Last updated: 2020-01-20

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Knit directory: 20170327_Psen2S4Ter_RNASeq/

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Introduction

This document simply provides the bash code used for running the pre-processing and alignment.

Basic Script

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
  

Kallisto

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                  

─ Packages ───────────────────────────────────────────────────────────────────
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 stringi       1.4.5   2020-01-11 [2] CRAN (R 3.6.2)
 stringr       1.4.0   2019-02-10 [2] CRAN (R 3.6.0)
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 usethis       1.5.1   2019-07-04 [2] CRAN (R 3.6.1)
 withr         2.1.2   2018-03-15 [2] CRAN (R 3.6.0)
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 xfun          0.12    2020-01-13 [2] CRAN (R 3.6.2)
 yaml          2.2.0   2018-07-25 [2] CRAN (R 3.6.0)

[1] /home/steveped/R/x86_64-pc-linux-gnu-library/3.6
[2] /usr/local/lib/R/site-library
[3] /usr/lib/R/site-library
[4] /usr/lib/R/library