After noticing that the initial MEGAN6 taxonomic assignments for our combined C.bairdi NanoPore data from 20200917 revealed a high number of bases assigned to E.canceri and Aquifex sp., I decided to explore the taxonomic breakdown of just the individual samples to see which of the samples was contributing to these taxonomic assignments most.

Ran the muscle (no Hematodinium infection; (20102558-2729-Q7) NanoPore sequencing data through DIAMOND BLASTx (on Mox) and subsequent output conversion to the MEGAN6 RMA6 format (on swoose due to program Java X11 requirement which is not functional on Mox) to obtain taxonomic assignments.

SBATCH script (GitHub):

20200928_cbai_diamond_blastx_nanopore_20102558-2729_Q7.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=cbai_blastx_DIAMOND_nanopore_20102558-2729_Q7
## Allocation Definition
#SBATCH --account=coenv
#SBATCH --partition=coenv
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=10-00:00:00
## Memory per node
#SBATCH --mem=200G
##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/20200928_cbai_diamond_blastx_nanopore_20102558-2729_Q7

# Script to run DIAMOND BLASTx on 20102558-2729 quality filtered (Q7) C.bairdi NanoPore reads
# from 20200928 using the --long-reads option
# for subsequent import into MEGAN6 to evaluate reads taxonomically.

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

# Input FastQ file
fastq=/gscratch/srlab/sam/data/C_bairdi/DNAseq/20200928_cbai_nanopore_20102558-2729_quality-7.fastq

# DIAMOND Output filename prefix
prefix=20200928_cbai_nanopore_20102558-2729_Q7

# Set number of CPUs to use
threads=28

# Program paths
diamond=/gscratch/srlab/programs/diamond-2.0.4/diamond

# DIAMOND NCBI nr database with taxonomy dumps
dmnd_db=/gscratch/srlab/blastdbs/ncbi-nr-20190925/nr.dmnd


###################################################################################
# Exit script if any command fails
set -e

# Load Python Mox module for Python module availability

module load intel-python3_2017

# SegFault fix?
export THREADS_DAEMON_MODEL=1


# Inititalize arrays
programs_array=()


# Programs array
programs_array=("${diamond}")


md5sum "${fastq}" > fastq_checksums.md5


# Run DIAMOND with blastx
# Output format 6 produces a standard BLAST tab-delimited file
# Run DIAMOND with blastx
# Output format 100 produces a DAA binary file for use with MEGAN
${diamond} blastx \
--long-reads \
--db ${dmnd_db} \
--query "${fastq}" \
--out "${prefix}".blastx.daa \
--outfmt 100 \
--top 5 \
--block-size 8.0 \
--index-chunks 1 \
--threads ${threads}

# Capture program options
for program in "${!programs_array[@]}"
do
    {
  echo "Program options for ${programs_array[program]}: "
    echo ""
    ${programs_array[program]} help
    echo ""
    echo ""
    echo "----------------------------------------------"
    echo ""
    echo ""
} &>> program_options.log || true
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

Bash script (GitHub) for daa2rma on Emu:

20200928_cbai_nanopore_20102558-2729-Q7_diamond_blastx_daa2rma.sh

#!/bin/bash

# Script to run MEGAN6 daa2rma on DIAMOND DAA files from
# 20200928_cbai_diamond_blastx_nanopore_20102558-2729_Q7.
# Utilizes the --longReads option

# Requires MEGAN mapping file from:
# http://ab.inf.uni-tuebingen.de/data/software/megan6/download


# MEGAN mapping file
megan_map=/home/sam/data/databases/MEGAN6/megan-map-Jul2020-2.db

# Programs array
declare -A programs_array
programs_array=(
[daa2rma]="/home/shared/megan_6.19.9/tools/daa2rma"
)

threads=16

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

# Exit script if any command fails
set -e


## Run daa2rma

# Capture start "time"
# Uses builtin bash variable called ${SECONDS}
start=${SECONDS}

for daa in *.daa
do
  start_loop=${SECONDS}
  sample_name=$(basename --suffix ".blastx.daa" "${daa}")

  echo "Now processing ${sample_name}.daa2rma.rma6"
  echo ""

  # Run daa2rma with long reads option
  ${programs_array[daa2rma]} \
  --in "${daa}" \
  --longReads \
  --mapDB ${megan_map} \
  --out "${sample_name}".daa2rma.rma6 \
  --threads ${threads} \
  2>&1 | tee --append daa2rma_log.txt

  end_loop=${SECONDS}
  loop_runtime=$((end_loop-start_loop))


  echo "Finished processing ${sample_name}.daa2rma.rma6 in ${loop_runtime} seconds."
  echo ""

done

# Caputure end "time"
end=${SECONDS}

runtime=$((end-start))

# Print daa2rma runtime, in seconds

{
  echo ""
  echo "---------------------"
  echo ""
  echo "Total runtime was: ${runtime} seconds"
} >> daa2rma_log.txt

# Capture program options
for program in "${!programs_array[@]}"
do
    {
  echo "Program options for ${program}: "
    echo ""
    ${programs_array[$program]} --help
    echo ""
    echo ""
    echo "----------------------------------------------"
    echo ""
    echo ""
} &>> program_options.log || true
done

# Document programs in PATH
{
date
echo ""
echo "System PATH:"
echo ""
printf "%0.s-" {1..10}
echo "${PATH}" | tr : \\n
} >> system_path.log

RESULTS

Pretty quick, a little over 17mins:

DIAMOND BLASTx and MEGAN daa2rma conversion for 20102558-2729-Q7 runtime

DIAMOND BLASTx Output folder:

20200928_cbai_nanopore_20102558-2729-Q7_diamond_blastx_daa2rma/
- DIAMOND BLASTx DAA file:
  - 20200928_cbai_nanopore_20102558-2729-Q7_diamond_blastx_daa2rma/20200928_cbai_nanopore_20102558-2729_Q7.blastx.daa (9.3MB)
- RMA6 file:
  - 20200928_cbai_nanopore_20102558-2729-Q7_diamond_blastx_daa2rma/20200928_cbai_nanopore_20102558-2729_Q7.daa2rma.rma6 (8.1MB)

Taxonomic tree

20102558-2729-Q7 MEGAN6 taxonomic assignments

Alrighty, we see both E.canceri and Aquifex sp. taxonomic assignments (and, of course Arthropoda). Surprisingly, there’re a relatively low number of bases assigned to Arthropoda (~1Mbp) and nearly the same assigned to E.canceri (~900kbp).

I’ve also highlighted the SAR supergroup (this is where we’d find Alveolata/Hematodinium), just to show that there’s essentially no assignments there. Now, need to see what the other sample looks like…

UPDATE 20201006: Taxonomic comparison between the two samples is here.