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metagenomics_pipeline.sh
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#!/bin/bash
#SBATCH --nodes=1
#SBATCH --cpus-per-task=24
#SBATCH --partition normal
#SBATCH --time=4:00:00
#SBATCH --mem=140G
#SBATCH --job-name=metagenomics_pipeline
#SBATCH --error ./log/%x_%j.err.txt
#SBATCH --output ./log/%x_%j.out.txt
## Parameters ############################################################################
R1_base=`basename $1 .fastq.gz`
R2_base=`basename $2 .fastq.gz`
READ_LENGTH=75
TMP_DIR=./tmp/${R1_base}
OUT_DIR=./output/${R1_base}
mkdir "$TMP_DIR"
mkdir "$OUT_DIR"
mkdir "$OUT_DIR"/alignment
mkdir "$OUT_DIR"/fastqc
mkdir "$OUT_DIR"/arriba
mkdir "$OUT_DIR"/kraken
mkdir "$OUT_DIR"/bracken
mkdir "$OUT_DIR"/bracken/report
mkdir "$OUT_DIR"/centrifuge
mkdir "$OUT_DIR"/scripts
BOWTIE_INDEX=/scratch/tmp/thomachr/metagenomics/bowtie_index/GRCh38_noalt_as
DB_VIRAL=/scratch/tmp/thomachr/references/kraken/k2_viral_20230314
DB_STANDARD=/scratch/tmp/thomachr/references/kraken/k2_standard_20230314
DB_EUPATH=/scratch/tmp/thomachr/references/kraken/k2_eupathdb48_20230407
DB_NT=/scratch/tmp/thomachr/references/kraken/k2_nt_20230502
CENTRIFUGE_INDEX=/scratch/tmp/thomachr/software/centrifuge-1.0.4-beta/indices/nt
BLAST_DB=/scratch/tmp/thomachr/references/blast/nt
BASE_DIR=/scratch/tmp/thomachr/arriba/arriba_v2.4.0
STAR_INDEX_DIR=$BASE_DIR/STAR_index_GRCh37viral_GENCODE19
ANNOTATION_GTF=$BASE_DIR/GENCODE19.gtf
ASSEMBLY_FA=$BASE_DIR/GRCh37viral.fa
BLACKLIST_TSV=$BASE_DIR/database/blacklist_hg19_hs37d5_GRCh37_v2.4.0.tsv.gz
KNOWN_FUSIONS_TSV=$BASE_DIR/database/known_fusions_hg19_hs37d5_GRCh37_v2.4.0.tsv.gz
TAGS_TSV="$KNOWN_FUSIONS_TSV"
PROTEIN_DOMAINS_GFF3=$BASE_DIR/database/protein_domains_hg19_hs37d5_GRCh37_v2.4.0.gff3
THREADS_KRAKEN=8
THREADS=24
export PATH=/scratch/tmp/thomachr/software/MEGAHIT-1.2.9-Linux-x86_64-static/bin:$PATH
cp ./scripts/metagenome_report.Rmd "$OUT_DIR"/scripts/metagenome_report.Rmd
cp ./scripts/metagenome_report.R "$OUT_DIR"/scripts/metagenome_report.R
##########################################################################################
## TRIM ##################################################################################
##########################################################################################
if [ -f ""$TMP_DIR"/${R1_base}.trimmed.fq.gz" ]; then
echo "Trimming for ${R1_base} already performed."
else
/scratch/tmp/thomachr/software/fqtrim-0.9.7/fqtrim -l 30 -D -p $THREADS -q 20 -P33 \
-r "$OUT_DIR"/alignment/fqtrim_report.txt \
-A -o trimmed.fq.gz --outdir "$TMP_DIR" "$1","$2"
fi
##########################################################################################
## FASTQC ################################################################################
##########################################################################################
if [ -f "${OUT_DIR}/fastqc/${R1_base}_fastqc.html" ]; then
echo "Fastqc for ${R1_base} already performed."
