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Snakefile
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shell.executable("/bin/bash")
shell.prefix("source $HOME/.bashrc; ")
configfile: "config.yaml"
PREFIX = config['raw_reads']
SUFFIX = config['r1_ext']
IDS = os.listdir(f'{PREFIX}')
bwa_threads = config['bwa_threads']
cdhit_mem = config['cdhit_mem']
cdhit_threads = config['cdhit_threads']
cov_split = config['cov_split']
db = config['db']
dia_threads = config['dia_threads']
diamond_db = config['diamond_db']
kofamscan_split = config['kofamscan_split']
kofamscan_threads = config['kofamscan_threads']
max_qc = config['max_qc']
mega_kmers = config['mega_kmers']
mega_len = config['mega_len']
mega_mem = config['mega_mem']
mega_threads = config['mega_threads']
min_pid = config['min_pid']
min_qc = config['min_qc']
minlen = config['minlen']
mmseq_db = config['mmseq_db']
mmseq_keggs_threads = config['mmseq_keggs_threads']
mmseq_nohit_threads = config['mmseq_nohit_threads']
outdir = config['outdir']
overlap = config['overlap']
combined_bdg = config['combined_bdg']
polyg = config['polyg']
qual = config['qual']
r1_ext = config['r1_ext']
r1ad = config['r1ad']
r2_ext = config['r2_ext']
r2ad = config['r2ad']
raw_reads = config['raw_reads']
split_num = config['split_num']
tmp_cat = config['tmp_cat']
trim_threads = config['trim_threads']
unmapped_threads = config['unmapped_threads']
kallisto = config['kallisto']
kallisto_threads = config['kallisto_threads']
nohit = config['nohit']
splitting_multiples = config['splitting_multiples']
grouping_multiples = config['grouping_multiples']
evenness_yes = config['evenness_yes']
evenness_no = config['evenness_no']
evenness_pid = config['evenness_pid']
checking_fqs = config['checking_fqs']
not_checking_fqs = config['not_checking_fqs']
#default
rule all:
input:
expand(f"{outdir}Touch/trim_{{id}}", id=IDS),
expand(f"{outdir}assemblies/{{id}}/{{id}}.contigs.fa", id=IDS),
expand(f"{outdir}Prodigal_Outputs/filtered_aminos/{{id}}/{{id}}.faa", id=IDS),
expand(f"{outdir}Abundance/{{id}}/{{id}}_bwa", id=IDS),
"scripts/BamDeal-0.24/bin/BamDeal_Linux",
expand(f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage", id=IDS),
f"{db}ko_list",
expand(f"{outdir}Kofamscan/{{id}}/{{id}}", id=IDS),
f"{outdir}Results/KOunts_Kofamscan.csv",
f"{outdir}Touch/cdhit",
f"{outdir}Touch/split_kegg",
expand("{outdir}Touch/mmseq_kegg_{split}", outdir=config["outdir"],split=config["split"]),
f"{outdir}Touch/mmseq_nohit",
f"{outdir}Results/Number_of_clusters.csv",
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_KOunt", id=IDS),
f"{outdir}Touch/kegg_fasta",
expand(f"{outdir}Touch/nohit_fa_{{id}}", id=IDS),
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohitp_KOunt", id=IDS),
expand(f"{outdir}Touch/{{id}}_nohit_fastq", id=IDS),
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohit_KOunts", id=IDS),
expand(f"{outdir}kallisto/{{id}}_kallisto_still_missing_KOunt", id=IDS),
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_unmapped_KOunt", id=IDS),
expand(f"{outdir}kallisto/{{id}}_unmapped_kallisto_still_missing_KOunt", id=IDS),
expand(f"{outdir}Results/All_KOunts_nohit_unmapped_default.csv", id=IDS)
#not performing protein clustering
rule all_without_clustering:
input:
expand(f"{outdir}Touch/trim_{{id}}", id=IDS),
expand(f"{outdir}assemblies/{{id}}/{{id}}.contigs.fa", id=IDS),
expand(f"{outdir}Prodigal_Outputs/filtered_aminos/{{id}}/{{id}}.faa", id=IDS),
expand(f"{outdir}Abundance/{{id}}/{{id}}_bwa", id=IDS),
"scripts/BamDeal-0.24/bin/BamDeal_Linux",
expand(f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage", id=IDS),
f"{db}ko_list",
expand(f"{outdir}Kofamscan/{{id}}/{{id}}", id=IDS),
f"{outdir}Results/KOunts_Kofamscan_without_clustering.csv",
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_noc_KOunt", id=IDS),
expand(f"{outdir}Touch/nohit_fa_{{id}}_noc", id=IDS),
expand(f"{outdir}Touch/{{id}}_nohit_fastq_noc", id=IDS),
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohit_KOunts_noc", id=IDS),
expand(f"{outdir}kallisto/{{id}}_kallisto_still_missing_KOunt_noc", id=IDS),
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_unmapped_KOunt", id=IDS),
expand(f"{outdir}kallisto/{{id}}_unmapped_kallisto_still_missing_KOunt", id=IDS),
expand(f"{outdir}Results/All_KOunts_nohit_unmapped_no_clustering.csv", id=IDS)
#without protein clustering and the reference database
rule all_without_reference:
input:
expand(f"{outdir}Touch/trim_{{id}}", id=IDS),
expand(f"{outdir}assemblies/{{id}}/{{id}}.contigs.fa", id=IDS),
expand(f"{outdir}Prodigal_Outputs/filtered_aminos/{{id}}/{{id}}.faa", id=IDS),
expand(f"{outdir}Abundance/{{id}}/{{id}}_bwa", id=IDS),
"scripts/BamDeal-0.24/bin/BamDeal_Linux",
expand(f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage_noref", id=IDS),
f"{db}ko_list",
expand(f"{outdir}Kofamscan/{{id}}/{{id}}", id=IDS),
f"{outdir}Results/All_KOunts_without_reference.csv",
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_noref_KOunt", id=IDS)
#not performing protein clustering or calculating RNA abundance
rule all_without_RNA:
input:
expand(f"{outdir}Touch/trim_{{id}}", id=IDS),
expand(f"{outdir}assemblies/{{id}}/{{id}}.contigs.fa", id=IDS),
expand(f"{outdir}Prodigal_Outputs/filtered_aminos/{{id}}/{{id}}.faa", id=IDS),
expand(f"{outdir}Abundance/{{id}}/{{id}}_bwa", id=IDS),
"scripts/BamDeal-0.24/bin/BamDeal_Linux",
expand(f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage", id=IDS),
f"{db}ko_list",
expand(f"{outdir}Kofamscan/{{id}}/{{id}}", id=IDS),
f"{outdir}Results/KOunts_Kofamscan_without_clustering.csv",
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_noc_KOunt", id=IDS),
expand(f"{outdir}Touch/nohit_fa_without_RNA_{{id}}", id=IDS),
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohit_without_RNA_KOunts", id=IDS),
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohitp_KOunt_norna", id=IDS),
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_unmapped_KOunt_no_RNA", id=IDS),
expand(f"{outdir}Results/All_KOunts_without_RNA.csv", id=IDS),
expand(f"{outdir}Touch/{{id}}_nohit_fastq_without_RNA", id=IDS)
rule trim:
input:
r1 = f'{raw_reads}{{id}}/{{id}}{r1_ext}',
r2 = f'{raw_reads}{{id}}/{{id}}{r2_ext}',
output:
f'{outdir}Touch/trim_{{id}}',
params:
trim = f'{outdir}trimmed',
trim_id = f'{outdir}trimmed/{{id}}',
threads=f'{trim_threads}',
r1_adapter = f'{r1ad}',
r2_adapter = f'{r2ad}',
polyG = f'{polyg}',
quality = f'{qual}',
min = f'{minlen}',
over = f'{overlap}',
