a/docs/output.md +++ b/docs/output.md @@ -674,7 +674,6 @@ The plot on the left hand side shows the standard PC plot - notice the variable ![MultiQC - Bracken top species plot](images/bracken-top-n-plot.png) - ### MultiQC
From 102152e5b07b1fa9d7f65c913e388db9bc6bc2c4 Mon Sep 17 00:00:00 2001 From: Ezra Greenberg Date: Thu, 15 Aug 2024 16:32:32 +0000 Subject: [PATCH 20/36] Change to --contaminant_screening param --- .../nf-core/bracken/bracken/nextflow.config | 2 +- .../nf-core/kraken2/kraken2/nextflow.config | 2 +- nextflow.config | 2 +- nextflow_schema.json | 20 +++++----- .../utils_nfcore_rnaseq_pipeline/main.nf | 39 +++++++------------ workflows/rnaseq/main.nf | 9 ++--- 6 files changed, 29 insertions(+), 45 deletions(-) diff --git a/modules/nf-core/bracken/bracken/nextflow.config b/modules/nf-core/bracken/bracken/nextflow.config index e6d396652..052a75482 100644 --- a/modules/nf-core/bracken/bracken/nextflow.config +++ b/modules/nf-core/bracken/bracken/nextflow.config @@ -1,5 +1,5 @@ if (!params.skip_alignment && !params.skip_qc) { - if (params.kraken_db && !params.skip_bracken) { + if (params.contaminant_screening == 'kraken2_bracken') { process { withName: 'BRACKEN' { ext.args = "-l ${params.bracken_precision}" diff --git a/modules/nf-core/kraken2/kraken2/nextflow.config b/modules/nf-core/kraken2/kraken2/nextflow.config index 0070ad9fd..f4375210f 100644 --- a/modules/nf-core/kraken2/kraken2/nextflow.config +++ b/modules/nf-core/kraken2/kraken2/nextflow.config @@ -1,5 +1,5 @@ if (!params.skip_alignment && !params.skip_qc) { - if (params.kraken_db) { + if (params.contaminant_screening in ['kraken2', 'kraken2_bracken']) { process { withName: 'KRAKEN2' { ext.args = "--confidence 0.05 --minimum-hit-groups 3" diff --git a/nextflow.config b/nextflow.config index 33cb94f49..4a66931dd 100644 --- a/nextflow.config +++ b/nextflow.config @@ -89,8 +89,8 @@ params { skip_preseq = true skip_dupradar = false skip_qualimap = false + contaminant_screening = null kraken_db = null - skip_bracken = false save_kraken_assignments = false save_kraken_unassigned = false bracken_precision = "S" diff --git a/nextflow_schema.json b/nextflow_schema.json index 436f4c4ad..b524e762f 100644 --- a/nextflow_schema.json +++ b/nextflow_schema.json @@ -552,18 +552,24 @@ "fa_icon": "fas fa-chart-pie", "description": "Specify the RSeQC modules to run." }, + "contaminant_screening": { + "type": "string", + "description": "Tool to use for detecting contaminants in unaligned reads - available options are 'kraken2' and 'kraken2_bracken'", + "fa_icon": "fas fa-virus-slash", + "enum": ["kraken2", "kraken2_bracken"] + }, "kraken_db": { "type": "string", - "description": "Run Kraken2 and Bracken with provided database on unaligned reads.", - "help_text": "See [pre-built Kraken/Bracken databases](https://benlangmead.github.io/aws-indexes/k2). This option must be ran with `--save_unaligned`", - "fa_icon": "fas fa-bacteria" + "description": "Database when using Kraken2/Bracken for contaminant screening.", + "help_text": "See [pre-built Kraken2/Bracken databases](https://benlangmead.github.io/aws-indexes/k2).", + "fa_icon": "fas fa-fish" }, "bracken_precision": { "type": "string", "default": "S", "fa_icon": "fas fa-tree", "description": "Taxonomic level for Bracken abundance estimations.", - "help_text": "Options are D, P, C, O, F, G, S." + "enum": ["D", "P", "C", "O", "F", "G", "S"] } } }, @@ -662,12 +668,6 @@ "fa_icon": "fas fa-fast-forward", "description": "Skip DESeq2 PCA and heatmap plotting." }, - "skip_bracken": { - "type": "boolean", - "description": "Skip Bracken.", - "fa_icon": "fas fa-fast-forward", - "help_text": "If running Kraken2, skips Bracken post-processing." - }, "skip_multiqc": { "type": "boolean", "description": "Skip MultiQC.", diff --git a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf index 3f5765272..0e64e4807 100644 --- a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf +++ b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf @@ -256,28 +256,24 @@ def validateInputParameters() { } } - // Check that unaligned reads are saved when using Kraken2 - if (params.kraken_db) { - if (!params.save_unaligned) { - error("Please provide --save_unaligned when using Kraken2 to save unaligned reads for analysis.") - } - + //General checks for if contaminant screening is used + if (params.contaminant_screening) { if (params.aligner == 'star_rsem') { - error("Kraken2 cannot be used with --aligner star_rsem since unaligned reads are not saved. Please use --aligner star_salmon or --aligner hisat2.") + error("Contaminant screeneing cannot be done with --aligner star_rsem since unaligned reads are not saved. Please use --aligner star_salmon or --aligner hisat2.") } } - //Check that Kraken/Bracken parameters are not provided without the --kraken_db - if (!params.kraken_db) { - + // Check that Kraken/Bracken database provided if using kraken2/bracken + if (params.contaminant_screening in ['kraken2', 'kraken2_bracken']) { + if (!params.kraken_db) { + error("Contaminant screening set to kraken2 but not database is provided. Please provide a database with the --kraken_db option.") + } + //Check that Kraken/Bracken parameters are not provided when Kraken2 is not being used + } else { if (!params.bracken_precision.equals('S')) { brackenPrecisionWithoutKrakenDBWarn() } - if (params.skip_bracken) { - skipBrackenWithoutKrackenWarn() - } - if (params.save_kraken_assignments || params.save_kraken_unassigned) { krakenArgumentsWithoutKrakenDBWarn() } @@ -498,22 +494,13 @@ def additionaFastaIndexWarn(index) { "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" } -// -// Print a warning if using '--skip_bracken' without '--kraken_db' -// -def skipBrackenWithoutKrackenWarn() { - log.warn "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n" + - " '--skip_bracken' parameter has been provided without '--kraken_db'.\n" + - " Neither Bracken nor Kraken2 will run.\n" + - "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" -} - // // Print a warning if --save_kraken_assignments or --save_kraken_unassigned is provided without --kraken_db // def krakenArgumentsWithoutKrakenDBWarn() { log.warn "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n" + - " '--save_kraken_assignments' or '--save_kraken_unassigned' parameters have been provided without '--kraken_db'.\n" + + " '--save_kraken_assignments' or '--save_kraken_unassigned' parameters\n" + + " have been provided when contaminant screening is not set to Kraken2.\n" + " Kraken2 is not being run so neither will be saved.\n" + "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" } @@ -523,7 +510,7 @@ def krakenArgumentsWithoutKrakenDBWarn() { /// def brackenPrecisionWithoutKrakenDBWarn() { log.warn "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n" + - " '--bracken-precision' parameter has been provided without '--kraken_db'.\n" + + " '--bracken-precision' parameter has been provided without Kraken2 contaminant screening.\n" + " Bracken will not run so precision will not be set.\n" + "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" } diff --git a/workflows/rnaseq/main.nf b/workflows/rnaseq/main.nf index 323db5d02..1100dfbbf 100755 --- a/workflows/rnaseq/main.nf +++ b/workflows/rnaseq/main.nf @@ -640,7 +640,7 @@ workflow RNASEQ { ch_multiqc_files = ch_multiqc_files.mix(ch_fail_strand_multiqc.collectFile(name: 'fail_strand_check_mqc.tsv')) } - if (params.kraken_db) { + if (params.contaminant_screening in ['kraken2', 'kraken2_bracken'] ) { KRAKEN2 ( ch_unaligned_sequences, params.kraken_db, @@ -650,12 +650,9 @@ workflow RNASEQ { ch_kraken_reports = KRAKEN2.out.report ch_versions = ch_versions.mix(KRAKEN2.out.versions) - // Only put Kraken2 output in MultiQC if Bracken is not run - if (params.skip_bracken) { + if (params.contaminant_screening == 'kraken2') { ch_multiqc_files = ch_multiqc_files.mix(KRAKEN2.out.report.collect{it[1]}) - } - - if (!params.skip_bracken) { + } else if (params.contaminant_screening == 'kraken2_bracken') BRACKEN ( ch_kraken_reports, params.kraken_db From 6dd752109e68ebb142bccc48a822593ad37743f4 Mon Sep 17 00:00:00 2001 From: Ezra Greenberg Date: Thu, 15 Aug 2024 19:27:03 +0000 Subject: [PATCH 21/36] Fixing save unaligned default --- modules.json | 3 +- .../nf-core/hisat2/align/hisat2-align.diff | 56 +++++++++++++++++++ modules/nf-core/hisat2/align/main.nf | 6 +- subworkflows/local/align_star/nextflow.config | 2 +- 4 files changed, 62 insertions(+), 5 deletions(-) create mode 100644 modules/nf-core/hisat2/align/hisat2-align.diff diff --git a/modules.json b/modules.json index 089f9f21e..c84770ba7 100644 --- a/modules.json +++ b/modules.json @@ -73,7 +73,8 @@ "hisat2/align": { "branch": "master", "git_sha": "2c6b1144ed58b6184ad58fc4e6b6a90219b4bf4f", - "installed_by": ["fastq_align_hisat2"] + "installed_by": ["fastq_align_hisat2"], + "patch": "modules/nf-core/hisat2/align/hisat2-align.diff" }, "hisat2/build": { "branch": "master", diff --git a/modules/nf-core/hisat2/align/hisat2-align.diff b/modules/nf-core/hisat2/align/hisat2-align.diff new file mode 100644 index 000000000..6086520f6 --- /dev/null +++ b/modules/nf-core/hisat2/align/hisat2-align.diff @@ -0,0 +1,56 @@ +Changes in module 'nf-core/hisat2/align' +--- modules/nf-core/hisat2/align/main.nf ++++ modules/nf-core/hisat2/align/main.nf +@@ -36,7 +36,7 @@ + ss = "$splicesites" ? "--known-splicesite-infile $splicesites" : '' + def seq_center = params.seq_center ? "--rg-id ${prefix} --rg SM:$prefix --rg CN:${params.seq_center.replaceAll('\\s','_')}" : "--rg-id ${prefix} --rg SM:$prefix" + if (meta.single_end) { +- def unaligned = params.save_unaligned ? "--un-gz ${prefix}.unmapped.fastq.gz" : '' ++ def unaligned = params.save_unaligned || params.contaminant_screening ? "--un-gz ${prefix}.unmapped.fastq.gz" : '' + """ + INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'` + hisat2 \\ +@@ -58,7 +58,7 @@ + END_VERSIONS + """ + } else { +- def unaligned = params.save_unaligned ? "--un-conc-gz ${prefix}.unmapped.fastq.gz" : '' ++ def unaligned = params.save_unaligned || params.contaminant_screening ? "--un-conc-gz ${prefix}.unmapped.fastq.gz" : '' + """ + INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'` + hisat2 \\ +@@ -93,7 +93,7 @@ + + stub: + def prefix = task.ext.prefix ?: "${meta.id}" +- def unaligned = params.save_unaligned ? "echo '' | gzip > ${prefix}.unmapped_1.fastq.gz \n echo '' | gzip > ${prefix}.unmapped_2.fastq.gz" : '' ++ def unaligned = params.save_unaligned || params.contaminant_screening ? "echo '' | gzip > ${prefix}.unmapped_1.fastq.gz \n echo '' | gzip > ${prefix}.unmapped_2.fastq.gz" : '' + def VERSION = '2.2.1' // WARN: Version information not provided by tool on CLI. Please update this string when bumping container versions. + """ + ${unaligned} + +--- modules/nf-core/hisat2/align/tests/main.nf.test ++++ modules/nf-core/hisat2/align/tests/main.nf.test +@@ -3,12 +3,6 @@ + name "Test Process HISAT2_ALIGN" + script "../main.nf" + process "HISAT2_ALIGN" +- tag "modules" +- tag "modules_nfcore" +- tag "hisat2" +- tag "hisat2/align" +- tag "hisat2/build" +- tag "hisat2/extractsplicesites" + + test("Single-End") { + + +--- modules/nf-core/hisat2/align/tests/tags.yml ++++ /dev/null +@@ -1,4 +0,0 @@ +-hisat2/align: +- - modules/nf-core/hisat2/align/** +- - modules/nf-core/hisat2/build/** +- - modules/nf-core/hisat2/extractsplicesites/** + +************************************************************ diff --git a/modules/nf-core/hisat2/align/main.nf b/modules/nf-core/hisat2/align/main.nf index ea186f62a..a8d1880c5 100644 --- a/modules/nf-core/hisat2/align/main.nf +++ b/modules/nf-core/hisat2/align/main.nf @@ -36,7 +36,7 @@ process HISAT2_ALIGN { ss = "$splicesites" ? "--known-splicesite-infile $splicesites" : '' def seq_center = params.seq_center ? "--rg-id ${prefix} --rg SM:$prefix --rg CN:${params.seq_center.replaceAll('\\s','_')}" : "--rg-id ${prefix} --rg SM:$prefix" if (meta.single_end) { - def unaligned = params.save_unaligned ? "--un-gz ${prefix}.unmapped.fastq.gz" : '' + def unaligned = params.save_unaligned || params.contaminant_screening ? "--un-gz ${prefix}.unmapped.fastq.gz" : '' """ INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'` hisat2 \\ @@ -58,7 +58,7 @@ process HISAT2_ALIGN { END_VERSIONS """ } else { - def unaligned = params.save_unaligned ? "--un-conc-gz ${prefix}.unmapped.fastq.gz" : '' + def unaligned = params.save_unaligned || params.contaminant_screening ? "--un-conc-gz ${prefix}.unmapped.fastq.gz" : '' """ INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'` hisat2 \\ @@ -93,7 +93,7 @@ process HISAT2_ALIGN { stub: def prefix = task.ext.prefix ?: "${meta.id}" - def unaligned = params.save_unaligned ? "echo '' | gzip > ${prefix}.unmapped_1.fastq.gz \n echo '' | gzip > ${prefix}.unmapped_2.fastq.gz" : '' + def unaligned = params.save_unaligned || params.contaminant_screening ? "echo '' | gzip > ${prefix}.unmapped_1.fastq.gz \n echo '' | gzip > ${prefix}.unmapped_2.fastq.gz" : '' def VERSION = '2.2.1' // WARN: Version information not provided by tool on CLI. Please update this string when bumping container versions. """ ${unaligned} diff --git a/subworkflows/local/align_star/nextflow.config b/subworkflows/local/align_star/nextflow.config index 119eb6e4a..511ff6e63 100644 --- a/subworkflows/local/align_star/nextflow.config +++ b/subworkflows/local/align_star/nextflow.config @@ -22,7 +22,7 @@ if (!params.skip_alignment && params.aligner == 'star_salmon') { --outSAMattributes NH HI AS NM MD --quantTranscriptomeBan Singleend --outSAMstrandField intronMotif - ${params.save_unaligned ? '--outReadsUnmapped Fastx' : ''} + ${params.save_unaligned || params.contaminant_screening ? '--outReadsUnmapped Fastx' : ''} """.trim()) // Consolidate the extra arguments From fdd85ad0926f0bb78ea87d0ce61a731ae3b90fe3 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Thu, 15 Aug 2024 17:53:51 -0400 Subject: [PATCH 22/36] Debugging --- subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf | 7 +++---- workflows/rnaseq/main.nf | 2 +- 2 files changed, 4 insertions(+), 5 deletions(-) diff --git a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf index 0e64e4807..2b556516c 100644 --- a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf +++ b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf @@ -274,7 +274,7 @@ def validateInputParameters() { brackenPrecisionWithoutKrakenDBWarn() } - if (params.save_kraken_assignments || params.save_kraken_unassigned) { + if (params.save_kraken_assignments || params.save_kraken_unassigned || params.kraken_db) { krakenArgumentsWithoutKrakenDBWarn() } } @@ -499,9 +499,8 @@ def additionaFastaIndexWarn(index) { // def krakenArgumentsWithoutKrakenDBWarn() { log.warn "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n" + - " '--save_kraken_assignments' or '--save_kraken_unassigned' parameters\n" + - " have been provided when contaminant screening is not set to Kraken2.