Calling PacBio Methylation

Small pipeline to call methylation in Streptococcus suis using standard PacBio tools

Goal

This pipeline was constructed to call m6A and m4C methylation in Streptococcus suis mutants. These mutants should differ in their methylation status, which we wanted to investigate using PacBio. For an introduction on identification of methylation using PacBio data, see this Nature Methods paper.

⚠️ The pipeline was uploaded to GitHub to be able to retrace our steps and as a rough guide for future analyses. This pipeline is not intended as a tool which others can easily apply to their own data ⚠️

Requirements

This pipeline has the following requirements:

• Snakemake and conda are available on your system
• ipdSummary and MultiMotifMaker are installed on your system. This is not possible through conda. See https://www.pacb.com/support/software-downloads/ for ipdSummary (part of smrttools) and https://github.com/bioinfomaticsCSU/MultiMotifMaker for MultiMotifMaker. Update paths to executables as appropriate.
• PacBio read data is available in fasta format in the directory pacbio_fa and paired-end Illumina data is available in gzipped fastq format in the directory illumina_fastq
• Enough computational power. The analysis was originally run on a 96 GB RAM computing node with 16 CPUs. This worked alright.

Unicycler and pbmm2 will be installed through conda, integrated in Snakemake.

Methods

This pipeline combines several tools to end up with motifs associated with particular methylation. The tools and their functions are:

Tool	Purpose	Outputs	Link
Unicycler	Hybrid assembly of complete genome	Assembly fasta (asembly.fasta) and some quality control files	https://github.com/rrwick/Unicycler
pbmm2	Mapping of PacBio reads to assembly	Bam file ("native PacBio" format)	https://github.com/PacificBiosciences/pbmm2
ipdSummary	Identification of m6A, m4C and unknown modifications from mapped reads	Modifications file in GFF3 format and an exhaustive overview of kinetics per nt in csv format	https://github.com/PacificBiosciences/kineticsTools
MultiMotifMaker	Identification of motifs associated with modifications	Csv file containing motifs	https://github.com/bioinfomaticsCSU/MultiMotifMaker

The final csv file containing motifs is the main output.