This Rmd file will download raw WGBS-seq FastQs for A.pulchra and evaluate them using FastQC and MultiQC (Ewels et al. 2016).
This allows usage of Bash variables across R Markdown chunks.
{
echo "#### Assign Variables ####"
echo ""
echo "# Data directories"
echo 'export deep_dive_expression_dir=/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/deep-dive-expression'
echo 'export output_dir_top=${deep_dive_expression_dir}/D-Apul/output/00.00-D-Apul-WGBS-reads-FastQC-MultiQC'
echo 'export raw_fastqc_dir=${output_dir_top}/raw-fastqc'
echo 'export raw_reads_dir=${deep_dive_expression_dir}/D-Apul/data/raw-fastqs'
echo 'export raw_reads_url="https://owl.fish.washington.edu/nightingales/E5-coral-deep-dive-expression/genohub2216545/"'
echo ""
echo "# Paths to programs"
echo 'export fastqc=/home/shared/FastQC-0.12.1/fastqc'
echo 'export multiqc=/home/sam/programs/mambaforge/bin/multiqc'
echo ""
echo "# Set FastQ filename patterns"
echo "export fastq_pattern='*.fastq.gz'"
echo "export R1_fastq_pattern='*_R1_*.fastq.gz'"
echo "export R2_fastq_pattern='*_R2_*.fastq.gz'"
echo ""
echo "# Set number of CPUs to use"
echo 'export threads=40'
echo ""
echo "## Inititalize arrays"
echo 'export fastq_array_R1=()'
echo 'export fastq_array_R2=()'
echo 'export raw_fastqs_array=()'
echo 'export R1_names_array=()'
echo 'export R2_names_array=()'
echo ""
echo "# Programs associative array"
echo "declare -A programs_array"
echo "programs_array=("
echo '[fastqc]="${fastqc}" \'
echo '[multiqc]="${multiqc}" \'
echo ")"
echo ""
echo "# Print formatting"
echo 'export line="--------------------------------------------------------"'
echo ""
} > .bashvars
cat .bashvars#### Assign Variables ####
# Data directories
export deep_dive_expression_dir=/home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/deep-dive-expression
export output_dir_top=${deep_dive_expression_dir}/D-Apul/output/00.00-D-Apul-WGBS-reads-FastQC-MultiQC
export raw_fastqc_dir=${output_dir_top}/raw-fastqc
export raw_reads_dir=${deep_dive_expression_dir}/D-Apul/data/raw-fastqs
export raw_reads_url="https://owl.fish.washington.edu/nightingales/E5-coral-deep-dive-expression/genohub2216545/"
# Paths to programs
export fastqc=/home/shared/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'
# 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=(
[fastqc]="${fastqc}" \
[multiqc]="${multiqc}" \
)
# Print formatting
export line="--------------------------------------------------------"Reads are downloaded from https://owl.fish.washington.edu/nightingales/E5-coral-deep-dive-expression/genohub2216545/
Since sequencing included multiple species, the code will also parse only those that are A.pulchra.
The --cut-dirs 3 command cuts the preceding directory structure (i.e.Β nightingales/E5-coral-deep-dive-expression/genohub2216545/) so that we just end up with the reads.
# Load bash variables into memory
source .bashvars
head ${deep_dive_expression_dir}/M-multi-species/data/e5_deep_dive_WGBS_metadata.csv |
column -t -s","Number Species Timepoint Collection Date Site colony_id Extraction Date Extraction notebook post DNA (ng/uL) Volume eluted Total DNA (ng) Primer Date Prepped Library concentration (ng/uL) bp peak Library volume (uL) Prep notebook post Notes
403 Pocillopora tuahiniensis TP2 20200305 Site1 POC-48 20211129 https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-11-29-20211129-RNA-DNA-extractions-from-E5-project.md 29.1 90 2619 25 20240613 8.67 253 14 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md Labeled as POC-55 in KXT extraction post
413 Acropora pulchra TP2 20200305 Site1 ACR-178 20210902 https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-09-02-20210902-RNA-DNA-extractions-from-E5-project.md 19.9 90 1791 26 20240613 9.64 269 14 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md
417 Pocillopora tuahiniensis TP2 20200305 Site1 POC-57 20211020 https://kterpis.github.io/Putnam_Lab_Notebook/20211020-RNA-DNA-extractions-from-E5-project/ 51.4 90 4626 27 20240613 9.3 256 14 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md Labeled as POC-238 in KXT extraction post
423 Acropora pulchra TP2 20200305 Site1 ACR-150 20210903 https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-09-03-20210903-RNA-DNA-extractions-from-E5-project.md 17.7 90 1593 29 20240613 8.61 267 14 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md Labeled as POR-75 in KXT extraction post