\n" + - " Kraken2 is not being run so neither will be saved.\n" + + " 'Kraken2 related arguments have been provided without setting contaminant\n" + + " screening to Kraken2. Kraken2 is not being run so these will not be used.\n" + "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" } diff --git a/workflows/rnaseq/main.nf b/workflows/rnaseq/main.nf index 1100dfbbf..a6725ec7f 100755 --- a/workflows/rnaseq/main.nf +++ b/workflows/rnaseq/main.nf @@ -652,7 +652,7 @@ workflow RNASEQ { if (params.contaminant_screening == 'kraken2') { ch_multiqc_files = ch_multiqc_files.mix(KRAKEN2.out.report.collect{it[1]}) - } else if (params.contaminant_screening == 'kraken2_bracken') + } else if (params.contaminant_screening == 'kraken2_bracken') { BRACKEN ( ch_kraken_reports, params.kraken_db From c0099d4a36980bb994651d203c4846f7776c9832 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Thu, 15 Aug 2024 17:55:14 -0400 Subject: [PATCH 23/36] Update usage --- docs/usage.md | 4 ++++ 1 file changed, 4 insertions(+) diff --git a/docs/usage.md b/docs/usage.md index b613ab79d..cbf752a89 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -296,6 +296,10 @@ Notes: By default, the input GTF file will be filtered to ensure that sequence names correspond to those in the genome fasta file, and to remove rows with empty transcript identifiers. Filtering can be bypassed completely where you are confident it is not necessary, using the `--skip_gtf_filter` parameter. If you just want to skip the 'transcript_id' checking component of the GTF filtering script used in the pipeline this can be disabled specifically using the `--skip_gtf_transcript_filter` parameter. +## Contamination screening options + +The pipeline provides the option to scan unaligned reads for contamination from other species by using [Kraken2](https://ccb.jhu.edu/software/kraken2/) with or without corrections from [Bracken](https://ccb.jhu.edu/software/bracken/). As Bracken is not a particularly expensive algorithm, we recommend using it to correct the abundance estimations from Kraken. An important note is that Kraken2 is [sensitive to the database](https://doi.org/10.1099/mgen.0.000949) that it is used with. It is [particularly important](https://doi.org/10.1128/mbio.01607-23) to ensure that the host genome is part of the database, and if you are particularly concerned about specific contaminants, it may be worthwhile to use a smaller database that contains primarily those contaminants rather than the full standard database. Various pre-built databases can be found [here](https://benlangmead.github.io/aws-indexes/k2) and instructions for building a custom database can be found at the [Kraken2 documentation](https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown). Genomes of contaminants detected in previous sequencing experiments can be found on the [OpenContami website](https://openlooper.hgc.jp/opencontami/help/help_oct.php). Additionally, note that while one of the primary strengths of Kraken2 is that it can detect loaw abundance contaminants in a sample, false positives can also occur. If a very low number of reads of some contaminating species is detected, these results should be treated with caution. + ## Running the pipeline The typical command for running the pipeline is as follows: From c25aa50f58f39e13b863420dd63afef68d0893c3 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Thu, 15 Aug 2024 17:55:30 -0400 Subject: [PATCH 24/36] Provide motivation for Kraken2 parameters --- modules/nf-core/kraken2/kraken2/nextflow.config | 2 ++ 1 file changed, 2 insertions(+) diff --git a/modules/nf-core/kraken2/kraken2/nextflow.config b/modules/nf-core/kraken2/kraken2/nextflow.config index f4375210f..421b2a1e0 100644 --- a/modules/nf-core/kraken2/kraken2/nextflow.config +++ b/modules/nf-core/kraken2/kraken2/nextflow.config @@ -2,6 +2,8 @@ if (!params.skip_alignment && !params.skip_qc) { if (params.contaminant_screening in ['kraken2', 'kraken2_bracken']) { process { withName: 'KRAKEN2' { + // See https://doi.org/10.1080/19490976.2024.2323235 for the confidence level + // and https://doi.org/10.1038/s41596-022-00738-y for the minimum-hit-groups ext.args = "--confidence 0.05 --minimum-hit-groups 3" publishDir = [ path: { "${params.outdir}/kraken2/kraken_reports" }, From 0063c2df38deeee0c7e0bf96ec50455f696ea94e Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Fri, 16 Aug 2024 13:23:14 -0400 Subject: [PATCH 25/36] Fix typo --- docs/usage.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/docs/usage.md b/docs/usage.md index cbf752a89..a86387066 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -298,7 +298,7 @@ By default, the input GTF file will be filtered to ensure that sequence names co ## Contamination screening options -The pipeline provides the option to scan unaligned reads for contamination from other species by using [Kraken2](https://ccb.jhu.edu/software/kraken2/) with or without corrections from [Bracken](https://ccb.jhu.edu/software/bracken/). As Bracken is not a particularly expensive algorithm, we recommend using it to correct the abundance estimations from Kraken. An important note is that Kraken2 is [sensitive to the database](https://doi.org/10.1099/mgen.0.000949) that it is used with. It is [particularly important](https://doi.org/10.1128/mbio.01607-23) to ensure that the host genome is part of the database, and if you are particularly concerned about specific contaminants, it may be worthwhile to use a smaller database that contains primarily those contaminants rather than the full standard database. Various pre-built databases can be found [here](https://benlangmead.github.io/aws-indexes/k2) and instructions for building a custom database can be found at the [Kraken2 documentation](https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown). Genomes of contaminants detected in previous sequencing experiments can be found on the [OpenContami website](https://openlooper.hgc.jp/opencontami/help/help_oct.php). Additionally, note that while one of the primary strengths of Kraken2 is that it can detect loaw abundance contaminants in a sample, false positives can also occur. If a very low number of reads of some contaminating species is detected, these results should be treated with caution. +The pipeline provides the option to scan unaligned reads for contamination from other species by using [Kraken2](https://ccb.jhu.edu/software/kraken2/) with or without corrections from [Bracken](https://ccb.jhu.edu/software/bracken/). As Bracken is not a particularly expensive algorithm, we recommend using it to correct the abundance estimations from Kraken. An important note is that Kraken2 is [sensitive to the database](https://doi.org/10.1099/mgen.0.000949) that it is used with. It is [particularly important](https://doi.org/10.1128/mbio.01607-23) to ensure that the host genome is part of the database, and if you are particularly concerned about specific contaminants, it may be worthwhile to use a smaller database that contains primarily those contaminants rather than the full standard database. Various pre-built databases can be found [here](https://benlangmead.github.io/aws-indexes/k2) and instructions for building a custom database can be found at the [Kraken2 documentation](https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown). Genomes of contaminants detected in previous sequencing experiments can be found on the [OpenContami website](https://openlooper.hgc.jp/opencontami/help/help_oct.php). Additionally, note that while one of the primary strengths of Kraken2 is that it can detect low abundance contaminants in a sample, false positives can also occur. 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Output files -- `kraken2/kraken_reports` +- `/contaminants/kraken2/kraken_reports` - `*.kraken2.report.txt`: Classification of unaligned reads in the Kraken report format. See the [kraken2 manual](https://github.com/DerrickWood/kraken2/wiki/Manual#output-formats) for more details - `*.classified*.fastq.gz` If `--save_kraken_alignments`, outputs fastq file for each sample with each classified read annotated with taxonomic identification from Kraken2. - `*.unclassified*.fastq.gz` If `save_kraken_unassigned`, outputs fastq file with all reads that were not classified by Kraken2. -- `bracken/` +- `/contaminants/bracken/` - `*.kraken2.report_bracken.txt`: Kraken-style reports of the Bracken abundance estimate results. See the [kraken2 manual](https://github.com/DerrickWood/kraken2/wiki/Manual#output-formats) for more details. - `*.tsv` Summary of estimated reads for each taxon member at the given classification level and what corrections were made from Kraken2. diff --git a/modules/nf-core/bracken/bracken/nextflow.config b/modules/nf-core/bracken/bracken/nextflow.config index 052a75482..975a9793a 100644 --- a/modules/nf-core/bracken/bracken/nextflow.config +++ b/modules/nf-core/bracken/bracken/nextflow.config @@ -4,7 +4,7 @@ if (!params.skip_alignment && !params.skip_qc) { withName: 'BRACKEN' { ext.args = "-l ${params.bracken_precision}" publishDir = [ - path: { "${params.outdir}/bracken" }, + path: { "${params.outdir}/${params.aligner}/contaminants/bracken" }, mode: params.publish_dir_mode ] } diff --git a/modules/nf-core/kraken2/kraken2/nextflow.config b/modules/nf-core/kraken2/kraken2/nextflow.config index 421b2a1e0..7289bd9a1 100644 --- a/modules/nf-core/kraken2/kraken2/nextflow.config +++ b/modules/nf-core/kraken2/kraken2/nextflow.config @@ -6,7 +6,7 @@ if (!params.skip_alignment && !params.skip_qc) { // and https://doi.org/10.1038/s41596-022-00738-y for the minimum-hit-groups ext.args = "--confidence 0.05 --minimum-hit-groups 3" publishDir = [ - path: { "${params.outdir}/kraken2/kraken_reports" }, + path: { "${params.outdir}/${params.aligner}/contaminants/kraken2/kraken_reports" }, mode: params.publish_dir_mode ] } From 69f2d0a847ea9a8cba5e5483133ea9e55a6073ca Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Tue, 10 Sep 2024 10:18:13 -0400 Subject: [PATCH 30/36] Update Changelog --- CHANGELOG.md | 63 ++++++++++++++++++++++++++++++++++++++++++++++++---- 1 file changed, 59 insertions(+), 4 deletions(-) diff --git a/CHANGELOG.md b/CHANGELOG.md index 061a10cb6..0dbb374c5 100644 --- a/CHANGELOG.md +++ b/CHANGELOG.md @@ -3,7 +3,44 @@ The format is based on [Keep a Changelog](https://keepachangelog.com/en/1.0.0/) and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0.html). -## v3.15.0dev - [date] +## v3.16.0dev - [date] + +Special thanks to the following for their contributions to the release: + +Thank you to everyone else that has contributed by reporting bugs, enhancements or in any other way, shape or form. + +### Enhancements & fixes + +- [PR #1351](https://github.com/nf-core/rnaseq/pull/1351) - Adding Kraken2/Bracken on unaligned reads as an additional quality control step to detect sample contamination + +### Parameters +| Old parameter | New parameter | +| ------------------ | `--contaminant_screening` | +| | `--kraken_db` | +| | `--save_kraken_assignments` | +| | `--save_kraken_unassigned` | +| | `--bracken_precision` | + +> **NB:** Parameter has been **updated** if both old and new parameter information is present. +> **NB:** Parameter has been **added** if just the new parameter information is present. +> **NB:** Parameter has been **removed** if new parameter information isn't present. + +### Software dependencies + +| Dependency | Old version | New version | +| -------------- | ----------- | ----------- | +| `Kraken2` | ----------- | 2.1.3 | +| `Bracken` | ----------- | 2.9 | + +> **NB:** Dependency has been **updated** if both old and new version information is present. +> +> **NB:** Dependency has been **added** if just the new version information is present. +> +> **NB:** Dependency has been **removed** if new version information isn't present. + +### Credits + +## [[3.15.0](https://github.com/nf-core/rnaseq/releases/tag/3.15.0)] - 2024-09-04 ### Credits @@ -17,6 +54,7 @@ Special thanks to the following for their contributions to the release: - [Luke Zappia](https://github.com/lazappi) - [Matthias Zepper](https://github.com/MatthiasZepper) - [Maxime Garcia](https://github.com/maxulysse) +- [Pieter Moris](https://github.com/pmoris) - [Rob Syme](https://github.com/robsyme) - [Thomas Danhorn](https://github.com/tdanhorn) @@ -24,7 +62,7 @@ Thank you to everyone else that has contributed by reporting bugs, enhancements ### Enhancements & fixes -- [PR #1351](https://github.com/nf-core/rnaseq/pull/1351) - Adding Kraken2/Bracken on unaligned reads as an additional quality control step to detect sample contamination +- [PR #1180](https://github.com/nf-core/rnaseq/pull/1180) - Bump pipeline version to 3.15.0dev - [PR #1186](https://github.com/nf-core/rnaseq/pull/1186) - Properly update qualimap/rnaseq module (ie not patch) - [PR #1197](https://github.com/nf-core/rnaseq/pull/1197) - Delete lib directory and replace with utils\_\* subworkflows - [PR #1199](https://github.com/nf-core/rnaseq/pull/1199) - Replace modules.config with more modular config files per module/subworkflow/workflow @@ -75,7 +113,7 @@ Thank you to everyone else that has contributed by reporting bugs, enhancements - [PR #1272](https://github.com/nf-core/rnaseq/pull/1272) - Simple pipeline level nf-tests - [PR #1274](https://github.com/nf-core/rnaseq/pull/1274) - Update bam_markduplicates_picard subworkflow - [PR #1278](https://github.com/nf-core/rnaseq/pull/1278) - Delocalise pseudo quant workflow -- [PR #1279](https://github.com/nf-core/rnaseq/pull/1279) - Add psueudoaligner pipeline level tests to test suite +- [PR #1279](https://github.com/nf-core/rnaseq/pull/1279) - Add pseudoaligner pipeline level tests to test suite - [PR #1280](https://github.com/nf-core/rnaseq/pull/1280) - Reorganise pipeline level tests into flat directory structure - [PR #1282](https://github.com/nf-core/rnaseq/pull/1282) - Fix CHANGELOG error - [PR #1283](https://github.com/nf-core/rnaseq/pull/1283) - Add output files to nf-test snapshot @@ -103,13 +141,29 @@ Thank you to everyone else that has contributed by reporting bugs, enhancements - [PR #1330](https://github.com/nf-core/rnaseq/pull/1330) - Update all nf-core/modules and subworkflows - [PR #1331](https://github.com/nf-core/rnaseq/pull/1331) - Adding stubs for local modules - [PR #1334](https://github.com/nf-core/rnaseq/pull/1334) - Update all nf-core/modules and subworkflows with stubs -- [PR #1336](https://github.com/nf-core/rnaseq/pull/1334) - Use nf-core/setup-nf-test to install nf-test from cache during CI/CD +- [PR #1335](https://github.com/nf-core/rnaseq/pull/1335) - Adding stubs at all levels +- [PR #1336](https://github.com/nf-core/rnaseq/pull/1336) - Use nf-core/setup-nf-test to install nf-test from cache during CI/CD - [PR #1340](https://github.com/nf-core/rnaseq/pull/1340) - Remove out-of-date Azure specific guidance - [PR #1341](https://github.com/nf-core/rnaseq/pull/1341) - Add rename in the MultiQC report for samples without techreps - [PR #1342](https://github.com/nf-core/rnaseq/pull/1342) - Factor out preprocessing - [PR #1345](https://github.com/nf-core/rnaseq/pull/1345) - Fix preprocessing call - [PR #1350](https://github.com/nf-core/rnaseq/pull/1350) - Reduce resource usage for sort process in bedtools/genomecov +- [PR #1352](https://github.com/nf-core/rnaseq/pull/1352) - Assorted fixes to MultiQC usage - [PR #1353](https://github.com/nf-core/rnaseq/pull/1353) - Correct conditional for salmon indexing in preprocessing workflow +- [PR #1355](https://github.com/nf-core/rnaseq/pull/1355) - Make all curves on subway map better looking, and all lines now have the same width +- [PR #1357](https://github.com/nf-core/rnaseq/pull/1357) - Fix anchor issue in multiqc +- [PR #1358](https://github.com/nf-core/rnaseq/pull/1358) - Update test profiles to restore a static URI for megatests +- [PR #1359](https://github.com/nf-core/rnaseq/pull/1359) - Update MultiQC and revert unnecessary workaround +- [PR #1360](https://github.com/nf-core/rnaseq/pull/1360) - More complete snapshots for pipeline level tests +- [PR #1361](https://github.com/nf-core/rnaseq/pull/1361) - Animate subway map +- [PR #1362](https://github.com/nf-core/rnaseq/pull/1362) - Move multiqc module prefix for nf-test to module +- [PR #1363](https://github.com/nf-core/rnaseq/pull/1363) - Minor updates of nf-core modules and subworkflows +- [PR #1363](https://github.com/nf-core/rnaseq/pull/1363) - Update dupradar script +- [PR #1366](https://github.com/nf-core/rnaseq/pull/1366) - Clarify docs on different tximport count files +- [PR #1367](https://github.com/nf-core/rnaseq/pull/1367) - Clarify design formula and blind dispersion estimation +- [PR #1370](https://github.com/nf-core/rnaseq/pull/1370) - Bump versions for 3.15.0 +- [PR #1371](https://github.com/nf-core/rnaseq/pull/1371) - Apply Maxime's CHANGELOG edits +- [PR #1372](https://github.com/nf-core/rnaseq/pull/1372) - Bump tximeta/tximport for gene table row names fix ### Parameters @@ -135,6 +189,7 @@ Thank you to everyone else that has contributed by reporting bugs, enhancements | `samtools` | 1.17 | 1.20 | | `sortmerna` | 4.3.4 | 4.3.6 | | `umi_tools` | 1.14 | 1.15 | +| `untar` | 1.3 | 1.34 | > **NB:** Dependency has been **updated** if both old and new version information is present. > From 68b9a2161883ebc462e0ca5b9370d50a1a0dc652 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Tue, 10 Sep 2024 10:20:55 -0400 Subject: [PATCH 31/36] Another changelog fix --- CHANGELOG.md | 2 -- 1 file changed, 2 deletions(-) diff --git a/CHANGELOG.md b/CHANGELOG.md index 0dbb374c5..2b85818b0 100644 --- a/CHANGELOG.md +++ b/CHANGELOG.md @@ -38,8 +38,6 @@ Thank you to everyone else that has contributed by reporting bugs, enhancements > > **NB:** Dependency has been **removed** if new version information isn't present. -### Credits - ## [[3.15.0](https://github.com/nf-core/rnaseq/releases/tag/3.15.0)] - 2024-09-04 ### Credits From b4332d919cf67f6dfcad140178cba205ec4b9954 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Tue, 10 Sep 2024 19:42:35 -0400 Subject: [PATCH 32/36] Linting fix --- CHANGELOG.md | 22 ++++++++++++---------- 1 file changed, 12 insertions(+), 10 deletions(-) diff --git a/CHANGELOG.md b/CHANGELOG.md index 2b85818b0..0ce92667b 100644 --- a/CHANGELOG.md +++ b/CHANGELOG.md @@ -14,12 +14,14 @@ Thank you to everyone else that has contributed by reporting bugs, enhancements - [PR #1351](https://github.com/nf-core/rnaseq/pull/1351) - Adding Kraken2/Bracken on unaligned reads as an additional quality control step to detect sample contamination ### Parameters -| Old parameter | New parameter | -| ------------------ | `--contaminant_screening` | -| | `--kraken_db` | -| | `--save_kraken_assignments` | -| | `--save_kraken_unassigned` | -| | `--bracken_precision` | + +| Old parameter | New parameter | +| ------------- | --------------------------- | +| | `--contaminant_screening` | +| | `--kraken_db` | +| | `--save_kraken_assignments` | +| | `--save_kraken_unassigned` | +| | `--bracken_precision` | > **NB:** Parameter has been **updated** if both old and new parameter information is present. > **NB:** Parameter has been **added** if just the new parameter information is present. @@ -27,10 +29,10 @@ Thank you to everyone else that has contributed by reporting bugs, enhancements ### Software dependencies -| Dependency | Old version | New version | -| -------------- | ----------- | ----------- | -| `Kraken2` | ----------- | 2.1.3 | -| `Bracken` | ----------- | 2.9 | +| Dependency | Old version | New version | +| ---------- | ----------- | ----------- | +| `Kraken2` | ----------- | 2.1.3 | +| `Bracken` | ----------- | 2.9 | > **NB:** Dependency has been **updated** if both old and new version information is present. > From bc193dfde41e649e62307746ef95ecc940998c24 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Thu, 19 Sep 2024 11:13:38 -0400 Subject: [PATCH 33/36] Merge conflcits --- CHANGELOG.md | 28 ++ CITATIONS.md | 8 + README.md | 1 + docs/images/bracken-top-n-plot.png | Bin 0 -> 55992 bytes docs/images/nf-core-rnaseq_metro_map_grey.png | Bin 291676 -> 302024 bytes docs/images/nf-core-rnaseq_metro_map_grey.svg | 414 ++++++++++-------- docs/output.md | 22 +- docs/usage.md | 8 + modules.json | 13 +- .../nf-core/bracken/bracken/environment.yml | 7 + modules/nf-core/bracken/bracken/main.nf | 55 +++ modules/nf-core/bracken/bracken/meta.yml | 51 +++ .../nf-core/bracken/bracken/nextflow.config | 13 + .../bracken/bracken/tests/genus_test.config | 5 + .../bracken/bracken/tests/main.nf.test | 167 +++++++ .../bracken/bracken/tests/main.nf.test.snap | 210 +++++++++ .../bracken/bracken/tests/nextflow.config | 5 + .../nf-core/bracken/bracken/tests/tags.yml | 2 + .../nf-core/hisat2/align/hisat2-align.diff | 56 +++ modules/nf-core/hisat2/align/main.nf | 7 +- .../nf-core/kraken2/kraken2/environment.yml | 9 + modules/nf-core/kraken2/kraken2/main.nf | 85 ++++ modules/nf-core/kraken2/kraken2/meta.yml | 78 ++++ .../nf-core/kraken2/kraken2/nextflow.config | 15 + .../kraken2/kraken2/tests/main.nf.test | 143 ++++++ .../kraken2/kraken2/tests/main.nf.test.snap | 74 ++++ .../nf-core/kraken2/kraken2/tests/tags.yml | 3 + nextflow.config | 5 + nextflow_schema.json | 32 ++ subworkflows/local/align_star/nextflow.config | 2 +- .../utils_nfcore_rnaseq_pipeline/main.nf | 43 ++ .../rnaseq/assets/multiqc/multiqc_config.yml | 8 + workflows/rnaseq/main.nf | 59 ++- workflows/rnaseq/nextflow.config | 2 + 34 files changed, 1429 insertions(+), 201 deletions(-) create mode 100644 docs/images/bracken-top-n-plot.png create mode 100644 modules/nf-core/bracken/bracken/environment.yml create mode 100644 modules/nf-core/bracken/bracken/main.nf create mode 100644 modules/nf-core/bracken/bracken/meta.yml create mode 100644 modules/nf-core/bracken/bracken/nextflow.config create mode 100644 modules/nf-core/bracken/bracken/tests/genus_test.config create mode 100644 modules/nf-core/bracken/bracken/tests/main.nf.test create mode 100644 modules/nf-core/bracken/bracken/tests/main.nf.test.snap create mode 100644 modules/nf-core/bracken/bracken/tests/nextflow.config create mode 100644 modules/nf-core/bracken/bracken/tests/tags.yml create mode 100644 modules/nf-core/hisat2/align/hisat2-align.diff create mode 100644 modules/nf-core/kraken2/kraken2/environment.yml create mode 100644 modules/nf-core/kraken2/kraken2/main.nf create mode 100644 modules/nf-core/kraken2/kraken2/meta.yml create mode 100644 modules/nf-core/kraken2/kraken2/nextflow.config create mode 100644 modules/nf-core/kraken2/kraken2/tests/main.nf.test create mode 100644 modules/nf-core/kraken2/kraken2/tests/main.nf.test.snap create mode 100644 modules/nf-core/kraken2/kraken2/tests/tags.yml diff --git a/CHANGELOG.md b/CHANGELOG.md index 916c78035..e9c90c34c 100644 --- a/CHANGELOG.md +++ b/CHANGELOG.md @@ -7,8 +7,36 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0 ### Enhancements & fixes +- [PR #1351](https://github.com/nf-core/rnaseq/pull/1351) - Adding Kraken2/Bracken on unaligned reads as an additional quality control step to detect sample contamination - [PR #1186](https://github.com/nf-core/rnaseq/pull/1186) - Bump pipeline version to 3.16.0dev +### Parameters + +| Old parameter | New parameter | +| ------------- | --------------------------- | +| | `--contaminant_screening` | +| | `--kraken_db` | +| | `--save_kraken_assignments` | +| | `--save_kraken_unassigned` | +| | `--bracken_precision` | + +> **NB:** Parameter has been **updated** if both old and new parameter information is present. +> **NB:** Parameter has been **added** if just the new parameter information is present. +> **NB:** Parameter has been **removed** if new parameter information isn't present. + +### Software dependencies + +| Dependency | Old version | New version | +| ---------- | ----------- | ----------- | +| `Kraken2` | ----------- | 2.1.3 | +| `Bracken` | ----------- | 2.9 | + +> **NB:** Dependency has been **updated** if both old and new version information is present. +> +> **NB:** Dependency has been **added** if just the new version information is present. +> +> **NB:** Dependency has been **removed** if new version information isn't present. + ## [[3.15.1](https://github.com/nf-core/rnaseq/releases/tag/3.15.1)] - 2024-09-16 ### Enhancements & fixes diff --git a/CITATIONS.md b/CITATIONS.md index 48a60b8e4..5eaeea7f8 100644 --- a/CITATIONS.md +++ b/CITATIONS.md @@ -16,6 +16,10 @@ > Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010 Mar 15;26(6):841-2. doi: 10.1093/bioinformatics/btq033. Epub 2010 Jan 28. PubMed PMID: 20110278; PubMed Central PMCID: PMC2832824. +- [Bracken](https://doi.org/10.7717/peerj-cs.104) + + > Lu, J., Breitwieser, F. P., Thielen, P., & Salzberg, S. L. (2017). Bracken: estimating species abundance in metagenomics data. PeerJ. Computer Science, 3(e104), e104. https://doi.org/10.7717/peerj-cs.104 + - [fastp](https://www.ncbi.nlm.nih.gov/pubmed/30423086/) > Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018 Sep 1;34(17):i884-i890. doi: 10.1093/bioinformatics/bty560. PubMed PMID: 30423086; PubMed Central PMCID: PMC6129281. @@ -38,6 +42,10 @@ > Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019 Aug;37(8):907-915. doi: 10.1038/s41587-019-0201-4. Epub 2019 Aug 2. PubMed PMID: 31375807. +- [Kraken2](https://doi.org/10.1186/s13059-019-1891-0) + + > Wood, D. E., Lu, J., & Langmead, B. (2019). Improved metagenomic analysis with Kraken 2. Genome Biology, 20(1), 257. https://doi.org/10.1186/s13059-019-1891-0 + - [MultiQC](https://pubmed.ncbi.nlm.nih.gov/27312411/) > Ewels P, Magnusson M, Lundin S, Käller M. MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016 Oct 1;32(19):3047-8. doi: 10.1093/bioinformatics/btw354. Epub 2016 Jun 16. PubMed PMID: 27312411; PubMed Central PMCID: PMC5039924. diff --git a/README.md b/README.md index c5f58478e..01da6cf8b 100644 --- a/README.md +++ b/README.md @@ -46,6 +46,7 @@ 3. [`dupRadar`](https://bioconductor.org/packages/release/bioc/html/dupRadar.html) 4. [`Preseq`](http://smithlabresearch.org/software/preseq/) 5. [`DESeq2`](https://bioconductor.org/packages/release/bioc/html/DESeq2.html) + 6. [`Kraken2`](https://ccb.jhu.edu/software/kraken2/) -> [`Bracken`](https://ccb.jhu.edu/software/bracken/) on unaligned sequences; _optional_ 15. Pseudoalignment and quantification ([`Salmon`](https://combine-lab.github.io/salmon/) or ['Kallisto'](https://pachterlab.github.io/kallisto/); _optional_) 16. 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sodipodi:docname="nf-core-rnaseq_metro_map_grey.svg" inkscape:export-filename="nf-core-rnaseq_metro_map_grey.png" inkscape:export-xdpi="89" @@ -2775,24 +2775,39 @@ id="stop23-7" />RSeQC(multiple modules)Kraken2/Bracken5RSeQC(multiple modules)PreseqPreseqdupRadarDESeq2(PCA only)MultiQCQualimaprnaseqHTMLHTMLHTMLdupRadarMultiQCQualimaprnaseqDESeq2(PCA only)MultiQC - DESeq2 sample similarity plot

+### Kraken2/Bracken + +
+Output files + +- `/contaminants/kraken2/kraken_reports` + - `*.kraken2.report.txt`: Classification of unaligned reads in the Kraken report format. See the [kraken2 manual](https://github.com/DerrickWood/kraken2/wiki/Manual#output-formats) for more details + - `*.classified*.fastq.gz` If `--save_kraken_alignments`, outputs fastq file for each sample with each classified read annotated with taxonomic identification from Kraken2. + - `*.unclassified*.fastq.gz` If `save_kraken_unassigned`, outputs fastq file with all reads that were not classified by Kraken2. +- `/contaminants/bracken/` + - `*.kraken2.report_bracken.txt`: Kraken-style reports of the Bracken abundance estimate results. See the [kraken2 manual](https://github.com/DerrickWood/kraken2/wiki/Manual#output-formats) for more details. + - `*.tsv` Summary of estimated reads for each taxon member at the given classification level and what corrections were made from Kraken2. + +
+ +[Kraken2](https://ccb.jhu.edu/software/kraken2/) is a taxonomic classification tool that uses k-mer matches paired with a lowest common ancestory (LCA) algorithm to classify species reads. [Bracken](https://ccb.jhu.edu/software/bracken/) is a statistical method to generate abundance estimates based off of the Kraken2 output. These algorithms are run on unaligned sequences to detect potential contamination of samples. MultiQC reports the top 5 taxon members detected at the level of classification used for Bracken, with toggles available for higher taxonomic levels. If Bracken is skipped, MultiQC will report the top 5 species detected by Kraken2. + +![MultiQC - Bracken top species plot](images/bracken-top-n-plot.png) + ### MultiQC
@@ -675,7 +695,7 @@ Results generated by MultiQC collate pipeline QC from supported tools i.e. FastQ ### Pseudoalignment -The principal output files are the same between Salmon and Kallsto: +The principal output files are the same between Salmon and Kallisto:
