Code for trimming and QCing RNAseq data, to be used on Pacific cod RNAseq data, raw reads found here
FastQC/MultiQC assessment of raw and flexbar-trimmed sequences of
Inputs:
*.fastq.gz)Outputs:
FastQC HTML reports for raw and trimmed reads.
Trimmed reads: *flexbar_trim.fastq.gz
(info about library prep/sequencing)
This allows usage of Bash variables (e.g. paths to common directories) across R Markdown chunks.
{
echo "#### Assign Variables ####"
echo ""
echo "# Data directories"
echo 'export cod_dir=/home/shared/8TB_HDD_02/shedurkin/project-cod-temperature'
echo 'export output_dir_top=${cod_dir}/output/05-cod-RNAseq-trimming'
echo 'export raw_fastqc_dir=${output_dir_top}/raw-fastqc'
echo 'export raw_reads_dir=${cod_dir}/data/05-cod-RNAseq-trimming/raw-reads'
echo 'export raw_reads_url="https://owl.fish.washington.edu/nightingales/G_macrocephalus/30-943133806/"'
echo 'export trimmed_fastqc_dir=${output_dir_top}/trimmed-fastqc'
echo 'export trimmed_reads_dir=${output_dir_top}/trimmed-reads'
echo ""
echo "# Paths to programs"
echo 'export fastqc=/home/shared/FastQC-0.12.1/fastqc'
echo 'export multiqc=/home/sam/programs/mambaforge/bin/multiqc'
echo 'export flexbar=/home/shared/flexbar-3.5.0-linux/flexbar'
echo ""
echo "# Set FastQ filename patterns"
echo "export fastq_pattern='*.fastq.gz'"
echo "export R1_fastq_pattern='*_R1_*.fastq.gz'"
echo "export R2_fastq_pattern='*_R2_*.fastq.gz'"
echo ""
echo "# Set number of CPUs to use"
echo 'export threads=20'
echo ""
echo "# Input/output files"
echo 'export fastq_checksums=input_fastq_checksums.md5'
echo 'export trimmed_checksums=trimmed_fastq_checksums.md5'
echo 'export NEB_adapters_fasta=NEB-adapters.fasta'
echo ""
echo "## Illumina adapters"
echo 'export first_adapter="AGATCGGAAGAGCACACGTCTGAACTCCAGTCA"'
echo 'export second_adapter="AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT"'
echo ""
echo "## Inititalize arrays"
echo 'export fastq_array_R1=()'
echo 'export fastq_array_R2=()'
echo 'export raw_fastqs_array=()'
echo 'export R1_names_array=()'
echo 'export R2_names_array=()'
echo 'export trimmed_fastqs_array=()'
echo ""
echo "# Programs associative array"
echo "declare -A programs_array"
echo "programs_array=("
echo '[fastqc]="${fastqc}" \'
echo '[multiqc]="${multiqc}" \'
echo '[flexbar]="${flexbar}"'
echo ")"
} > .bashvars
cat .bashvars#### Assign Variables ####
# Data directories
export cod_dir=/home/shared/8TB_HDD_02/shedurkin/project-cod-temperature
export output_dir_top=${cod_dir}/output/05-cod-RNAseq-trimming
export raw_fastqc_dir=${output_dir_top}/raw-fastqc
export raw_reads_dir=${cod_dir}/data/05-cod-RNAseq-trimming/raw-reads
export raw_reads_url="https://owl.fish.washington.edu/nightingales/G_macrocephalus/30-943133806/"
export trimmed_fastqc_dir=${output_dir_top}/trimmed-fastqc
export trimmed_reads_dir=${output_dir_top}/trimmed-reads
# Paths to programs
export fastqc=/home/shared/FastQC-0.12.1/fastqc
export multiqc=/home/sam/programs/mambaforge/bin/multiqc
export flexbar=/home/shared/flexbar-3.5.0-linux/flexbar
# Set FastQ filename patterns
export fastq_pattern='*.fastq.gz'
export R1_fastq_pattern='*_R1_*.fastq.gz'
export R2_fastq_pattern='*_R2_*.fastq.gz'
# Set number of CPUs to use
export threads=20
# Input/output files
export fastq_checksums=input_fastq_checksums.md5
export trimmed_checksums=trimmed_fastq_checksums.md5
export NEB_adapters_fasta=NEB-adapters.fasta
## Illumina adapters
export first_adapter="AGATCGGAAGAGCACACGTCTGAACTCCAGTCA"
export second_adapter="AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT"
## Inititalize arrays
export fastq_array_R1=()
export fastq_array_R2=()
export raw_fastqs_array=()
export R1_names_array=()
export R2_names_array=()
export trimmed_fastqs_array=()
# Programs associative array
declare -A programs_array
programs_array=(
[fastqc]="${fastqc}" \
[multiqc]="${multiqc}" \
[flexbar]="${flexbar}"
)Reads are downloaded from:
The --cut-dirs 3 command cuts the preceding directory structure (i.e. nightingales/P_meandrina/30-852430235/) so that we just end up with
the reads.