r1 = f'{outdir}trimmed/{{id}}/{{id}}.trimmed.R1.fastq.gz',
r2 = f'{outdir}trimmed/{{id}}/{{id}}.trimmed.R2.fastq.gz',
tmp_r1 = f'{outdir}trimmed/{{id}}/{{id}}.trimmed_tmp.R1.fastq.gz',
tmp_r2 = f'{outdir}trimmed/{{id}}/{{id}}.trimmed_tmp.R2.fastq.gz',
checking_fqs=f"{checking_fqs}",
not_checking_fqs=f"{not_checking_fqs}"
conda: "envs/fastp_utils_KOunt.yaml"
shell:
'''
mkdir -p {params.trim}
mkdir -p {params.trim_id}
fastp -i {input.r1} -o {params.r1} -I {input.r2} -O {params.r2} --adapter_sequence {params.r1_adapter} --adapter_sequence_r2 {params.r2_adapter} {params.quality} -l {params.min} -w {params.threads} --overlap_len_require {params.over} {params.polyG} --json /dev/null --html /dev/null
zcat {params.r1} | perl -pe 's/ .*// if /^@/' | sed '/\/1/! s/^@.*/&\/1/' | gzip > {params.tmp_r1}
zcat {params.r2} | perl -pe 's/ .*// if /^@/' | sed '/\/2/! s/^@.*/&\/2/' | gzip > {params.tmp_r2}
mv {params.tmp_r1} {params.r1}
mv {params.tmp_r2} {params.r2}
{params.checking_fqs} if fastq_info {params.r1} {params.r2}; then touch {output}; else echo "read names not unique or fastqs invalid"; fi #include if checking read ids
{params.not_checking_fqs} touch {output} #include if not checking read ids
'''
rule megahit:
input: f'{outdir}Touch/trim_{{id}}'
params:
r1 = f'{outdir}trimmed/{{id}}/{{id}}.trimmed.R1.fastq.gz',
r2 = f'{outdir}trimmed/{{id}}/{{id}}.trimmed.R2.fastq.gz',
outdir=f'{outdir}megahit/{{id}}/',
mega=f'{outdir}megahit',
memory=f'{mega_mem}',
kmers=f'{mega_kmers}',
len=f'{mega_len}',
threads=f'{mega_threads}'
output:
final=f'{outdir}assemblies/{{id}}/{{id}}.contigs.fa'
conda: "envs/megahit_KOunt.yaml"
shell:
'''
mkdir -p {params.mega}
megahit -m {params.memory} --kmin-1pass --k-list {params.kmers} --min-contig-len {params.len} -t {params.threads} -1 {params.r1} -2 {params.r2} -o {params.outdir}
mv {params.outdir}final.contigs.fa {output.final}
./scripts/add_sample_name.sh {output.final} {wildcards.id}
rm -r {params.outdir}
'''
rule prodigal:
input: f'{outdir}assemblies/{{id}}/{{id}}.contigs.fa'
params:
output=f'{outdir}Prodigal_Outputs/{{id}}/'
output:
filtered_nuc=f'{outdir}Prodigal_Outputs/filtered_nucleotides/{{id}}/{{id}}.fa',
filtered_aa=f'{outdir}Prodigal_Outputs/filtered_aminos/{{id}}/{{id}}.faa',
filtered_gff=f'{outdir}Prodigal_Outputs/filtered_gffs/{{id}}/{{id}}.gff'
conda: "envs/prodigal_KOunt.yaml"
shell:
'''
mkdir -p {params.output}
prodigal -i {input} -p meta -o {params.output}{wildcards.id}.gff -d {params.output}{wildcards.id}.fa -a {params.output}{wildcards.id}.faa -f gff
awk '/^>/ {{P=index($0,"partial=00")}} {{if(P) print}} ' {params.output}{wildcards.id}.fa > {output.filtered_nuc}
awk '/^>/ {{P=index($0,"partial=00")}} {{if(P) print}} ' {params.output}{wildcards.id}.faa > {output.filtered_aa}
sed -i 's/ .*$//' {output.filtered_nuc}
sed -i 's/ .*$//' {output.filtered_aa}
awk '!/^>/ {{ printf "%s", $0; n="\\n" }} /^>/ {{ print n $0; n="" }} END {{ printf "%s", n }}' {output.filtered_nuc} > {output.filtered_nuc}_tmp && mv {output.filtered_nuc}_tmp {output.filtered_nuc}
awk '!/^>/ {{ printf "%s", $0; n="\\n" }} /^>/ {{ print n $0; n="" }} END {{ printf "%s", n }}' {output.filtered_aa} > {output.filtered_aa}_tmp && mv {output.filtered_aa}_tmp {output.filtered_aa}
awk '{{P=index($0,"partial=00")}} {{if(P) print}}' {params.output}{wildcards.id}.gff | sed 's/ID=[^_]*_/ID=/g' > {output.filtered_gff}
rm -r {params.output}
'''
rule bwa:
input:
fasta=f"{outdir}assemblies/{{id}}/{{id}}.contigs.fa",
touch=f"{outdir}Touch/trim_{{id}}"
output:
touchfile=f"{outdir}Abundance/{{id}}/{{id}}_bwa"
params:
r1 = f"{outdir}trimmed/{{id}}/{{id}}.trimmed.R1.fastq.gz",
r2 = f"{outdir}trimmed/{{id}}/{{id}}.trimmed.R2.fastq.gz",
bam = f"{outdir}Abundance/{{id}}/{{id}}.bam",
threads=f"{bwa_threads}"
conda: "envs/bwa_map_KOunt.yaml"
shell:
'''
bwa index {input.fasta}
bwa mem -t {params.threads} {input.fasta} {params.r1} {params.r2} | samtools view -bS - > {params.bam}
rm {input.fasta}.*
touch {output.touchfile}
'''
rule bamdeal:
output:
bamdeal="scripts/BamDeal-0.24/bin/BamDeal_Linux"
params:
download="https://github.com/BGI-shenzhen/BamDeal/archive/v0.24.tar.gz",
bamdeal_location="scripts/"
shell:
'''
wget -P {params.bamdeal_location} {params.download}
tar -C {params.bamdeal_location} -zxvf {params.bamdeal_location}v0.24.tar.gz
chmod 755 {output.bamdeal}
rm {params.bamdeal_location}v0.24.tar.gz
'''
rule coverage:
input:
filtered_gff=f"{outdir}Prodigal_Outputs/filtered_gffs/{{id}}/{{id}}.gff",
bamdeal="scripts/BamDeal-0.24/bin/BamDeal_Linux",
touchfile=f"{outdir}Abundance/{{id}}/{{id}}_bwa"
output:
coverage=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage"
params:
bam=f"{outdir}Abundance/{{id}}/{{id}}.bam",
wd=f"{outdir}Abundance/{{id}}/{{id}}",
split=f"{cov_split}",
abun=f"{outdir}Abundance/",
out=f"{outdir}Abundance_Outputs/{{id}}/",
stats=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_stats",
gff=f"{outdir}Prodigal_Outputs/filtered_gffs/",
mapped=f"{outdir}Abundance/{{id}}/{{id}}_mapped_reads",
evenness_yes=f"{evenness_yes}",
evenness=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_evenness"
conda: "envs/coverage_KOunt.yaml"
shell:
'''
mkdir -p {params.wd}
mkdir -p {params.out}
awk '{{print $1}}' {input.filtered_gff} | sort | uniq > {params.wd}_contigs
split -l {params.split} {params.wd}_contigs {params.wd}
for file in {params.wd}??; do awk -v file=$file '{{print $1 "\t" file}}' $file | awk -F'/|\t' '{{print $1 "\t" $NF}}'; done > {params.wd}_bedtools_list
{input.bamdeal} modify bamAssign -i {params.bam} -a {params.wd}_bedtools_list -q 0 -o {params.wd}
for file in {params.wd}??; do ./scripts/bedtools.sh $file {params.gff} {params.wd}; done > {output.coverage}
{params.evenness_yes} for file in {params.wd}??; do ./scripts/evenness.sh $file {params.wd}; done
{params.evenness_yes} cat {params.wd}??_evenness > {params.evenness}
{params.evenness_yes} rm {params.wd}??_evenness
bedtools bamtobed -i {params.bam} > {params.bam}.bed
rm {params.bam}
LC_ALL=C sort -k1,1 -k2,2n {params.bam}.bed > {params.bam}.bed_sorted
LC_ALL=C sort -k1,1 -k4,4n {input.filtered_gff} > {input.filtered_gff}_sorted
bedtools intersect -a {input.filtered_gff}_sorted -b {params.bam}.bed_sorted -wb -sorted | awk '{{print $13}}' | sort -S 50% | uniq > {params.mapped}
rm {input.filtered_gff}_sorted
rm {params.bam}.bed_sorted
rm -r {params.wd}
rm {params.wd}_contigs
rm {params.wd}_bedtools_list
rm {params.wd}*coverage
rm {params.wd}*gff
rm {params.wd}??
