Per this GitHub Issue, Steven wanted me to download all of the SRA data (RNA-seq and WGBS-seq) from NCBI BioProject PRJNA744403 and run QC on the data.

Prior to QC, I had to get the data:

1. Acquired accessions list file:

   

   Resulting file looks like this:

    Run accession
    SRR15101687
    SRR15101688
    SRR15101689
    SRR15101690
    SRR15101691
    SRR15101692
    SRR15101693
    SRR15101694
    SRR15101695
   

2. Downloaded associated SRA runs (done on Mox using a build node):

    time \
    /gscratch/srlab/programs/sratoolkit.2.11.3-centos_linux64/bin/prefetch \
    --option-file /gscratch/srlab/sam/data/NCBI-BioProject-PRJNA744403-coral_metagenomics/SraAccList-PRJNA744403.txt
   

   NOTE: This puts all files in this directory:

    /gscratch/srlab/data/ncbi/sra
   

   Took a bit over 2hrs to download all the files:

   

3. Converted .sra files to FastQ (done on Mox using a build node):

    for file in *.sra; do /gscratch/srlab/programs/sratoolkit.2.11.3-centos_linux64/bin/fasterq-dump "${file}"; done
   

4. Moved FastQ files to species-specific directories and library-specific (i.e. BS-seq or RNA-seq) directories.

5. gzip-ed all FastQ files. This is needed for significant space savings, but also needed for some potential downstream pipelines we’re considering running.

6. Generated MD5 checksums for all files.

Had to run QC/trimming SLURM script for coral SRA data (BioProject PRJNA74403) a couple of times. Seemed like fastp was trimming data (i.e. output reports indicate average read length after filtering is ~140bp, but the resulting graphs of read lengths after trimming still show 150bp…). Additionally, the first ~20bp looked rough, so decided to add a hard 20bp trim from 5’ end of all reads…

FastQC, fastp, and MultiQC were run on Mox.

SBATCH script (GitHub):

• 20230119-coral-fastqc-fastp-multiqc-PRJNA744403.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=20230119-coral-fastqc-fastp-multiqc-PRJNA744403
## Allocation Definition
#SBATCH --account=srlab
#SBATCH --partition=srlab
## Resources
## Nodes
#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 --mail-user=samwhite@uw.edu
## Specify the working directory for this job
#SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20230119-coral-fastqc-fastp-multiqc-PRJNA744403

### FastQC and fastp trimming coral metagenome SRA BioProject PRJNA744403 sequencing data.

### fastp expects input FastQ files to be in format: *_R[12].fastq.gz



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

## Assign Variables

# Set FastQ filename patterns
fastq_pattern='*.fastq.gz'
R1_fastq_pattern='*_1*.fastq.gz'
R2_fastq_pattern='*_2*.fastq.gz'

# Set species array
species_array=(P_grandis P_meandrina)

# Set number of CPUs to use
threads=40

# Set working directory
working_dir=$(pwd)

# Input/output files
trimmed_checksums=trimmed_fastq_checksums.md5
fastq_checksums=input_fastq_checksums.md5

# Data directories
bsseq_dir="BS-seq"
rnaseq_dir="RNA-seq"


## Inititalize arrays
raw_fastqs_array=()
R1_names_array=()
R2_names_array=()

# Paths to programs
fastp=/gscratch/srlab/programs/fastp.0.23.1
fastqc=/gscratch/srlab/programs/fastqc_v0.11.9/fastqc
multiqc=/gscratch/srlab/programs/anaconda3/bin/multiqc

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

# Sync raw FastQ files to working directory
# Uses "P_[gm]*" to get only files from
# P_grandis and P_meandrina directories
echo ""
echo "Transferring files via rsync..."
rsync --archive \
--verbose \
--progress \
--files-from=<(find ../../data/P_[gm]* -name "*.fastq.gz") \
../../ .
echo ""
echo "File transfer complete."
echo ""





for species in "${species_array[@]}"
do

  for library in "${bsseq_dir}" "${rnaseq_dir}"
  do

    ## Re-inititalize arrays
    fastq_array_R1=()
    fastq_array_R2=()
    raw_fastqs_array=()
    R1_names_array=()
    R2_names_array=()
    trimmed_fastq_array=()

    # Change to bisulfite data directory
    cd "${working_dir}/data/${species}/${library}"

    echo ""
    echo "Moving to ${working_dir}/data/${species}/${library}"
    echo ""

    # Create trimmed subdirectory
    mkdir --parents trimmed

    ### Run FastQC ###

    ### NOTE: Do NOT quote raw_fastqc_list

    # Set FastQC output directory
    output_dir=$(pwd)

    # Create array of raw FastQs
    raw_fastqs_array=("${fastq_pattern}")

    # Pass array contents to new variable as space-delimited list
    raw_fastqc_list=$(echo "${raw_fastqs_array[*]}")

    echo "Beginning FastQC on raw reads..."
    echo ""

    # Run FastQC
    ${programs_array[fastqc]} \
    --threads ${threads} \
    --outdir ${output_dir} \
    ${raw_fastqc_list}

    echo ""
    echo "FastQC on raw reads complete!"
    echo ""

    ### END FASTQC ###

    # 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


    # Create MD5 checksums for raw FastQs
    for fastq in ${fastq_pattern}
    do
      echo "Generating checksum for ${fastq}"
      md5sum "${fastq}" | tee --append ${fastq_checksums}
      echo ""
    done

