RNAseq Alignments – Trimmed S.salar RNAseq to GCF_000233375.1_ICSASG_v2_genomic.fa Using Hisat2 on Mox

This is a continuation of addressing Shelly Trigg’s (regarding some Salmo salar RNAseq data) request (GitHub Issue) to trim (completed 20201029), perform genome alignment, and transcriptome alignment.

Ran HISAT2 with the trimmed FastQ files from 20201029 with the following options:

  • --dta: This stands for “downstream transcriptome alignment”. Since we’ll be performing a subsequent alignment using the transcriptome using StringTie, I decided to add this option.
  • --new-summary: This creates a summary file that can be easily read by MultiQC.

This was run on Mox.

SBATCH script (GitHub):

#!/bin/bash ## Job Name #SBATCH --job-name=20201103_ssal_RNAseq_hisat2_alignment ## Allocation Definition #SBATCH --account=coenv #SBATCH --partition=coenv ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-00:00:00 ## Memory per node #SBATCH --mem=200G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20201103_ssal_RNAseq_hisat2_alignment ### S.salar RNAseq Hisat2 alignment. ### Uses fastp-trimmed FastQ files from 20201029. ### Uses GCF_000233375.1_ICSASG_v2_genomic.fa as reference, ### created by Shelly Trigg. ### This is a subset of the NCBI RefSeq GCF_000233375.1_ICSASG_v2_genomic.fna. ### Includes only "chromosome" sequence entries. ################################################################################### # These variables need to be set by user ## Assign Variables # Set number of CPUs to use threads=27 # Input/output files fastq_checksums=fastq_checksums.md5 fastq_dir="/gscratch/srlab/sam/data/S_salar/RNAseq/" genome_fasta="/gscratch/srlab/sam/data/S_salar/genomes/GCF_000233375.1_ICSASG_v2_genomic.fa" genome_index_name="GCF_000233375.1_ICSASG_v2" # Paths to programs hisat2_dir="/gscratch/srlab/programs/hisat2-2.1.0" hisat2="${hisat2_dir}/hisat2" hisat2_build="${hisat2_dir}/hisat2-build" samtools="/gscratch/srlab/programs/samtools-1.10/samtools" ## Inititalize arrays fastq_array_R1=() fastq_array_R2=() names_array=() # Programs associative array declare -A programs_array programs_array=( [hisat2]="${hisat2}" \ [hisat2-build]="${hisat2_build}" [samtools_index]="${samtools} index" \ [samtools_sort]="${samtools} sort" \ [samtools_view]="${samtools} view" ) ################################################################################### # Exit script if any command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # Capture date timestamp=$(date +%Y%m%d) # Create array of fastq R1 files for fastq in "${fastq_dir}"*_1.fastp-trim.20201029.fq.gz do fastq_array_R1+=("${fastq}") # Create array of sample names ## Uses parameter substitution to strip leading path from filename ## Uses awk to parse out sample name from filename names_array+=($(echo "${fastq#${fastq_dir}}" | awk -F"[_]" '{print $1 "_" $2}')) done # Create array of fastq R2 files for fastq in "${fastq_dir}"*_2.fastp-trim.20201029.fq.gz do fastq_array_R2+=("${fastq}") done # Build Hisat2 reference index "${programs_array[hisat2-build]}" \ "${genome_fasta}" \ "${genome_index_name}" \ -p "${threads}" \ 2> hisat2_build.err # Hisat2 alignments for index in "${!fastq_array_R1[@]}" do # Get current sample name sample_name=$(echo "${names_array[index]}") # Run Hisat2 # Sets --dta which tailors output for downstream transcriptome assemblers (e.g. Stringtie) # Sets --new-summary option for use with MultiQC "${programs_array[hisat2]}" \ -x "${genome_index_name}" \ --dta \ --new-summary \ -1 "${fastq_array_R1[index]}" \ -2 "${fastq_array_R2[index]}" \ -S "${sample_name}".sam \ 2> "${sample_name}"_hisat2.err # Sort SAM files, convert to BAM ${programs_array[samtools_view]} \ -@ "${threads}" \ -Su "${sample_name}".sam \ | ${programs_array[samtools_sort]} - \ -@ "${threads}" \ -o "${sample_name}".sorted.bam # Index sorted BAM file ${programs_array[samtools_index]} "${sample_name}".sorted.bam done # Create list of fastq files used in analysis ## Uses parameter substitution to strip leading path from filename for fastq in "${fastq_dir}"*fastp-trim.20201029.fq.gz do echo "${fastq#${fastq_dir}}" >> fastq.list.txt md5sum "${fastq}" >> ${fastq_checksums} done # Capture program options for program in "${!programs_array[@]}" do { echo "Program options for ${program}: " echo "" # Handle samtools help menus if [[ "${program}" == "samtools_index" ]] \ || [[ "${program}" == "samtools_sort" ]] \ || [[ "${program}" == "samtools_view" ]] then ${programs_array[$program]} fi ${programs_array[$program]} -h echo "" echo "" echo "