FastQ Trimming and QC - C.virginica Larval BS-seq Data from Lotterhos Lab and Part of CEABIGR Project Using fastp on Mox

BSseq
Lotterhos
CEABIGR
Crassostrea virginica
Eastern oyster
fastp
mox
2022
Miscellaneous
Author

Sam White

Published

August 29, 2022

Warning

The trimmming described below is probably incomplete and should be ignored.

Updated, good trimming is available here:

We had some old Crassostrea virginica (Eastern oyster) larval/zygote BS-seq data from the Lotterhos Lab that’s part of the CEABiGR Workshop/Project (GitHub Repo) and Steven asked that I QC/trim it in this GitHub Issue.

The original data/experiment is described in this repo (NOTE: this might be a private repo…):

Metadata for the files processed in this notebook:

Some notes:

I trimmed with fastp and performed trimming QC summary using MultiQC. The job was run on Mox.

See RESULTS section for lists of files processed

SBATCH script (GitHub):

#!/bin/bash
## Job Name
#SBATCH --job-name=20220826-cvir-larvae_zygote-RNAseq-fastp_trimming
## 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/20220826-cvir-larvae_zygote-BSseq-fastp_trimming


### Lotterhos C.virginica larvae and gonad BSseq trimming using fastp, and MultiQC.

### Expects input FastQ files to be in format: EF03-EM04-Larvae_R1_001.fastq.gz



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

# Set FastQ filename patterns
fastq_pattern='*.fastq.gz'
R1_fastq_pattern='*R1*.fastq.gz'
R2_fastq_pattern='*R2*.fastq.gz'

# Set number of CPUs to use
threads=40

# Input/output files
## Raw reads directory
raw_reads_dir=/gscratch/scrubbed/samwhite/data/C_virginica/BSseq

## checksum files
trimmed_checksums=trimmed-fastq-checksums.md5
raw_fastq_checksums=raw-fastq-checksums.md5
input_fastq_checksums=input-fastq-checksums.md5

# Paths to programs
fastp=/gscratch/srlab/programs/fastp-0.20.0/fastp
multiqc=/gscratch/srlab/programs/anaconda3/bin/multiqc

## Inititalize arrays
fastq_array_R1=()
fastq_array_R2=()
R1_names_array=()
R2_names_array=()


# Programs associative array
declare -A programs_array
programs_array=(
[fastp]="${fastp}" \
[multiqc]="${multiqc}"
)


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

# Exit script if any command fails
set -e

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

# Capture date
timestamp=$(date +%Y%m%d)

# Concatenate FastQ files from 1st and 2nd runs
# Do NOT quote fastq_pattern variable
# Will rsync all first run FastQs to working directory first,
# as there are missing second run FastQs; making concatenation process more complicated
# than I want to deal with.
for first_run_fastq in "${raw_reads_dir}"2018OALarvae_DNAm_discovery/${fastq_pattern}
do
  echo "Generating checksums for raw input FastQs..."

  # Strip full path to just get filename.
  first_run_fastq_name="${first_run_fastq##*/}"

  # Determine MD5 checksum
  md5sum "${first_run_fastq}" | tee --append "${raw_fastq_checksums}"
  echo ""

  # Rsync FastQ
  echo "Rsyncing ${first_run_fastq} to working directory."
  rsync -aP "${first_run_fastq}" .
  echo "Finished rsyncing ${first_run_fastq}."
  echo ""

  # Process second run and concatenate with corresponding FastQ from first run
  # Do NOT quote fastq_pattern variable
  for second_run_fastq in "${raw_reads_dir}"2018OALarvae_DNAm_discovery/second_lane/${fastq_pattern}
  do

    # Strip full path to just get filename.
    second_run_fastq_name="${second_run_fastq##*/}"

    # Concatenate FastQs with same filenames
    if [[ "${first_run_fastq_name}" == "${second_run_fastq_name}" ]]; then
      echo "Concatenating ${first_run_fastq_name} with ${second_run_fastq} to ${first_run_fastq_name}"
      echo ""
      cat "${second_run_fastq}" >> "${first_run_fastq_name}"
    fi
  done
  echo "Generating checksums for concatenated FastQs..."
  md5sum "${first_run_fastq_name}" | tee --append "${input_fastq_checksums}"
  echo ""
done

# Generate MD5 checksums for second run of FastQs
# Do NOT quote fastq_pattern variable
for second_run_fastq in "${raw_reads_dir}"2018OALarvae_DNAm_discovery/${fastq_pattern}
do
  echo "Generating checksums for second run raw input FastQs..."
    # Determine MD5 checksum
    md5sum "${second_run_fastq}" | tee --append "${raw_fastq_checksums}"
    echo ""
done

echo ""
echo "FastQ concatenation complete."
echo ""

# Create arrays of fastq R1 files and sample names
# Do NOT quote R1_fastq_pattern variable

for fastq in ${R1_fastq_pattern}
do
  fastq_array_R1+=("${fastq}")

  # Use parameter substitution to remove all text up to and including last "." from
  # right side of string.
  R1_names_array+=("${fastq%%.*}")
done

# Create array of fastq R2 files
# Do NOT quote R2_fastq_pattern variable
for fastq in ${R2_fastq_pattern}
do
  fastq_array_R2+=("${fastq}")

  # Use parameter substitution to remove all text up to and including last "." from
  # right side of string.
  R2_names_array+=(${fastq%%.*})
done



# Run fastp on files
# Adds JSON report output for downstream usage by MultiQC

echo "Beginning fastp trimming."
echo ""

for index in "${!fastq_array_R1[@]}"
do
  R1_sample_name="${R1_names_array[index]}"
  R2_sample_name="${R2_names_array[index]}"
  ${fastp} \
  --in1 ${fastq_array_R1[index]} \
  --in2 ${fastq_array_R2[index]} \
  --detect_adapter_for_pe \
  --thread ${threads} \
  --html "${R1_sample_name}".fastp-trim."${timestamp}".report.html \
  --json "${R1_sample_name}".fastp-trim."${timestamp}".report.json \
  --out1 "${R1_sample_name}".fastp-trim."${timestamp}".fq.gz \
  --out2 "${R2_sample_name}".fastp-trim."${timestamp}".fq.gz

  # Generate md5 checksums for newly trimmed files
  {
      md5sum "${R1_sample_name}".fastp-trim."${timestamp}".fq.gz
      md5sum "${R2_sample_name}".fastp-trim."${timestamp}".fq.gz
  } >> "${trimmed_checksums}"
  
  # Remove original FastQ files
  echo ""
  echo " Removing ${fastq_array_R1[index]} and ${fastq_array_R2[index]}."
  rm "${fastq_array_R1[index]}" "${fastq_array_R2[index]}"
done

echo "fastp trimming complete."
echo ""

# Run MultiQC
echo "Beginning MultiQC..."
echo ""
${multiqc} .
echo ""
echo "MultiQC complete."
echo ""

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

# Capture program options
if [[ "${#programs_array[@]}" -gt 0 ]]; then
  echo "Logging program options..."
  for program in "${!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
fi


# 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 surprisingly long, ~13hrs:

Screencap of Mox runtime for fastp trimming of C.virginica larval/zygote FastQs from inbox

Output folder: