Yesterday, I produced a bedgraph file of our Olympia oyster RNAseq data coverage using our Olurida_v081 genome.

I decided that I wanted to use the Olurida_v080 version instead (or, in addtion to?), as the Olurida_v080 version has not been size restricted (the Olurida v081 version is only contigs >1000bp). I feel like we could miss some important regions, so wanted to run this analysis using all of the genome data we currently have available. Additionally, this will be consistent with [my previous Bismark (DNA methylation analysis)(2018/09/13/dna-methylation-analysis-olympia-oyster-whole-genome-bsseq-bismark-pipeline-comparison.html).

Used HISAT2 on our HPC Mox node to align our RNAseq reads to our Olurida_v080 genome assembly:

• Olurida_v080.fa

SBATCH script file:

• 20180926_oly_RNAseq_bedgraphs.sh

NOTE: For brevity sake, I have not listed all of the input RNAseq files below. Please see the full script, which is linked above.

<code>
#!/bin/bash
## Job Name
#SBATCH --job-name=20180926_oly_hisat2
## Allocation Definition 
#SBATCH --account=srlab
#SBATCH --partition=srlab
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=5-00:00:00
## 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/20180926_oly_RNAseq_genome_hisat2_bedgraph

# 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


# Set genome assembly path
oly_genome_path=/gscratch/srlab/sam/data/O_lurida/oly_genome_assemblies

# Set sorted transcriptome assembly bam file
oly_transcriptome_bam=20180926_Olurida_v080.sorted.bam

# Set hisat2 basename
hisat2_basename=Olurida_v080

# Set program paths
## hisat2
hisat2=/gscratch/srlab/programs/hisat2-2.1.0

## bedtools
bedtools=/gscratch/srlab/programs/bedtools-2.27.1/bin

## samtools
stools=/gscratch/srlab/programs/samtools-1.9/samtools

# Build hisat2 genome index
${hisat2}/hisat2-build \
-f ${oly_genome_path}/Olurida_v080.fa \
Olurida_v080 \
-p 28

# Align reads to oly genome assembly
${hisat2}/hisat2 \
--threads 28 \
-x "${hisat2_basename}" \
-q \
-1 \
-2 \
-S 20180926_"${hisat2_basename}".sam

# Convert SAM file to BAM
"${stools}" view \
--threads 28 \
-b 20180926_"${hisat2_basename}".sam > 20180926_"${hisat2_basename}".bam

# Sort BAM
"${stools}" sort \
--threads 28 \
20180926_"${hisat2_basename}".bam \
-o 20180926_"${hisat2_basename}".sorted.bam

# Index for use in IGV
##-@ specifies thread count; --thread option not available in samtools index
"${stools}" index \
-@ 28 \
20180926_"${hisat2_basename}".sorted.bam


# Create bedgraph
## Reports depth at each position (-bg in bedgraph format) and report regions with zero coverage (-a).
## Screens for portions of reads coming from exons (-split).
## Add genome browser track line to header of bedgraph file.
${bedtools}/genomeCoverageBed \
-ibam ${oly_transcriptome_bam} \
-bga \
-split \
-trackline \
> 20180926_oly_RNAseq.bedgraph
</code>

The script performs the following functions:

• Genome indexing

• RNAseq alignment to genome

• Convert SAM to BAM

• Sort and index BAM

• Determine RNAseq coverage

---

RESULTS

Output folder:

• 20180926_oly_RNAseq_genome_hisat2_bedgraph/

Bedgraph file (1.9GB):

• 20180926_oly_RNAseq_genome_hisat2_bedgraph/20180926_oly_RNAseq.bedgraph

Loaded in to IGV to verify things looked OK: