Following the genome prep did an alignment. This was preceded by an assessment of min_score variable. Selection was based on high alignment rate with minimal non CpG methylation.

Based on https://marineomics.github.io/FUN_02_DNA_methylation.html

Alignment

# Set variables
reads_dir="../data/"
genome_folder="../data/"
output_dir="../output/01.2-bismark"
score_min="L,0,-1.0"  # Single value for score_min

# Get the list of sample files and corresponding sample names
for file in ${reads_dir}*_R1.fastp-trim.fq.gz; do
    sample_name=$(basename "$file" "_R1.fastp-trim.fq.gz")
    
    echo "Running Bismark for sample ${sample_name} with score_min ${score_min}"

    
    # Run Bismark alignment
    /home/shared/Bismark-0.24.0/bismark \
        --path_to_bowtie2 /home/shared/bowtie2-2.4.4-linux-x86_64 \
        -genome ${genome_folder} \
        -p 8 \
        -score_min ${score_min} \
        -1 ${reads_dir}${sample_name}_R1.fastp-trim.fq.gz \
        -2 ${reads_dir}${sample_name}_R2.fastp-trim.fq.gz \
        -o ${output_dir} \
        --basename ${sample_name} \
        2> "${output_dir}/${sample_name}-bismark_summary.txt"
done

deduplication

find ../output/01.2-bismark/*.bam | \
xargs -n 1 basename -s .bam | \
parallel -j 8 /home/shared/Bismark-0.24.0/deduplicate_bismark \
--bam \
--paired \
--output_dir ../output/09-meth-quant \
../output/01.2-bismark/{}.bam

Methylation extraction

find ../output/09-meth-quant/*deduplicated.bam | xargs -n 1 -I{} /home/shared/Bismark-0.24.0/bismark_methylation_extractor --bedGraph --counts --comprehensive --merge_non_CpG --multicore 24 --buffer_size 75% --output ../output/09-meth-quant {}

Methylation call

find ../output/09-meth-quant/*deduplicated.bismark.cov.gz | \
xargs -n 1 basename -s _pe.deduplicated.bismark.cov.gz | \
parallel -j 48 /home/shared/Bismark-0.24.0/coverage2cytosine \
--genome_folder ../data/ \
-o ../output/09-meth-quant/{} \
--merge_CpG \
--zero_based \
../output/09-meth-quant/{}_pe.deduplicated.bismark.cov.gz

This resulted in CpG_report.merged_CpG_evidence.cov files..

Sorted bams

deduplicated sorted bams were also sorted..

find *.bam | \
xargs basename -s .bam | \
xargs -I{} /home/shared/samtools-1.12/samtools \
sort --threads 48 {}.bam \
-o {}.sorted.bam

--- title: "Mytilus DNA methylation calls" description: "associated with contaminants" categories: [mytilus, methylation, bismark] #citation: date: 12-19-2024 image: http://gannet.fish.washington.edu/seashell/snaps/Monosnap_project-mytilus-methylation__RStudio_Server_2024-12-20_04-34-37.png # finding a good image author: - name: Steven Roberts url: orcid: 0000-0001-8302-1138 affiliation: Professor, UW - School of Aquatic and Fishery Sciences affiliation-url: https://robertslab.info #url: # self-defined draft: false # setting this to `true` will prevent your post from appearing on your listing page until you're ready! format: html: code-fold: FALSE code-tools: true code-copy: true highlight-style: github code-overflow: wrap --- Following the genome prep did an alignment. This was preceded by an assessment of min_score variable. Selection was based on high alignment rate with minimal non CpG methylation. Based on <https://marineomics.github.io/FUN_02_DNA_methylation.html> # Alignment ``` bash # Set variables reads_dir="../data/" genome_folder="../data/" output_dir="../output/01.2-bismark" score_min="L,0,-1.0" # Single value for score_min # Get the list of sample files and corresponding sample names for file in ${reads_dir}*_R1.fastp-trim.fq.gz; do sample_name=$(basename "$file" "_R1.fastp-trim.fq.gz") echo "Running Bismark for sample ${sample_name} with score_min ${score_min}" # Run Bismark alignment /home/shared/Bismark-0.24.0/bismark \ --path_to_bowtie2 /home/shared/bowtie2-2.4.4-linux-x86_64 \ -genome ${genome_folder} \ -p 8 \ -score_min ${score_min} \ -1 ${reads_dir}${sample_name}_R1.fastp-trim.fq.gz \ -2 ${reads_dir}${sample_name}_R2.fastp-trim.fq.gz \ -o ${output_dir} \ --basename ${sample_name} \ 2> "${output_dir}/${sample_name}-bismark_summary.txt" done ``` # deduplication ``` bash find ../output/01.2-bismark/*.bam | \ xargs -n 1 basename -s .bam | \ parallel -j 8 /home/shared/Bismark-0.24.0/deduplicate_bismark \ --bam \ --paired \ --output_dir ../output/09-meth-quant \ ../output/01.2-bismark/{}.bam ``` # Methylation extraction ``` bash find ../output/09-meth-quant/*deduplicated.bam | xargs -n 1 -I{} /home/shared/Bismark-0.24.0/bismark_methylation_extractor --bedGraph --counts --comprehensive --merge_non_CpG --multicore 24 --buffer_size 75% --output ../output/09-meth-quant {} ``` # Methylation call ``` bash find ../output/09-meth-quant/*deduplicated.bismark.cov.gz | \ xargs -n 1 basename -s _pe.deduplicated.bismark.cov.gz | \ parallel -j 48 /home/shared/Bismark-0.24.0/coverage2cytosine \ --genome_folder ../data/ \ -o ../output/09-meth-quant/{} \ --merge_CpG \ --zero_based \ ../output/09-meth-quant/{}_pe.deduplicated.bismark.cov.gz ``` This resulted in CpG_report.merged_CpG_evidence.cov files.. # Sorted bams deduplicated sorted bams were also sorted.. ``` bash find *.bam | \ xargs basename -s .bam | \ xargs -I{} /home/shared/samtools-1.12/samtools \ sort --threads 48 {}.bam \ -o {}.sorted.bam ```