Is it possible to use HiFi reads to call base modifications?

Base modifications can be inferred from per-base pulse width (PW) and inter-pulse duration (IPD) kinetics. Running ccs with --hifi-kinetics generates averaged kinetic information for polished reads, independently for both strands of the insert. Forward is defined with respect to the orientation represented in SEQ and is considered to be the native orientation. As with other PacBio-specific tags, aligners will not re-orient these fields.

Minor cases exist where a certain orientation may get filtered out entirely from a ZMW, preventing valid values from being passed for that record. In these cases, empty lists will be passed for the respective record/orientation and number of passes will be set to zero.

In order to facilitate the use of HiFi reads with base modifications workflows, we have added an executable in pbtk called ccs-kinetics-bystrandify which creates a pseudo --by-strand BAM with corresponding pw and ip tags that imitates a normal, unaligned subreads BAM. You can install it from Bioconda by calling conda install pbtk.

Please see the BAM output documentation for all available kinetics tags.

What about single-strand reads?

Starting with ccs v6.4.0, single-strand reads are correctly annotated with HiFi kinetics in the corresponding pw and ip tags. Combining --by-strand --hifi-kinetics is no longer prohibited. This is an experimental feature. If something does not work, please let us know!

What about mixed single- and double-strand read BAM files?

Starting with ccs v6.4.0, combining --hd-finder --hifi-kinetics will produce correct annotations for single-strand and double-strand reads. If your single-strand reads have the HiFi kinetic tags (fi, fp, etc) please process the output BAM with the latest ccs-kinetics-bystrandify from the pbtk conda package, it will properly split reads, even for older ccs v6.3.0 files.