    ### RUN MULTIQC ###
    echo "Beginning MultiQC..."
    echo ""
    ${programs_array[multiqc]} .
    echo ""
    echo "MultiQC complete."
    echo ""

    ### END MULTIQC ###


    ### RUN FASTP ###

    # 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 \
      --trim_poly_g \
      --trim_front1 20 \
      --trim_front2 20 \
      --thread ${threads} \
      --html trimmed/"SRA-${R1_sample_name%_*}.${species}.fastp-trim.${timestamp}".report.html \
      --json trimmed/"SRA-${R1_sample_name%_*}.${species}.fastp-trim.${timestamp}".report.json \
      --out1 trimmed/"SRA-${R1_sample_name}.${species}.fastp-trim.${timestamp}".fq.gz \
      --out2 trimmed/"SRA-${R2_sample_name}.${species}.fastp-trim.${timestamp}".fq.gz

      ### END FASTP ###


      # Generate md5 checksums for newly trimmed files
      {
          md5sum trimmed/"SRA-${R1_sample_name}.${species}.fastp-trim.${timestamp}".fq.gz
          md5sum trimmed/"SRA-${R2_sample_name}.${species}.fastp-trim.${timestamp}".fq.gz
      } >> trimmed/"${trimmed_checksums}"
    done

    ### RUN FASTQC ###

    ### NOTE: Do NOT quote ${trimmed_fastqc_list}

    # Change to trimmed directory
    cd trimmed

    # Set FastQC output directory
    output_dir=$(pwd)

    # Create array of trimmed FastQs
    trimmed_fastq_array=(*fastp-trim*.fq.gz)

    # Pass array contents to new variable as space-delimited list
    trimmed_fastqc_list=$(echo "${trimmed_fastq_array[*]}")

    # Run FastQC
    echo "Beginning FastQC on trimmed reads..."
    echo ""
    ${programs_array[fastqc]} \
    --threads ${threads} \
    --outdir ${output_dir} \
    ${trimmed_fastqc_list}

    echo ""
    echo "FastQC on trimmed reads complete!"
    echo ""

    ### END FASTQC ###

    ### RUN MULTIQC ###
    echo "Beginning MultiQC..."
    echo ""
    ${programs_array[multiqc]} .
    echo ""
    echo "MultiQC complete."
    echo ""

    ### END MULTIQC ###
  done

done

# Return to top directory
cd "${working_dir}"

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

# 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 ~6.25hrs:

Due to the large number of files, along with attempts to keep things semi-organized, please refer to the directory-tree first.

To cut right to the chase, the following four files can be downloaded and then used with wget to download all of the described FastQ files.

P.grandis BS-seq raw FastQs

• 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_grandis/BS-seq/pgrandis.bs-seq.raw.download.list.txt

P.grandis BS-seq trimmed FastQs

• 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_grandis/BS-seq/trimmed/pgrandis.bs-seq.trimmed.download.list.txt

P.grandis RNA-seq raw FastQs

• 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_grandis/RNA-seq/pgrandis.rna-seq.raw.download.list.txt

P.grandis RNA-seq trimmed FastQs

• 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_grandis/RNA-seq/trimmed/pgrandis.rna-seq.trimmed.download.list.txt

P.meandrina BS-seq raw FastQs

• 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_meandrina/BS-seq/pmeandrina.bs-seq.raw.download.list.txt

P.meandrina BS-seq trimmed FastQs

• 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_meandrina/BS-seq/trimmed/pmeandrina.bs-seq.trimmed.download.list.txt

P.meandrina RNA-seq raw FastQs

• 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_meandrina/RNA-seq/pmeandrina.rna-seq.raw.download.list.txt

P.meandrina RNA-seq trimmed FastQs

• 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_meandrina/RNA-seq/trimmed/pmeandrina.rna-seq.trimmed.download.list.txt

Example of how to use these files:

wget --input-file=pgrandis.bs-seq.raw.download.list.txt

This will download all of the P.grandis BS-seq raw FastQ files and an MD5 checksum file for verifying data after transfer.

Output folder:

• 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/

  • Directory tree (text)

    • 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/directory-tree.txt

  • NCBI BioProject PRJNA744403 Metadata (CSV)

    • 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/metadata-SraAccList-PRJNA744403.csv

  • P.grandis BS-seq raw reads MultiQC report (HTML):

    • 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_grandis/BS-seq/multiqc_report.html

  • P.grandis BS-seq trimmed reads MultiQC report (HTML):

    • 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_grandis/BS-seq/trimmed/multiqc_report.html

  • P.grandis RNA-seq raw reads MultiQC report (HTML):

    • 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_grandis/RNA-seq/multiqc_report.html

  • P.grandis RNA-seq trimmed reads MultiQC report (HTML):

    • 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_grandis/RNA-seq/trimmed/multiqc_report.html

  • P.meandrina BS-seq raw reads MultiQC report (HTML):

    • 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_meandrina/BS-seq/multiqc_report.html

  • P.meandrina BS-seq trimmed reads MultiQC report (HTML):

    • 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_meandrina/BS-seq/trimmed/multiqc_report.html

  • P.meandrina RNA-seq raw reads MultiQC report (HTML):

    • 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_meandrina/RNA-seq/multiqc_report.html

  • P.meandrina RNA-seq trimmed reads MultiQC report (HTML):

    • 20230119-coral-fastqc-fastp-multiqc-PRJNA744403/data/P_meandrina/RNA-seq/trimmed/multiqc_report.html