Bedgraph - Olympia oyster transcriptome (FAIL)

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=‘ {’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:

• 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.