ccsmethphase

Methylation phasing using PacBio CCS reads

Contents

• Installation
• Demo data
• Input
• Usage
• Output
• Acknowledgements
• TODO

Installation

Recommended Hardware requirements: 128 GB RAM, 40 CPU processors, 4 TB disk storage, >=8 GB GPU

Recommended OS: Linux (Ubuntu 16.04, CentOS 7, etc.)

• (1) Install conda from Conda if neeeded.

• (2) Install nextflow (version>=21.10.6).

# create a new environment and install nextflow in it
conda create -n nextflow -c conda-forge -c bioconda nextflow

# OR, install nextflow in an existing environment
conda install -c conda-forge -c bioconda nextflow

• (3) Download ccsmethphase from github.

git clone https://github.com/PengNi/ccsmethphase.git

• (4) Install Docker or Singularity if needed.

# e.g., install singularity using conda
conda install -c conda-forge singularity 

• (5) [optional] Install graphviz.

conda install -c conda-forge graphviz

Demo data

Check ccsmethphase/demo for demo data:

• hg002.chr20_demo.hifi.bam: HG002 demo hifi reads which are aligned to human genome chr20:10000000-10100000.
• chr20_demo.fa: reference sequence of human chr20:10000000-10100000.
• hg002_bsseq_chr20_demo.bed: HG002 BS-seq results of region chr20:10000000-10100000.
• input_sheet.tsv: Information of the demo PacBio bam file hg002.chr20_demo.hifi.bam.

Input

ccsmethphase takes files of PacBio reads (subreads.bam or hifi.bam), and a reference genome as input.

The information of PacBio reads files should be organized into a tsv file, like input_sheet.tsv in demo data:

Group_ID	Sample_ID	Type	Path
G1	HG002_demo	hifi	./demo/hg002.chr20_demo.hifi.bam
G1	HG002_demo	hifi	path of another flowcell bam file for HG002_demo
G1	HG003	hifi	path of a bam file for HG003

• Group_ID: For group comparation, values can be like control, case, or anything else.
• Sample_ID: The name of the sample sequenced.
• Type: Data type, should be hifi or subreads.
• Path: Path of a (flowcell) bam file sequenced using the sample. Absolute path recommended.

NOTE: ccsmethphase can be run with conda, docker, and singularity by setting -profile. If you are using -profile conda to run this workflow, ccsmeth models should be set as input too. Check ccsmeth to get ccsmeth 5mCpG models.

Usage

Parameters (to be completed):

--dsname:   job name
--input:    the tsv file containing information of PacBio reads files
--genome:   file path of the reference genome
--include_all_ctgs: "true" or "false". default false, means only [chr][1-22,X,Y] included.
-profile:   conda/docker/singularity, test

Example 1. Run with singularity (recommended)

If it is the first time you run with singularity (e.g. using -profile singularity), the following cmd will cache the dafault singularity image (--singularity_name and/or --clair3_singularity_name) to the --singularity_cache directory (default: local_singularity_cache) first. There will be .img file(s) in the --singularity_cache directory.

NOTE: If you are using relative paths of bam files in input_sheet.tsv, make sure the relative paths are the right relative paths to the directory you launch the workflow.

For the example data:

# activate nextflow environment
conda activate nextflow

cd /path/to/ccsmethphase
nextflow run main.nf \
    --dsname test \
    -profile singularity,test

The above command is equal to the command following:

nextflow run /path/to/ccsmethphase \
    --dsname test \
    --genome /path/to/ccsmethphase/demo/chr20_demo.fa \
    --input /path/to/ccsmethphase/demo/input_sheet.tsv \
    --include_all_ctgs true \
    --max_cpus 8 \
    --max_memory "12.GB" \
    --max_time "6.h" \
    -profile singularity

Try the following command to enable GPU:

CUDA_VISIBLE_DEVICES=0 nextflow run /path/to/ccsmethphase \
    --dsname test \
    --genome /path/to/ccsmethphase/demo/chr20_demo.fa \
    --input /path/to/ccsmethphase/demo/input_sheet.tsv \
    --include_all_ctgs true \
    -profile singularity

The downloaded singularity .img file(s) can be re-used, without being downloaded again:

nextflow run /path/to/ccsmethphase \
    --dsname test2 \
    --genome /path/to/some/other/genome/fa \
    --input /path/to/some/other/input_sheet.tsv \
    -profile singularity \
    --singularity_cache /path/to/local_singularity_cache

Try -resume to re-run a modified/failed job to save time:

nextflow run /path/to/ccsmethphase \
    --dsname test \
    --genome /path/to/ccsmethphase/demo/chr20_demo.fa \
    --input /path/to/ccsmethphase/demo/input_sheet.tsv \
    --include_all_ctgs true \
    -profile singularity \
    -resume

Possible issues:

1. no space left on device ERROR

This may be caused by limited space of the SINGULARITY_TMPDIR (default /tmp) dir. Try setting SINGULARITY_TMPDIR to a disk that have enough space in current environment:

export SINGULARITY_TMPDIR=/path/to/another/dir

Output

The output directory should look like the following:

ccsmethphase_results/
├── pipeline_info
└── test
    ├── bam
    │   ├── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.SNV_PASS_whatshap.bam
    │   └── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.SNV_PASS_whatshap.bam.bai
    ├── diff_methyl
    │   ├── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.SNV_PASS_whatshap.freq.aggregate.hp_callDML.txt
    │   ├── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.SNV_PASS_whatshap.freq.aggregate.hp_callDMR.autosomes_cf0.2.bed
    │   └── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.SNV_PASS_whatshap.freq.aggregate.hp_callDMR.txt
    ├── mods_freq
    │   ├── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.SNV_PASS_whatshap.freq.aggregate.all.bed
    │   ├── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.SNV_PASS_whatshap.freq.aggregate.hp1.bed
    │   └── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.SNV_PASS_whatshap.freq.aggregate.hp2.bed
    └── vcf
        ├── clair3_called
        │   ├── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.clair3_merge.vcf.gz
        │   └── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.clair3_merge.vcf.gz.tbi
        └── whatshap_phased
            ├── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.clair3_merge.SNV_PASS_whatshap.vcf.gz
            └── G1.HG002_demo.hifi.ccsmeth.modbam.pbmm2.merged_size1.clair3_merge.SNV_PASS_whatshap.vcf.gz.tbi

• pipeline_info: Information of the workflow execution
• test: directory to save the results of ccsmethphase, name set by --dsname
  • bam: modbam files
  • diff_methyl: DMLs and DMRs generated by CCS
  • mods_freq: bedmethyl files, site-level methylation frequencies
  • vcf: VCF files for SNVs
    • clair3_called: SNVs generated by clair3
    • whatshap_phsed: Phased SNVs by whatshap

Acknowledgements

• Some code were referenced from nanome and nf-core.
• Code for ASM detection using DSS were referenced from NanoMethPhase of Akbari et al.

TODO

• input format -> group_id type(hifi/subreads) file_abs_path
• have tested docker on cpu, singularity on cpu/gpu/cpu-in-gpu-machine; did not test docker on gpu/cpu-in-gpu-machine yet
• complete --help/-h option
• default model in docker container
• add quality-control/statistics process for ccs data?
• add DSS-2.44.0, update docker/env.yml/readme/image
• report-summary (Rmarkdown->html?)
• add test profile?