else
ml palma/2019a FastQC/0.11.9-Java-11
fastqc --extract -t $THREADS -o "$OUT_DIR"/fastqc "$1" "$2"
fastqc --extract -t $THREADS -o "$OUT_DIR"/fastqc "$TMP_DIR"/*.trimmed.fq.gz
fi
##########################################################################################
## Host removal ##########################################################################
##########################################################################################
if [ -f "${TMP_DIR}/${R1_base}_nonhuman_reads.fastq.gz" ]; then
echo "Host removal for ${R1_base} already performed."
else
ml palma/2020b GCC/10.2.0 Bowtie2/2.4.2
bowtie2 -x $BOWTIE_INDEX -p $THREADS -1 "$TMP_DIR"/${R1_base}.trimmed.fq.gz \
-2 "$TMP_DIR"/${R2_base}.trimmed.fq.gz \
--un-conc-gz "$TMP_DIR"/${R1_base}_nonhuman_reads \
-S "$TMP_DIR"/${R1_base}_host_alignment.sam
mv "$TMP_DIR"/${R1_base}_nonhuman_reads.1 "$TMP_DIR"/${R1_base}_nonhuman_reads.fastq.gz
mv "$TMP_DIR"/${R1_base}_nonhuman_reads.2 "$TMP_DIR"/${R2_base}_nonhuman_reads.fastq.gz
fi
echo $(zcat "$1"|wc -l)/4|bc > "$OUT_DIR"/alignment/total_reads.txt
echo $(zcat "$TMP_DIR"/${R1_base}_nonhuman_reads.fastq.gz|wc -l)/4|bc > "$OUT_DIR"/alignment/non_human_reads.txt
##########################################################################################
## Kraken2 ###############################################################################
##########################################################################################
if [ -f "${OUT_DIR}/kraken/${R1_base}.kraken.standard.txt" ]; then
echo "Kraken output for ${R1_base} already exists."
else
module unload
ml palma/2021b GCC/11.2.0 OpenMPI/4.1.1 Kraken2/2.1.2 Bracken/2.7
kraken2 \
--db $DB_STANDARD \
--threads $THREADS_KRAKEN \
--minimum-hit-groups 3 \
--use-names \
--report-minimizer-data \
--output "$TMP_DIR"/${R1_base}.kraken \
--report "$OUT_DIR"/kraken/${R1_base}.kraken.standard.txt \
--paired "$TMP_DIR"/${R1_base}_nonhuman_reads.fastq.gz \
"$TMP_DIR"/${R2_base}_nonhuman_reads.fastq.gz
kraken2 \
--db $DB_VIRAL \
--threads $THREADS_KRAKEN \
--minimum-hit-groups 3 \
--use-names \
--report-minimizer-data \
--output "$TMP_DIR"/${R1_base}.kraken \
--report "$OUT_DIR"/kraken/${R1_base}.kraken.viral.txt \
--paired "$TMP_DIR"/${R1_base}_nonhuman_reads.fastq.gz \
"$TMP_DIR"/${R2_base}_nonhuman_reads.fastq.gz
kraken2 \
--db $DB_EUPATH \
--threads $THREADS_KRAKEN \
--minimum-hit-groups 3 \
--use-names \
--report-minimizer-data \
--output "$TMP_DIR"/${R1_base}.kraken \
--report "$OUT_DIR"/kraken/${R1_base}.kraken.eupath.txt \
--paired "$TMP_DIR"/${R1_base}_nonhuman_reads.fastq.gz \
"$TMP_DIR"/${R2_base}_nonhuman_reads.fastq.gz
fi
if [ -f "${OUT_DIR}/bracken/${R1_base}.standard_genus.txt" ]; then
echo "Bracken output for ${R1_base} already exists."
else
# Standard Database
bracken \
-d $DB_STANDARD \
-i "$OUT_DIR"/kraken/${R1_base}.kraken.standard.txt \
-r $READ_LENGTH \
-l G \
-t 10 \
-o "$OUT_DIR"/bracken/${R1_base}.standard_genus.txt \
-w "$OUT_DIR"/bracken/report/${R1_base}.standard_genus.report.txt
bracken \
-d $DB_STANDARD \
-i "$OUT_DIR"/kraken/${R1_base}.kraken.standard.txt \
-r $READ_LENGTH \
-l S \
-t 10 \
-o "$OUT_DIR"/bracken/${R1_base}.standard_species.txt \
-w "$OUT_DIR"/bracken/report/${R1_base}.standard_species.report.txt
# Viral Database
bracken \
-d $DB_VIRAL \
-i "$OUT_DIR"/kraken/${R1_base}.kraken.viral.txt \
-r $READ_LENGTH \
-l G \
-t 10 \
-o "$OUT_DIR"/bracken/${R1_base}.viral_genus.txt \
-w "$OUT_DIR"/bracken/report/${R1_base}.viral_genus.report.txt
bracken \
-d $DB_VIRAL \
-i "$OUT_DIR"/kraken/${R1_base}.kraken.viral.txt \
-r $READ_LENGTH \
-l S \
-t 10 \
-o "$OUT_DIR"/bracken/${R1_base}.viral_species.txt \
-w "$OUT_DIR"/bracken/report/${R1_base}.viral_species.report.txt
# EUPATHDB48 Database
bracken \
-d $DB_EUPATH \
-i "$OUT_DIR"/kraken/${R1_base}.kraken.eupath.txt \
-r $READ_LENGTH \
-l G \
-t 10 \
-o "$OUT_DIR"/bracken/${R1_base}.eupath_genus.txt \
-w "$OUT_DIR"/bracken/report/${R1_base}.eupath_genus.report.txt
bracken \
-d $DB_EUPATH \
-i "$OUT_DIR"/kraken/${R1_base}.kraken.eupath.txt \
-r $READ_LENGTH \
-l S \
-t 10 \
-o "$OUT_DIR"/bracken/${R1_base}.eupath_species.txt \
-w "$OUT_DIR"/bracken/report/${R1_base}.eupath_species.report.txt
fi
##########################################################################################
## Centrifuge ############################################################################
##########################################################################################
if [ -f ""$OUT_DIR"/centrifuge/${R1_base}.centrifuge.txt" ]; then
echo "Centrifuge output for ${R1_base} already exists."
else
module unload
ml palma/2019b GCC/8.3.0 OpenMPI/3.1.4 HISAT2/2.2.1
export PATH=/scratch/tmp/thomachr/software/centrifuge-1.0.4:$PATH
centrifuge -q \
-1 "$TMP_DIR"/${R1_base}_nonhuman_reads.fastq.gz \
-2 "$TMP_DIR"/${R2_base}_nonhuman_reads.fastq.gz \
--report-file "$OUT_DIR"/centrifuge/${R1_base}.centrifuge.txt \
--threads $THREADS \
-x $CENTRIFUGE_INDEX
fi
##########################################################################################
## Arriba ################################################################################
##########################################################################################
if [ -f ""$OUT_DIR"/arriba/${R1_base}virus_expression1.tsv" ]; then
echo "Arriba analysis for ${R1_base} already exists."
else
module unload
ml palma/2019a GCC/8.2.0-2.31.1 OpenMPI/3.1.3 SAMtools/1.9
/scratch/tmp/thomachr/software/STAR-2.7.10b/source/STAR \
--runThreadN "$THREADS" \
--outFileNamePrefix "$TMP_DIR"/ \
--genomeDir "$STAR_INDEX_DIR" \
--genomeLoad NoSharedMemory \
--readFilesIn "$TMP_DIR"/${R1_base}_nonhuman_reads.fastq.gz "$TMP_DIR"/${R2_base}_nonhuman_reads.fastq.gz \
--readFilesCommand zcat \
--outStd BAM_Unsorted \
--outSAMtype BAM Unsorted \
--outSAMunmapped Within \
--outBAMcompression 0 \
--outFilterMultimapNmax 50 \
--peOverlapNbasesMin 10 \
--alignSplicedMateMapLminOverLmate 0.5 \
--alignSJstitchMismatchNmax 5 -1 5 5 \
--chimSegmentMin 10 \
--chimOutType WithinBAM HardClip \
--chimJunctionOverhangMin 10 \
--chimScoreDropMax 30 \
--chimScoreJunctionNonGTAG 0 \
--chimScoreSeparation 1 \
--chimSegmentReadGapMax 3 \
--chimMultimapNmax 50 |
tee "$TMP_DIR"/${R1_base}_Aligned.out.bam |
"$BASE_DIR/arriba" \
-x /dev/stdin -I \
-o "$OUT_DIR"/arriba/${R1_base}fusions.tsv \
-a "$ASSEMBLY_FA" -g "$ANNOTATION_GTF" -b "$BLACKLIST_TSV" \
-k "$KNOWN_FUSIONS_TSV" -t "$TAGS_TSV" -p "$PROTEIN_DOMAINS_GFF3"
samtools sort -@ "$THREADS" -m $((40000/THREADS))M -T tmp \
-O bam "$TMP_DIR"/${R1_base}_Aligned.out.bam > "$TMP_DIR"/${R1_base}_Aligned.sortedByCoord.out.bam
rm -f "$TMP_DIR"/${R1_base}_Aligned.out.bam
samtools index "$TMP_DIR"/${R1_base}_Aligned.sortedByCoord.out.bam
$BASE_DIR/scripts/quantify_virus_expression.sh "$TMP_DIR"/${R1_base}_Aligned.sortedByCoord.out.bam \
"$OUT_DIR"/arriba/${R1_base}virus_expression.tsv
fi
##########################################################################################
## Report ################################################################################
##########################################################################################
module unload
ml palma/2020b GCC/10.2.0 OpenMPI/4.0.5 R/4.0.3 Pandoc/2.10
Rscript ./output/${R1_base}/scripts/metagenome_report.R ./output/${R1_base}/scripts
##########################################################################################
## De novo assembly ######################################################################
##########################################################################################
if [ -f ""$OUT_DIR"/assembly/${R1_base}.contigs.fa" ]; then
echo "De novo assambly for ${R1_base} already exists."
else
megahit -1 "$TMP_DIR"/${R1_base}_nonhuman_reads.fastq.gz \
-2 "$TMP_DIR"/${R2_base}_nonhuman_reads.fastq.gz \
-o "$OUT_DIR"/assembly --min-contig-len 1000 \
--out-prefix ${R1_base} -t $THREADS
fi
rm -R "$OUT_DIR"/assembly/intermediate_contigs/*
##########################################################################################
## Kraken of de novo assembled contigs ###################################################
##########################################################################################
if [ -f "$OUT_DIR"/assembly/${R1_base}.kraken.report.txt ]; then
echo "Kraken output for ${R1_base} (de novo assembly) already exists."
else
module unload
ml palma/2021b GCC/11.2.0 OpenMPI/4.1.1 Kraken2/2.1.2 Bracken/2.7
kraken2 \
--db $DB_STANDARD \
--threads $THREADS_KRAKEN \
--minimum-hit-groups 3 \
--use-names \
--report-minimizer-data \
--output "$OUT_DIR"/assembly/${R1_base}.kraken.output.txt \
--report "$OUT_DIR"/assembly/${R1_base}.kraken.report.txt \
"$OUT_DIR"/assembly/${R1_base}.contigs.fa
fi
##########################################################################################
## Centrifuge of de novo assembled contigs ###############################################
##########################################################################################
if [ -f "$OUT_DIR"/assembly/${R1_base}.centrifuge.txt ]; then
echo "Centrifuge output for ${R1_base} (de novo assembly) already exists."
else
module unload
ml palma/2019b GCC/8.3.0 OpenMPI/3.1.4 HISAT2/2.2.1
export PATH=/scratch/tmp/thomachr/software/centrifuge-1.0.4:$PATH
centrifuge -f \
--report-file "$OUT_DIR"/assembly/${R1_base}.centrifuge.report.txt \
--threads $THREADS \
-S "$OUT_DIR"/assembly/${R1_base}.centrifuge.txt \
-x $CENTRIFUGE_INDEX \
"$OUT_DIR"/assembly/${R1_base}.contigs.fa
fi
##########################################################################################
## Report ################################################################################
##########################################################################################
module unload
ml palma/2020b GCC/10.2.0 OpenMPI/4.0.5 R/4.0.3 Pandoc/2.10
Rscript ./output/${R1_base}/scripts/metagenome_report.R ./output/${R1_base}/scripts