Two-pass alignment improves novel splice junction quantification.

ABSTRACT: Discovery of novel splicing from RNA sequence data remains a critical and exciting focus of transcriptomics, but reduced alignment power impedes expression quantification of novel splice junctions.Here, we profile performance characteristics of two-pass alignment, which separates splice junction discovery from quantification. Per sample, across a variety of transcriptome sequencing datasets, two-pass alignment improved quantification of at least 94% of simulated novel splice junctions, and provided as much as 1.7-fold deeper median read depth over those splice junctions. We further demonstrate that two-pass alignment works by increasing alignment of reads to splice junctions by short lengths, and that potential alignment errors are readily identifiable by simple classification. Taken together, two-pass alignment promises to advance quantification and discovery of novel splicing [e-mail], [e-mail]-pass alignment was implemented here as sequential alignment, genome indexing, and re-alignment steps with STAR. Full parameters are provided in Supplementary Table 2.Supplementary data are available at Bioinformatics online.

SUBMITTER: Veeneman BA 

PROVIDER: S-EPMC5006238 | biostudies-other | 2016 Jan

REPOSITORIES: biostudies-other