'''
rule coverage_without_reference:
input:
filtered_gff=f"{outdir}Prodigal_Outputs/filtered_gffs/{{id}}/{{id}}.gff",
bamdeal="scripts/BamDeal-0.24/bin/BamDeal_Linux",
touchfile=f"{outdir}Abundance/{{id}}/{{id}}_bwa"
output:
coverage=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage_noref"
params:
bam=f"{outdir}Abundance/{{id}}/{{id}}.bam",
wd=f"{outdir}Abundance/{{id}}/{{id}}",
split=f"{cov_split}",
abun=f"{outdir}Abundance/",
out=f"{outdir}Abundance_Outputs/{{id}}/",
stats=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_stats",
gff=f"{outdir}Prodigal_Outputs/filtered_gffs/",
evenness_yes=f"{evenness_yes}",
evenness=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_evenness"
conda: "envs/coverage_KOunt.yaml"
shell:
'''
mkdir -p {params.wd}
mkdir -p {params.out}
awk '{{print $1}}' {input.filtered_gff} | sort | uniq > {params.wd}_contigs
split -l {params.split} {params.wd}_contigs {params.wd}
for file in {params.wd}??; do awk -v file=$file '{{print $1 "\t" file}}' $file | awk -F'/|\t' '{{print $1 "\t" $NF}}'; done > {params.wd}_bedtools_list
{input.bamdeal} modify bamAssign -i {params.bam} -a {params.wd}_bedtools_list -q 0 -o {params.wd}
for file in {params.wd}??; do ./scripts/bedtools.sh $file {params.gff} {params.wd}; done > {output.coverage}
{params.evenness_yes} for file in {params.wd}??; do ./scripts/evenness.sh $file {params.wd}; done
{params.evenness_yes} cat {params.wd}??_evenness > {params.evenness}
{params.evenness_yes} rm {params.wd}??_evenness
rm {params.bam}
rm -r {params.wd}
rm {params.wd}_contigs
rm {params.wd}_bedtools_list
rm {params.wd}*coverage
rm {params.wd}*gff
rm {params.wd}??
'''
rule kegg_db:
output:
profiles=directory(f"{db}profiles/"),
list=f"{db}ko_list",
touch=f"{db}touch"
params:
profiles="ftp://ftp.genome.jp/pub/db/kofam/profiles.tar.gz",
ko_list="ftp://ftp.genome.jp/pub/db/kofam/ko_list.gz",
location=f"{db}"
shell:
'''
touch {output.touch}
wget -P {params.location} {params.profiles}
wget -P {params.location} {params.ko_list}
tar -C {params.location} -xf {params.location}/profiles.tar.gz
gunzip {params.location}/ko_list.gz
rm {params.location}/profiles.tar.gz
'''
rule kofamscan:
input:
filtered_aa=f"{outdir}Prodigal_Outputs/filtered_aminos/{{id}}/{{id}}.faa",
touch=f"{db}touch"
params:
profiles=f"{db}profiles/",
list=f"{db}ko_list",
tmp=f"{outdir}Kofamscan/tmp_{{id}}",
threads=f"{kofamscan_threads}"
output:
ko=f"{outdir}Kofamscan/{{id}}/{{id}}"
conda: "envs/kofamscan_hmmr_KOunt.yaml"
shell:
'''
exec_annotation --cpu {params.threads} -p {params.profiles} -k {params.list} -o {output.ko} --tmp-dir {params.tmp} {input.filtered_aa}
rm -r {params.tmp}
'''
rule kofamscan_results:
input:
ko=f"{outdir}Kofamscan/{{id}}/{{id}}",
coverage=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage",
filtered_aa=f"{outdir}Prodigal_Outputs/filtered_aminos/{{id}}/{{id}}.faa"
params:
split_keggs=f"{outdir}CDHit/",
splitting_multiples=f"{splitting_multiples}",
grouping_multiples=f"{grouping_multiples}",
evenness_yes=f"{evenness_yes}",
evenness_no=f"{evenness_no}",
evenness=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_evenness",
pid=f"{evenness_pid}"
output:
out=f"{outdir}Kofamscan/Results/{{id}}/{{id}}",
KOunt=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_KOunt",
nohit=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohit"
shell:
'''
grep '^\*' {input.ko} | awk '{{print $2 "\t" $3}}' > {output.out}
{params.splitting_multiples}{params.evenness_no}./scripts/splitting_multiples.sh {input.coverage} {output.out} {output.out} #include if splitting multiples and not using evenness
{params.splitting_multiples}{params.evenness_yes}./scripts/splitting_multiples_evenness.sh {input.coverage} {output.out} {output.out} {params.evenness} {params.pid} #include if splitting multiples and using evenness
{params.grouping_multiples}awk '{{print $1}}' {output.out} | sort | uniq -d > {output.out}_duplicates #include if grouping multiples
{params.grouping_multiples}cat {output.out} | parallel --colsep '\t' ./scripts/multiples.sh {{1}} {output.out} {output.out}_duplicates | sort | uniq > {output.out}_multiples #include if grouping multiples
{params.grouping_multiples}awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {output.out}_multiples {input.coverage} | awk 'BEGIN{{OFS=FS}} $3 == "" {{$3 = "NoHit"}} 1' > {output.out}_coverage #include if grouping multiples
{params.grouping_multiples}rm {output.out}_duplicates #include if grouping multiples
{params.grouping_multiples}rm {output.out}_multiples #include if grouping multiples
{params.grouping_multiples}{params.evenness_no}awk '{{print $2 "\t" $3}}' {output.out}_coverage | awk '{{a[$2]+=$1}}END{{for(i in a) print i,a[i]}}' > {output.KOunt} #include if grouping multiples and not using evenness
{params.grouping_multiples}{params.evenness_yes}awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {params.evenness} {output.out}_coverage | gawk -v pid="{params.pid}" '$4>pid' > {output.out}_coverage && awk '{{print $2 "\t" $3}}' {output.out}_coverage | awk '{{a[$2]+=$1}}END{{for(i in a) print i,a[i]}}' > {output.KOunt} #include if grouping multiples and using evenness
mkdir -p {params.split_keggs}
awk '{{print $3}}' {output.out}_coverage > {output.out}_coverage_tmp && while read l; do mkdir -p {params.split_keggs}/"$l"; done < {output.out}_coverage_tmp && rm {output.out}_coverage_tmp
awk '{{print $1>f"{params.split_keggs}" $3 "/{wildcards.id}_" $3}}' {output.out}_coverage
for file in {params.split_keggs}*/{wildcards.id}_*; do ./scripts/extracting_genes.sh $file {input.filtered_aa}; done
grep NoHit {output.out}_coverage | awk '{{print $1}}' > {output.nohit}
rm {output.out}_coverage
'''
rule kofamscan_results_no_clustering:
input:
ko=f"{outdir}Kofamscan/{{id}}/{{id}}",
coverage=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage"
params:
splitting_multiples=f"{splitting_multiples}",
grouping_multiples=f"{grouping_multiples}",
evenness_yes=f"{evenness_yes}",
evenness_no=f"{evenness_no}",
evenness=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_evenness",
pid=f"{evenness_pid}"
output:
out=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_noc",
KOunt=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_noc_KOunt",
nohit=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohit_noc"
shell:
'''
grep '^\*' {input.ko} | awk '{{print $2 "\t" $3}}' > {output.out}
{params.splitting_multiples}{params.evenness_no}./scripts/splitting_multiples.sh {input.coverage} {output.out} {output.out} #include if splitting multiples and not using evenness
{params.splitting_multiples}{params.evenness_yes}./scripts/splitting_multiples_evenness.sh {input.coverage} {output.out} {output.out} {params.evenness} {params.pid} #include if splitting multiples and using evenness
{params.grouping_multiples}awk '{{print $1}}' {output.out} | sort | uniq -d > {output.out}_duplicates #include if grouping multiples
{params.grouping_multiples}cat {output.out} | parallel --colsep '\t' ./scripts/multiples.sh {{1}} {output.out} {output.out}_duplicates | sort | uniq > {output.out}_multiples #include if grouping multiples
{params.grouping_multiples}awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {output.out}_multiples {input.coverage} | awk 'BEGIN{{OFS=FS}} $3 == "" {{$3 = "NoHit"}} 1' > {output.out}_coverage #include if grouping multiples
{params.grouping_multiples}rm {output.out}_duplicates #include if grouping multiples
{params.grouping_multiples}rm {output.out}_multiples #include if grouping multiples
{params.grouping_multiples}{params.evenness_no}awk '{{print $2 "\t" $3}}' {output.out}_coverage | awk '{{a[$2]+=$1}}END{{for(i in a) print i,a[i]}}' > {output.KOunt} #include if grouping multiples and not using evenness
{params.grouping_multiples}{params.evenness_yes}awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {params.evenness} {output.out}_coverage | gawk -v pid="{params.pid}" '$4>pid' > {output.out}_coverage && awk '{{print $2 "\t" $3}}' {output.out}_coverage | awk '{{a[$2]+=$1}}END{{for(i in a) print i,a[i]}}' > {output.KOunt} #include if grouping multiples and using evenness
grep NoHit {output.out}_coverage | awk '{{print $1}}' > {output.nohit}
rm {output.out}_coverage
'''
rule kofamscan_results_noref:
input:
ko=f"{outdir}Kofamscan/{{id}}/{{id}}",
coverage=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage_noref"
params:
splitting_multiples=f"{splitting_multiples}",
grouping_multiples=f"{grouping_multiples}",
evenness_yes=f"{evenness_yes}",
evenness_no=f"{evenness_no}",
evenness=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_evenness",
pid=f"{evenness_pid}"
output:
out=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_noref",
KOunt=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_noref_KOunt"
shell:
'''
grep '^\*' {input.ko} | awk '{{print $2 "\t" $3}}' > {output.out}
{params.splitting_multiples}{params.evenness_no}./scripts/splitting_multiples.sh {input.coverage} {output.out} {output.out} #include if splitting multiples and not using evenness
{params.splitting_multiples}{params.evenness_yes}./scripts/splitting_multiples_evenness.sh {input.coverage} {output.out} {output.out} {params.evenness} {params.pid} #include if splitting multiples and using evenness
{params.grouping_multiples}awk '{{print $1}}' {output.out} | sort | uniq -d > {output.out}_duplicates #include if grouping multiples
{params.grouping_multiples}cat {output.out} | parallel --colsep '\t' ./scripts/multiples.sh {{1}} {output.out} {output.out}_duplicates | sort | uniq > {output.out}_multiples #include if grouping multiples
{params.grouping_multiples}awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {output.out}_multiples {input.coverage} | awk 'BEGIN{{OFS=FS}} $3 == "" {{$3 = "NoHit"}} 1' > {output.out}_coverage #include if grouping multiples
{params.grouping_multiples}rm {output.out}_duplicates #include if grouping multiples
{params.grouping_multiples}rm {output.out}_multiples #include if grouping multiples
{params.grouping_multiples}{params.evenness_no}awk '{{print $2 "\t" $3}}' {output.out}_coverage | awk '{{a[$2]+=$1}}END{{for(i in a) print i,a[i]}}' > {output.KOunt} #include if grouping multiples and not using evenness
{params.grouping_multiples}{params.evenness_yes}awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {params.evenness} {output.out}_coverage | gawk -v pid="{params.pid}" '$4>pid' > {output.out}_coverage && awk '{{print $2 "\t" $3}}' {output.out}_coverage | awk '{{a[$2]+=$1}}END{{for(i in a) print i,a[i]}}' > {output.KOunt} #include if grouping multiples and using evenness
rm {output.out}_coverage
'''
rule abundance_results:
input:
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_KOunt", id=IDS)
params:
tmp=f"{outdir}Kofamscan/tmp/",
list=f"{db}ko_list",
kegglist=f"{outdir}kegglist"
output:
results=f"{outdir}Results/KOunts_Kofamscan.csv"
