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:



    Job Name

    #SBATCH –job-name=20180924_oly_bedgraphs

    Allocation Definition

    #SBATCH –account=srlab #SBATCH –partition=srlab



    #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 –

    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


    Set indexed genome assembly file


    Set sorted transcriptome assembly bam file


    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

    -i ${oly_transcriptome} \


    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.

    -i ${PWD}/20180924_oly_RNAseq.bed
    -g Olurida_v081.fa.fai.genome
    -trackline \

    20180924_oly_RNAseq.bed </code>

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


Output folder:

Indexed and formatted genome file:

Bedgraph file (for IGV):

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

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

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.