Last updated: 2020-01-20
Checks: 6 1
Knit directory: 20170327_Psen2S4Ter_RNASeq/
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This document simply provides the bash code used for running the pre-processing and alignment.
cat(readLines("code/runPipeline.sh"), sep = "\n")#!/bin/bash
#SBATCH -p batch
#SBATCH -N 1
#SBATCH -n 12
#SBATCH --time=2:00:00
#SBATCH --mem=32GB
#SBATCH -o /data/biohub/20170327_Psen2S4Ter_RNASeq/slurm/%x_%j.out
#SBATCH -e /data/biohub/20170327_Psen2S4Ter_RNASeq/slurm/%x_%j.err
#SBATCH --mail-type=END
#SBATCH --mail-type=FAIL
#SBATCH --mail-user=stephen.pederson@adelaide.edu.au
## Clean run of the PSEN2 data.
## Cores
CORES=12
## Modules
module load FastQC/0.11.7
module load STAR/2.7.0d-foss-2016b
module load SAMtools/1.3.1-GCC-5.3.0-binutils-2.25
module load cutadapt/1.14-foss-2016b-Python-2.7.13
module load Subread/1.5.2-foss-2016b
## Function for checking directories
checkAndMake () {
echo "Checking if $1 exists"
if [[ ! -d $1 ]]
then
echo "Creating $1"
mkdir -p $1
fi
if [[ -d $1 ]]
then
echo "Found $1"
else
echo "$1 could not be created or found"
exit 1
fi
}
## Directories
PROJROOT=/data/biohub/20170327_Psen2S4Ter_RNASeq/data
REFS=/data/biorefs/reference_genomes/ensembl-release-98/danio_rerio/
if [[ ! -d ${REFS} ]]
then
echo "Couldn't find ${REFS}"
exit 1
fi
GTF=${REFS}/Danio_rerio.GRCz11.98.chr.gtf.gz
if [[ ! -f ${GTF} ]]
then
echo "Couldn't find ${GTF}"
exit 1
fi
# Raw Data
RAWDIR=${PROJROOT}/0_rawData
checkAndMake ${RAWDIR}
checkAndMake ${RAWDIR}/FastQC
## Trimmed
TRIMDIR=${PROJROOT}/1_trimmedData
checkAndMake ${TRIMDIR}/fastq
checkAndMake ${TRIMDIR}/FastQC
checkAndMake ${TRIMDIR}/log
## Aligned
ALIGNDIR=${PROJROOT}/2_alignedData
checkAndMake ${ALIGNDIR}
checkAndMake ${ALIGNDIR}/bam
checkAndMake ${ALIGNDIR}/FastQC
checkAndMake ${ALIGNDIR}/log
checkAndMake ${ALIGNDIR}/featureCounts
echo "All directories checked and created"
##----------------------------------------------------------------------------##
## Initial FastQC ##
##----------------------------------------------------------------------------##
fastqc -t ${CORES} -o ${RAWDIR}/FastQC --noextract ${RAWDIR}/fastq/*fastq.gz
##----------------------------------------------------------------------------##
## Trimming ##
##----------------------------------------------------------------------------##
for R1 in ${RAWDIR}/fastq/*R1.fastq.gz
do
R2=${R1%_R1.fastq.gz}_R2.fastq.gz
echo -e "The R1 file should be ${R1}"
echo -e "The R2 file should be ${R2}"
## Create output filenames
out1=${TRIMDIR}/fastq/$(basename $R1)
out2=${TRIMDIR}/fastq/$(basename $R2)
BNAME=${TRIMDIR}/fastq/$(basename ${R1%_1.fq.gz})
echo -e "Output file 1 will be ${out1}"
echo -e "Output file 2 will be ${out2}"
echo -e "Trimming:\t${BNAME}"
LOG=${TRIMDIR}/log/$(basename ${BNAME}).info
echo -e "Trimming info will be written to ${LOG}"
cutadapt \
-a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC \
-A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT \
-o ${out1} \
-p ${out2} \
-m 35 \
--trim-n \
--max-n=1 \
--nextseq-trim=30 \
${R1} \
${R2} > ${LOG}
done
fastqc -t ${CORES} -o ${TRIMDIR}/FastQC --noextract ${TRIMDIR}/fastq/*fastq.gz
##----------------------------------------------------------------------------##
## STAR Alignment ##
##----------------------------------------------------------------------------##
