We want to generate an additional Tanner crab (Chionoecetes bairdi) transcriptome, per this GitHub issue, to generate an additional C.bairdi transcriptome. This has come about due to the release of the genome of a very closely related crab species, Chionoecetes opilio (Snow crab).

I used DIAMOND BLASTx along with the Snow crab genome protein FastA (8.7MB) from NCBI (Acc: GCA_016584305.1).

NOTE: Since this is geared toward just identifying matching reads, the BLASTx output format will only contain the query ID. There will be one BLASTx output file for each corresponding input FastQ file.

SBATCH script (GitHub):

• 20210312_cbai-vs-copi_diamond_blastx.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=20210312_cbai-vs-copi_diamond_blastx
## Allocation Definition
#SBATCH --account=srlab
#SBATCH --partition=srlab
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=20-00:00:00
## Memory per node
#SBATCH --mem=120G
##turn on e-mail notification
#SBATCH --mail-type=ALL
#SBATCH --mail-user=samwhite@uw.edu
## Specify the working directory for this job
#SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20210312_cbai-vs-copi_diamond_blastx

## Script for running BLASTx (using DIAMOND) with all of our C.bairdi RNAseq data to-date.
## BLASTx against C.opilio _(snow crab) NCBI protein FastA
## Output will be in standard BLAST output format 6, but only query ID.
## Output will be used to extract just reads with matches to to C.opilio genome,
## for downstream transcriptome assembly

###################################################################################
# These variables need to be set by user

# FastQ directory
reads_dir=/gscratch/srlab/sam/data/C_bairdi/RNAseq

# DIAMOND database
dmnd=/gscratch/srlab/sam/data/C_opilio/blastdbs/GCA_016584305.1_ASM1658430v1_protein.dmnd

# Programs array
declare -A programs_array
programs_array=(
[diamond]="/gscratch/srlab/programs/diamond-0.9.29/diamond"
)

# FastQ array
fastq_array=(${reads_dir}/*fastp-trim*.fq.gz)


###################################################################################

# Exit script if any command fails
set -e

# Load Python Mox module for Python module availability
module load intel-python3_2017


# BLASTx FastQ files
for fastq in "${!fastq_array[@]}"
do

  # Remove path from transcriptome using parameter substitution
  fastq_name="${fastq_array[$fastq]##*/}"

  # Generate checksums for reference
  echo ""
  echo "Generating checksum for ${fastq_array[$fastq]}."
  md5sum "${fastq_array[$fastq]}">> fastq.checksums.md5
  echo "Completed checksum for ${fastq_array[$fastq]}."
  echo ""

  # Run DIAMOND with blastx
  # Output format 6 query only returns a single query ID per match
  # block-size and index-chunks are computing resource optimatization paraeters
  ${programs_array[diamond]} blastx \
  --db ${dmnd} \
  --query "${fastq_array[$fastq]}" \
  --out "${fastq_name}".blastx.outfmt6-query \
  --outfmt 6 qseqid \
  --evalue 1e-4 \
  --max-target-seqs 1 \
  --max-hsps 1 \
  --block-size 15.0 \
  --index-chunks 4
done


###################################################################################

# Capture program options
echo "Logging program options..."
for program in "${!programs_array[@]}"
do
	{
  echo "Program options for ${program}: "
	echo ""
  # Handle samtools help menus
  if [[ "${program}" == "samtools_index" ]] \
  || [[ "${program}" == "samtools_sort" ]] \
  || [[ "${program}" == "samtools_view" ]]
  then
    ${programs_array[$program]}

  # Handle DIAMOND BLAST menu
  elif [[ "${program}" == "diamond" ]]; then
    ${programs_array[$program]} help

  # Handle NCBI BLASTx menu
  elif [[ "${program}" == "blastx" ]]; then
    ${programs_array[$program]} -help
  fi
	${programs_array[$program]} -h
	echo ""
	echo ""
	echo "----------------------------------------------"
	echo ""
	echo ""
} &>> program_options.log || true

  # If MultiQC is in programs_array, copy the config file to this directory.
  if [[ "${program}" == "multiqc" ]]; then
  	cp --preserve ~/.multiqc_config.yaml multiqc_config.yaml
  fi
done

# Document programs in PATH (primarily for program version ID)
{
  date
  echo ""
  echo "System PATH for $SLURM_JOB_ID"
  echo ""
  printf "%0.s-" {1..10}
  echo "${PATH}" | tr : \\n
} >> system_path.log

echo "Finished logging system PATH"

---

RESULTS

Runtime was surprisingly long, considering how fast DIAMOND BLASTx has run on other samples. It took nearly 7hrs to complete:

Output folder:

• 20210312_cbai-vs-copi_diamond_blastx/

  • Input FastQ list/MD5 checksums:

    • fastq.checksums.md5

Due to the large number of output files, I will not link to each of them here. Please browse the output folder linked above.