Methylation Extraction - C.virginica CEASMALLR WGBS Using Bismark

2024
Crassostrea virginica
Eastern oyster
ceasmallr
wgbs
BS-seq
Bismark
methylation extraction
Author

Sam White

Published

November 26, 2024

INTRO

This notebook is part of the ceasmallr (GitHub repo) project, comparing DNA methylation in Crassostrea virginica (Eastern oyster) larvae and zygotes, via whole-genome bisulfite sequencing (WGBS).

Deduplication was previously performed on 20241125 and now needed to Bismark methylation extraction (Bismark GitHub).

Note

This notebook was copied from knitted markdown from 02.20-bismark-methylation-extraction.Rmd (commit 92cf9be).

1 DESCRIPTION

This notebook is a continuation of using Bismark (Krueger and Andrews 2011) to perform methylation analysis. This step will perform methylation extraction.

Results will be summarized by MultiQC (Ewels et al. 2016).

1.1 Inputs

This notebook relies on deduplicated BAMs generated by 02.10-bismark-deduplication.Rmd.

1.2 Outputs

  • *.CpG_report.merged_CpG_evidence.cov.gz: CpG position and counts merged from all strands. Generated by coverage2cytosine.

  • *.deduplicated.bedGraph.gz: Bedgraph file with CpG loci and percent methylation.

  • *.deduplicated.bedGraph.gz.bismark.zero.cov: Extension of *deduplicated.bedGraph.gz which includes two additional columns, count methylated and count unmethylated, in that order.

  • *.deduplicated.bismark.cov.gz: 1-based counting version of *.deduplicated.bedGraph.gz.bismark.zero.cov. E.g. loci are at 49 49, instead of 48 49.

  • *.deduplicated.CpG_report.txt.gz: Report on all CpGs. <chromosome> <0-based start> <methylation status, +/-> <count_methyldated> <CG> <trinucleotide_context>

  • *.deduplicated.cytosine_context_summary.txt: Cytosine context summary. See file headers for info.

  • *.deduplicated.M-bias.txt: Methylation bias table. See file headers for info.

  • *.deduplicated_splitting_report.txt: Summary methylation report in a human-readable format.

  • CH*.deduplicated.txt.gz: Strand-specific methylation status for each read in CHH or CHG context.

  • CpG*.deduplicated.txt.gz: Strand-specific methylation status for each read in CpG context.

  • multiqc_report.html: A summary report of the alignment results generated by MultiQC, in HTML format.

Due to the large file sizes of most of the output files, these cannot be hosted in the ceasmallr GitHub repo. As such these files are available for download here:

2 Create a Bash variables file

This allows usage of Bash variables across R Markdown chunks.

{
echo "#### Assign Variables ####"
echo ""

echo "# Data directories"
echo 'export repo_dir=/home/shared/8TB_HDD_01/sam/gitrepos/ceasmallr'
echo 'export output_dir_top=${repo_dir}/output/02.20-bismark-methylation-extraction'
echo 'export deduplication_dir="${repo_dir}/output/02.10-bismark-deduplication"'
echo 'export genome_dir="${repo_dir}/data/Cvirginica_v300/"'
echo ""


echo "# Paths to programs"
echo 'export programs_dir="/home/shared"'
echo 'export bismark_dir="${programs_dir}/Bismark-0.24.0"'
echo 'export bowtie2_dir="${programs_dir}/bowtie2-2.4.4-linux-x86_64"'
echo 'export multiqc="/home/sam/programs/mambaforge/bin/multiqc"'
echo 'export samtools_dir="${programs_dir}/samtools-1.12"'
echo ""

echo "# Set number of CPUs to use"
echo 'export threads=40'


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

cat .bashvars
#### Assign Variables ####

# Data directories
export repo_dir=/home/shared/8TB_HDD_01/sam/gitrepos/ceasmallr
export output_dir_top=${repo_dir}/output/02.20-bismark-methylation-extraction
export deduplication_dir="${repo_dir}/output/02.10-bismark-deduplication"
export genome_dir="${repo_dir}/data/Cvirginica_v300/"

# Paths to programs
export programs_dir="/home/shared"
export bismark_dir="${programs_dir}/Bismark-0.24.0"
export bowtie2_dir="${programs_dir}/bowtie2-2.4.4-linux-x86_64"
export multiqc="/home/sam/programs/mambaforge/bin/multiqc"
export samtools_dir="${programs_dir}/samtools-1.12"