shell:
'''
mkdir -p {params.tmp}
if [ ! -e {params.kegglist} ]; then cat {input} | awk '{{print $1}}' | sort | uniq > {params.kegglist}; fi
for i in {input}; do cp $i {params.tmp}; done
./scripts/abundance_matrix.sh {params.tmp} {params.list} {params.kegglist} > {output.results}
rm -r {params.tmp}
if [ -e {outdir}Touch/kegg_fasta ]; then rm {params.kegglist}; fi
'''
rule abundance_results_noc:
input:
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_noc_KOunt", id=IDS)
params:
tmp=f"{outdir}Kofamscan/tmp/",
list=f"{db}ko_list",
kegglist=f"{outdir}kegglist"
output:
results=f"{outdir}Results/KOunts_Kofamscan_without_clustering.csv"
shell:
'''
mkdir -p {params.tmp}
if [ ! -e {params.kegglist} ]; then cat {input} | awk '{{print $1}}' | sort | uniq > {params.kegglist}; fi
for i in {input}; do cp $i {params.tmp}; done
./scripts/abundance_matrix_noc.sh {params.tmp} {params.list} {params.kegglist} > {output.results}
rm -r {params.tmp}
if [ -e {outdir}Touch/kegg_fasta ]; then rm {params.kegglist}; fi
'''
rule abundance_results_noref:
input:
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_noref_KOunt", id=IDS)
params:
tmp=f"{outdir}Kofamscan/tmp/",
list=f"{db}ko_list",
kegglist=f"{outdir}kegglist"
output:
results=f"{outdir}Results/All_KOunts_without_reference.csv"
shell:
'''
mkdir -p {params.tmp}
if [ ! -e {params.kegglist} ]; then cat {input} | awk '{{print $1}}' | sort | uniq > {params.kegglist}; fi
for i in {input}; do cp $i {params.tmp}; done
./scripts/abundance_matrix_noref.sh {params.tmp} {params.list} {params.kegglist} > {output.results}
rm -r {params.tmp}
if [ -e {outdir}Touch/kegg_fasta ]; then rm {params.kegglist}; fi
'''
rule kegg_fastas:
input:
expand(f"{outdir}Kofamscan/Results/{{id}}/{{id}}_KOunt", id=IDS)
params:
split_dir=f"{outdir}CDHit/",
cd=f"{outdir}CD-Hit_Outputs",
cdhit=f"{outdir}CD-Hit_Outputs/By_Sample/",
mega=f"{outdir}megahit",
results=f"{outdir}Kofamscan/Results/all_KOunts.csv",
kegglist=f"{outdir}kegglist"
output: f"{outdir}Touch/kegg_fasta"
shell:
'''
mkdir -p {params.cd}
mkdir -p {params.cdhit}
if [ ! -e {params.kegglist} ]; then cat {input} | awk '{{print $1}}' | sort | uniq > {params.kegglist}; fi
cat {params.kegglist} | parallel "cat {params.split_dir}{{1}}/*faa > {params.cdhit}{{1}}.faa"
rm -r {params.split_dir}
if [ -e {params.mega} ]; then rm -r {params.mega}; fi
touch {output}
if [ -e {params.results} ]; then rm {params.kegglist}; fi
'''
rule cdhit:
input:
f"{outdir}Touch/kegg_fasta"
params:
cdhit=f"{outdir}CD-Hit_Outputs/By_Sample/",
memory=f"{cdhit_mem}",
threads=f"{cdhit_threads}"
output: f"{outdir}Touch/cdhit"
conda: "envs/cdhit_KOunt.yaml"
shell:
'''
for file in {params.cdhit}*faa; do cd-hit -i $file -o "$file"_1.0 -c 1.0 -d 0 -M {params.memory} -T {params.threads}; done
for file in {params.cdhit}*clstr; do clstr2txt.pl $file | awk '{{print $2 "\t" $1}}' > "$file".txt; done
for file in {params.cdhit}*clstr; do clstr_rep.pl $file > "$file"_reps; done
touch {output}
'''
rule split_keggs:
input: f"{outdir}Touch/cdhit"
output: f"{outdir}Touch/split_kegg"
params:
splitting_multiples=f"{splitting_multiples}",
grouping_multiples=f"{grouping_multiples}",
sample=f"{outdir}CD-Hit_Outputs/By_Sample/",
num=f"{split_num}",
lists=f"{outdir}lists"
shell:
'''
mkdir -p {params.lists}
(cd {params.sample} && ls K*_1.0) > keggslist
{params.grouping_multiples}(cd {params.sample} && ls Multiple*1.0) >> keggslist #include if grouping multiples
split -d --number=l/{params.num} keggslist {params.lists}/keggslist
rm keggslist
touch {output}
'''
rule mmseq_keggs:
input:
keggs=f"{outdir}Touch/split_kegg",
cdhit=f"{outdir}Touch/cdhit"
params:
cdhit=f"{outdir}CD-Hit_Outputs/By_Sample/",
list="{outdir}lists/keggslist{split}",
mm=f"{outdir}MMSeq_Outputs/",
mmseq=f"{outdir}MMSeq_Outputs/By_Sample/",
threads=f"{mmseq_keggs_threads}"
output: "{outdir}Touch/mmseq_kegg_{split}"
conda: "envs/mmseqs2_KOunt.yaml"
shell:
'''
mkdir -p {params.mm}
mkdir -p {params.mmseq}
while read file; do ./scripts/mmseq_keggs.sh $file {params.cdhit} {params.mmseq}; done < {params.list}
while read file; do ./scripts/count_mmseq_clusters.sh {params.mmseq}"$file"_0.9_all; done < {params.list}
while read file; do ./scripts/count_mmseq_clusters.sh {params.mmseq}"$file"_0.5_all; done < {params.list}
rm {params.list}
touch {output}
'''
rule mmseq_nohit:
input:
cdhit=f"{outdir}Touch/cdhit"
params:
cdhit=f"{outdir}CD-Hit_Outputs/By_Sample/",
mm=f"{outdir}MMSeq_Outputs/",
mmseq=f"{outdir}MMSeq_Outputs/By_Sample/",
threads=mmseq_nohit_threads
output: f"{outdir}Touch/mmseq_nohit"
conda: "envs/mmseqs2_KOunt.yaml"
shell:
'''
mkdir -p {params.mm}
mkdir -p {params.mmseq}
./scripts/mmseq_keggs.sh NoHit.faa_1.0 {params.cdhit} {params.mmseq}
./scripts/count_mmseq_clusters.sh {params.mmseq}NoHit.faa_1.0_0.9_all
./scripts/count_mmseq_clusters.sh {params.mmseq}NoHit.faa_1.0_0.5_all
touch {output}
'''
rule mmseq_cluster_count:
input:
keggs=expand("{outdir}Touch/mmseq_kegg_{split}", outdir=config["outdir"], split=config['split']),
nohit=f"{outdir}Touch/mmseq_nohit"
params:
mmseq=f"{outdir}MMSeq_Outputs/By_Sample/",
list=f"{outdir}lists/"
output:f"{outdir}Results/Number_of_clusters.csv"
shell:
'''
echo -e "KEGG,Number of clusters,Number of singletons,Number with multiple genes,Number of proteins\n$(cat {params.mmseq}*txt)" > {output}
rm {params.mmseq}*txt
rm -r {params.list}
'''
rule nohit_fastas:
input:
nohit=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohit",