# Load bash variables into memory
source .bashvars
wget \
--directory-prefix ${raw_reads_dir} \
--recursive \
--no-check-certificate \
--continue \
--cut-dirs 3 \
--no-host-directories \
--no-parent \
--quiet \
--accept ${fastq_pattern} ${raw_reads_url}
ls -lh "${raw_reads_dir}"# Load bash variables into memory
source .bashvars
cd "${raw_reads_dir}"
md5sum checksums.md5 --check# Load bash variables into memory
source .bashvars
############ RUN FASTQC ############
# Create array of raw FastQs
raw_fastqs_array=(${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
${programs_array[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 ""
${programs_array[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 ${raw_fastqc_dir}total 71M
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Notes: - Not enough starting material: 19, 41, 48, 98, 129, 149, - RIN < 6: 10, 13, 37, 57-S, 58-S - DV200 < 70: 19, 41, 48, 98, 129, 149, 57-S
RNA Integrity Number (RIN) is a measure of RNA quality ranging from 1 - 10, with 1 representing highly degraded RNA and 10 representing intact RNA. Most researchers aim for RIN values of > 8, but Azenta considers anything > 6 acceptable. It is also generally assumed that RIN is representative of mRNA quality. It’s worth noting there is some debate surrounding the accuracy of RIN as a measure of RNA integrity – see ScienceDirect’s summary of RIN
DV200 is another measure of RNA quality that represents the percentage of RNA fragments of >200 nucleotides in length. A recent study suggested that DV200 may be a more meaningful assessment of mRNA quality than RIN (Matsubara et al 2020) for NGS, but both are still commonly used.
# Load bash variables into memory
source .bashvars
# Change to directory with raw reads
cd "${raw_reads_dir}"
# 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 FLEXBAR ############
# Uses parameter substitution (e.g. ${R1_sample_name%%_*})to rm the _R[12]
# Uses built-in adapter presets (https://github.com/seqan/flexbar/wiki/Manual#adapter-removal-presets)
# --adapter-preset TruSeq: several adapter presets for Illumina libraries are included in Flexbar
# --adapter-pair-overlap ON: Recommended by NEB sRNA kit
# --qtrim-threshold 25: Minimum quality
# --qtrim-format i1.8: Sets sequencer as Illumina
# --target: Sets file naming patterns
# --zip-output GZ: Sets type of compression. GZ = gzip
#
# Run flexbar on files
echo "Beginning flexbar trimming."
echo ""
time \
for index in "${!fastq_array_R1[@]}"
do
R1_sample_name="${R1_names_array[index]}"
R2_sample_name="${R2_names_array[index]}"
# Begin flexbar trimming
${programs_array[flexbar]} \
--reads ${fastq_array_R1[index]} \
--reads2 ${fastq_array_R2[index]} \
--adapter-preset "TruSeq" \
--adapter-pair-overlap ON \
--qtrim-format i1.8 \
--qtrim-threshold 25 \
--threads ${threads} \
--target "${trimmed_reads_dir}/${R1_sample_name%%_*}.flexbar_trim.R" \
--zip-output GZ
# Move to trimmed directory
# This is done so checksums file doesn't include excess path
cd ${trimmed_reads_dir}
# Generate md5 checksums for newly trimmed files
{
md5sum "${R1_sample_name%%_*}.flexbar_trim.R_1.fastq.gz"
md5sum "${R2_sample_name%%_*}.flexbar_trim.R_2.fastq.gz"
} >> "${trimmed_checksums}"
# Change back to to raw reads directory
cd "${raw_reads_dir}"
done
echo ""
echo "flexbar trimming complete."
echo ""
echo "Trimmed FastQs MD5 checksums:"
echo ""
cat "${trimmed_reads_dir}/${trimmed_checksums}"
############ END FLEXBAR ############# Load bash variables into memory
source .bashvars
############ RUN FASTQC ############
### NOTE: Do NOT quote raw_fastqc_list
# Create array of trimmed FastQs
trimmed_fastqs_array=(${trimmed_reads_dir}/${fastq_pattern})
# Pass array contents to new variable as space-delimited list
trimmed_fastqc_list=$(echo "${trimmed_fastqs_array[*]}")
echo "Beginning FastQC on raw reads..."
echo ""
# Run FastQC
${programs_array[fastqc]} \
--threads ${threads} \
--outdir ${trimmed_fastqc_dir} \
--quiet \
${trimmed_fastqc_list}
echo "FastQC on trimmed reads complete!"
echo ""
############ END FASTQC ############
############ RUN MULTIQC ############
echo "Beginning MultiQC on raw FastQC..."
echo ""
${programs_array[multiqc]} ${trimmed_fastqc_dir} -o ${trimmed_fastqc_dir}
echo ""
echo "MultiQC on trimmed FastQs complete."
echo ""
############ END MULTIQC ############
echo "Removing FastQC zip files."
echo ""
rm ${trimmed_fastqc_dir}/*.zip
echo "FastQC zip files removed."
echo ""# View directory contents
ls -lh ${trimmed_fastqc_dir}total 71M
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