Sam’s Notebook: Genome Annotation – O.lurida 20190709-v081 Hisat2 Transcript Isoform Index

Last week I re-annotated our Olurida_v081 genome using tissue-specific transcriptomes. The MAKER annotations don’t yield transcript isoforms, so this is the first part of the process in identifying/annotating different isoforms within the transcriptome.

Essentially, the steps below (which is what was done here) are needed to prepare files for use with Stringtie:

  1. Create GTF file (basically a GFF specifically for use with transcripts – thus the “T” in GTF) from input GFF file. Done with GFF utilities software.
  2. Identify splice sites and exons in newly-created GTF. Done with Hisat2 software.
  3. Create a Hisat2 reference index that utilizes the GTF. Done with Hisat2 software.

This was run on Mox.

The SBATCH script has a bunch of leftover extraneous steps that aren’t relevant to this step of the annotation process; specifically the FastQ manipulation steps. This is due to a copy/paste from a previous Hisat2 run that I neglected to edit out and I didn’t want to edit the script after I actually ran it, so have left it in here.

SBATCH script (GitHub):

 #!/bin/bash ## Job Name #SBATCH --job-name=oly_hisat2 ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=25-00:00:00 ## Memory per node #SBATCH --mem=500G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite@uw.edu ## Specify the working directory for this job #SBATCH --workdir=/gscratch/scrubbed/samwhite/outputs/20190716_hisat2-build_20190709-olur_v081 # Exit script if any command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # Document programs in PATH (primarily for program version ID) date >> system_path.log echo "" >> system_path.log echo "System PATH for $SLURM_JOB_ID" >> system_path.log echo "" >> system_path.log printf "%0.s-" {1..10} >> system_path.log echo "${PATH}" | tr : \\n >> system_path.log threads=27 genome_index_name="20190709-Olurida_v081" # Paths to programs gffread="/gscratch/srlab/programs/gffread-0.11.4.Linux_x86_64/gffread" hisat2_dir="/gscratch/srlab/programs/hisat2-2.1.0" hisat2_build="${hisat2_dir}/hisat2-build" hisat2_exons="${hisat2_dir}/hisat2_extract_exons.py" hisat2_splice_sites="${hisat2_dir}/hisat2_extract_splice_sites.py" # Input/output files fastq_dir="/gscratch/srlab/sam/data/O_lurida/RNAseq/" genome_dir="/gscratch/srlab/sam/data/O_lurida/genomes/Olurida_v081" genome_gff="${genome_dir}/20190709-Olurida_v081_genome_snap02.all.renamed.putative_function.domain_added.gff" exons="hisat2_exons.tab" genome_fasta="${genome_dir}/Olurida_v081.fa" splice_sites="hisat2_splice_sites.tab" transcripts_gtf="20190709-Olurida_v081_genome_snap02.all.renamed.putative_function.domain_added.gtf" ## Inititalize arrays fastq_array_R1=() fastq_array_R2=() # Create array of fastq R1 files for fastq in "${fastq_dir}"/*R1*.gz do fastq_array_R1+=("${fastq}") done # Create array of fastq R2 files for fastq in "${fastq_dir}"/*R2*.gz do fastq_array_R2+=("${fastq}") done # Create array of sample names ## Uses parameter substitution to strip leading path from filename ## Uses awk to parse out sample name from filename for R1_fastq in "${fastq_dir}"/*R1*.gz do names_array+=($(echo "${R1_fastq#${fastq_dir}}" | awk -F"[_.]" '{print $1 "_" $5}')) done # Create list of fastq files used in analysis ## Uses parameter substitution to strip leading path from filename for fastq in "${fastq_dir}"*.gz do echo "${fastq#${fastq_dir}}" >> fastq.list.txt done # Create transcipts GTF from genome FastA "${gffread}" -T \ "${genome_gff}" \ -o "${transcripts_gtf}" # Create Hisat2 exons tab file "${hisat2_exons}" \ "${transcripts_gtf}" \ > "${exons}" # Create Hisate2 splice sites tab file "${hisat2_splice_sites}" \ "${transcripts_gtf}" \ > "${splice_sites}" # Build Hisat2 reference index using splice sites and exons "${hisat2_build}" \ "${genome_fasta}" \ "${genome_index_name}" \ --exon "${exons}" \ --ss "${splice_sites}" \ -p "${threads}" \ 2> hisat2_build.err # Copy Hisat2 index files to my data directory rsync -av "${genome_index_name}"*.ht2 "${genome_dir}"