faa=f"{outdir}Prodigal_Outputs/filtered_aminos/{{id}}/{{id}}.faa",
fa=f"{outdir}Prodigal_Outputs/filtered_nucleotides/{{id}}/{{id}}.fa"
params:
faa=f"{outdir}diamond/NoHit_{{id}}.faa",
fa=f"{outdir}diamond/NoHit_{{id}}.fa",
wd=f"{outdir}diamond/"
output:f"{outdir}Touch/nohit_fa_{{id}}"
shell:
'''
mkdir -p {params.wd}
./scripts/extracting_fastas.sh {input.nohit} {input.faa} > {params.faa}
./scripts/extracting_fastas.sh {input.nohit} {input.fa} > {params.fa}
touch {output}
'''
rule nohit_fastas_noclustering:
input:
nohit=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohit_noc",
faa=f"{outdir}Prodigal_Outputs/filtered_aminos/{{id}}/{{id}}.faa",
fa=f"{outdir}Prodigal_Outputs/filtered_nucleotides/{{id}}/{{id}}.fa"
params:
faa=f"{outdir}diamond/NoHit_{{id}}.faa",
fa=f"{outdir}diamond/NoHit_{{id}}.fa",
wd=f"{outdir}diamond/"
output:f"{outdir}Touch/nohit_fa_{{id}}_noc"
shell:
'''
mkdir -p {params.wd}
./scripts/extracting_fastas.sh {input.nohit} {input.faa} > {params.faa}
./scripts/extracting_fastas.sh {input.nohit} {input.fa} > {params.fa}
touch {output}
'''
rule nohit_fastas_without_RNA:
input:
nohit=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohit_noc",
faa=f"{outdir}Prodigal_Outputs/filtered_aminos/{{id}}/{{id}}.faa"
params:
faa=f"{outdir}diamond/NoHit_{{id}}.faa",
fa=f"{outdir}diamond/NoHit_{{id}}.fa",
wd=f"{outdir}diamond/"
output:f"{outdir}Touch/nohit_fa_without_RNA_{{id}}"
shell:
'''
mkdir -p {params.wd}
./scripts/extracting_fastas.sh {input.nohit} {input.faa} > {params.faa}
touch {output}
'''
rule diamond_search:
input:
nohit=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohit",
cov=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage",
touch=f"{outdir}Touch/nohit_fa_{{id}}"
output:
raw=f"{outdir}diamond/NoHit_{{id}}",
fil=f"{outdir}diamond/{{id}}/{{id}}_filtered",
best=f"{outdir}diamond/NoHit_{{id}}_besthits",
ko=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohitp_ko",
KOunt=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohitp_KOunt",
no=f"{outdir}diamond/still_nohit_{{id}}",
mmseq=f"{outdir}MMSeq_Outputs/{{id}}/nohitp_{{id}}"
params:
faa=f"{outdir}diamond/NoHit_{{id}}.faa",
fa=f"{outdir}diamond/NoHit_{{id}}.fa",
rna=f"{mmseq_db}",
of="6 qseqid sseqid pident qlen slen qstart qend sstart send evalue",
ofm="query,target,pident,qlen,tlen,qstart,qend,tstart,tend,evalue",
db=f"{diamond_db}",
tmp=f"{outdir}diamond/{{id}}_tmp",
cat=f"{outdir}diamond/NoHit_{{id}}_cat",
min_qc=f"{min_qc}",
max_qc=f"{max_qc}",
min_pid=f"{min_pid}",
threads=f"{dia_threads}"
conda: "envs/diamond_bedtools_mmseq2_KOunt.yaml"
shell:
'''
diamond blastp --query {params.faa} --threads {params.threads} --outfmt {params.of} -d {params.db} > {output.raw}
mmseqs easy-search {params.fa} {params.rna} {output.mmseq} {params.tmp} --threads {params.threads} --search-type 3 --format-output {params.ofm}
cat {output.raw} {output.mmseq} > {params.cat}
rm -r {params.tmp}
awk -F '\t' '{{ print $0 "\t" ($4/$5) * 100 }}' {params.cat} | gawk -F '\t' '$NF>{params.min_qc} {{print $0}}' | gawk -F '\t' '$NF<{params.max_qc} {{print $0}}' | gawk -F '\t' '$3>{params.min_pid} {{print $0}}' > {output.fil}
awk '! a[$1]++' {output.fil} > {output.fil}_tmp
awk '{{print $1 "," $3 "," $10 "\t" $2}}' {output.fil}_tmp > {output.fil}_tmp.1
awk '{{print $1 "," $3 "," $10 "\t" $2}}' {output.fil} > {output.fil}.1
awk 'NR==FNR{{a[$1];next}}$1 in a' {output.fil}_tmp.1 {output.fil}.1 | tr ',' '\t' > {output.best}
rm {output.fil}_tmp.1
rm {output.fil}.1
awk -F '_' '{{print $0 "\t" $NF}}' {output.best} | awk '{{print $1 "\t" $NF}}' | sort | uniq > {output.ko}
awk '{{print $1}}' {output.ko} | uniq -c | awk '{{print $2 "\t" $1}}' > {output.ko}_count
awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {input.cov} {output.ko} | awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {output.ko}_count - | awk '{{print $1 "\t" $2 "\t" ($3/$4)}}' > {output.ko}_coverage
awk '{{a[$2]+=$3}}END{{for(i in a) print i,a[i]}}' {output.ko}_coverage | awk -F'-' '{{print $0 "\t" NF-1}}' | awk -v OFMT='%.5f' '{{print $1"\t",($2/$3)}}' | cut -d- -f2- | awk '{{gsub(/-/,"\t"$2",")}}1' | tr ',' '\n' | awk -v OFMT='%.5f' '{{a[$1]+=$2}}END{{for(i in a) print i,a[i]}}' > {output.KOunt}
awk '{{print $1}}' {output.ko}_coverage > {output.ko}_coverage_tmp
cat {input.nohit} {output.ko}_coverage_tmp | sort | uniq -u > {output.no}
rm {output.ko}_coverage_tmp
rm {output.ko}_coverage
rm {output.ko}_count
'''
rule diamond_search_noclustering:
input:
nohit=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohit_noc",
cov=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage",
touch=f"{outdir}Touch/nohit_fa_{{id}}_noc"
output:
raw=f"{outdir}diamond/NoHit_{{id}}_noc",
fil=f"{outdir}diamond/{{id}}/{{id}}_filtered_noc",
best=f"{outdir}diamond/NoHit_{{id}}_besthits_noc",
ko=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohitp_ko_noc",
KOunt=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohitp_KOunt_noc",
no=f"{outdir}diamond/still_nohit_{{id}}_noc",
mmseq=f"{outdir}MMSeq_Outputs/{{id}}/nohitp_{{id}}_noc"
params:
faa=f"{outdir}diamond/NoHit_{{id}}.faa",
fa=f"{outdir}diamond/NoHit_{{id}}.fa",
rna=f"{mmseq_db}",
of="6 qseqid sseqid pident qlen slen qstart qend sstart send evalue",
ofm="query,target,pident,qlen,tlen,qstart,qend,tstart,tend,evalue",
db=f"{diamond_db}",
tmp=f"{outdir}diamond/{{id}}_tmp",
cat=f"{outdir}diamond/NoHit_{{id}}_cat",