# Set number of CPUs to use
export threads=40
# Print formatting
export line="--------------------------------------------------------"

3 Methylation Extraction

# Load bash variables into memory
source .bashvars

cd "${deduplication_dir}"

${bismark_dir}/bismark_methylation_extractor \
--bedGraph \
--counts \
--scaffolds \
--parallel 10 \
--output_dir ${output_dir_top} \
--samtools_path ${samtools_dir} \
--gzip \
--paired-end \
--comprehensive \
--merge_non_CpG \
--zero_based \
--buffer_size 75% \
--cytosine_report \
--genome_folder ${genome_dir} \
*deduplicated.bam

4 Cytosine coverage

# Load bash variables into memory
source .bashvars

cd "${output_dir_top}"

find *deduplicated.bismark.cov.gz \
| xargs basename -s _bismark_bt2_pe.deduplicated.bismark.cov.gz \
| xargs -I{} ${bismark_dir}/coverage2cytosine \
--genome_folder ${genome_dir} \
-o {} \
--merge_CpG \
--zero_based \
--gzip \
{}_bismark_bt2_pe.deduplicated.bismark.cov.gz

5 Peek at output files

Glance at output file formats, using CF01-CM01-Zygote files as example.

# Load bash variables into memory
source .bashvars

cd "${output_dir_top}"

for file in *CF01-CM01-Zygote*; do
  if [[ -f "$file" ]]; then
    if [[ "$file" == *.gz ]]; then
      echo "<===== $file ====>"
      echo ""
      zcat "$file" | head | column -t -s $'\t'
      echo ""
      echo "${line}"
      echo ""
    else
      echo "<===== $file ====>"
      echo ""
      echo "$file"
      head "$file" | column -t -s $'\t'
      echo ""
      echo "${line}"
      echo ""
    fi
  fi
done
<===== CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.bedGraph.gz ====>

track type=bedGraph
NC_007175.2          48   49   2.08333333333333
NC_007175.2          49   50   0
NC_007175.2          50   51   1.92307692307692
NC_007175.2          51   52   0
NC_007175.2          87   88   0
NC_007175.2          88   89   0
NC_007175.2          146  147  0
NC_007175.2          147  148  1.75438596491228
NC_007175.2          148  149  0

--------------------------------------------------------

<===== CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.bedGraph.gz.bismark.zero.cov ====>

CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.bedGraph.gz.bismark.zero.cov
NC_007175.2  48   49   2.08333333333333  1  47
NC_007175.2  49   50   0                 0  2
NC_007175.2  50   51   1.92307692307692  1  51
NC_007175.2  51   52   0                 0  1
NC_007175.2  87   88   0                 0  104
NC_007175.2  88   89   0                 0  33
NC_007175.2  146  147  0                 0  116
NC_007175.2  147  148  1.75438596491228  1  56
NC_007175.2  148  149  0                 0  1
NC_007175.2  192  193  0                 0  51

--------------------------------------------------------

<===== CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.bismark.cov.gz ====>

NC_007175.2  49   49   2.08333333333333  1  47
NC_007175.2  50   50   0                 0  2
NC_007175.2  51   51   1.92307692307692  1  51
NC_007175.2  52   52   0                 0  1
NC_007175.2  88   88   0                 0  104
NC_007175.2  89   89   0                 0  33
NC_007175.2  147  147  0                 0  116
NC_007175.2  148  148  1.75438596491228  1  56
NC_007175.2  149  149  0                 0  1
NC_007175.2  193  193  0                 0  51

--------------------------------------------------------

<===== CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.CpG_report.txt.gz ====>

NC_007175.2  48   +  1  47   CG  CGC
NC_007175.2  49   -  0  2    CG  CGA
NC_007175.2  50   +  1  51   CG  CGG
NC_007175.2  51   -  0  1    CG  CGC
NC_007175.2  87   +  0  104  CG  CGT
NC_007175.2  88   -  0  33   CG  CGT
NC_007175.2  146  +  0  116  CG  CGG
NC_007175.2  147  -  1  56   CG  CGA
NC_007175.2  192  +  0  51   CG  CGC
NC_007175.2  193  -  0  39   CG  CGA

--------------------------------------------------------

<===== CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.cytosine_context_summary.txt ====>

CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.cytosine_context_summary.txt
upstream  C-context  full context  count methylated  count unmethylated  percent methylation
A         CAA        ACAA          66                446                 12.89
C         CAA        CCAA          66                479                 12.11
G         CAA        GCAA          19                198                 8.76
T         CAA        TCAA          59                526                 10.09
A         CAC        ACAC          16                118                 11.94
C         CAC        CCAC          7                 69                  9.21
G         CAC        GCAC          9                 38                  19.15
T         CAC        TCAC          15                120                 11.11
A         CAG        ACAG          142               1235                10.31

--------------------------------------------------------

<===== CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.M-bias.txt ====>

CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.M-bias.txt
CpG context (R1)
================
position          count methylated  count unmethylated  % methylation  coverage
1                 9383              45046               17.24          54429
2                 9270              45432               16.95          54702
3                 9184              45622               16.76          54806
4                 9457              45750               17.13          55207
5                 10083             48122               17.32          58205
6                 10548             48157               17.97          58705
7                 10617             48528               17.95          59145

--------------------------------------------------------

<===== CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated_splitting_report.txt ====>

CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated_splitting_report.txt
CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.bam
Parameters used to extract methylation information:
Bismark Extractor Version: v0.24.0
Bismark result file: paired-end (SAM format)
Output specified: comprehensive
No overlapping methylation calls specified
Methylation in CHG and CHH context will be merged into "non-CpG context" output

--------------------------------------------------------

<===== CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED.CpG_report.merged_CpG_evidence.cov.gz ====>

NC_007175.2  48   50   2.000000  1  49
NC_007175.2  50   52   1.886792  1  52
NC_007175.2  87   89   0.000000  0  137
NC_007175.2  146  148  0.578035  1  172
NC_007175.2  192  194  0.000000  0  90
NC_007175.2  245  247  2.898551  2  67
NC_007175.2  256  258  4.166667  5  115
NC_007175.2  263  265  0.000000  0  155
NC_007175.2  265  267  1.142857  2  173
NC_007175.2  331  333  0.546448  1  182

--------------------------------------------------------

<===== CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED.CpG_report.txt.gz ====>

NC_007175.2  48   +  1  47   CG  CGC
NC_007175.2  49   -  0  2    CG  CGA
NC_007175.2  50   +  1  51   CG  CGG
NC_007175.2  51   -  0  1    CG  CGC
NC_007175.2  87   +  0  104  CG  CGT
NC_007175.2  88   -  0  33   CG  CGT
NC_007175.2  146  +  0  116  CG  CGG
NC_007175.2  147  -  1  56   CG  CGA
NC_007175.2  192  +  0  51   CG  CGC
NC_007175.2  193  -  0  39   CG  CGA

--------------------------------------------------------

<===== CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED.cytosine_context_summary.txt ====>

CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED.cytosine_context_summary.txt
upstream  C-context  full context  count methylated  count unmethylated  percent methylation
A         CAA        ACAA          66                446                 12.89
C         CAA        CCAA          66                479                 12.11
G         CAA        GCAA          19                198                 8.76
T         CAA        TCAA          59                526                 10.09
A         CAC        ACAC          16                118                 11.94
C         CAC        CCAC          7                 69                  9.21
G         CAC        GCAC          9                 38                  19.15
T         CAC        TCAC          15                120                 11.11
A         CAG        ACAG          142               1235                10.31

--------------------------------------------------------

<===== CpG_context_CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.txt.gz ====>

Bismark methylation extractor version v0.24.0
GWNJ-1012:512:GW210315000:4:1101:21884:1094_1:N:0:TTATAACC+TCGATATC  -  NC_035785.1  24407680  z
GWNJ-1012:512:GW210315000:4:1101:11776:1219_1:N:0:TTATAACC+TCGATATC  -  NC_035780.1  32653438  z
GWNJ-1012:512:GW210315000:4:1101:20699:1141_1:N:0:TTATAACC+TCGATATC  -  NC_035788.1  57095043  z
GWNJ-1012:512:GW210315000:4:1101:20699:1141_1:N:0:TTATAACC+TCGATATC  -  NC_035788.1  57095087  z
GWNJ-1012:512:GW210315000:4:1101:20699:1141_1:N:0:TTATAACC+TCGATATC  -  NC_035788.1  57095119  z
GWNJ-1012:512:GW210315000:4:1101:20699:1141_1:N:0:TTATAACC+TCGATATC  -  NC_035788.1  57095138  z
GWNJ-1012:512:GW210315000:4:1101:9372:1595_1:N:0:TTATAACC+TCGATATC   +  NC_035781.1  2018694   Z
GWNJ-1012:512:GW210315000:4:1101:15402:1642_1:N:0:TTATAACC+TCGATATC  +  NC_035783.1  49860452  Z
GWNJ-1012:512:GW210315000:4:1101:9372:1595_1:N:0:TTATAACC+TCGATATC   +  NC_035781.1  2018682   Z

