Last week, I ran all of our Q7-filtered C.baird NanoPore reads through MEGAN6 to evaluate the taxonomic breakdown (on 20200917) and noticed that there were a large quantity of bases assigned to E.canceri (a known microsporidian agent of infection in crabs) and Aquifex sp. (a genus of thermophylic bacteria), in addition to the expected Arthropoda assignments. Notably, Alveolata assignments were remarkably low.

Since our NanoPore data was a combination of an uninfected sample and a Hematodinium-infected sample, I decided to find out which of the two samples was contributing to the E.canceri and Aquifex sp. assignments. To do so, first I need to generate a singular Q7-filtered FastQ using NanoFilt.

This set was the uninfected muscle sample:

Job was run on Mox.

SBATCH script (GitHub):

20200928_cbai_nanofilt_Q7_20102558-2729_nanopore-data.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=cbai_nanofilt_Q7_20102558-2729_nanopore-data
## 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_nanofilt_Q7_20102558-2729_nanopore-data




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

# Load Anaconda
# Uknown why this is needed, but Anaconda will not run if this line is not included.
. "/gscratch/srlab/programs/anaconda3/etc/profile.d/conda.sh"


# Activate the NanoPlot Anaconda environment
conda activate nanofilt_2.6.0_env


# Declare array
raw_reads_dir_array=()

# Paths to reads
raw_reads_dir_array=(
"/gscratch/srlab/sam/data/C_bairdi/DNAseq/ont_FAL58500_04bb4d86_20102558-2729" \
"/gscratch/srlab/sam/data/C_bairdi/DNAseq/ont_FAL58500_94244ffd_20102558-2729"
)

# FastQ concatenation filename
fastq_cat=20200928_cbai_nanopore_20102558-2729.fastq

fastq_filtered=20200928_cbai_nanopore_20102558-2729_quality-7.fastq

# Paths to programs
nanofilt=NanoFilt

# Set mean quality filter (integer)
quality=7

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


# Exit script if any command fails
set -e

# Inititalize array
programs_array=()

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


# Loop through NanoPore data directories
# to run NanoPlot, FastQC, and MultiQC
for directory in "${raw_reads_dir_array[@]}"
do

  # Find all FastQ files and concatenate into singel file
  while IFS= read -r -d '' filename
  do
    # Concatenate all FastQ files into single file
    # for NanoFilt and generate MD5 checksums
    echo "Now concatenating ${filename} to ${fastq_cat}..."
    cat "${filename}" >> ${fastq_cat}
    echo "Concatenation of ${filename} to ${fastq_cat} complete."

    # Create checksums file
    echo "Now generating checksum for ${filename}..."
    echo ""
    md5sum "${filename}" >> fastq_checksums.md5
    echo "Checksum for ${filename} complete."
    echo ""

  done < <(find "${directory}" -name "*.fastq" -type f -print0)

done

# Generate MD5 checksum for concatenated FastQ file
echo "Now generating checksum for ${fastq_cat}..."
echo ""
md5sum "${fastq_cat}" >> fastq_checksums.md5
echo "checksum for ${fastq_cat} complete."
echo ""

# Run NanoFilt
## Sets readtype to 1D (default)
## Filters on mean quality >= 7 (ONT "standard")
## FYI: seems to require piping stdin (i.e. cat fastq |)to NanoFilt...
echo "Running ${programs_array[nanofilt]}"
echo ""
cat ${fastq_cat} \
| ${programs_array[nanofilt]} \
--readtype 1D \
--quality ${quality} \
> ${fastq_filtered}
echo "${programs_array[nanofilt]} complete."
echo ""

# Generate MD5 checksum for concatenated FastQ file
echo "Now generating checksum for ${fastq_filtered}..."
echo ""
md5sum "${fastq_filtered}" >> fastq_checksums.md5
echo "checksum for ${fastq_filtered} complete."
echo ""

# Capture program options
for program in "${!programs_array[@]}"
do
    {
  echo "Program options for ${programs_array[program]}: "
    echo ""
    ${programs_array[program]} -h
    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

RESULTS

Runtime was very fast, 43s:

NanoFilt runtime on mox for 20102558-2729

Output folder:

20200928_cbai_nanofilt_Q7_20102558-2729_nanopore-data/

Q7 Filtered FastQ file (148MB):

20200928_cbai_nanofilt_Q7_20102558-2729_nanopore-data/20200928_cbai_nanopore_20102558-2729_quality-7.fastq
- MD5 checksum:
  - eb439470cbc765052b4b190e837fcdd7

Will get taxonomy assignments using MEGAN6.