INTRO
The remainder of RNA-seq data for Andy Dittman at NOAA came in on 20260715 (Notebook entry). The data is the remainder of the RNA-seq data, after an initial run of a subset of samples to see how the data looked and to make sure it was of good quality. The initial GitHub Issue indicated that the RNA Integrity Number (RIN) for these samples was not ideal, however, the initial FastQC/MultiQC (notebook entry) and subsequent trimming (notebook entry) showed the data was good. I ran FastQC and MultiQC on these new raw reads to assess their quality.
Analysis was run on Hyak using the following Roberts Lab Apptainer image:
srlab-R4.4-bioinformatics-container-c3d3116.sif(built on 20260410)
RESULTS
Output directory:
MultiQC Report (HTML):
Overall, the FastQC and MultiQC reports indicate that the raw reads are of good quality, with quality being consistent for what we usually see with RNA-seq data. Will proceed with quality trimming and adapter removal using FastP, and then re-run FastQC and MultiQC on the trimmed reads.
Code is below.
1 SETUP
1.1 Libraries
library(knitr)
knitr::opts_chunk$set(
echo = TRUE, # Display code chunks
eval = TRUE, # Evaluate code chunks
warning = FALSE, # Hide warnings
message = FALSE, # Hide messages
comment = "", # Prevents appending '##' to beginning of lines in code output
results = 'hold' # Holds output so it's all printed together after code chunk
)2 VARIABLES
# DIRECTORIES
raw_reads_dir <- "/mmfs1/gscratch/scrubbed/samwhite/data/dittman_grc_rnaseq_1"
output_dir <- "/mmfs1/gscratch/scrubbed/samwhite/outputs/20260806-FastQC-MultiQC-dittman-grc-rnaseq/"
# FILES
fastq_pattern="*.fastq.gz"
# SETTINGS
threads <- "50"
# Export these as environment variables for bash chunks.
Sys.setenv(
fastq_pattern = fastq_pattern,
raw_reads_dir = raw_reads_dir,
output_dir = output_dir,
threads = threads
)3 FASTQC/MULTIQC
# Make output directory if it doesn't exist
mkdir --parents "${raw_reads_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### NOTE: Do NOT quote raw_fastqc_list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_reads_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_reads_dir} \
--interactive \
-o ${raw_reads_dir}
echo ""
echo "MultiQC on raw FastQs complete."
echo ""
############ END MULTIQC ############
echo "Removing FastQC zip files."
echo ""
rm ${raw_reads_dir}/*.zip
echo "FastQC zip files removed."
echo ""Beginning FastQC on raw reads...
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
application/gzip
FastQC on raw reads complete!
Beginning MultiQC on raw FastQC...
/// MultiQC 🔍 v1.24.1
version_check | MultiQC Version v1.35 now available!
file_search | Search path: /mmfs1/gscratch/scrubbed/samwhite/data/dittman_grc_rnaseq_1
fastqc | Found 155 reports
write_results | Data : /mmfs1/gscratch/scrubbed/samwhite/data/dittman_grc_rnaseq_1/multiqc_data
write_results | Report : /mmfs1/gscratch/scrubbed/samwhite/data/dittman_grc_rnaseq_1/multiqc_report.html
multiqc | MultiQC complete
MultiQC on raw FastQs complete.
Removing FastQC zip files.
FastQC zip files removed.