CStone: A de novo transcriptome assembler for short-read data that identifies non-chimeric contigs based on underlying graph structure

PLoS Comput Biol. 2021 Nov 23;17(11):e1009631. doi: 10.1371/journal.pcbi.1009631. eCollection 2021 Nov.

Abstract

With the exponential growth of sequence information stored over the last decade, including that of de novo assembled contigs from RNA-Seq experiments, quantification of chimeric sequences has become essential when assembling read data. In transcriptomics, de novo assembled chimeras can closely resemble underlying transcripts, but patterns such as those seen between co-evolving sites, or mapped read counts, become obscured. We have created a de Bruijn based de novo assembler for RNA-Seq data that utilizes a classification system to describe the complexity of underlying graphs from which contigs are created. Each contig is labelled with one of three levels, indicating whether or not ambiguous paths exist. A by-product of this is information on the range of complexity of the underlying gene families present. As a demonstration of CStones ability to assemble high-quality contigs, and to label them in this manner, both simulated and real data were used. For simulated data, ten million read pairs were generated from cDNA libraries representing four species, Drosophila melanogaster, Panthera pardus, Rattus norvegicus and Serinus canaria. These were assembled using CStone, Trinity and rnaSPAdes; the latter two being high-quality, well established, de novo assembers. For real data, two RNA-Seq datasets, each consisting of ≈30 million read pairs, representing two adult D. melanogaster whole-body samples were used. The contigs that CStone produced were comparable in quality to those of Trinity and rnaSPAdes in terms of length, sequence identity of aligned regions and the range of cDNA transcripts represented, whilst providing additional information on chimerism. Here we describe the details of CStones assembly and classification process, and propose that similar classification systems can be incorporated into other de novo assembly tools. Within a related side study, we explore the effects that chimera's within reference sets have on the identification of differentially expression genes. CStone is available at: https://sourceforge.net/projects/cstone/.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chimerism
  • DNA, Complementary / genetics
  • Datasets as Topic
  • Sequence Analysis, RNA / methods
  • Software
  • Transcriptome*

Substances

  • DNA, Complementary

Grants and funding

This work was funded by National Funds through FCT (Fundação para a Ciência e a Tecnologia) and FEDER through the Operational Programme for Competitiveness Factors (COMPETE), via a project awarded to JA, under the references POCI-01-0145-FEDER-029115 and PTDC/BIA-EVL/29115/2017. RL's post doctoral position was supported by this project under POCI-01-0145-FEDER-029115. The website of the Portuguese Foundation for Science and Technology is: https://www.fct.pt. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The website of the Portuguese Foundation for Science and Technology is: https://www.fct.pt The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.