Detect Base Modifications with SMRT Sequencing

Base modification detection and motif analysis are included in SMRT® Analysis v1.3.3 (and higher) as fully automated data analysis workflows. The pre-release tools described on this page can still be used, but they are no longer necessary for standard analyses.

PacBio® has developed some pre-release tools to look at DNA modification patterns. The initial tools support analysis of changes in single molecule kinetics between a case and a control:

R-kinetics project provides an R vignette that describes how to perform basic kinetic analyses using the R suite of tools provided by Pacific Biosciences. GitHub project: R-Kinetics
Documentation: R-Kinetics documentation (PDF)

kineticsTools is a Python tool that will process a reference FASTA plus the associated aligned reads HDF5 file and output modification and motif analysis in the same formats used by SMRT Portal. Unlike SMRT Portal it does not use the base call HDF5 file, but the post-alignment HDF5 file. Also included is a PDF description of the analysis method used. GitHub project: kineticsTools Documentation: kineticsTools documentation

MotifMaker is a command-line Java program that will analysis your FASTA file and the modifications.gff file output by kineticsTools or SMRT Portal to find methylated motifs within a genome. It is designed for bacterial methylome analysis. This program runs quickly and is also a good way to iterate on methylome analysis with different thresholds than used by default in SMRT Portal. GitHub project: MotifMaker Documentation found on the project home page.

motif-finding is an R vignette that walks a user through analysis of methylated motifs. It allows more flexible visualization and analysis than with MotifMaker or SMRT Portal for users comfortable with the R environment. GitHub project: motif-finding Documentation: motif-finding documentation (PDF)