Output files diff --git a/docs/usage.md b/docs/usage.md index b613ab79d..cd42a399b 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -296,6 +296,14 @@ Notes: By default, the input GTF file will be filtered to ensure that sequence names correspond to those in the genome fasta file, and to remove rows with empty transcript identifiers. Filtering can be bypassed completely where you are confident it is not necessary, using the `--skip_gtf_filter` parameter. If you just want to skip the 'transcript_id' checking component of the GTF filtering script used in the pipeline this can be disabled specifically using the `--skip_gtf_transcript_filter` parameter. +## Contamination screening options + +The pipeline provides the option to scan unaligned reads for contamination from other species using [Kraken2](https://ccb.jhu.edu/software/kraken2/), with the possibility of applying corrections from [Bracken](https://ccb.jhu.edu/software/bracken/). Since running Bracken is not computationally expensive, we recommend always using it to refine the abundance estimates generated by Kraken2. + +It is important to note that the accuracy of Kraken2 is [highly dependent on the database](https://doi.org/10.1099/mgen.0.000949) used. Specifically, it is [crucial](https://doi.org/10.1128/mbio.01607-23) to ensure that the host genome is included in the database. If you are particularly concerned about certain contaminants, it may be beneficial to use a smaller, more focused database containing primarily those contaminants instead of the full standard database. Various pre-built databases [are available for download](https://benlangmead.github.io/aws-indexes/k2), and instructions for building a custom database can be found in the [Kraken2 documentation](https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown). Additionally, genomes of contaminants detected in previous sequencing experiments are available on the [OpenContami website](https://openlooper.hgc.jp/opencontami/help/help_oct.php). + +While Kraken2 is capable of detecting low-abundance contaminants in a sample, false positives can occur. Therefore, if only a very small number of reads from a contaminating species are detected, these results should be interpreted with caution. + ## Running the pipeline The typical command for running the pipeline is as follows: diff --git a/modules.json b/modules.json index 30090ab26..4c9ba76f1 100644 --- a/modules.json +++ b/modules.json @@ -15,6 +15,11 @@ "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", "installed_by": ["modules"] }, + "bracken/bracken": { + "branch": "master", + "git_sha": "c214fad97b328eb6d6233f779be9ba44814a9136", + "installed_by": ["modules"] + }, "cat/fastq": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", @@ -68,7 +73,8 @@ "hisat2/align": { "branch": "master", "git_sha": "ad30f90cfc383dfaa505771d24f9e292c53157ab", - "installed_by": ["fastq_align_hisat2"] + "installed_by": ["fastq_align_hisat2"], + "patch": "modules/nf-core/hisat2/align/hisat2-align.diff" }, "hisat2/build": { "branch": "master", @@ -90,6 +96,11 @@ "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", "installed_by": ["modules", "quantify_pseudo_alignment"] }, + "kraken2/kraken2": { + "branch": "master", + "git_sha": "a13d5d945742a60bbef6e5c177e81cda540f75dc", + "installed_by": ["modules"] + }, "multiqc": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", diff --git a/modules/nf-core/bracken/bracken/environment.yml b/modules/nf-core/bracken/bracken/environment.yml new file mode 100644 index 000000000..6eb5b1b9a --- /dev/null +++ b/modules/nf-core/bracken/bracken/environment.yml @@ -0,0 +1,7 @@ +name: bracken_bracken +channels: + - conda-forge + - bioconda + - defaults +dependencies: + - "bioconda::bracken=2.9" diff --git a/modules/nf-core/bracken/bracken/main.nf b/modules/nf-core/bracken/bracken/main.nf new file mode 100644 index 000000000..e3d32fb21 --- /dev/null +++ b/modules/nf-core/bracken/bracken/main.nf @@ -0,0 +1,55 @@ +process BRACKEN_BRACKEN { + tag "$meta.id" + label 'process_low' + + conda "${moduleDir}/environment.yml" + container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ? + 'https://depot.galaxyproject.org/singularity/bracken:2.9--py38h2494328_0': + 'biocontainers/bracken:2.9--py38h2494328_0' }" + + input: + tuple val(meta), path(kraken_report) + path database + + output: + tuple val(meta), path(bracken_report) , emit: reports + tuple val(meta), path(bracken_kraken_style_report), emit: txt + path "versions.yml" , emit: versions + + when: + task.ext.when == null || task.ext.when + + script: + def args = task.ext.args ?: "" + def prefix = task.ext.prefix ?: "${meta.id}" + bracken_report = "${prefix}.tsv" + bracken_kraken_style_report = "${prefix}.kraken2.report_bracken.txt" + """ + bracken \\ + ${args} \\ + -d '${database}' \\ + -i '${kraken_report}' \\ + -o '${bracken_report}' \\ + -w '${bracken_kraken_style_report}' + + cat <<-END_VERSIONS > versions.yml + "${task.process}": + bracken: \$(echo \$(bracken -v) | cut -f2 -d'v') + END_VERSIONS + """ + + stub: + def args = task.ext.args ?: '' + def prefix = task.ext.prefix ?: "${meta.id}" + bracken_report = "${prefix}.tsv" + bracken_kraken_style_report = "${prefix}.kraken2.report_bracken.txt" + """ + touch ${prefix}.tsv + touch ${bracken_kraken_style_report} + + cat <<-END_VERSIONS > versions.yml + "${task.process}": + bracken: \$(echo \$(bracken -v) | cut -f2 -d'v') + END_VERSIONS + """ +} diff --git a/modules/nf-core/bracken/bracken/meta.yml b/modules/nf-core/bracken/bracken/meta.yml new file mode 100644 index 000000000..b7ff4489f --- /dev/null +++ b/modules/nf-core/bracken/bracken/meta.yml @@ -0,0 +1,51 @@ +name: bracken_bracken +description: Re-estimate taxonomic abundance of metagenomic samples analyzed by kraken. +keywords: + - bracken + - metagenomics + - abundance + - kraken2 +tools: + - bracken: + description: Bracken (Bayesian Reestimation of Abundance with KrakEN) is a highly accurate statistical method that computes the abundance of species in DNA sequences from a metagenomics sample. + homepage: https://ccb.jhu.edu/software/bracken/ + documentation: https://ccb.jhu.edu/software/bracken/index.shtml?t=manual + tool_dev_url: https://github.com/jenniferlu717/Bracken + doi: "10.7717/peerj-cs.104" + licence: ["GPL v3"] +input: + - meta: + type: map + description: | + Groovy Map containing sample information + e.g. [ id:'test', single_end:false ] + - kraken_report: + type: file + description: TSV file with six columns coming from kraken2 output + pattern: "*.{tsv}" + - database: + type: file + description: Directory containing the kraken2/Bracken files for analysis + pattern: "*" +output: + - meta: + type: map + description: | + Groovy Map containing sample information + e.g. [ id:'test', single_end:false ] + - versions: + type: file + description: File containing software versions + pattern: "versions.yml" + - reports: + type: file + description: TSV output report of the re-estimated abundances + pattern: "*.{tsv}" + - txt: + type: file + description: TXT file of bracken corrected results of Kraken2 report output + pattern: "*.txt" +authors: + - "@Midnighter" +maintainers: + - "@Midnighter" diff --git a/modules/nf-core/bracken/bracken/nextflow.config b/modules/nf-core/bracken/bracken/nextflow.config new file mode 100644 index 000000000..975a9793a --- /dev/null +++ b/modules/nf-core/bracken/bracken/nextflow.config @@ -0,0 +1,13 @@ +if (!params.skip_alignment && !params.skip_qc) { + if (params.contaminant_screening == 'kraken2_bracken') { + process { + withName: 'BRACKEN' { + ext.args = "-l ${params.bracken_precision}" + publishDir = [ + path: { "${params.outdir}/${params.aligner}/contaminants/bracken" }, + mode: params.publish_dir_mode + ] + } + } + } +} \ No newline at end of file diff --git a/modules/nf-core/bracken/bracken/tests/genus_test.config b/modules/nf-core/bracken/bracken/tests/genus_test.config new file mode 100644 index 000000000..bc5f63ae0 --- /dev/null +++ b/modules/nf-core/bracken/bracken/tests/genus_test.config @@ -0,0 +1,5 @@ +process { + withName: BRACKEN_BRACKEN { + ext.args = "-l G -t 10 -r 150" + } +} diff --git a/modules/nf-core/bracken/bracken/tests/main.nf.test b/modules/nf-core/bracken/bracken/tests/main.nf.test new file mode 100644 index 000000000..9d2105ded --- /dev/null +++ b/modules/nf-core/bracken/bracken/tests/main.nf.test @@ -0,0 +1,167 @@ +nextflow_process { + + name "Test Process BRACKEN_BRACKEN" + script "../main.nf" + process "BRACKEN_BRACKEN" + + tag "modules" + tag "modules_nfcore" + tag "bracken" + tag "bracken/bracken" + tag "kraken2/kraken2" + tag "untar" + + setup { + run ("UNTAR") { + script "../../../untar/main.nf" + process { + """ + input[0] = [[],file(params.modules_testdata_base_path + 'genomics/sarscov2/genome/db/kraken2_bracken.tar.gz', checkIfExists: true)] + """ + } + } + } + + test("sarscov2 - single-end - fastq") { + + config "./nextflow.config" + + setup { + run("KRAKEN2_KRAKEN2") { + script "../../../kraken2/kraken2/main.nf" + process { + """ + input[0] = [[id: 'test', single_end: true], file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_1.fastq.gz', checkIfExists: true)] + input[1] = UNTAR.out.untar.map{it[1]} + input[2] = false + input[3] = false + """ + } + } + } + + when { + process { + """ + input[0] = KRAKEN2_KRAKEN2.out.report + input[1] = UNTAR.out.untar.map{it[1]} + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot(process.out).match() } + ) + } + + } + + test("sarscov2 - paired-end - fastq - genus config") { + + config "./genus_test.config" + + setup { + run("KRAKEN2_KRAKEN2") { + script "../../../kraken2/kraken2/main.nf" + process { + """ + input[0] = [ + [id: 'test', single_end: false], + [ + file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_1.fastq.gz', checkIfExists: true), + file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_2.fastq.gz', checkIfExists: true) + ] + ] + input[1] = UNTAR.out.untar.map{it[1]} + input[2] = false + input[3] = false + """ + } + } + } + + when { + process { + """ + input[0] = KRAKEN2_KRAKEN2.out.report + input[1] = UNTAR.out.untar.map{it[1]} + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot(process.out).match() } + ) + } + + } + + test("sarscov2 - paired-end - fastq") { + + config "./nextflow.config" + + setup { + run("KRAKEN2_KRAKEN2") { + script "../../../kraken2/kraken2/main.nf" + process { + """ + input[0] = [ + [id: 'test', single_end: false], + [ + file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_1.fastq.gz', checkIfExists: true), + file(params.modules_testdata_base_path + 'genomics/sarscov2/illumina/fastq/test_2.fastq.gz', checkIfExists: true) + ] + ] + input[1] = UNTAR.out.untar.map{it[1]} + input[2] = false + input[3] = false + """ + } + } + } + + when { + process { + """ + input[0] = KRAKEN2_KRAKEN2.out.report + input[1] = UNTAR.out.untar.map{it[1]} + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot(process.out).match() } + ) + } + + } + + test("sarscov2 - stub - fastq") { + + options "-stub" + + when { + process { + """ + input[0] = [[id: 'test'],file(params.modules_testdata_base_path + 'generic/txt/hello.txt', checkIfExists: true)] + input[1] = UNTAR.out.untar.map{it[1]} + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot(process.out).match() } + ) + } + + } + +} diff --git a/modules/nf-core/bracken/bracken/tests/main.nf.test.snap b/modules/nf-core/bracken/bracken/tests/main.nf.test.snap new file mode 100644 index 000000000..c97a4c71b --- /dev/null +++ b/modules/nf-core/bracken/bracken/tests/main.nf.test.snap @@ -0,0 +1,210 @@ +{ + "sarscov2 - single-end - fastq": { + "content": [ + { + "0": [ + [ + { + "id": "test", + "single_end": true + }, + "test.tsv:md5,4a21ae14ff8a0311d55f139af5247838" + ] + ], + "1": [ + [ + { + "id": "test", + "single_end": true + }, + "test.kraken2.report_bracken.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + ] + ], + "2": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ], + "reports": [ + [ + { + "id": "test", + "single_end": true + }, + "test.tsv:md5,4a21ae14ff8a0311d55f139af5247838" + ] + ], + "txt": [ + [ + { + "id": "test", + "single_end": true + }, + "test.kraken2.report_bracken.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + ] + ], + "versions": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ] + } + ], + "meta": { + "nf-test": "0.9.0", + "nextflow": "24.04.4" + }, + "timestamp": "2024-08-06T11:35:03.995620397" + }, + "sarscov2 - paired-end - fastq - genus config": { + "content": [ + { + "0": [ + [ + { + "id": "test", + "single_end": false + }, + "test.tsv:md5,f609b09d6edb5ebc1ea1435d1dd46cde" + ] + ], + "1": [ + [ + { + "id": "test", + "single_end": false + }, + "test.kraken2.report_bracken.txt:md5,2ce58814420a3690da1f08e10e8d3a30" + ] + ], + "2": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ], + "reports": [ + [ + { + "id": "test", + "single_end": false + }, + "test.tsv:md5,f609b09d6edb5ebc1ea1435d1dd46cde" + ] + ], + "txt": [ + [ + { + "id": "test", + "single_end": false + }, + "test.kraken2.report_bracken.txt:md5,2ce58814420a3690da1f08e10e8d3a30" + ] + ], + "versions": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ] + } + ], + "meta": { + "nf-test": "0.9.0", + "nextflow": "24.04.4" + }, + "timestamp": "2024-08-06T12:13:33.399680181" + }, + "sarscov2 - paired-end - fastq": { + "content": [ + { + "0": [ + [ + { + "id": "test", + "single_end": false + }, + "test.tsv:md5,4a21ae14ff8a0311d55f139af5247838" + ] + ], + "1": [ + [ + { + "id": "test", + "single_end": false + }, + "test.kraken2.report_bracken.