Abstract
Background Transcriptome assembly from RNA-sequencing data in species without a reliable reference genome has to be performed de novo, but studies have shown that de novo methods often have inadequate ability to reconstruct transcript isoforms. We address this issue by constructing an assembly pipeline whose main purpose is to produce a comprehensive set of transcript isoforms.
Results We present the de novo transcript isoform assembler ClusTrast, which clusters a set of guiding contigs by similarity, aligns short reads to the guiding contigs, and assembles each clustered set of short reads individually. We tested ClusTrast on datasets from six eukaryotic species, and showed that ClusTrast reconstructed more expressed known isoforms than any of the other tested de novo assemblers, at a moderate reduction in precision. For recall, ClusTrast was on top in the lower end of expression levels (<15% percentile) for all tested datasets, and over the entire range for almost all datasets. Reference transcripts were often (35–69% for the six datasets) reconstructed to at least 95% of their length by ClusTrast, and more than half of reference transcripts (58–81%) were reconstructed with contigs that exhibited polymorphism, measuring on a subset of reliably predicted contigs.
Conclusion We suggest that ClusTrast can be a useful tool for studying isoforms in species without a reliable reference genome, in particular when the goal is to produce a comprehensive transcriptome set with polymorphic variants.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
westrin{at}kth.se, wk{at}warrenwk.com, olofem{at}kth.se
We have tried to make it clearer that the purpose of ClusTrast is to produce a comprehensive set of transcripts, and that it is not a suitable tool when specificity is the focus. The revision includes more analyses of the performance of the assemblers, e.g. the difference between true positive sets as defined by SQANTI and CRBB (3.1.3) and an investigation of polymorphisms and alternative splicing in the reconstructed transcriptomes (novel section 3.1.5). The description of our method, ClusTrast, has been moved from the Results chapter to the Implementation chapter (formerly Materials and Methods). A new chapter, Conclusion, is introduced. One of the datasets in our original submission, poplar, turned out to be a mixed dataset with also a fungus included. Thus, we have removed that data set from the main manuscript, and we use another poplar dataset instead.
