01.00-D-Apul-RNAseq-trimming-fastp-FastQC-MultiQC

---

This Rmd file trims A.pulchra RNA-seq files using fastp (Chen 2023), followed by quality checks with FastQC and MultiQC(Ewels et al. 2016).

Expects input files formatted like so: <number>--<sample_name>_R[12]_001.fastq.gz

All trimmed FastQs produced by this script are here:

01.00-D-Apul-RNAseq-trimming-fastp-FastQC-MultiQC/trimmed-fastqs/

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 timeseries_dir=/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular'
echo 'export output_dir_top=${timeseries_dir}/D-Apul/output/01.00-D-Apul-RNAseq-trimming-fastp-FastQC-MultiQC'
echo 'export raw_reads_dir=${timeseries_dir}/D-Apul/data/raw-fastqs'
echo 'export raw_reads_url="https://owl.fish.washington.edu/nightingales/E5-coral-time-series/30-1047560508/"'
echo 'export trimmed_fastqs_dir=${output_dir_top}/trimmed-fastqs'
echo 'export trimmed_fastqc_dir=${output_dir_top}/trimmed-fastqc'
echo ""

echo "# Paths to programs"
echo 'export programs_dir="/home/shared"'
echo 'export fastp="${programs_dir}/fastp"'
echo 'export fastqc=${programs_dir}/FastQC-0.12.1/fastqc'
echo 'export multiqc=/home/sam/programs/mambaforge/bin/multiqc'
echo ""

echo "# Set FastQ filename patterns"
echo "export fastq_pattern='*.fastq.gz'"
echo "export R1_fastq_pattern='*_R1_*.fastq.gz'"
echo "export R2_fastq_pattern='*_R2_*.fastq.gz'"
echo "export trimmed_fastq_pattern='*fastp-trim.fq.gz'"
echo ""

echo "# Set number of CPUs to use"
echo 'export threads=40'
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 "# Programs associative array"
echo "declare -A programs_array"
echo "programs_array=("
echo '[fastp]="${fastp}" \'
echo '[fastqc]="${fastqc}" \'
echo '[multiqc]="${multiqc}" \'
echo ")"
echo ""

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

cat .bashvars

#### Assign Variables ####

# Data directories
export timeseries_dir=/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular
export output_dir_top=${timeseries_dir}/D-Apul/output/01.00-D-Apul-RNAseq-trimming-fastp-FastQC-MultiQC
export raw_reads_dir=${timeseries_dir}/D-Apul/data/raw-fastqs
export raw_reads_url="https://owl.fish.washington.edu/nightingales/E5-coral-time-series/30-1047560508/"
export trimmed_fastqs_dir=${output_dir_top}/trimmed-fastqs
export trimmed_fastqc_dir=${output_dir_top}/trimmed-fastqc

# Paths to programs
export programs_dir="/home/shared"
export fastp="${programs_dir}/fastp"
export fastqc=${programs_dir}/FastQC-0.12.1/fastqc
export multiqc=/home/sam/programs/mambaforge/bin/multiqc

# Set FastQ filename patterns
export fastq_pattern='*.fastq.gz'
export R1_fastq_pattern='*_R1_*.fastq.gz'
export R2_fastq_pattern='*_R2_*.fastq.gz'
export trimmed_fastq_pattern='*fastp-trim.fq.gz'

# Set number of CPUs to use
export threads=40

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

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

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

If needed, download raw RNA-seq.

Change eval=FALSE to eval=TRUE to execute the next two chunks to download RNA-seq and then verify MD5 checksums.

# Load bash variables into memory
source .bashvars

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

# Create list of only A.pulchra sample names
sample_list=$(awk -F "," 'NR > 2 {print $5"\t"$6}' ${timeseries_dir}/data/rna_metadata.csv | awk -F"[\t-]" '$2 == "ACR" {print $1}')

echo ""
echo "${line}"
echo ""
echo "Sample list:"
echo ""
echo "${sample_list}"
echo ""
echo "${line}"
echo ""


# Use printf to format each item for use in wget
formatted_list=$(printf "*%s*," ${sample_list})

# Remove the trailing comma and append *.md5
formatted_list="${formatted_list%,},*.md5"

# Output the final wget command
echo ""
echo "${line}"
echo ""
echo "Formatted wget accept list:"
echo ""
echo "wget --accept=\"$formatted_list\""
echo ""
echo "${line}"
echo ""

# Run wget to retrieve FastQs and MD5 files
wget \
--directory-prefix ${raw_reads_dir} \
--recursive \
--no-check-certificate \
--continue \
--cut-dirs 3 \
--no-host-directories \
--no-parent \
--quiet \
--accept=\"$formatted_list\" ${raw_reads_url}

ls -lh "${raw_reads_dir}"

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 *.md5
do
  md5sum --check "${file}"
done

2 Fastp Trimming

fastp (Chen 2023) is set to auto-detect Illumina adapters, as well as trim the first 20bp from each read, as past experience shows these first 20bp are more inconsistent than the remainder of the read length.

# Load bash variables into memory
source .bashvars

# Make output directories, if it doesn't exist
mkdir --parents "${trimmed_fastqs_dir}"

# Change to raw reads directory
cd "${raw_reads_dir}"

# Create arrays of fastq R1 files and sample names
for fastq in ${R1_fastq_pattern}
do
  fastq_array_R1+=("${fastq}")
  R1_names_array+=("$(echo "${fastq}" | awk -F"_" '{print $1}')")
done

# Create array of fastq R2 files
for fastq in ${R2_fastq_pattern}
do
  fastq_array_R2+=("${fastq}")
  R2_names_array+=("$(echo "${fastq}" | awk -F"_" '{print $1}')")
done

# Create list of fastq files used in analysis
# Create MD5 checksum for reference
if [ ! -f "${output_dir_top}"/raw-fastq-checksums.md5 ]; then
for fastq in *.gz
  do
    md5sum ${fastq} >> "${output_dir_top}"/raw-fastq-checksums.md5
  done
fi

# Run fastp on files
# Adds JSON report output for downstream usage by MultiQC
for index in "${!fastq_array_R1[@]}"
do
  R1_sample_name=$(echo "${R1_names_array[index]}")
  R2_sample_name=$(echo "${R2_names_array[index]}")
  ${fastp} \
  --in1 ${fastq_array_R1[index]} \
  --in2 ${fastq_array_R2[index]} \
  --detect_adapter_for_pe \
  --trim_front1 20 \
  --trim_front2 20 \
  --thread ${threads} \
  --html "${trimmed_fastqs_dir}"/"${R1_sample_name}".fastp-trim.report.html \
  --json "${trimmed_fastqs_dir}"/"${R1_sample_name}".fastp-trim.report.json \
  --out1 "${trimmed_fastqs_dir}"/"${R1_sample_name}"_R1_001.fastp-trim.fq.gz \
  --out2 "${trimmed_fastqs_dir}"/"${R2_sample_name}"_R2_001.fastp-trim.fq.gz \
  2>> "${trimmed_fastqs_dir}"/fastp.stderr

  # Generate md5 checksums for newly trimmed files
  cd "${trimmed_fastqs_dir}"
  md5sum "${R1_sample_name}"_R1_001.fastp-trim.fq.gz > "${R1_sample_name}"_R1_001.fastp-trim.fq.gz.md5
  md5sum "${R2_sample_name}"_R2_001.fastp-trim.fq.gz > "${R2_sample_name}"_R2_001.fastp-trim.fq.gz.md5
  cd -
done

3 Quality Check with FastQC and MultiQC

# Load bash variables into memory
source .bashvars

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

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


# Create array of trimmed FastQs
trimmed_fastqs_array=(${trimmed_fastqs_dir}/${trimmed_fastq_pattern})

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

echo "Beginning FastQC on trimmed reads..."
echo ""

# Run FastQC
### NOTE: Do NOT quote raw_fastqc_list
${programs_array[fastqc]} \
--threads ${threads} \
--outdir ${trimmed_fastqs_dir} \
--quiet \
${trimmed_fastqc_list}

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

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

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

${programs_array[multiqc]} ${trimmed_fastqs_dir} -o ${trimmed_fastqs_dir}

echo ""
echo "MultiQC on trimmed FastQs complete."
echo ""

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

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

Beginning FastQC on trimmed reads...

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FastQC on trimmed reads complete!

Beginning MultiQC on trimmed FastQC...


  /// MultiQC 🔍 | v1.14

|           multiqc | MultiQC Version v1.25.1 now available!
|           multiqc | Search path : /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/output/01.00-D-Apul-RNAseq-trimming-fastp-FastQC-MultiQC/trimmed-fastqs
|         searching | ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 100% 450/450  
|            fastqc | Found 88 reports
|           multiqc | Compressing plot data
|           multiqc | Previous MultiQC output found! Adjusting filenames..
|           multiqc | Use -f or --force to overwrite existing reports instead
|           multiqc | Report      : ../output/01.00-D-Apul-RNAseq-trimming-fastp-FastQC-MultiQC/trimmed-fastqs/multiqc_report_2.html
|           multiqc | Data        : ../output/01.00-D-Apul-RNAseq-trimming-fastp-FastQC-MultiQC/trimmed-fastqs/multiqc_data_2
|           multiqc | MultiQC complete
|           multiqc | 1 flat-image plot used in the report due to large sample numbers
|           multiqc | To force interactive plots, use the '--interactive' flag. 
See the documentation.

MultiQC on trimmed FastQs complete.

Removing FastQC zip files.

FastQC zip files removed.

Chen, Shifu. 2023. “Ultrafast One-Pass FASTQ Data Preprocessing, Quality Control, and Deduplication Using Fastp.” iMeta 2 (2). https://doi.org/10.1002/imt2.107.

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.