min_qc=f"{min_qc}",
max_qc=f"{max_qc}",
min_pid=f"{min_pid}",
threads=f"{dia_threads}"
conda: "envs/diamond_bedtools_mmseq2_KOunt.yaml"
shell:
'''
diamond blastp --query {params.faa} --threads {params.threads} --outfmt {params.of} -d {params.db} > {output.raw}
mmseqs easy-search {params.fa} {params.rna} {output.mmseq} {params.tmp} --threads {params.threads} --search-type 3 --format-output {params.ofm}
cat {output.raw} {output.mmseq} > {params.cat}
rm -r {params.tmp}
awk -F '\t' '{{ print $0 "\t" ($4/$5) * 100 }}' {params.cat} | gawk -F '\t' '$NF>{params.min_qc} {{print $0}}' | gawk -F '\t' '$NF<{params.max_qc} {{print $0}}' | gawk -F '\t' '$3>{params.min_pid} {{print $0}}' > {output.fil}
awk '! a[$1]++' {output.fil} > {output.fil}_tmp
awk '{{print $1 "," $3 "," $10 "\t" $2}}' {output.fil}_tmp > {output.fil}_tmp.1
awk '{{print $1 "," $3 "," $10 "\t" $2}}' {output.fil} > {output.fil}.1
awk 'NR==FNR{{a[$1];next}}$1 in a' {output.fil}_tmp.1 {output.fil}.1 | tr ',' '\t' > {output.best}
rm {output.fil}_tmp.1
rm {output.fil}.1
awk -F '_' '{{print $0 "\t" $NF}}' {output.best} | awk '{{print $1 "\t" $NF}}' | sort | uniq > {output.ko}
awk '{{print $1}}' {output.ko} | uniq -c | awk '{{print $2 "\t" $1}}' > {output.ko}_count
awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {input.cov} {output.ko} | awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {output.ko}_count - | awk '{{print $1 "\t" $2 "\t" ($3/$4)}}' > {output.ko}_coverage
awk '{{a[$2]+=$3}}END{{for(i in a) print i,a[i]}}' {output.ko}_coverage | awk -F'-' '{{print $0 "\t" NF-1}}' | awk -v OFMT='%.5f' '{{print $1"\t",($2/$3)}}' | cut -d- -f2- | awk '{{gsub(/-/,"\t"$2",")}}1' | tr ',' '\n' | awk -v OFMT='%.5f' '{{a[$1]+=$2}}END{{for(i in a) print i,a[i]}}' > {output.KOunt}
awk '{{print $1}}' {output.ko}_coverage > {output.ko}_coverage_tmp
cat {input.nohit} {output.ko}_coverage_tmp | sort | uniq -u > {output.no}
rm {output.ko}_coverage_tmp
rm {output.ko}_coverage
rm {output.ko}_count
'''
rule diamond_search_without_RNA:
input:
nohit=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohit_noc",
cov=f"{outdir}Abundance_Outputs/{{id}}/{{id}}_coverage",
touch=f"{outdir}Touch/nohit_fa_without_RNA_{{id}}"
output:
raw=f"{outdir}diamond/NoHit_{{id}}_norna",
fil=f"{outdir}diamond/{{id}}/{{id}}_filtered_norna",
best=f"{outdir}diamond/NoHit_{{id}}_besthits_norna",
ko=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohitp_ko_norna",
KOunt=f"{outdir}Kofamscan/Results/{{id}}/{{id}}_nohitp_KOunt_norna",
no=f"{outdir}diamond/still_nohit_{{id}}_norna"
params:
faa=f"{outdir}diamond/NoHit_{{id}}.faa",
rna=f"{mmseq_db}",
of="6 qseqid sseqid pident qlen slen qstart qend sstart send evalue",
db=f"{diamond_db}",
tmp=f"{outdir}diamond/{{id}}_tmp",
cat=f"{outdir}diamond/NoHit_{{id}}_cat",
min_qc=f"{min_qc}",
max_qc=f"{max_qc}",
min_pid=f"{min_pid}",
threads=f"{dia_threads}"
conda: "envs/diamond_bedtools_mmseq2_KOunt.yaml"
shell:
'''
diamond blastp --query {params.faa} --threads {params.threads} --outfmt {params.of} -d {params.db} > {output.raw}
awk -F '\t' '{{ print $0 "\t" ($4/$5) * 100 }}' {output.raw} | gawk -F '\t' '$NF>{params.min_qc} {{print $0}}' | gawk -F '\t' '$NF<{params.max_qc} {{print $0}}' | gawk -F '\t' '$3>{params.min_pid} {{print $0}}' > {output.fil}
awk '! a[$1]++' {output.fil} > {output.fil}_tmp
awk '{{print $1 "," $3 "," $10 "\t" $2}}' {output.fil}_tmp > {output.fil}_tmp.1
awk '{{print $1 "," $3 "," $10 "\t" $2}}' {output.fil} > {output.fil}.1
awk 'NR==FNR{{a[$1];next}}$1 in a' {output.fil}_tmp.1 {output.fil}.1 | tr ',' '\t' > {output.best}
rm {output.fil}_tmp.1
rm {output.fil}.1
awk -F '_' '{{print $0 "\t" $NF}}' {output.best} | awk '{{print $1 "\t" $NF}}' | sort | uniq > {output.ko}
awk '{{print $1}}' {output.ko} | uniq -c | awk '{{print $2 "\t" $1}}' > {output.ko}_count
awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {input.cov} {output.ko} | awk 'NR==FNR{{a[$1]=$2;next}}{{print $0,a[$1]}}' {output.ko}_count - | awk '{{print $1 "\t" $2 "\t" ($3/$4)}}' > {output.ko}_coverage
awk '{{a[$2]+=$3}}END{{for(i in a) print i,a[i]}}' {output.ko}_coverage | awk -F'-' '{{print $0 "\t" NF-1}}' | awk -v OFMT='%.5f' '{{print $1"\t",($2/$3)}}' | cut -d- -f2- | awk '{{gsub(/-/,"\t"$2",")}}1' | tr ',' '\n' | awk -v OFMT='%.5f' '{{a[$1]+=$2}}END{{for(i in a) print i,a[i]}}' > {output.KOunt}
awk '{{print $1}}' {output.ko}_coverage > {output.ko}_coverage_tmp
cat {input.nohit} {output.ko}_coverage_tmp | sort | uniq -u > {output.no}
rm {output.ko}_coverage_tmp
rm {output.ko}_coverage
rm {output.ko}_count
'''
rule nohit_fastq:
input:
touchfile=f"{outdir}Abundance/{{id}}/{{id}}_bwa",
nohit=f"{outdir}diamond/still_nohit_{{id}}",
gff=f"{outdir}Prodigal_Outputs/filtered_gffs/{{id}}/{{id}}.gff"
params:
bed=f"{outdir}diamond/{{id}}.bed",
nobed=f"{outdir}nohit/RNA_{{id}}_missing.bed",
bam=f"{outdir}Abundance/{{id}}/{{id}}.bam.bed",
fastq=f"{outdir}nohit/RNA_{{id}}_missing.fastq.gz",
reads=f"{outdir}nohit/RNA_{{id}}_missing_reads",
r1 = f"{outdir}trimmed/{{id}}/{{id}}.trimmed.R1.fastq.gz",
r2 = f"{outdir}trimmed/{{id}}/{{id}}.trimmed.R2.fastq.gz",
nohit=f"{outdir}nohit"
conda: "envs/bedtools_seqtk_KOunt.yaml"
output: f"{outdir}Touch/{{id}}_nohit_fastq"
shell:
'''
mkdir -p {params.nohit}
awk -F '=|\t|;' '{{print $1 "_" $10 "\t" ($4-1) "\t" $5}}' {input.gff} > {params.bed}
awk 'NR==FNR{{a[$1]=$2;b[$1]=$3;next}}{{print $0 "\t" a[$1] "\t" b[$1]}}' {params.bed} {input.nohit} > {params.nobed}_tmp
