Snakemake workflow: snakemake-methylseq

A Snakemake workflow that enables data processing of methylation sequencing platforms e.g bisulfite sequencing and enzymatic-methyl sequencing.

Workflow

Quality Control

1. FastQC
2. Fastp
3. MultiQC

Alignment

1. Two aligners are used here;
   • bwa-meth + samtools
   • abismal + samtools
2. VerifyBamID2

Methylation Analysis

Two methods are employed;

1. MethylDackel
   • Integrates well with bwa-meth
2. DMNTools
   • Integrates well with abismal

Expected Output

Quality Control

<path-to-output-folder>\
|   <project-name>\qc\
|       fastqc\
|           <sample>_<read>_fastqc.html
|           <sample>_<read>_fastqc.zip
|       fastp\
|           <sample>_<read>.trimmed_fastqc.html
|           <sample>_<read>.trimmed_fastqc.html
|           <sample>_1.trimmed.fastq
|           <sample>_2.trimmed.fastq
|           <sample>_u1.fastq
|           <sample>_u2.fastq
|           <sample>.merged.fastq
|           <sample>.failed.fastq
|           <sample>.html
|           <sample>.json
|       multiqc\
|           trimmed\
|                multiqc_data\
|                multiqc_report.html
|           untrimmed\
|                multiqc_data\
|                multiqc_report.html

Alignment

1. bwa-meth + samtools + VerifyBamID2
<path-to-output-folder>\
|   <project-name>\alignment\bwa\
|       unsorted/<sample>.sam
|       sorted/
|            <sample>.bam
|            <sample>.bai
|            <sample>.ancestry
|            <sample>.selfsm
|           /picard
|                <sample>.bam
|                <sample>.bam.bai
|                <sample>.metrics.txt

2. abismal + samtools + VerifyBamID2
<path-to-output-folder>\
|   <project-name>\alignment\abismal
|       <sample>.bam
|       <sample>.sorted.bam
|       <sample>.sorted.bai
|       <sample>.filtered.bam
|       stats\
|           <sample>.metrics.yaml
|           <sample>.filtered.metrics.yaml
|       verify_bam_id\
|           <sample>.ancestry
|           <sample>.selfsm

Methylation Analysis

1. DNMTools
<path-to-output-folder>\
|   <project-name>\methyl\dnmtools\
|       <sample>.bsrate
|       <sample>_single_base.meth
|       <sample>_symmetric.meth
|       <sample>_global.meth
|       <sample>.hmr
|       <sample>.hypermr
|       <sample>.epiread
|       <sample>.entropy.meth
|       <sample>.avg.meth

2. MethylDackel - To be completed

Reference files

• Genome (hg38)
• Canonical cis-Regulatory elements

Usage

Set-up working conda environment conda create --name <envname> --file requirements.txt NB: - The above step asumes you have conda already installed. - This workflow was built on Ubuntu20.04. Other platforms were not tested.

launch the pipeline form within the <snakemake-methylseq>\<workflow> directory

Test

All Rules: bash run_test.sh all

1. Quality Control bash run.sh qc

2. Alignment

   • bwa-meth: bash run.sh alignment bwa
   • abismal: bash run.sh alignment abismal

3. Methylation Analysis

   • DNMTools: bash run.sh methyl dnmtools
   • MethylDackel: bash run.sh methyl methyldackel

Run workflows

1. Quality Control bash run.sh qc - <num_cores>

2. Alignment

   • bwa-meth: bash run.sh alignment bwa
   • abismal: bash run.sh alignment abismal

3. Methylation Analysis

   • DNMTools: bash run.sh methyl dnmtools
   • MethylDackel: bash run.sh methyl methyldackel

TODO

• Add script to download and index reference files
• Add script to download svd_mu for VerifyBamID2
• Fix MethylDackel environment for to complete analysis
• Work on the format conversion between abismal and bwa-meth
• Fix errors from rules entropy and avg_meth_level_region
• Provide PC plots for contamination of samples
• Add notes on updating the config file
• Add documentation on rules and their purpose