--------------------------------------------------------

<===== Non_CpG_context_CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.txt.gz ====>

Bismark methylation extractor version v0.24.0
GWNJ-1012:512:GW210315000:4:1101:11776:1219_1:N:0:TTATAACC+TCGATATC  -  NC_035780.1  32653467  x
GWNJ-1012:512:GW210315000:4:1101:11776:1219_1:N:0:TTATAACC+TCGATATC  -  NC_035780.1  32653466  h
GWNJ-1012:512:GW210315000:4:1101:11776:1219_1:N:0:TTATAACC+TCGATATC  -  NC_035780.1  32653465  h
GWNJ-1012:512:GW210315000:4:1101:11776:1219_1:N:0:TTATAACC+TCGATATC  -  NC_035780.1  32653464  h
GWNJ-1012:512:GW210315000:4:1101:11776:1219_1:N:0:TTATAACC+TCGATATC  -  NC_035780.1  32653461  x
GWNJ-1012:512:GW210315000:4:1101:11776:1219_1:N:0:TTATAACC+TCGATATC  -  NC_035780.1  32653455  h
GWNJ-1012:512:GW210315000:4:1101:11776:1219_1:N:0:TTATAACC+TCGATATC  -  NC_035780.1  32653454  h
GWNJ-1012:512:GW210315000:4:1101:11776:1219_1:N:0:TTATAACC+TCGATATC  -  NC_035780.1  32653450  h
GWNJ-1012:512:GW210315000:4:1101:20699:1141_1:N:0:TTATAACC+TCGATATC  +  NC_035788.1  57095003  X

--------------------------------------------------------

6 List all outputs

# Load bash variables into memory
source .bashvars

cd "${output_dir_top}"