rm {params.bed}
awk '{{print $1}}' {params.nobed}_tmp | rev | cut -d "_" -f 2- | rev > {params.nobed}_tmp.1
awk '{{print $2 "\t" $3}}' {params.nobed}_tmp > {params.nobed}_tmp.2
paste {params.nobed}_tmp.1 {params.nobed}_tmp.2 > {params.nobed}
rm {params.nobed}_tmp
rm {params.nobed}_tmp.1
rm {params.nobed}_tmp.2
bedtools intersect -a {params.nobed} -b {params.bam} -bed -wb | awk '{{print $7}}' | sort | uniq > {params.reads}
rm {params.nobed}
rm {params.bam}
gawk -F '/' '$NF==1' {params.reads} > {params.reads}_R1
gawk -F '/' '$NF==2' {params.reads} > {params.reads}_R2
zcat {params.r1} | seqtk subseq - {params.reads}_R1 > {params.fastq}_R1
zcat {params.r2} | seqtk subseq - {params.reads}_R2 > {params.fastq}_R2
gzip {params.fastq}_R1
gzip {params.fastq}_R2
rm {params.reads}_R1
rm {params.reads}_R2
cat {params.fastq}_R1.gz {params.fastq}_R2.gz > {params.fastq}
rm {params.fastq}_R1.gz
rm {params.fastq}_R2.gz
touch {output}
'''
rule nohit_fastq_without_RNA:
input:
touchfile=f"{outdir}Abundance/{{id}}/{{id}}_bwa",
nohit=f"{outdir}diamond/still_nohit_{{id}}_norna",
gff=f"{outdir}Prodigal_Outputs/filtered_gffs/{{id}}/{{id}}.gff"
params:
bed=f"{outdir}diamond/{{id}}.bed",
nobed=f"{outdir}diamond/no_RNA_{{id}}_missing.bed",
bam=f"{outdir}Abundance/{{id}}/{{id}}.bam.bed",
fastq=f"{outdir}diamond/no_RNA_{{id}}_missing.fastq.gz",
reads=f"{outdir}diamond/no_RNA_{{id}}_missing_reads",
r1 = f"{outdir}trimmed/{{id}}/{{id}}.trimmed.R1.fastq.gz",
r2 = f"{outdir}trimmed/{{id}}/{{id}}.trimmed.R2.fastq.gz",
nohit=f"{outdir}nohit"
conda: "envs/bedtools_seqtk_KOunt.yaml"
output: f"{outdir}Touch/{{id}}_nohit_fastq_without_RNA"
shell:
'''
mkdir -p {params.nohit}
awk -F '=|\t|;' '{{print $1 "_" $10 "\t" ($4-1) "\t" $5}}' {input.gff} > {params.bed}
awk 'NR==FNR{{a[$1]=$2;b[$1]=$3;next}}{{print $0 "\t" a[$1] "\t" b[$1]}}' {params.bed} {input.nohit} > {params.nobed}_tmp
rm {params.bed}
awk '{{print $1}}' {params.nobed}_tmp | rev | cut -d "_" -f 2- | rev > {params.nobed}_tmp.1
awk '{{print $2 "\t" $3}}' {params.nobed}_tmp > {params.nobed}_tmp.2
paste {params.nobed}_tmp.1 {params.nobed}_tmp.2 > {params.nobed}
rm {params.nobed}_tmp
rm {params.nobed}_tmp.1
rm {params.nobed}_tmp.2
bedtools intersect -a {params.nobed} -b {params.bam} -bed -wb | awk '{{print $7}}' | sort | uniq > {params.reads}
rm {params.nobed}
rm {params.bam}
gawk -F '/' '$NF==1' {params.reads} > {params.reads}_R1
gawk -F '/' '$NF==2' {params.reads} > {params.reads}_R2
zcat {params.r1} | seqtk subseq - {params.reads}_R1 > {params.fastq}_R1
zcat {params.r2} | seqtk subseq - {params.reads}_R2 > {params.fastq}_R2
gzip {params.fastq}_R1
gzip {params.fastq}_R2
rm {params.reads}_R1
rm {params.reads}_R2
cat {params.fastq}_R1.gz {params.fastq}_R2.gz > {params.fastq}
rm {params.fastq}_R1.gz
rm {params.fastq}_R2.gz
touch {output}
'''
rule nohit_fastq_noclustering:
input:
touchfile=f"{outdir}Abundance/{{id}}/{{id}}_bwa",
nohit=f"{outdir}diamond/still_nohit_{{id}}_noc",
gff=f"{outdir}Prodigal_Outputs/filtered_gffs/{{id}}/{{id}}.gff"
params:
bed=f"{outdir}diamond/{{id}}.bed",
nobed=f"{outdir}nohit/RNA_{{id}}_missing.bed",
bam=f"{outdir}Abundance/{{id}}/{{id}}.bam.bed",
fastq=f"{outdir}nohit/RNA_{{id}}_missing.fastq.gz",
reads=f"{outdir}nohit/RNA_{{id}}_missing_reads",
r1 = f"{outdir}trimmed/{{id}}/{{id}}.trimmed.R1.fastq.gz",
r2 = f"{outdir}trimmed/{{id}}/{{id}}.trimmed.R2.fastq.gz",
nohit=f"{outdir}nohit"
conda: "envs/bedtools_seqtk_KOunt.yaml"
output: f"{outdir}Touch/{{id}}_nohit_fastq_noc"
shell:
'''
mkdir -p {params.nohit}
awk -F '=|\t|;' '{{print $1 "_" $10 "\t" ($4-1) "\t" $5}}' {input.gff} > {params.bed}
awk 'NR==FNR{{a[$1]=$2;b[$1]=$3;next}}{{print $0 "\t" a[$1] "\t" b[$1]}}' {params.bed} {input.nohit} > {params.nobed}_tmp
rm {params.bed}
awk '{{print $1}}' {params.nobed}_tmp | rev | cut -d "_" -f 2- | rev > {params.nobed}_tmp.1
awk '{{print $2 "\t" $3}}' {params.nobed}_tmp > {params.nobed}_tmp.2
paste {params.nobed}_tmp.1 {params.nobed}_tmp.2 > {params.nobed}
rm {params.nobed}_tmp
rm {params.nobed}_tmp.1
rm {params.nobed}_tmp.2
bedtools intersect -a {params.nobed} -b {params.bam} -bed -wb | awk '{{print $7}}' | sort | uniq > {params.reads}
rm {params.nobed}
rm {params.bam}
gawk -F '/' '$NF==1' {params.reads} > {params.reads}_R1
gawk -F '/' '$NF==2' {params.reads} > {params.reads}_R2
zcat {params.r1} | seqtk subseq - {params.reads}_R1 > {params.fastq}_R1
zcat {params.r2} | seqtk subseq - {params.reads}_R2 > {params.fastq}_R2
gzip {params.fastq}_R1
gzip {params.fastq}_R2
rm {params.reads}_R1
rm {params.reads}_R2
cat {params.fastq}_R1.gz {params.fastq}_R2.gz > {params.fastq}
rm {params.fastq}_R1.gz
rm {params.fastq}_R2.gz
touch {output}
'''
rule nohit_annotate_reads:
input:
touch=f"{outdir}Touch/{{id}}_nohit_fastq",
db=f"{diamond_db}",
rna=f"{mmseq_db}",
bg=f"{combined_bdg}"
params:
fq=f"{outdir}nohit/RNA_{{id}}_missing.fastq.gz",
of="6 qseqid sseqid pident qlen slen qstart qend sstart send evalue",
ofm="query,target,pident,qlen,tlen,qstart,qend,tstart,tend,evalue",
wd=f"{outdir}Abundance/{{id}}/nohit/",
tmp=f"{outdir}diamond/{{id}}_reads_tmp",
cat=f"{outdir}diamond/NoHit_{{id}}_cat",
nohit=f"{outdir}diamond/{{id}}/{{id}}_hits_nohit",
mmseq=f"{outdir}MMSeq_Outputs/{{id}}/nohit_reads_{{id}}",
reads=f"{outdir}nohit/RNA_{{id}}_missing_reads",
missing=f"{outdir}nohit/RNA_{{id}}_still_missing_reads",
threads=f"{nohit}"
conda: "envs/diamond_bedtools_mmseq2_KOunt.yaml"
output: