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:

• 20180924_oly_RNAseq_bedgraphs.sh

  #!/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 </code>

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

• Olurida_v081.fa

---

RESULTS:

Output folder:

• 20180924_oly_RNAseq_bedgraphs/

Indexed and formatted genome file:

• Olurida_v081.fa.fai.genome

Bedgraph file (for IGV):

• 20180924_oly_RNAseq.bed

---

This doesn’t appear to have worked properly. Here’s a view of the bedgraph file:

<code>
track type=bedGraph
Contig0 0   116746  0
Contig1 0   87411   0
Contig2 0   139250  0
Contig3 0   141657  0
Contig4 0   95692   0
Contig5 0   130522  0
Contig6 0   94893   0
Contig7 0   109667  0
Contig8 0   95943   0
</code>

I’d expect multiple entries for each contig (ideally), indicating start/stop positions for where transcripts align within a given contig. However, this appears to simply be a list of all the genome contigs and their lengths (Start=0, Stop=n).

I would expect to see something li

I’ll look into this further and see where this pipeline goes wrong.