## Aligning, filtering and sorting
for R1 in ${TRIMDIR}/fastq/*R1.fastq.gz
do
BNAME=$(basename ${R1%_R1.fastq.gz})
R2=${R1%_R1.fastq.gz}_R2.fastq.gz
echo -e "STAR will align:\t${R1}"
echo -e "STAR will also align:\t${R2}"
STAR \
--runThreadN ${CORES} \
--genomeDir ${REFS}/star \
--readFilesIn ${R1} ${R2} \
--readFilesCommand gunzip -c \
--outFileNamePrefix ${ALIGNDIR}/bam/${BNAME} \
--outSAMtype BAM SortedByCoordinate
done
## Move the log files into their own folder
mv ${ALIGNDIR}/bam/*out ${ALIGNDIR}/log
mv ${ALIGNDIR}/bam/*tab ${ALIGNDIR}/log
## Fastqc and indexing
for BAM in ${ALIGNDIR}/bam/*.bam
do
fastqc -t ${CORES} -f bam_mapped -o ${ALIGNDIR}/FastQC --noextract ${BAM}
samtools index ${BAM}
done
##----------------------------------------------------------------------------##
## featureCounts ##
##----------------------------------------------------------------------------##
## Feature Counts - obtaining all sorted bam files
sampleList=`find ${ALIGNDIR}/bam -name "*out.bam" | tr '\n' ' '`
## Extract gtf for featureCounts
zcat ${GTF} > temp.gtf
## Running featureCounts on the sorted bam files
featureCounts -Q 10 \
-s 2 \
-T ${CORES} \
-p \
--fracOverlap 1 \
-a temp.gtf \
-o ${ALIGNDIR}/featureCounts/counts.out ${sampleList}
## Remove the temporary gtf
rm temp.gtf
## Storing the output in a single file
cut -f1,7- ${ALIGNDIR}/featureCounts/counts.out | \
sed 1d > ${ALIGNDIR}/featureCounts/genes.out
##----------------------------------------------------------------------------##
## kallisto ##
##----------------------------------------------------------------------------##
## Aligning, filtering and sorting
for R1 in ${TRIMDIR}/fastq/*R1.fastq.gz
do
sbatch ${PROJROOT}/bash/singleKallisto.sh ${R1}
done
The final step of the above script is to call an instance of the following for each sample.
cat(readLines("code/singleKallisto.sh"), sep = "\n")#!/bin/bash
#SBATCH -p batch
#SBATCH -N 1
#SBATCH -n 1
#SBATCH --time=2:00:00
#SBATCH --mem=4GB
#SBATCH -o /data/biohub/20170327_Psen2S4Ter_RNASeq/slurm/%x_%j.out
#SBATCH -e /data/biohub/20170327_Psen2S4Ter_RNASeq/slurm/%x_%j.err
#SBATCH --mail-type=END
#SBATCH --mail-type=FAIL
#SBATCH --mail-user=stephen.pederson@adelaide.edu.au
# Load modules
module load kallisto/0.43.1-foss-2016b
## Reference Files
REFS=/data/biorefs/reference_genomes/ensembl-release-98/danio_rerio/
IDX=/${REFS}/kallisto/Danio_rerio.GRCz11.cdna.primary_assembly.psen2S4Ter.idx
## Directories
PROJROOT=/data/biohub/20170327_Psen2S4Ter_RNASeq/data
## Setup for kallisto output
ALIGNDIR=${PROJROOT}/3_kallisto
## Now organise the input files
F1=$1
F2=${F1%_R1.fastq.gz}_R2.fastq.gz
## Organise the output files
OUTDIR=${ALIGNDIR}/$(basename ${F1%_R1.fastq.gz})
echo -e "Creating ${OUTDIR}"
mkdir -p ${OUTDIR}
echo -e "Currently aligning:\n\t${F1}\n\t${F2}"
echo -e "Output will be written to ${OUTDIR}"
kallisto quant \
-b 50 \
--rf-stranded \
-t 1 \
-i ${IDX} \
-o ${OUTDIR} \
${F1} ${F2}
devtools::session_info()─ Session info ───────────────────────────────────────────────────────────────
setting value
version R version 3.6.2 (2019-12-12)
os Ubuntu 18.04.3 LTS
system x86_64, linux-gnu
ui X11
language en_AU:en
collate en_AU.UTF-8
ctype en_AU.UTF-8
tz Australia/Adelaide
date 2020-01-20
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