INTRO

This is part of Chris’ project-mytilus-methylation(GitHub repo). Data was received from Psomagen on 20241101 (Notebook entry). I performed the initial QC using FastQC and MultiQC. I’ll add this info to Nightingales (Google Sheet).

A full backup of this repo, including large files is available here:

https://gannet.fish.washington.edu/Atumefaciens/gitrepos/project-mytilus-methylation/

Note

This notebook is markdown knitted from 00.00-WGBSseq-reads-FastQC-MultiQC.Rmd (commit b352e66).

This Rmd file will download raw WGBSseq FastQs and evaluate them using FastQC and MultiQC(Ewels et al. 2016).

1 Create a Bash variables file

This allows usage of Bash variables across R Markdown chunks.

{
echo "#### Assign Variables ####"
echo ""

echo "# Data directories"
echo 'export output_dir_top="../output/00.00-WGBSseq-reads-FastQC-MultiQC"'
echo 'export raw_fastqc_dir="${output_dir_top}/raw-fastqc"'
echo 'export raw_reads_dir="../data/raw-wgbs"'
echo 'export raw_reads_url="https://owl.fish.washington.edu/nightingales/M_trossulus/"'
echo ""


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

echo "# Set number of CPUs to use"
echo 'export threads=50'
echo ""


echo "## Inititalize arrays"
echo 'export fastq_array_R1=()'
echo 'export fastq_array_R2=()'
echo 'export raw_fastqs_array=()'
echo 'export R1_names_array=()'
echo 'export R2_names_array=()'
echo ""

echo "# Print formatting"
echo 'export line="--------------------------------------------------------"'
echo ""
} > .bashvars

cat .bashvars

#### Assign Variables ####

# Data directories
export output_dir_top="../output/00.00-WGBSseq-reads-FastQC-MultiQC"
export raw_fastqc_dir="${output_dir_top}/raw-fastqc"
export raw_reads_dir="../data/raw-wgbs"
export raw_reads_url="https://owl.fish.washington.edu/nightingales/M_trossulus/"

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

# Set number of CPUs to use
export threads=50

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

# Print formatting
export line="--------------------------------------------------------"

2 Download raw FastQs

2.1 Download raw reads

Reads are downloaded from https://owl.fish.washington.edu/nightingales/M_trossulus/

# Load bash variables into memory
source .bashvars

# Make output directory if it doesn't exist
mkdir --parents ${raw_reads_dir}

# Run wget to retrieve FastQs and MD5 files
wget \
--directory-prefix ${raw_reads_dir} \
--recursive \
--no-check-certificate \
--continue \
--cut-dirs 3 \
--no-host-directories \
--no-parent \
--quiet \
--level=1 \
--accept "[0-9]M*.fastq.gz,[0-9]M*.fastq.gz.md5sum" \
${raw_reads_url}

2.2 Verify raw read checksums

# Load bash variables into memory
source .bashvars

cd "${raw_reads_dir}"

# Checksums file contains other files, so this just looks for the sRNAseq files.
for file in *.md5sum
do
  md5sum --check "${file}"
done

3 FastQC/MultiQC on raw reads

# Load bash variables into memory
source .bashvars

# Make output directory if it doesn't exist
mkdir --parents "${raw_fastqc_dir}"

############ RUN FASTQC ############


# Create array of trimmed FastQs
raw_fastqs_array=(${raw_reads_dir}/${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
### NOTE: Do NOT quote raw_fastqc_list
fastqc \
--threads ${threads} \
--outdir ${raw_fastqc_dir} \
--quiet \
${raw_fastqc_list}

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

############ END FASTQC ############

############ RUN MULTIQC ############
echo "Beginning MultiQC on raw FastQC..."
echo ""

multiqc ${raw_fastqc_dir} -o ${raw_fastqc_dir}

echo ""
echo "MultiQC on raw FastQs complete."
echo ""

############ END MULTIQC ############

echo "Removing FastQC zip files."
echo ""
rm ${raw_fastqc_dir}/*.zip
echo "FastQC zip files removed."
echo ""

# View directory contents
ls -lh ${raw_fastqc_dir}

Ewels, Philip, Måns Magnusson, Sverker Lundin, and Max Käller. 2016. “MultiQC: Summarize Analysis Results for Multiple Tools and Samples in a Single Report.” Bioinformatics 32 (19): 3047–48. https://doi.org/10.1093/bioinformatics/btw354.