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + ] + ], + "2": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ], + "reports": [ + [ + { + "id": "test", + "single_end": false + }, + "test.tsv:md5,4a21ae14ff8a0311d55f139af5247838" + ] + ], + "txt": [ + [ + { + "id": "test", + "single_end": false + }, + "test.kraken2.report_bracken.txt:md5,ca0fbeedc4353b5fdd081688823a33df" + ] + ], + "versions": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ] + } + ], + "meta": { + "nf-test": "0.9.0", + "nextflow": "24.04.4" + }, + "timestamp": "2024-08-06T12:09:15.465609745" + }, + "sarscov2 - stub - fastq": { + "content": [ + { + "0": [ + [ + { + "id": "test" + }, + "test.tsv:md5,d41d8cd98f00b204e9800998ecf8427e" + ] + ], + "1": [ + [ + { + "id": "test" + }, + "test.kraken2.report_bracken.txt:md5,d41d8cd98f00b204e9800998ecf8427e" + ] + ], + "2": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ], + "reports": [ + [ + { + "id": "test" + }, + "test.tsv:md5,d41d8cd98f00b204e9800998ecf8427e" + ] + ], + "txt": [ + [ + { + "id": "test" + }, + "test.kraken2.report_bracken.txt:md5,d41d8cd98f00b204e9800998ecf8427e" + ] + ], + "versions": [ + "versions.yml:md5,599fbbf4c1cd5851022a98788f2afdba" + ] + } + ], + "meta": { + "nf-test": "0.9.0", + "nextflow": "24.04.4" + }, + "timestamp": "2024-08-06T11:35:42.965471207" + } +} \ No newline at end of file diff --git a/modules/nf-core/bracken/bracken/tests/nextflow.config b/modules/nf-core/bracken/bracken/tests/nextflow.config new file mode 100644 index 000000000..b550b7489 --- /dev/null +++ b/modules/nf-core/bracken/bracken/tests/nextflow.config @@ -0,0 +1,5 @@ +process { + withName: BRACKEN_BRACKEN { + ext.args = "-l S -t 10 -r 150" + } +} diff --git a/modules/nf-core/bracken/bracken/tests/tags.yml b/modules/nf-core/bracken/bracken/tests/tags.yml new file mode 100644 index 000000000..6a2cbd384 --- /dev/null +++ b/modules/nf-core/bracken/bracken/tests/tags.yml @@ -0,0 +1,2 @@ +bracken/bracken: + - "modules/nf-core/bracken/bracken/**" diff --git a/modules/nf-core/hisat2/align/hisat2-align.diff b/modules/nf-core/hisat2/align/hisat2-align.diff new file mode 100644 index 000000000..6086520f6 --- /dev/null +++ b/modules/nf-core/hisat2/align/hisat2-align.diff @@ -0,0 +1,56 @@ +Changes in module 'nf-core/hisat2/align' +--- modules/nf-core/hisat2/align/main.nf ++++ modules/nf-core/hisat2/align/main.nf +@@ -36,7 +36,7 @@ + ss = "$splicesites" ? "--known-splicesite-infile $splicesites" : '' + def seq_center = params.seq_center ? "--rg-id ${prefix} --rg SM:$prefix --rg CN:${params.seq_center.replaceAll('\\s','_')}" : "--rg-id ${prefix} --rg SM:$prefix" + if (meta.single_end) { +- def unaligned = params.save_unaligned ? "--un-gz ${prefix}.unmapped.fastq.gz" : '' ++ def unaligned = params.save_unaligned || params.contaminant_screening ? "--un-gz ${prefix}.unmapped.fastq.gz" : '' + """ + INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'` + hisat2 \\ +@@ -58,7 +58,7 @@ + END_VERSIONS + """ + } else { +- def unaligned = params.save_unaligned ? "--un-conc-gz ${prefix}.unmapped.fastq.gz" : '' ++ def unaligned = params.save_unaligned || params.contaminant_screening ? "--un-conc-gz ${prefix}.unmapped.fastq.gz" : '' + """ + INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'` + hisat2 \\ +@@ -93,7 +93,7 @@ + + stub: + def prefix = task.ext.prefix ?: "${meta.id}" +- def unaligned = params.save_unaligned ? "echo '' | gzip > ${prefix}.unmapped_1.fastq.gz \n echo '' | gzip > ${prefix}.unmapped_2.fastq.gz" : '' ++ def unaligned = params.save_unaligned || params.contaminant_screening ? "echo '' | gzip > ${prefix}.unmapped_1.fastq.gz \n echo '' | gzip > ${prefix}.unmapped_2.fastq.gz" : '' + def VERSION = '2.2.1' // WARN: Version information not provided by tool on CLI. Please update this string when bumping container versions. + """ + ${unaligned} + +--- modules/nf-core/hisat2/align/tests/main.nf.test ++++ modules/nf-core/hisat2/align/tests/main.nf.test +@@ -3,12 +3,6 @@ + name "Test Process HISAT2_ALIGN" + script "../main.nf" + process "HISAT2_ALIGN" +- tag "modules" +- tag "modules_nfcore" +- tag "hisat2" +- tag "hisat2/align" +- tag "hisat2/build" +- tag "hisat2/extractsplicesites" + + test("Single-End") { + + +--- modules/nf-core/hisat2/align/tests/tags.yml ++++ /dev/null +@@ -1,4 +0,0 @@ +-hisat2/align: +- - modules/nf-core/hisat2/align/** +- - modules/nf-core/hisat2/build/** +- - modules/nf-core/hisat2/extractsplicesites/** + +************************************************************ diff --git a/modules/nf-core/hisat2/align/main.nf b/modules/nf-core/hisat2/align/main.nf index f45f9bccb..464a28e2b 100644 --- a/modules/nf-core/hisat2/align/main.nf +++ b/modules/nf-core/hisat2/align/main.nf @@ -34,7 +34,7 @@ process HISAT2_ALIGN { ss = "$splicesites" ? "--known-splicesite-infile $splicesites" : '' def seq_center = params.seq_center ? "--rg-id ${prefix} --rg SM:$prefix --rg CN:${params.seq_center.replaceAll('\\s','_')}" : "--rg-id ${prefix} --rg SM:$prefix" if (meta.single_end) { - def unaligned = params.save_unaligned ? "--un-gz ${prefix}.unmapped.fastq.gz" : '' + def unaligned = params.save_unaligned || params.contaminant_screening ? "--un-gz ${prefix}.unmapped.fastq.gz" : '' """ INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'` hisat2 \\ @@ -56,7 +56,7 @@ process HISAT2_ALIGN { END_VERSIONS """ } else { - def unaligned = params.save_unaligned ? "--un-conc-gz ${prefix}.unmapped.fastq.gz" : '' + def unaligned = params.save_unaligned || params.contaminant_screening ? "--un-conc-gz ${prefix}.unmapped.fastq.gz" : '' """ INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'` hisat2 \\ @@ -91,7 +91,8 @@ process HISAT2_ALIGN { stub: def prefix = task.ext.prefix ?: "${meta.id}" - def unaligned = params.save_unaligned ? "echo '' | gzip > ${prefix}.unmapped_1.fastq.gz \n echo '' | gzip > ${prefix}.unmapped_2.fastq.gz" : '' + def unaligned = params.save_unaligned || params.contaminant_screening ? "echo '' | gzip > ${prefix}.unmapped_1.fastq.gz \n echo '' | gzip > ${prefix}.unmapped_2.fastq.gz" : '' + def VERSION = '2.2.1' // WARN: Version information not provided by tool on CLI. Please update this string when bumping container versions. """ ${unaligned} diff --git a/modules/nf-core/kraken2/kraken2/environment.yml b/modules/nf-core/kraken2/kraken2/environment.yml new file mode 100644 index 000000000..0c067feeb --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/environment.yml @@ -0,0 +1,9 @@ +name: kraken2_kraken2 +channels: + - conda-forge + - bioconda + - defaults +dependencies: + - "bioconda::kraken2=2.1.3" + - "coreutils=9.4" + - "pigz=2.8" diff --git a/modules/nf-core/kraken2/kraken2/main.nf b/modules/nf-core/kraken2/kraken2/main.nf new file mode 100644 index 000000000..364a6fe24 --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/main.nf @@ -0,0 +1,85 @@ +process KRAKEN2_KRAKEN2 { + tag "$meta.id" + label 'process_high' + + conda "${moduleDir}/environment.yml" + container "${ workflow.containerEngine == 'singularity' && !task.ext.singularity_pull_docker_container ? + 'https://depot.galaxyproject.org/singularity/mulled-v2-8706a1dd73c6cc426e12dd4dd33a5e917b3989ae:c8cbdc8ff4101e6745f8ede6eb5261ef98bdaff4-0' : + 'biocontainers/mulled-v2-8706a1dd73c6cc426e12dd4dd33a5e917b3989ae:c8cbdc8ff4101e6745f8ede6eb5261ef98bdaff4-0' }" + + input: + tuple val(meta), path(reads) + path db + val save_output_fastqs + val save_reads_assignment + + output: + tuple val(meta), path('*.classified{.,_}*') , optional:true, emit: classified_reads_fastq + tuple val(meta), path('*.unclassified{.,_}*') , optional:true, emit: unclassified_reads_fastq + tuple val(meta), path('*classifiedreads.txt') , optional:true, emit: classified_reads_assignment + tuple val(meta), path('*report.txt') , emit: report + path "versions.yml" , emit: versions + + when: + task.ext.when == null || task.ext.when + + script: + def args = task.ext.args ?: '' + def prefix = task.ext.prefix ?: "${meta.id}" + def paired = meta.single_end ? "" : "--paired" + def classified = meta.single_end ? "${prefix}.classified.fastq" : "${prefix}.classified#.fastq" + def unclassified = meta.single_end ? "${prefix}.unclassified.fastq" : "${prefix}.unclassified#.fastq" + def classified_option = save_output_fastqs ? "--classified-out ${classified}" : "" + def unclassified_option = save_output_fastqs ? "--unclassified-out ${unclassified}" : "" + def readclassification_option = save_reads_assignment ? "--output ${prefix}.kraken2.classifiedreads.txt" : "--output /dev/null" + def compress_reads_command = save_output_fastqs ? "pigz -p $task.cpus *.fastq" : "" + + """ + kraken2 \\ + --db $db \\ + --threads $task.cpus \\ + --report ${prefix}.kraken2.report.txt \\ + --gzip-compressed \\ + $unclassified_option \\ + $classified_option \\ + $readclassification_option \\ + $paired \\ + $args \\ + $reads + + $compress_reads_command + + cat <<-END_VERSIONS > versions.yml + "${task.process}": + kraken2: \$(echo \$(kraken2 --version 2>&1) | sed 's/^.*Kraken version //; s/ .*\$//') + pigz: \$( pigz --version 2>&1 | sed 's/pigz //g' ) + END_VERSIONS + """ + + stub: + def args = task.ext.args ?: '' + def prefix = task.ext.prefix ?: "${meta.id}" + def paired = meta.single_end ? "" : "--paired" + def classified = meta.single_end ? "${prefix}.classified.fastq.gz" : "${prefix}.classified_1.fastq.gz ${prefix}.classified_2.fastq.gz" + def unclassified = meta.single_end ? "${prefix}.unclassified.fastq.gz" : "${prefix}.unclassified_1.fastq.gz ${prefix}.unclassified_2.fastq.gz" + def readclassification_option = save_reads_assignment ? "--output ${prefix}.kraken2.classifiedreads.txt" : "--output /dev/null" + def compress_reads_command = save_output_fastqs ? "pigz -p $task.cpus *.fastq" : "" + + """ + touch ${prefix}.kraken2.report.txt + if [ "$save_output_fastqs" == "true" ]; then + touch $classified + touch $unclassified + fi + if [ "$save_reads_assignment" == "true" ]; then + touch ${prefix}.kraken2.classifiedreads.txt + fi + + cat <<-END_VERSIONS > versions.yml + "${task.process}": + kraken2: \$(echo \$(kraken2 --version 2>&1) | sed 's/^.*Kraken version //; s/ .*\$//') + pigz: \$( pigz --version 2>&1 | sed 's/pigz //g' ) + END_VERSIONS + """ + +} diff --git a/modules/nf-core/kraken2/kraken2/meta.yml b/modules/nf-core/kraken2/kraken2/meta.yml new file mode 100644 index 000000000..7909ffe7e --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/meta.yml @@ -0,0 +1,78 @@ +name: kraken2_kraken2 +description: Classifies metagenomic sequence data +keywords: + - classify + - metagenomics + - fastq + - db +tools: + - kraken2: + description: | + Kraken2 is a taxonomic sequence classifier that assigns taxonomic labels to sequence reads + homepage: https://ccb.jhu.edu/software/kraken2/ + documentation: https://github.com/DerrickWood/kraken2/wiki/Manual + doi: 10.1186/s13059-019-1891-0 + licence: ["MIT"] +input: + - meta: + type: map + description: | + Groovy Map containing sample information + e.g. [ id:'test', single_end:false ] + - reads: + type: file + description: | + List of input FastQ files of size 1 and 2 for single-end and paired-end data, + respectively. + - db: + type: directory + description: Kraken2 database + - save_output_fastqs: + type: string + description: | + If true, optional commands are added to save classified and unclassified reads + as fastq files + - save_reads_assignment: + type: string + description: | + If true, an optional command is added to save a file reporting the taxonomic + classification of each input read +output: + - meta: + type: map + description: | + Groovy Map containing sample information + e.g. [ id:'test', single_end:false ] + - classified_reads_fastq: + type: file + description: | + Reads classified as belonging to any of the taxa + on the Kraken2 database. + pattern: "*{fastq.gz}" + - unclassified_reads_fastq: + type: file + description: | + Reads not classified to any of the taxa + on the Kraken2 database. + pattern: "*{fastq.gz}" + - classified_reads_assignment: + type: file + description: | + Kraken2 output file indicating the taxonomic assignment of + each input read + - report: + type: file + description: | + Kraken2 report containing stats about classified + and not classifed reads. + pattern: "*.{report.txt}" + - versions: + type: file + description: File containing software versions + pattern: "versions.yml" +authors: + - "@joseespinosa" + - "@drpatelh" +maintainers: + - "@joseespinosa" + - "@drpatelh" diff --git a/modules/nf-core/kraken2/kraken2/nextflow.config b/modules/nf-core/kraken2/kraken2/nextflow.config new file mode 100644 index 000000000..7289bd9a1 --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/nextflow.config @@ -0,0 +1,15 @@ +if (!params.skip_alignment && !params.skip_qc) { + if (params.contaminant_screening in ['kraken2', 'kraken2_bracken']) { + process { + withName: 'KRAKEN2' { + // See https://doi.org/10.1080/19490976.2024.2323235 for the confidence level + // and https://doi.org/10.1038/s41596-022-00738-y for the minimum-hit-groups + ext.args = "--confidence 0.05 --minimum-hit-groups 3" + publishDir = [ + path: { "${params.outdir}/${params.aligner}/contaminants/kraken2/kraken_reports" }, + mode: params.publish_dir_mode + ] + } + } + } +} \ No newline at end of file diff --git a/modules/nf-core/kraken2/kraken2/tests/main.nf.test b/modules/nf-core/kraken2/kraken2/tests/main.nf.test new file mode 100644 index 000000000..c0843df29 --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/tests/main.nf.test @@ -0,0 +1,143 @@ +nextflow_process { + name "Test Process KRAKEN2_KRAKEN2" + script "../main.nf" + process "KRAKEN2_KRAKEN2" + tag "modules" + tag "modules_nfcore" + tag "untar" + tag "kraken2" + tag "kraken2/kraken2" + + setup { + run("UNTAR") { + script "modules/nf-core/untar/main.nf" + process { + """ + input[0] = Channel.of([ + [], + file( + params.modules_testdata_base_path + "genomics/sarscov2/genome/db/kraken2.tar.gz", + checkIfExists: true + ) + ]) + """ + } + } + } + + test("sarscov2 illumina single end [fastq]") { + when { + process { + """ + input[0] = [ + [ id:'test', single_end:true ], // meta map + [ file( + params.modules_testdata_base_path + "genomics/sarscov2/illumina/fastq/test_1.fastq.gz", + checkIfExists: true + )] + ] + input[1] = UNTAR.out.untar.map{ it[1] } + input[2] = true + input[3] = false + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot( + process.out.report, + process.out.versions, + ).match() + }, + { assert process.out.classified_reads_fastq.get(0).get(1) ==~ ".*/test.classified.fastq.gz" }, + { assert process.out.unclassified_reads_fastq.get(0).get(1) ==~ ".*/test.unclassified.fastq.gz" }, + ) + } + } + + test("sarscov2 illumina paired end [fastq]") { + when { + params { + outdir = "$outputDir" + } + + process { + """ + input[0] = [ + [ id:'test', single_end:false ], // meta map + [ + file( + params.modules_testdata_base_path + "genomics/sarscov2/illumina/fastq/test_1.fastq.gz", + checkIfExists: true + ), + file( + params.modules_testdata_base_path + "genomics/sarscov2/illumina/fastq/test_2.fastq.gz", + checkIfExists: true + ) + + ] + ] + input[1] = UNTAR.out.untar.map{ it[1] } + input[2] = true + input[3] = false + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot( + process.out.report, + process.out.versions, + ).match() + }, + { assert process.out.classified_reads_fastq.get(0).get(1).get(0) + ==~ ".*/test.classified_1.fastq.gz" }, + { assert process.out.classified_reads_fastq.get(0).get(1).get(1) + ==~ ".*/test.classified_2.fastq.gz" }, + { assert process.out.unclassified_reads_fastq.get(0).get(1).get(0) + ==~ ".*/test.unclassified_1.fastq.gz" }, + { assert process.out.unclassified_reads_fastq.get(0).get(1).get(1) + ==~ ".