427 Acropora pulchra TP2 20200305 Site1 ACR-145 20211012 https://kterpis.github.io/Putnam_Lab_Notebook/20211012-RNA-DNA-extractions-from-E5-project/ 19.6 90 1764 30 20240613 8.82 271 14 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md
439 Acropora pulchra TP2 20200305 Site1 ACR-173 20211102 https://kterpis.github.io/Putnam_Lab_Notebook/20211102-RNA-DNA-extractions-from-E5-project/ 19.9 90 1791 31 20240613 11.9 264 14 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md
467 Acropora pulchra TP2 20200305 Site1 ACR-140 20210921 https://github.com/Kterpis/Putnam_Lab_Notebook/blob/master/_posts/2021-09-21-20210921-RNA-DNA-extractions-from-E5-project.md 21.9 90 1971 32 20240613 16 270 14 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md DNA 1 and 2 Qubit readings are 27.6 and 27.4 but average is reported as 4.79 on github and on master sample sheet; JA re-qubited on 20240229 and got 21.9 ul/ng
471 Porites evermanni TP2 20200305 Site1 POR-76 20211015 https://kterpis.github.io/Putnam_Lab_Notebook/20211015-RNA-DNA-extractions-from-E5-project/ 2.7 90 243 33 20240613 5.25 245 14 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md
491 Porites evermanni TP2 20200305 Site1 POR-73 20211101 https://kterpis.github.io/Putnam_Lab_Notebook/20211101-RNA-DNA-extractions-from-E5-project/ 2.11 90 189.9 36 20240613 4.65 315 14 https://github.com/JillAshey/JillAshey_Putnam_Lab_Notebook/blob/master/_posts/2024-06-13-Zymo-Pico-Methyl-Seq-Library-Prep.md # 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 "," '$6 ~ /^ACR/ {print $1}' ${deep_dive_expression_dir}/M-multi-species/data/e5_deep_dive_WGBS_metadata.csv)
echo ""
echo "${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_*," ${sample_list})
# Remove the trailing comma
formatted_list=${formatted_list%,}
# 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
# Note: the --no-clobber command will skip re-downloading any files that are already present in the output directory
wget \
--directory-prefix ${raw_reads_dir} \
--recursive \
--no-check-certificate \
--continue \
--cut-dirs 3 \
--no-host-directories \
--no-parent \
--quiet \
--no-clobber \
--accept=${formatted_list} ${raw_reads_url}
ls -lh "${raw_reads_dir}"# Load bash variables into memory
source .bashvars
cd "${raw_reads_dir}"
# Checksums file contains other files, so this just looks for the RNAseq files.
for file in *.md5
do
md5sum --check "${file}"
done413_S1_R1_001.fastq.gz: OK
413_S1_R2_001.fastq.gz: OK
423_S2_R1_001.fastq.gz: OK
423_S2_R2_001.fastq.gz: OK
427_S3_R1_001.fastq.gz: OK
427_S3_R2_001.fastq.gz: OK
439_S4_R1_001.fastq.gz: OK
439_S4_R2_001.fastq.gz: OK
467_S5_R1_001.fastq.gz: OK
467_S5_R2_001.fastq.gz: OK# 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
${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 ""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
FastQC on raw reads complete!
Beginning MultiQC on raw FastQC...
/// MultiQC π | v1.14
| multiqc | MultiQC Version v1.27 now available!
| multiqc | Search path : /home/shared/8TB_HDD_01/sam/gitrepos/urol-e5/deep-dive-expression/D-Apul/output/00.00-D-Apul-WGBS-reads-FastQC-MultiQC/raw-fastqc
| searching | ββββββββββββββββββββββββββββββββββββββββ 100% 20/20
| fastqc | Found 10 reports
| multiqc | Compressing plot data
| multiqc | Report : ../output/00.00-D-Apul-WGBS-reads-FastQC-MultiQC/raw-fastqc/multiqc_report.html
| multiqc | Data : ../output/00.00-D-Apul-WGBS-reads-FastQC-MultiQC/raw-fastqc/multiqc_data
| multiqc | MultiQC complete
MultiQC on raw FastQs complete.
Removing FastQC zip files.
FastQC zip files removed.# Load bash variables into memory
source .bashvars
# View directory contents
ls -lh ${raw_fastqc_dir}
total 6.9M
-rw-r--r-- 1 sam sam 579K Feb 6 12:41 413_S1_R1_001_fastqc.html
-rw-r--r-- 1 sam sam 586K Feb 6 12:42 413_S1_R2_001_fastqc.html
-rw-r--r-- 1 sam sam 580K Feb 6 12:47 423_S2_R1_001_fastqc.html
-rw-r--r-- 1 sam sam 582K Feb 6 12:48 423_S2_R2_001_fastqc.html
-rw-r--r-- 1 sam sam 578K Feb 6 12:49 427_S3_R1_001_fastqc.html
-rw-r--r-- 1 sam sam 581K Feb 6 12:49 427_S3_R2_001_fastqc.html
-rw-r--r-- 1 sam sam 577K Feb 6 12:46 439_S4_R1_001_fastqc.html
-rw-r--r-- 1 sam sam 581K Feb 6 12:45 439_S4_R2_001_fastqc.html
-rw-r--r-- 1 sam sam 576K Feb 6 12:49 467_S5_R1_001_fastqc.html
-rw-r--r-- 1 sam sam 579K Feb 6 12:50 467_S5_R2_001_fastqc.html
drwxr-xr-x 2 sam sam 4.0K Feb 6 12:50 multiqc_data
-rw-r--r-- 1 sam sam 1.3M Feb 6 12:50 multiqc_report.html