Sam’s Notebook: Bedgraph – Olympia oyster transcriptome (FAIL)

0000-0002-2747-368X

Progress on generating bedgraphs from our Olympia oyster transcriptome continues.

Transcriptome assembly with Trinity completed 20180919.

Then, aligned the assembled transcriptome to our genome using Bowtie2.

Finally, I used BEDTools to convert the BAM to BED to bedgraph.

This required an initial indexing of our Olympia oyster genome FastA using samtools faidx tool.

SBATCH script file:

 #!/bin/bash ## Job Name #SBATCH --job-name=20180924_oly_bedgraphs ## 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/20180924_oly_RNAseq_bedgraphs # 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 FastA oly_genome_fasta=/gscratch/srlab/sam/data/O_lurida/oly_genome_assemblies/Olurida_v081.fa # Set indexed genome assembly file oly_genome_indexed=/gscratch/srlab/sam/data/O_lurida/oly_genome_assemblies/Olurida_v081.fa.fai # Set sorted transcriptome assembly bam file oly_transcriptome=/gscratch/scrubbed/samwhite/20180919_oly_transcriptome_bowtie2/20180919_Olurida_v081.sorted.bam # Set program paths bedtools=/gscratch/srlab/programs/bedtools-2.27.1/bin samtools=/gscratch/srlab/programs/samtools-1.9/samtools # Index genome FastA ${samtools} faidx ${oly_genome_fasta} # Format indexed genome for bedtools ## Requires only two columns: namelength awk -v OFS='\t' {'print $1,$2'} ${oly_genome_indexed} > Olurida_v081.fa.fai.genome # Create bed file ${bedtools}/bamToBed \ -i ${oly_transcriptome} \ > 20180924_oly_RNAseq.bam.bed # 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 \ -i ${PWD}/20180924_oly_RNAseq.bed \ -g Olurida_v081.fa.fai.genome \ -bga \ -split \ -trackline \ > 20180924_oly_RNAseq.bed 

Alignment was done using the following version of the Olympia oyster genome assembly: