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@uw.edu
## 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 "----------------------------------------------"
	echo ""
	echo ""
} &>> program_options.log || true

  # If MultiQC is in programs_array, copy the config file to this directory.
  if [[ "${program}" == "multiqc" ]]; then
  	cp --preserve ~/.multiqc_config.yaml "${timestamp}_multiqc_config.yaml"
  fi
done


# Document programs in PATH (primarily for program version ID)
{
date
echo ""
echo "System PATH for $SLURM_JOB_ID"
echo ""
printf "%0.s-" {1..10}
echo "${PATH}" | tr : \\n
} >> system_path.log

NOTE: I manually removed the SAM files that were generated during this process. This step was not included in the SBATCH script above because I didn’t realize the SAM files would remain after creating the (desired) BAM files. The SAM files were very large and not necessary for downstream analysis.


RESULTS

Runtime was close to 3hrs:

Cumulative HISAT2 runtime on Mox

Output folder:

BAM files for each alignment are linked below. The BAM files will be used for subsequent usage in StringTie. Additionally, each BAM file has an associated index file and a HISAT2 “error” file. This “error” file is actually the summary report of the alignment and will be used by MultiQC later on. In the future, I’ll try to remember to change the labelling for this…