*/test.unclassified_2.fastq.gz" }, + ) + } + } + + test("sarscov2 illumina single end [fastq] + save_reads_assignment") { + when { + params { + outdir = "$outputDir" + } + + process { + """ + input[0] = [ + [ id:'test', single_end:true ], // meta map + [ file( + params.modules_testdata_base_path + "genomics/sarscov2/illumina/fastq/test_1.fastq.gz", + checkIfExists: true + )] + ] + input[1] = UNTAR.out.untar.map{ it[1] } + input[2] = false + input[3] = true + """ + } + } + + then { + assertAll( + { assert process.success }, + { assert snapshot( + process.out.report, + process.out.classified_reads_assignment, + process.out.versions, + ).match() + }, + ) + } + } +} diff --git a/modules/nf-core/kraken2/kraken2/tests/main.nf.test.snap b/modules/nf-core/kraken2/kraken2/tests/main.nf.test.snap new file mode 100644 index 000000000..b432f878f --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/tests/main.nf.test.snap @@ -0,0 +1,74 @@ +{ + "sarscov2 illumina single end [fastq]": { + "content": [ + [ + [ + { + "id": "test", + "single_end": true + }, + "test.kraken2.report.txt:md5,4227755fe40478b8d7dc8634b489761e" + ] + ], + [ + "versions.yml:md5,79adf2ca1cfc625cb77e391b27142c43" + ] + ], + "meta": { + "nf-test": "0.8.4", + "nextflow": "23.10.1" + }, + "timestamp": "2024-04-04T18:47:03.745692" + }, + "sarscov2 illumina paired end [fastq]": { + "content": [ + [ + [ + { + "id": "test", + "single_end": false + }, + "test.kraken2.report.txt:md5,4227755fe40478b8d7dc8634b489761e" + ] + ], + [ + "versions.yml:md5,79adf2ca1cfc625cb77e391b27142c43" + ] + ], + "meta": { + "nf-test": "0.8.4", + "nextflow": "23.10.1" + }, + "timestamp": "2024-04-04T18:47:13.75649" + }, + "sarscov2 illumina single end [fastq] + save_reads_assignment": { + "content": [ + [ + [ + { + "id": "test", + "single_end": true + }, + "test.kraken2.report.txt:md5,4227755fe40478b8d7dc8634b489761e" + ] + ], + [ + [ + { + "id": "test", + "single_end": true + }, + "test.kraken2.classifiedreads.txt:md5,e7a90531f0d8d777316515c36fe4cae0" + ] + ], + [ + "versions.yml:md5,79adf2ca1cfc625cb77e391b27142c43" + ] + ], + "meta": { + "nf-test": "0.8.4", + "nextflow": "23.10.1" + }, + "timestamp": "2024-04-04T18:47:22.459465" + } +} \ No newline at end of file diff --git a/modules/nf-core/kraken2/kraken2/tests/tags.yml b/modules/nf-core/kraken2/kraken2/tests/tags.yml new file mode 100644 index 000000000..9ebfd7ab6 --- /dev/null +++ b/modules/nf-core/kraken2/kraken2/tests/tags.yml @@ -0,0 +1,3 @@ +kraken2/kraken2: + - modules/nf-core/kraken2/kraken2/** + - modules/nf-core/untar/** diff --git a/nextflow.config b/nextflow.config index d5e00c6f4..94481109e 100644 --- a/nextflow.config +++ b/nextflow.config @@ -89,6 +89,11 @@ params { skip_preseq = true skip_dupradar = false skip_qualimap = false + contaminant_screening = null + kraken_db = null + save_kraken_assignments = false + save_kraken_unassigned = false + bracken_precision = "S" skip_rseqc = false skip_biotype_qc = false skip_deseq2_qc = false diff --git a/nextflow_schema.json b/nextflow_schema.json index d89b6398e..e7cd6d468 100644 --- a/nextflow_schema.json +++ b/nextflow_schema.json @@ -518,6 +518,18 @@ "fa_icon": "fas fa-save", "description": "Where possible, save unaligned reads from either STAR, HISAT2 or Salmon to the results directory.", "help_text": "This may either be in the form of FastQ or BAM files depending on the options available for that particular tool." + }, + "save_kraken_assignments": { + "type": "boolean", + "fa_icon": "fas fa-save", + "description": "Save read-by-read assignments from Kraken2.", + "help_text": "`--kraken_db` parameter must be provided." + }, + "save_kraken_unassigned": { + "type": "boolean", + "fa_icon": "fas fa-save", + "description": "Save reads that were not given assignment from Kraken2.", + "help_text": "`--kraken_db` parameter must be provided." } } }, @@ -539,6 +551,26 @@ "default": "bam_stat,inner_distance,infer_experiment,junction_annotation,junction_saturation,read_distribution,read_duplication", "fa_icon": "fas fa-chart-pie", "description": "Specify the RSeQC modules to run." + }, + "contaminant_screening": { + "type": "string", + "description": "Tool to use for detecting contaminants in unaligned reads - available options are 'kraken2' and 'kraken2_bracken'", + "fa_icon": "fas fa-virus-slash", + "enum": ["kraken2", "kraken2_bracken"] + }, + "kraken_db": { + "type": "string", + "description": "Database when using Kraken2/Bracken for contaminant screening.", + "help_text": "See the usage tab for more information", + "fa_icon": "fas fa-fish" + }, + "bracken_precision": { + "type": "string", + "default": "S", + "fa_icon": "fas fa-tree", + "description": "Taxonomic level for Bracken abundance estimations.", + "help_text": "First letter of Domain / Phylum / Class / Order / Family / Genus / Species", + "enum": ["D", "P", "C", "O", "F", "G", "S"] } } }, diff --git a/subworkflows/local/align_star/nextflow.config b/subworkflows/local/align_star/nextflow.config index 119eb6e4a..511ff6e63 100644 --- a/subworkflows/local/align_star/nextflow.config +++ b/subworkflows/local/align_star/nextflow.config @@ -22,7 +22,7 @@ if (!params.skip_alignment && params.aligner == 'star_salmon') { --outSAMattributes NH HI AS NM MD --quantTranscriptomeBan Singleend --outSAMstrandField intronMotif - ${params.save_unaligned ? '--outReadsUnmapped Fastx' : ''} + ${params.save_unaligned || params.contaminant_screening ? '--outReadsUnmapped Fastx' : ''} """.trim()) // Consolidate the extra arguments diff --git a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf index b2b890479..5d55ce0a8 100644 --- a/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf +++ b/subworkflows/local/utils_nfcore_rnaseq_pipeline/main.nf @@ -256,6 +256,29 @@ def validateInputParameters() { } } + //General checks for if contaminant screening is used + if (params.contaminant_screening) { + if (params.aligner == 'star_rsem') { + error("Contaminant screeneing cannot be done with --aligner star_rsem since unaligned reads are not saved. Please use --aligner star_salmon or --aligner hisat2.") + } + } + + // Check that Kraken/Bracken database provided if using kraken2/bracken + if (params.contaminant_screening in ['kraken2', 'kraken2_bracken']) { + if (!params.kraken_db) { + error("Contaminant screening set to kraken2 but not database is provided. Please provide a database with the --kraken_db option.") + } + //Check that Kraken/Bracken parameters are not provided when Kraken2 is not being used + } else { + if (!params.bracken_precision.equals('S')) { + brackenPrecisionWithoutKrakenDBWarn() + } + + if (params.save_kraken_assignments || params.save_kraken_unassigned || params.kraken_db) { + krakenArgumentsWithoutKrakenDBWarn() + } + } + // Check which RSeQC modules we are running def valid_rseqc_modules = ['bam_stat', 'inner_distance', 'infer_experiment', 'junction_annotation', 'junction_saturation', 'read_distribution', 'read_duplication', 'tin'] def rseqc_modules = params.rseqc_modules ? params.rseqc_modules.split(',').collect{ it.trim().toLowerCase() } : [] @@ -471,6 +494,26 @@ def additionaFastaIndexWarn(index) { "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" } +// +// Print a warning if --save_kraken_assignments or --save_kraken_unassigned is provided without --kraken_db +// +def krakenArgumentsWithoutKrakenDBWarn() { + log.warn "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n" + + " 'Kraken2 related arguments have been provided without setting contaminant\n" + + " screening to Kraken2. Kraken2 is not being run so these will not be used.\n" + + "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" +} + +/// +/// Print a warning if --bracken-precision is provided without --kraken_db +/// +def brackenPrecisionWithoutKrakenDBWarn() { + log.warn "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~\n" + + " '--bracken-precision' parameter has been provided without Kraken2 contaminant screening.\n" + + " Bracken will not run so precision will not be set.\n" + + "~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~" +} + // // Function to generate an error if contigs in genome fasta file > 512 Mbp // diff --git a/workflows/rnaseq/assets/multiqc/multiqc_config.yml b/workflows/rnaseq/assets/multiqc/multiqc_config.yml index ff154e1cb..63fad09f1 100644 --- a/workflows/rnaseq/assets/multiqc/multiqc_config.yml +++ b/workflows/rnaseq/assets/multiqc/multiqc_config.yml @@ -93,6 +93,8 @@ run_modules: - preseq - rseqc - qualimap + - kraken + - bracken # Order of modules @@ -156,6 +158,12 @@ sp: samtools/idxstats: fn: "*.idxstats*" + kraken: + fn: "*report.txt" + + bracken: + fn: "*.kraken2.report_bracken.txt" + rseqc/bam_stat: fn: "*.bam_stat.txt" rseqc/gene_body_coverage: diff --git a/workflows/rnaseq/main.nf b/workflows/rnaseq/main.nf index 2ee8384f4..8a85cf845 100755 --- a/workflows/rnaseq/main.nf +++ b/workflows/rnaseq/main.nf @@ -33,13 +33,15 @@ include { methodsDescriptionText } from '../../subworkflows/local/utils_ // // MODULE: Installed directly from nf-core/modules // -include { DUPRADAR } from '../../modules/nf-core/dupradar' -include { SAMTOOLS_SORT } from '../../modules/nf-core/samtools/sort' -include { PRESEQ_LCEXTRAP } from '../../modules/nf-core/preseq/lcextrap' -include { QUALIMAP_RNASEQ } from '../../modules/nf-core/qualimap/rnaseq' -include { STRINGTIE_STRINGTIE } from '../../modules/nf-core/stringtie/stringtie' -include { SUBREAD_FEATURECOUNTS } from '../../modules/nf-core/subread/featurecounts' -include { MULTIQC } from '../../modules/nf-core/multiqc' +include { DUPRADAR } from '../../modules/nf-core/dupradar' +include { SAMTOOLS_SORT } from '../../modules/nf-core/samtools/sort' +include { PRESEQ_LCEXTRAP } from '../../modules/nf-core/preseq/lcextrap' +include { QUALIMAP_RNASEQ } from '../../modules/nf-core/qualimap/rnaseq' +include { STRINGTIE_STRINGTIE } from '../../modules/nf-core/stringtie/stringtie' +include { SUBREAD_FEATURECOUNTS } from '../../modules/nf-core/subread/featurecounts' +include { KRAKEN2_KRAKEN2 as KRAKEN2 } from '../../modules/nf-core/kraken2/kraken2/main' +include { BRACKEN_BRACKEN as BRACKEN } from '../../modules/nf-core/bracken/bracken/main' +include { MULTIQC } from '../../modules/nf-core/multiqc' include { UMITOOLS_PREPAREFORRSEM as UMITOOLS_PREPAREFORSALMON } from '../../modules/nf-core/umitools/prepareforrsem' include { BEDTOOLS_GENOMECOV as BEDTOOLS_GENOMECOV_FW } from '../../modules/nf-core/bedtools/genomecov' include { BEDTOOLS_GENOMECOV as BEDTOOLS_GENOMECOV_REV } from '../../modules/nf-core/bedtools/genomecov' @@ -171,9 +173,10 @@ workflow RNASEQ { // // SUBWORKFLOW: Alignment with STAR and gene/transcript quantification with Salmon // - ch_genome_bam = Channel.empty() - ch_genome_bam_index = Channel.empty() - ch_star_log = Channel.empty() + ch_genome_bam = Channel.empty() + ch_genome_bam_index = Channel.empty() + ch_star_log = Channel.empty() + ch_unaligned_sequences = Channel.empty() if (!params.skip_alignment && params.aligner == 'star_salmon') { // Check if an AWS iGenome has been provided to use the appropriate version of STAR def is_aws_igenome = false @@ -193,10 +196,11 @@ workflow RNASEQ { is_aws_igenome, ch_fasta.map { [ [:], it ] } ) - ch_genome_bam = ALIGN_STAR.out.bam - ch_genome_bam_index = ALIGN_STAR.out.bai - ch_transcriptome_bam = ALIGN_STAR.out.bam_transcript - ch_star_log = ALIGN_STAR.out.log_final + ch_genome_bam = ALIGN_STAR.out.bam + ch_genome_bam_index = ALIGN_STAR.out.bai + ch_transcriptome_bam = ALIGN_STAR.out.bam_transcript + ch_star_log = ALIGN_STAR.out.log_final + ch_unaligned_sequences = ALIGN_STAR.out.fastq ch_multiqc_files = ch_multiqc_files.mix(ALIGN_STAR.out.stats.collect{it[1]}) ch_multiqc_files = ch_multiqc_files.mix(ALIGN_STAR.out.flagstat.collect{it[1]}) ch_multiqc_files = ch_multiqc_files.mix(ALIGN_STAR.out.idxstats.collect{it[1]}) @@ -350,8 +354,9 @@ workflow RNASEQ { ch_splicesites.map { [ [:], it ] }, ch_fasta.map { [ [:], it ] } ) - ch_genome_bam = FASTQ_ALIGN_HISAT2.out.bam - ch_genome_bam_index = FASTQ_ALIGN_HISAT2.out.bai + ch_genome_bam = FASTQ_ALIGN_HISAT2.out.bam + ch_genome_bam_index = FASTQ_ALIGN_HISAT2.out.bai + ch_unaligned_sequences = FASTQ_ALIGN_HISAT2.out.fastq ch_multiqc_files = ch_multiqc_files.mix(FASTQ_ALIGN_HISAT2.out.stats.collect{it[1]}) ch_multiqc_files = ch_multiqc_files.mix(FASTQ_ALIGN_HISAT2.out.flagstat.collect{it[1]}) ch_multiqc_files = ch_multiqc_files.mix(FASTQ_ALIGN_HISAT2.out.idxstats.collect{it[1]}) @@ -647,6 +652,28 @@ workflow RNASEQ { ch_multiqc_files = ch_multiqc_files.mix(ch_fail_strand_multiqc.collectFile(name: 'fail_strand_check_mqc.tsv')) } + + if (params.contaminant_screening in ['kraken2', 'kraken2_bracken'] ) { + KRAKEN2 ( + ch_unaligned_sequences, + params.kraken_db, + params.save_kraken_assignments, + params.save_kraken_unassigned + ) + ch_kraken_reports = KRAKEN2.out.report + ch_versions = ch_versions.mix(KRAKEN2.out.versions) + + if (params.contaminant_screening == 'kraken2') { + ch_multiqc_files = ch_multiqc_files.mix(KRAKEN2.out.report.collect{it[1]}) + } else if (params.contaminant_screening == 'kraken2_bracken') { + BRACKEN ( + ch_kraken_reports, + params.kraken_db + ) + ch_versions = ch_versions.mix(BRACKEN.out.versions) + ch_multiqc_files = ch_multiqc_files.mix(BRACKEN.out.txt.collect{it[1]}) + } + } } // diff --git a/workflows/rnaseq/nextflow.config b/workflows/rnaseq/nextflow.config index c7eeac733..9cbf0cd30 100644 --- a/workflows/rnaseq/nextflow.config +++ b/workflows/rnaseq/nextflow.config @@ -8,6 +8,8 @@ includeConfig "../../modules/nf-core/qualimap/rnaseq/nextflow.config" includeConfig "../../modules/nf-core/sortmerna/nextflow.config" includeConfig "../../modules/nf-core/stringtie/stringtie/nextflow.config" includeConfig "../../modules/nf-core/subread/featurecounts/nextflow.config" +includeConfig "../../modules/nf-core/kraken2/kraken2/nextflow.config" +includeConfig "../../modules/nf-core/bracken/bracken/nextflow.config" includeConfig "../../subworkflows/local/align_star/nextflow.config" includeConfig "../../subworkflows/local/quantify_rsem/nextflow.config" includeConfig "../../subworkflows/nf-core/quantify_pseudo_alignment/nextflow.config" From 41bcd9c431f6c64387169ccb059c0c0a3dfbd152 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Thu, 19 Sep 2024 11:51:03 -0400 Subject: [PATCH 34/36] Update hisat2 patch --- modules.json | 286 +++++++++++++----- .../nf-core/hisat2/align/hisat2-align.diff | 9 +- 2 files changed, 223 insertions(+), 72 deletions(-) diff --git a/modules.json b/modules.json index 4c9ba76f1..82e0e34be 100644 --- a/modules.json +++ b/modules.json @@ -8,189 +8,272 @@ "bbmap/bbsplit": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["fastq_qc_trim_filter_setstrandedness", "modules"] + "installed_by": [ + "fastq_qc_trim_filter_setstrandedness", + "modules" + ] }, "bedtools/genomecov": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "bracken/bracken": { "branch": "master", "git_sha": "c214fad97b328eb6d6233f779be9ba44814a9136", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "cat/fastq": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["fastq_qc_trim_filter_setstrandedness", "modules"] + "installed_by": [ + "fastq_qc_trim_filter_setstrandedness", + "modules" + ] }, "custom/catadditionalfasta": { "branch": "master", "git_sha": "e743b2dea725bcfc4b76a209981808987332013a", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "custom/getchromsizes": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "custom/tx2gene": { "branch": "master", "git_sha": "e743b2dea725bcfc4b76a209981808987332013a", - "installed_by": ["modules", "quantify_pseudo_alignment"] + "installed_by": [ + "modules", + "quantify_pseudo_alignment" + ] }, "dupradar": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "fastp": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["fastq_fastqc_umitools_fastp", "modules"] + "installed_by": [ + "fastq_fastqc_umitools_fastp", + "modules" + ] }, "fastqc": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["fastq_fastqc_umitools_fastp", "fastq_fastqc_umitools_trimgalore"] + "installed_by": [ + "fastq_fastqc_umitools_fastp", + "fastq_fastqc_umitools_trimgalore" + ] }, "fq/subsample": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["fastq_subsample_fq_salmon", "modules"] + "installed_by": [ + "fastq_subsample_fq_salmon", + "modules" + ] }, "gffread": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "gunzip": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "hisat2/align": { "branch": "master", "git_sha": "ad30f90cfc383dfaa505771d24f9e292c53157ab", - "installed_by": ["fastq_align_hisat2"], + "installed_by": [ + "fastq_align_hisat2" + ], "patch": "modules/nf-core/hisat2/align/hisat2-align.diff" }, "hisat2/build": { "branch": "master", "git_sha": "ad30f90cfc383dfaa505771d24f9e292c53157ab", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "hisat2/extractsplicesites": { "branch": "master", "git_sha": "ad30f90cfc383dfaa505771d24f9e292c53157ab", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "kallisto/index": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "kallisto/quant": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules", "quantify_pseudo_alignment"] + "installed_by": [ + "modules", + "quantify_pseudo_alignment" + ] }, "kraken2/kraken2": { "branch": "master", "git_sha": "a13d5d945742a60bbef6e5c177e81cda540f75dc", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "multiqc": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "picard/markduplicates": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_markduplicates_picard"], + "installed_by": [ + "bam_markduplicates_picard" + ], "patch": "modules/nf-core/picard/markduplicates/picard-markduplicates.diff" }, "preseq/lcextrap": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "qualimap/rnaseq": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "rsem/calculateexpression": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "rsem/preparereference": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "rseqc/bamstat": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_rseqc"] + "installed_by": [ + "bam_rseqc" + ] }, "rseqc/inferexperiment": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_rseqc"] + "installed_by": [ + "bam_rseqc" + ] }, "rseqc/innerdistance": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_rseqc"] + "installed_by": [ + "bam_rseqc" + ] }, "rseqc/junctionannotation": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_rseqc"] + "installed_by": [ + "bam_rseqc" + ] }, "rseqc/junctionsaturation": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_rseqc"] + "installed_by": [ + "bam_rseqc" + ] }, "rseqc/readdistribution": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_rseqc"] + "installed_by": [ + "bam_rseqc" + ] }, "rseqc/readduplication": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_rseqc"] + "installed_by": [ + "bam_rseqc" + ] }, "rseqc/tin": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_rseqc"] + "installed_by": [ + "bam_rseqc" + ] }, "salmon/index": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["fastq_subsample_fq_salmon"] + "installed_by": [ + "fastq_subsample_fq_salmon" + ] }, "salmon/quant": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["fastq_subsample_fq_salmon", "modules", "quantify_pseudo_alignment"] + "installed_by": [ + "fastq_subsample_fq_salmon", + "modules", + "quantify_pseudo_alignment" + ] }, "samtools/flagstat": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_stats_samtools"] + "installed_by": [ + "bam_stats_samtools" + ] }, "samtools/idxstats": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_stats_samtools"] + "installed_by": [ + "bam_stats_samtools" + ] }, "samtools/index": { "branch": "master", @@ -204,82 +287,118 @@ "samtools/sort": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_sort_stats_samtools"] + "installed_by": [ + "bam_sort_stats_samtools" + ] }, "samtools/stats": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_stats_samtools"] + "installed_by": [ + "bam_stats_samtools" + ] }, "sortmerna": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["fastq_qc_trim_filter_setstrandedness", "modules"] + "installed_by": [ + "fastq_qc_trim_filter_setstrandedness", + "modules" + ] }, "star/align": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "star/genomegenerate": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "stringtie/stringtie": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "subread/featurecounts": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "summarizedexperiment/summarizedexperiment": { "branch": "master", "git_sha": "625fbbc0531fb1717349c7f1b1c677a3b8619767", - "installed_by": ["modules", "quantify_pseudo_alignment"] + "installed_by": [ + "modules", + "quantify_pseudo_alignment" + ] }, "trimgalore": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["fastq_fastqc_umitools_trimgalore"] + "installed_by": [ + "fastq_fastqc_umitools_trimgalore" + ] }, "tximeta/tximport": { "branch": "master", "git_sha": "625fbbc0531fb1717349c7f1b1c677a3b8619767", - "installed_by": ["modules", "quantify_pseudo_alignment"] + "installed_by": [ + "modules", + "quantify_pseudo_alignment" + ] }, "ucsc/bedclip": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bedgraph_bedclip_bedgraphtobigwig"] + "installed_by": [ + "bedgraph_bedclip_bedgraphtobigwig" + ] }, "ucsc/bedgraphtobigwig": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bedgraph_bedclip_bedgraphtobigwig"] + "installed_by": [ + "bedgraph_bedclip_bedgraphtobigwig" + ] }, "umitools/dedup": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["bam_dedup_stats_samtools_umitools"] + "installed_by": [ + "bam_dedup_stats_samtools_umitools" + ] }, "umitools/extract": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["fastq_fastqc_umitools_fastp", "fastq_fastqc_umitools_trimgalore"] + "installed_by": [ + "fastq_fastqc_umitools_fastp", + "fastq_fastqc_umitools_trimgalore" + ] }, "umitools/prepareforrsem": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] }, "untar": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": ["modules"] + "installed_by": [ + "modules" + ] } } }, @@ -288,22 +407,30 @@ "bam_dedup_stats_samtools_umitools": { "branch": "master", "git_sha": "46eca555142d6e597729fcb682adcc791796f514", - "installed_by": ["subworkflows"] + "installed_by": [ + "subworkflows" + ] }, "bam_markduplicates_picard": { "branch": "master", "git_sha": "46eca555142d6e597729fcb682adcc791796f514", - "installed_by": ["subworkflows"] + "installed_by": [ + "subworkflows" + ] }, "bam_rseqc": { "branch": "master", "git_sha": "0eacd714effe5aac1c1de26593873960b3346cab", - "installed_by": ["subworkflows"] + "installed_by": [ + "subworkflows" + ] }, "bam_sort_stats_samtools": { "branch": "master", "git_sha": "46eca555142d6e597729fcb682adcc791796f514", - "installed_by": ["fastq_align_hisat2"] + "installed_by": [ + "fastq_align_hisat2" + ] }, "bam_stats_samtools": { "branch": "master", @@ -317,55 +444,78 @@ "bedgraph_bedclip_bedgraphtobigwig": { "branch": "master", "git_sha": "0eacd714effe5aac1c1de26593873960b3346cab", - "installed_by": ["subworkflows"] + "installed_by": [ + "subworkflows" + ] }, "fastq_align_hisat2": { "branch": "master", "git_sha": "c789476080a150f87066ca3ed42a622339a26c0b", - "installed_by": ["subworkflows"] + "installed_by": [ + "subworkflows" + ] }, "fastq_fastqc_umitools_fastp": { "branch": "master", "git_sha": "46eca555142d6e597729fcb682adcc791796f514", - "installed_by": ["fastq_qc_trim_filter_setstrandedness", "subworkflows"] + "installed_by": [ + "fastq_qc_trim_filter_setstrandedness", + "subworkflows" + ] }, "fastq_fastqc_umitools_trimgalore": { "branch": "master", "git_sha": "b49b8992e5271ce427f3a7cdb29628fc58400eb5", - "installed_by": ["fastq_qc_trim_filter_setstrandedness", "subworkflows"] + "installed_by": [ + "fastq_qc_trim_filter_setstrandedness", + "subworkflows" + ] }, "fastq_qc_trim_filter_setstrandedness": { "branch": "master", "git_sha": "ee3b8b84da02320fff5f3f2dc1d6f9b72d7cd5af", - "installed_by": ["subworkflows"] + "installed_by": [ + "subworkflows" + ] }, "fastq_subsample_fq_salmon": { "branch": "master", "git_sha": "fbe341e9af0bb98533757f536b26d38507d31724", - "installed_by": ["fastq_qc_trim_filter_setstrandedness", "subworkflows"] + "installed_by": [ + "fastq_qc_trim_filter_setstrandedness", + "subworkflows" + ] }, "quantify_pseudo_alignment": { "branch": "master", "git_sha": "fbe341e9af0bb98533757f536b26d38507d31724", - "installed_by": ["subworkflows"] + "installed_by": [ + "subworkflows" + ] }, "utils_nextflow_pipeline": { "branch": "master", "git_sha": "d20fb2a9cc3e2835e9d067d1046a63252eb17352", - "installed_by": ["subworkflows"] + "installed_by": [ + "subworkflows" + ] }, "utils_nfcore_pipeline": { "branch": "master", "git_sha": "2fdce49d30c0254f76bc0f13c55c17455c1251ab", - "installed_by": ["subworkflows"] + "installed_by": [ + "subworkflows" + ] }, "utils_nfvalidation_plugin": { "branch": "master", "git_sha": "5caf7640a9ef1d18d765d55339be751bb0969dfa", - "installed_by": ["subworkflows"] + "installed_by": [ + "subworkflows" + ] } } } } } -} +} \ No newline at end of file diff --git a/modules/nf-core/hisat2/align/hisat2-align.diff b/modules/nf-core/hisat2/align/hisat2-align.diff index 6086520f6..2a64e4f4f 100644 --- a/modules/nf-core/hisat2/align/hisat2-align.diff +++ b/modules/nf-core/hisat2/align/hisat2-align.diff @@ -1,7 +1,7 @@ Changes in module 'nf-core/hisat2/align' --- modules/nf-core/hisat2/align/main.nf +++ modules/nf-core/hisat2/align/main.nf -@@ -36,7 +36,7 @@ +@@ -34,7 +34,7 @@ ss = "$splicesites" ? "--known-splicesite-infile $splicesites" : '' def seq_center = params.seq_center ? "--rg-id ${prefix} --rg SM:$prefix --rg CN:${params.seq_center.replaceAll('\\s','_')}" : "--rg-id ${prefix} --rg SM:$prefix" if (meta.single_end) { @@ -10,7 +10,7 @@ Changes in module 'nf-core/hisat2/align' """ INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'` hisat2 \\ -@@ -58,7 +58,7 @@ +@@ -56,7 +56,7 @@ END_VERSIONS """ } else { @@ -19,15 +19,16 @@ Changes in module 'nf-core/hisat2/align' """ INDEX=`find -L ./ -name "*.1.ht2" | sed 's/\\.1.ht2\$//'` hisat2 \\ -@@ -93,7 +93,7 @@ +@@ -91,7 +91,8 @@ stub: def prefix = task.ext.prefix ?: "${meta.id}" - def unaligned = params.save_unaligned ? "echo '' | gzip > ${prefix}.unmapped_1.fastq.gz \n echo '' | gzip > ${prefix}.unmapped_2.fastq.gz" : '' + def unaligned = params.save_unaligned || params.contaminant_screening ? "echo '' | gzip > ${prefix}.unmapped_1.fastq.gz \n echo '' | gzip > ${prefix}.unmapped_2.fastq.gz" : '' - def VERSION = '2.2.1' // WARN: Version information not provided by tool on CLI. Please update this string when bumping container versions. ++ def VERSION = '2.2.1' // WARN: Version information not provided by tool on CLI. Please update this string when bumping container versions. """ ${unaligned} + --- modules/nf-core/hisat2/align/tests/main.nf.test +++ modules/nf-core/hisat2/align/tests/main.nf.test From 3e0b3e96de08e740646d20bdf08c79ebde4b2c60 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Thu, 19 Sep 2024 11:52:30 -0400 Subject: [PATCH 35/36] (Hopefully) final linting fix --- modules.json | 286 ++++++++++++--------------------------------------- 1 file changed, 68 insertions(+), 218 deletions(-) diff --git a/modules.json b/modules.json index 82e0e34be..4c9ba76f1 100644 --- a/modules.json +++ b/modules.json @@ -8,272 +8,189 @@ "bbmap/bbsplit": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "fastq_qc_trim_filter_setstrandedness", - "modules" - ] + "installed_by": ["fastq_qc_trim_filter_setstrandedness", "modules"] }, "bedtools/genomecov": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "bracken/bracken": { "branch": "master", "git_sha": "c214fad97b328eb6d6233f779be9ba44814a9136", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "cat/fastq": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "fastq_qc_trim_filter_setstrandedness", - "modules" - ] + "installed_by": ["fastq_qc_trim_filter_setstrandedness", "modules"] }, "custom/catadditionalfasta": { "branch": "master", "git_sha": "e743b2dea725bcfc4b76a209981808987332013a", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "custom/getchromsizes": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "custom/tx2gene": { "branch": "master", "git_sha": "e743b2dea725bcfc4b76a209981808987332013a", - "installed_by": [ - "modules", - "quantify_pseudo_alignment" - ] + "installed_by": ["modules", "quantify_pseudo_alignment"] }, "dupradar": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "fastp": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "fastq_fastqc_umitools_fastp", - "modules" - ] + "installed_by": ["fastq_fastqc_umitools_fastp", "modules"] }, "fastqc": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "fastq_fastqc_umitools_fastp", - "fastq_fastqc_umitools_trimgalore" - ] + "installed_by": ["fastq_fastqc_umitools_fastp", "fastq_fastqc_umitools_trimgalore"] }, "fq/subsample": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "fastq_subsample_fq_salmon", - "modules" - ] + "installed_by": ["fastq_subsample_fq_salmon", "modules"] }, "gffread": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "gunzip": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "hisat2/align": { "branch": "master", "git_sha": "ad30f90cfc383dfaa505771d24f9e292c53157ab", - "installed_by": [ - "fastq_align_hisat2" - ], + "installed_by": ["fastq_align_hisat2"], "patch": "modules/nf-core/hisat2/align/hisat2-align.diff" }, "hisat2/build": { "branch": "master", "git_sha": "ad30f90cfc383dfaa505771d24f9e292c53157ab", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "hisat2/extractsplicesites": { "branch": "master", "git_sha": "ad30f90cfc383dfaa505771d24f9e292c53157ab", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "kallisto/index": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "kallisto/quant": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules", - "quantify_pseudo_alignment" - ] + "installed_by": ["modules", "quantify_pseudo_alignment"] }, "kraken2/kraken2": { "branch": "master", "git_sha": "a13d5d945742a60bbef6e5c177e81cda540f75dc", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "multiqc": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "picard/markduplicates": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_markduplicates_picard" - ], + "installed_by": ["bam_markduplicates_picard"], "patch": "modules/nf-core/picard/markduplicates/picard-markduplicates.diff" }, "preseq/lcextrap": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "qualimap/rnaseq": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "rsem/calculateexpression": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "rsem/preparereference": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "rseqc/bamstat": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_rseqc" - ] + "installed_by": ["bam_rseqc"] }, "rseqc/inferexperiment": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_rseqc" - ] + "installed_by": ["bam_rseqc"] }, "rseqc/innerdistance": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_rseqc" - ] + "installed_by": ["bam_rseqc"] }, "rseqc/junctionannotation": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_rseqc" - ] + "installed_by": ["bam_rseqc"] }, "rseqc/junctionsaturation": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_rseqc" - ] + "installed_by": ["bam_rseqc"] }, "rseqc/readdistribution": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_rseqc" - ] + "installed_by": ["bam_rseqc"] }, "rseqc/readduplication": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_rseqc" - ] + "installed_by": ["bam_rseqc"] }, "rseqc/tin": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_rseqc" - ] + "installed_by": ["bam_rseqc"] }, "salmon/index": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "fastq_subsample_fq_salmon" - ] + "installed_by": ["fastq_subsample_fq_salmon"] }, "salmon/quant": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "fastq_subsample_fq_salmon", - "modules", - "quantify_pseudo_alignment" - ] + "installed_by": ["fastq_subsample_fq_salmon", "modules", "quantify_pseudo_alignment"] }, "samtools/flagstat": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_stats_samtools" - ] + "installed_by": ["bam_stats_samtools"] }, "samtools/idxstats": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_stats_samtools" - ] + "installed_by": ["bam_stats_samtools"] }, "samtools/index": { "branch": "master", @@ -287,118 +204,82 @@ "samtools/sort": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_sort_stats_samtools" - ] + "installed_by": ["bam_sort_stats_samtools"] }, "samtools/stats": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_stats_samtools" - ] + "installed_by": ["bam_stats_samtools"] }, "sortmerna": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "fastq_qc_trim_filter_setstrandedness", - "modules" - ] + "installed_by": ["fastq_qc_trim_filter_setstrandedness", "modules"] }, "star/align": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "star/genomegenerate": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "stringtie/stringtie": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "subread/featurecounts": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "summarizedexperiment/summarizedexperiment": { "branch": "master", "git_sha": "625fbbc0531fb1717349c7f1b1c677a3b8619767", - "installed_by": [ - "modules", - "quantify_pseudo_alignment" - ] + "installed_by": ["modules", "quantify_pseudo_alignment"] }, "trimgalore": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "fastq_fastqc_umitools_trimgalore" - ] + "installed_by": ["fastq_fastqc_umitools_trimgalore"] }, "tximeta/tximport": { "branch": "master", "git_sha": "625fbbc0531fb1717349c7f1b1c677a3b8619767", - "installed_by": [ - "modules", - "quantify_pseudo_alignment" - ] + "installed_by": ["modules", "quantify_pseudo_alignment"] }, "ucsc/bedclip": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bedgraph_bedclip_bedgraphtobigwig" - ] + "installed_by": ["bedgraph_bedclip_bedgraphtobigwig"] }, "ucsc/bedgraphtobigwig": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bedgraph_bedclip_bedgraphtobigwig" - ] + "installed_by": ["bedgraph_bedclip_bedgraphtobigwig"] }, "umitools/dedup": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "bam_dedup_stats_samtools_umitools" - ] + "installed_by": ["bam_dedup_stats_samtools_umitools"] }, "umitools/extract": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "fastq_fastqc_umitools_fastp", - "fastq_fastqc_umitools_trimgalore" - ] + "installed_by": ["fastq_fastqc_umitools_fastp", "fastq_fastqc_umitools_trimgalore"] }, "umitools/prepareforrsem": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] }, "untar": { "branch": "master", "git_sha": "06c8865e36741e05ad32ef70ab3fac127486af48", - "installed_by": [ - "modules" - ] + "installed_by": ["modules"] } } }, @@ -407,30 +288,22 @@ "bam_dedup_stats_samtools_umitools": { "branch": "master", "git_sha": "46eca555142d6e597729fcb682adcc791796f514", - "installed_by": [ - "subworkflows" - ] + "installed_by": ["subworkflows"] }, "bam_markduplicates_picard": { "branch": "master", "git_sha": "46eca555142d6e597729fcb682adcc791796f514", - "installed_by": [ - "subworkflows" - ] + "installed_by": ["subworkflows"] }, "bam_rseqc": { "branch": "master", "git_sha": "0eacd714effe5aac1c1de26593873960b3346cab", - "installed_by": [ - "subworkflows" - ] + "installed_by": ["subworkflows"] }, "bam_sort_stats_samtools": { "branch": "master", "git_sha": "46eca555142d6e597729fcb682adcc791796f514", - "installed_by": [ - "fastq_align_hisat2" - ] + "installed_by": ["fastq_align_hisat2"] }, "bam_stats_samtools": { "branch": "master", @@ -444,78 +317,55 @@ "bedgraph_bedclip_bedgraphtobigwig": { "branch": "master", "git_sha": "0eacd714effe5aac1c1de26593873960b3346cab", - "installed_by": [ - "subworkflows" - ] + "installed_by": ["subworkflows"] }, "fastq_align_hisat2": { "branch": "master", "git_sha": "c789476080a150f87066ca3ed42a622339a26c0b", - "installed_by": [ - "subworkflows" - ] + "installed_by": ["subworkflows"] }, "fastq_fastqc_umitools_fastp": { "branch": "master", "git_sha": "46eca555142d6e597729fcb682adcc791796f514", - "installed_by": [ - "fastq_qc_trim_filter_setstrandedness", - "subworkflows" - ] + "installed_by": ["fastq_qc_trim_filter_setstrandedness", "subworkflows"] }, "fastq_fastqc_umitools_trimgalore": { "branch": "master", "git_sha": "b49b8992e5271ce427f3a7cdb29628fc58400eb5", - "installed_by": [ - "fastq_qc_trim_filter_setstrandedness", - "subworkflows" - ] + "installed_by": ["fastq_qc_trim_filter_setstrandedness", "subworkflows"] }, "fastq_qc_trim_filter_setstrandedness": { "branch": "master", "git_sha": "ee3b8b84da02320fff5f3f2dc1d6f9b72d7cd5af", - "installed_by": [ - "subworkflows" - ] + "installed_by": ["subworkflows"] }, "fastq_subsample_fq_salmon": { "branch": "master", "git_sha": "fbe341e9af0bb98533757f536b26d38507d31724", - "installed_by": [ - "fastq_qc_trim_filter_setstrandedness", - "subworkflows" - ] + "installed_by": ["fastq_qc_trim_filter_setstrandedness", "subworkflows"] }, "quantify_pseudo_alignment": { "branch": "master", "git_sha": "fbe341e9af0bb98533757f536b26d38507d31724", - "installed_by": [ - "subworkflows" - ] + "installed_by": ["subworkflows"] }, "utils_nextflow_pipeline": { "branch": "master", "git_sha": "d20fb2a9cc3e2835e9d067d1046a63252eb17352", - "installed_by": [ - "subworkflows" - ] + "installed_by": ["subworkflows"] }, "utils_nfcore_pipeline": { "branch": "master", "git_sha": "2fdce49d30c0254f76bc0f13c55c17455c1251ab", - "installed_by": [ - "subworkflows" - ] + "installed_by": ["subworkflows"] }, "utils_nfvalidation_plugin": { "branch": "master", "git_sha": "5caf7640a9ef1d18d765d55339be751bb0969dfa", - "installed_by": [ - "subworkflows" - ] + "installed_by": ["subworkflows"] } } } } } -} \ No newline at end of file +} From 02f65ab01b6f911516ae629762b7f9a81e6e0e17 Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Thu, 19 Sep 2024 12:15:03 -0400 Subject: [PATCH 36/36] Change PR number --- CHANGELOG.md | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/CHANGELOG.md b/CHANGELOG.md index e9c90c34c..6558baf06 100644 --- a/CHANGELOG.md +++ b/CHANGELOG.md @@ -7,7 +7,7 @@ and this project adheres to [Semantic Versioning](https://semver.org/spec/v2.0.0 ### Enhancements & fixes -- [PR #1351](https://github.com/nf-core/rnaseq/pull/1351) - Adding Kraken2/Bracken on unaligned reads as an additional quality control step to detect sample contamination +- [PR #1388](https://github.com/nf-core/rnaseq/pull/1351) - Adding Kraken2/Bracken on unaligned reads as an additional quality control step to detect sample contamination - [PR #1186](https://github.com/nf-core/rnaseq/pull/1186) - Bump pipeline version to 3.16.0dev ### Parameters

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zR}&jm{kHrg-P@Ua{y*RHwe`*a<1P5}EyLUV_xb<#r)=2J@8AEwkNHQ7|3AO2X}$mV iA^&?e|M%U=@^uYkzvlR?pMOZ<%h+hb$P**1H~cTIV!x&U diff --git a/docs/images/nf-core-rnaseq_metro_map_grey.svg b/docs/images/nf-core-rnaseq_metro_map_grey.svg index 46be07297..2eb57fe62 100644 --- a/docs/images/nf-core-rnaseq_metro_map_grey.svg +++ b/docs/images/nf-core-rnaseq_metro_map_grey.svg @@ -7,7 +7,7 @@ viewBox="0 0 646.4851 269.92565" version="1.1" id="svg8" - inkscape:version="1.3.2 (1:1.3.2+202311252150+091e20ef0f)" + inkscape:version="1.3.2 (091e20e, 2023-11-25)" sodipodi:docname="nf-core-rnaseq_metro_map_grey.svg" inkscape:export-filename="nf-core-rnaseq_metro_map_grey.png" inkscape:export-xdpi="89" @@ -21,7 +21,22 @@ xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:cc="http://creativecommons.org/ns#" xmlns:dc="http://purl.org/dc/elements/1.1/">catcatfastqfastqsubsamplefastq(fq)5RSeQC(multiple modules)PreseqdupRadarKraken2/BrackenDESeq2(PCA only)MultiQCQualimaprnaseqrnaseqSortMeRNAFastQCinferstrandedness(fq, Salmon)(Salmon)BBSplit Date: Mon, 19 Aug 2024 11:59:33 -0400 Subject: [PATCH 27/36] Update schema --- nextflow_schema.json | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/nextflow_schema.json b/nextflow_schema.json index b524e762f..a8c29c97f 100644 --- a/nextflow_schema.json +++ b/nextflow_schema.json @@ -561,7 +561,7 @@ "kraken_db": { "type": "string", "description": "Database when using Kraken2/Bracken for contaminant screening.", - "help_text": "See [pre-built Kraken2/Bracken databases](https://benlangmead.github.io/aws-indexes/k2).", + "help_text": "See the usage tab for more information", "fa_icon": "fas fa-fish" }, "bracken_precision": { @@ -569,6 +569,7 @@ "default": "S", "fa_icon": "fas fa-tree", "description": "Taxonomic level for Bracken abundance estimations.", + "help_text": "First letter of Domain / Phylum / Class / Order / Family / Genus / Species", "enum": ["D", "P", "C", "O", "F", "G", "S"] } } From 75a10f7f119da07108563a79c15efcc9596d9ae9 Mon Sep 17 00:00:00 2001 From: Ezra Greenberg <120955867+egreenberg7@users.noreply.github.com> Date: Mon, 19 Aug 2024 12:01:54 -0400 Subject: [PATCH 28/36] Update docs/usage.md Co-authored-by: Matthias Zepper <6963520+MatthiasZepper@users.noreply.github.com> --- docs/usage.md | 6 +++++- 1 file changed, 5 insertions(+), 1 deletion(-) diff --git a/docs/usage.md b/docs/usage.md index a86387066..cd42a399b 100644 --- a/docs/usage.md +++ b/docs/usage.md @@ -298,7 +298,11 @@ By default, the input GTF file will be filtered to ensure that sequence names co ## Contamination screening options -The pipeline provides the option to scan unaligned reads for contamination from other species by using [Kraken2](https://ccb.jhu.edu/software/kraken2/) with or without corrections from [Bracken](https://ccb.jhu.edu/software/bracken/). As Bracken is not a particularly expensive algorithm, we recommend using it to correct the abundance estimations from Kraken. An important note is that Kraken2 is [sensitive to the database](https://doi.org/10.1099/mgen.0.000949) that it is used with. It is [particularly important](https://doi.org/10.1128/mbio.01607-23) to ensure that the host genome is part of the database, and if you are particularly concerned about specific contaminants, it may be worthwhile to use a smaller database that contains primarily those contaminants rather than the full standard database. Various pre-built databases can be found [here](https://benlangmead.github.io/aws-indexes/k2) and instructions for building a custom database can be found at the [Kraken2 documentation](https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown). Genomes of contaminants detected in previous sequencing experiments can be found on the [OpenContami website](https://openlooper.hgc.jp/opencontami/help/help_oct.php). Additionally, note that while one of the primary strengths of Kraken2 is that it can detect low abundance contaminants in a sample, false positives can also occur. If a very low number of reads of some contaminating species is detected, these results should be treated with caution. +The pipeline provides the option to scan unaligned reads for contamination from other species using [Kraken2](https://ccb.jhu.edu/software/kraken2/), with the possibility of applying corrections from [Bracken](https://ccb.jhu.edu/software/bracken/). Since running Bracken is not computationally expensive, we recommend always using it to refine the abundance estimates generated by Kraken2. + +It is important to note that the accuracy of Kraken2 is [highly dependent on the database](https://doi.org/10.1099/mgen.0.000949) used. Specifically, it is [crucial](https://doi.org/10.1128/mbio.01607-23) to ensure that the host genome is included in the database. If you are particularly concerned about certain contaminants, it may be beneficial to use a smaller, more focused database containing primarily those contaminants instead of the full standard database. Various pre-built databases [are available for download](https://benlangmead.github.io/aws-indexes/k2), and instructions for building a custom database can be found in the [Kraken2 documentation](https://github.com/DerrickWood/kraken2/blob/master/docs/MANUAL.markdown). Additionally, genomes of contaminants detected in previous sequencing experiments are available on the [OpenContami website](https://openlooper.hgc.jp/opencontami/help/help_oct.php). + +While Kraken2 is capable of detecting low-abundance contaminants in a sample, false positives can occur. Therefore, if only a very small number of reads from a contaminating species are detected, these results should be interpreted with caution. ## Running the pipeline From cfc89458ad3ab931eb0dfd313a2d19dcf61308bb Mon Sep 17 00:00:00 2001 From: egreenberg7 Date: Mon, 19 Aug 2024 12:42:26 -0400 Subject: [PATCH 29/36] Change output directory for kraken2/bracken --- docs/output.md | 4 ++-- modules/nf-core/bracken/bracken/nextflow.config | 2 +- modules/nf-core/kraken2/kraken2/nextflow.config | 2 +- 3 files changed, 4 insertions(+), 4 deletions(-) diff --git a/docs/output.md b/docs/output.md index 144caabd5..068a5db17 100644 --- a/docs/output.md +++ b/docs/output.md @@ -660,11 +660,11 @@ The plot on the left hand side shows the standard PC plot - notice the variable