nf-methTRAP

This pipeline is primarily developed to extract methylation data from ONT reads. Recently we added support to process PacBio data too.

Trim, Repair, Align and Pileup modBam

From modBAM to methylBed

This pipeline takes modification basecalled ONT reads or PacBio HiFi reads predicted with 5mC (modBam) as input, align to assembly provided and then extract methylation calls into bed/ bedgraph format.

Require inputs:

• ONT/ PacBio modBAM
• reference genome for alignment

Work flow

ONT workflow:

1. trim and repair tags of input modBam (optional)

   • trim and repair workflow:
     1. sort modBam - samtools sort
     2. convert modBam to fastq - samtools fastq
     3. trim barcode and adapters - porechop
     4. convert trimmed modfastq to modBam - samtools import
     5. repair MM/ML tags of trimmed modBam - modkit repair

2. align to reference (plus sorting and indexing) - dorado aligner

   • include alignment summary - samtools flagstat

3. create bedMethyl - modkit pileup

4. create bedgraphs (optional)

PacBio workflow:

1. align to reference - minimap2 or pbmm2 (default)

   • minimap workflow:

     1. convert modBam to fastq - samtools convert
     2. alignment - minimap2
     3. sort and index - samtools sort
     4. alignment summary - samtools flagstat

   • pbmm2 workflow:

     1. alignment and sorting - pbmm2
     2. index - samtools index
     3. alignment summary - samtools flagstat

2. create bedMethyl - modkit pileup (default) or pb-CpG-tools

   • pileup with modkit pileup is default setting
   • 2 options for pb-CpG-tools:
     1. default using count (no need to give any parameters)
     2. or can set to using model (parameter settings check next section)
     3. prior to pileup, aligned reads are split based on F/R strands

3. create bedgraph (optional)

Usage

To run the pipeline with a samplesheet on biohpc_gen with charliecloud:

nextflow run nf-methTRAP --samplesheet 'path/to/sample_sheet.csv' \
                          --out './results' \
                          -profile biohpc_gen,charliecloud

Note: Porechop, Modkit and Dorado containers are hosted at the LRZ gitlab registry. This requires authentication, currently not handled by nextflow. These containers need to be pre-pulled. Example:

ch-image pull --auth gitlab.lrz.de:5005/beckerlab/container-playground/porechop_pigz:4ba2bef9

Parameters

Parameter	Effect
--samplesheet	Path to samplesheet
--out	Results directory, default: './results'
--no_trim	skip trim
--aligner	minimap2, default: pbmm2
--pileup_method	pbcpgtools, default: modkit
--model	parse --pileup-mode model to pb-CpG-tools, default: --pileup-mode count
--bedgraph	convert bed to bedgraph, compatible to methylScore input

Samplesheet

Samplesheet .csv with header:

sample,modBam,ref,method

Column	Content
sample	Name of the sample
modBam	Path to basecalled modBam file
ref	Path to assembly fasta file
method	specify ont / pacbio

Outputs

The outputs will be put into params.out, defaulting to ./results.

├── ont
│   │
│   ├── trim
│   │   ├── trimmed.fastq.gz
│   │   ├── trimmed.bam
│   │   └── trimmed.log
│   │
│   ├── repair
│   │   ├── repaired.bam
│   │   └── repaired.log
│   │
│   ├── alignment
│   │   ├── aligned.bam
│   │   ├── aligned.bai
│   │   ├── summary.txt
│   │   └── aligned.flagstat
│   │
│   ├── pileup/modkit
│   │   ├── pileup.bed
│   │   └── pileup.log
│   │
│   └── bedgraph
│       └── bedgraphs
│ 
│  
└── pacbio   
    │
    ├── aligned_minimap2/ aligned_pbmm2
    │   ├── aligned.bam
    │   ├── aligned.bai/csi
    │   └── aligned.flagstat
    │
    ├── pileup: modkit/pb_cpg_tools
    │   ├── pileup.bed
    │   ├── pileup.log
    │   └── pileup.bw (only pb_cpg_tools)
    │
    └── bedgraph
        └── bedgraphs

Output of bedgraph

1. bed2bedgraph

Convert bedMethyl tables to bedgraphs (compatible with mehtylScore), filter out positions with <5x coverage.

bedgraph format:

column	name
1	chrom
2	pos1
3	pos2
4	methylation percentage
5	modified base coverage
6	canonical base coverage

Changes to be made

1. default pacbio pileup tool -> pbcpgtool (model)
2. document which parameters & versions used for some tools
   • minimap
   • pileup settings (modkit & pbcpgtools)