ls -lh
total 93G
-rw-r--r-- 1 sam sam  21M Nov 26 11:17 CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.bedGraph.gz
-rw-r--r-- 1 sam sam 124M Nov 26 11:17 CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.bedGraph.gz.bismark.zero.cov
-rw-r--r-- 1 sam sam  18M Nov 26 11:17 CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.bismark.cov.gz
-rw-r--r-- 1 sam sam 107M Nov 26 11:27 CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.7K Dec  4 12:17 CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  20K Dec  4 12:17 CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.M-bias.txt
-rw-r--r-- 1 sam sam  926 Dec  4 12:17 CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated_splitting_report.txt
-rw-r--r-- 1 sam sam  21M Dec  4 12:17 CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED.CpG_report.merged_CpG_evidence.cov.gz
-rw-r--r-- 1 sam sam 107M Nov 27 07:59 CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.7K Dec  4 12:17 CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  90M Nov 26 11:43 CF01-CM02-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz
-rw-r--r-- 1 sam sam 551M Nov 26 11:43 CF01-CM02-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz.bismark.zero.cov
-rw-r--r-- 1 sam sam  89M Nov 26 11:43 CF01-CM02-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bismark.cov.gz
-rw-r--r-- 1 sam sam 126M Nov 26 11:54 CF01-CM02-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 CF01-CM02-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  23K Dec  4 12:17 CF01-CM02-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.M-bias.txt
-rw-r--r-- 1 sam sam  928 Dec  4 12:17 CF01-CM02-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated_splitting_report.txt
-rw-r--r-- 1 sam sam  72M Dec  4 12:17 CF01-CM02-Larvae_R1_001.fastp-trim.CpG_report.merged_CpG_evidence.cov.gz
-rw-r--r-- 1 sam sam 126M Nov 27 08:11 CF01-CM02-Larvae_R1_001.fastp-trim.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 CF01-CM02-Larvae_R1_001.fastp-trim.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  44M Nov 26 12:00 CF02-CM02-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz
-rw-r--r-- 1 sam sam 263M Nov 26 12:00 CF02-CM02-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz.bismark.zero.cov
-rw-r--r-- 1 sam sam  41M Nov 26 12:00 CF02-CM02-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bismark.cov.gz
-rw-r--r-- 1 sam sam 113M Nov 26 12:12 CF02-CM02-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 CF02-CM02-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  21K Dec  4 12:17 CF02-CM02-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.M-bias.txt
-rw-r--r-- 1 sam sam  926 Dec  4 12:17 CF02-CM02-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated_splitting_report.txt
-rw-r--r-- 1 sam sam  41M Dec  4 12:17 CF02-CM02-Zygote_R1_001.fastp-trim.CpG_report.merged_CpG_evidence.cov.gz
-rw-r--r-- 1 sam sam 113M Nov 27 08:22 CF02-CM02-Zygote_R1_001.fastp-trim.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 CF02-CM02-Zygote_R1_001.fastp-trim.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  65M Nov 26 12:20 CF03-CM03-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz
-rw-r--r-- 1 sam sam 395M Nov 26 12:20 CF03-CM03-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz.bismark.zero.cov
-rw-r--r-- 1 sam sam  61M Nov 26 12:20 CF03-CM03-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bismark.cov.gz
-rw-r--r-- 1 sam sam 118M Nov 26 12:32 CF03-CM03-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 CF03-CM03-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  22K Dec  4 12:17 CF03-CM03-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.M-bias.txt
-rw-r--r-- 1 sam sam  925 Dec  4 12:17 CF03-CM03-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated_splitting_report.txt
-rw-r--r-- 1 sam sam  57M Dec  4 12:17 CF03-CM03-Zygote_R1_001.fastp-trim.CpG_report.merged_CpG_evidence.cov.gz
-rw-r--r-- 1 sam sam 118M Nov 27 08:33 CF03-CM03-Zygote_R1_001.fastp-trim.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 CF03-CM03-Zygote_R1_001.fastp-trim.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  85M Nov 26 12:45 CF03-CM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz
-rw-r--r-- 1 sam sam 528M Nov 26 12:45 CF03-CM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz.bismark.zero.cov
-rw-r--r-- 1 sam sam  83M Nov 26 12:45 CF03-CM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bismark.cov.gz
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-rw-r--r-- 1 sam sam 112M Nov 27 13:22 EF07-EM01-Zygote_R1_001.fastp-trim.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.7K Dec  4 12:17 EF07-EM01-Zygote_R1_001.fastp-trim.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  88M Nov 26 21:38 EF07-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz
-rw-r--r-- 1 sam sam 547M Nov 26 21:38 EF07-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz.bismark.zero.cov
-rw-r--r-- 1 sam sam  87M Nov 26 21:38 EF07-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bismark.cov.gz
-rw-r--r-- 1 sam sam 124M Nov 26 21:50 EF07-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 EF07-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  23K Dec  4 12:17 EF07-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.M-bias.txt
-rw-r--r-- 1 sam sam  927 Dec  4 12:17 EF07-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated_splitting_report.txt
-rw-r--r-- 1 sam sam  71M Dec  4 12:17 EF07-EM03-Larvae_R1_001.fastp-trim.CpG_report.merged_CpG_evidence.cov.gz
-rw-r--r-- 1 sam sam 124M Nov 27 13:34 EF07-EM03-Larvae_R1_001.fastp-trim.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 EF07-EM03-Larvae_R1_001.fastp-trim.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  89M Nov 26 22:06 EF08-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz
-rw-r--r-- 1 sam sam 546M Nov 26 22:06 EF08-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz.bismark.zero.cov
-rw-r--r-- 1 sam sam  88M Nov 26 22:06 EF08-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bismark.cov.gz
-rw-r--r-- 1 sam sam 125M Nov 26 22:18 EF08-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 EF08-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  23K Dec  4 12:17 EF08-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.M-bias.txt
-rw-r--r-- 1 sam sam  927 Dec  4 12:17 EF08-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated_splitting_report.txt
-rw-r--r-- 1 sam sam  72M Dec  4 12:17 EF08-EM03-Larvae_R1_001.fastp-trim.CpG_report.merged_CpG_evidence.cov.gz
-rw-r--r-- 1 sam sam 125M Nov 27 13:46 EF08-EM03-Larvae_R1_001.fastp-trim.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 EF08-EM03-Larvae_R1_001.fastp-trim.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  84M Nov 26 22:31 EF08-EM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz
-rw-r--r-- 1 sam sam 517M Nov 26 22:31 EF08-EM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bedGraph.gz.bismark.zero.cov
-rw-r--r-- 1 sam sam  82M Nov 26 22:31 EF08-EM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bismark.cov.gz
-rw-r--r-- 1 sam sam 124M Nov 26 22:43 EF08-EM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 EF08-EM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.cytosine_context_summary.txt
-rw-r--r-- 1 sam sam  23K Dec  4 12:17 EF08-EM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.M-bias.txt
-rw-r--r-- 1 sam sam  928 Dec  4 12:17 EF08-EM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated_splitting_report.txt
-rw-r--r-- 1 sam sam  68M Dec  4 12:17 EF08-EM04-Larvae_R1_001.fastp-trim.CpG_report.merged_CpG_evidence.cov.gz
-rw-r--r-- 1 sam sam 124M Nov 27 13:57 EF08-EM04-Larvae_R1_001.fastp-trim.CpG_report.txt.gz
-rw-r--r-- 1 sam sam 1.8K Dec  4 12:17 EF08-EM04-Larvae_R1_001.fastp-trim.cytosine_context_summary.txt
drwxr-xr-x 2 sam sam 4.0K Dec  4 12:17 multiqc_data
-rw-r--r-- 1 sam sam 1.2M Dec  4 12:17 multiqc_report.html
-rw-r--r-- 1 sam sam 191M Nov 26 11:16 Non_CpG_context_CF01-CM01-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 2.6G Nov 26 11:37 Non_CpG_context_CF01-CM02-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 890M Nov 26 11:59 Non_CpG_context_CF02-CM02-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 1.3G Nov 26 12:18 Non_CpG_context_CF03-CM03-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 2.2G Nov 26 12:41 Non_CpG_context_CF03-CM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 2.4G Nov 26 13:07 Non_CpG_context_CF03-CM05-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 1.6G Nov 26 13:30 Non_CpG_context_CF04-CM04-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 3.4G Nov 26 13:58 Non_CpG_context_CF05-CM02-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 1.9G Nov 26 14:25 Non_CpG_context_CF05-CM05-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 1.6G Nov 26 14:47 Non_CpG_context_CF06-CM01-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 1.4G Nov 26 15:07 Non_CpG_context_CF06-CM02-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 992M Nov 26 15:25 Non_CpG_context_CF07-CM02-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 970M Nov 26 15:42 Non_CpG_context_CF08-CM03-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam  29M Nov 26 15:55 Non_CpG_context_CF08-CM04-Larvae_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam  27M Nov 26 16:05 Non_CpG_context_CF08-CM05-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 1.5G Nov 26 16:21 Non_CpG_context_EF01-EM01-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 1.6G Nov 26 16:42 Non_CpG_context_EF02-EM02-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 904M Nov 26 17:01 Non_CpG_context_EF03-EM03-Zygote_R1_001.fastp-trim.REPAIRED_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 2.3G Nov 26 17:23 Non_CpG_context_EF03-EM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 2.4G Nov 26 17:49 Non_CpG_context_EF03-EM05-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 485M Nov 26 18:08 Non_CpG_context_EF04-EM04-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 3.1G Nov 26 18:32 Non_CpG_context_EF04-EM05-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 1.9G Nov 26 18:58 Non_CpG_context_EF05-EM01-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 275M Nov 26 19:14 Non_CpG_context_EF05-EM05-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 3.4G Nov 26 19:39 Non_CpG_context_EF05-EM06-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 3.1G Nov 26 20:10 Non_CpG_context_EF06-EM01-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 2.3G Nov 26 20:38 Non_CpG_context_EF06-EM02-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 550M Nov 26 20:57 Non_CpG_context_EF06-EM06-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 653M Nov 26 21:12 Non_CpG_context_EF07-EM01-Zygote_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 2.4G Nov 26 21:33 Non_CpG_context_EF07-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 2.7G Nov 26 22:01 Non_CpG_context_EF08-EM03-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz
-rw-r--r-- 1 sam sam 2.1G Nov 26 22:27 Non_CpG_context_EF08-EM04-Larvae_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.txt.gz

7 Checksums

# Load bash variables into memory
source .bashvars

cd "${output_dir_top}"

for file in *; do
  if [ "${file}" != "checksums.md5" ]; then
    md5sum "${file}" >> checksums.md5
  fi
done

8 MultiQC

# Load bash variables into memory
source .bashvars

cd "${output_dir_top}"

${multiqc} .
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.
Krueger, Felix, and Simon R. Andrews. 2011. “Bismark: A Flexible Aligner and Methylation Caller for Bisulfite-Seq Applications.” Bioinformatics 27 (11): 1571–72. https://doi.org/10.1093/bioinformatics/btr167.