MGcount: a total RNA-seq quantification tool to address multi-mapping and multi-overlapping alignments ambiguity in non-coding transcripts

BMC Bioinformatics, vol. 23, pp. 39

Abstract

BACKGROUND: Total-RNA sequencing (total-RNA-seq) allows the simultaneous study of both the coding and the non-coding transcriptome. Yet, computational pipelines have traditionally focused on particular biotypes, making assumptions that are not fullfilled by total-RNA-seq datasets. Transcripts from distinct RNA biotypes vary in length, biogenesis, and function, can overlap in a genomic region, and may be present in the genome with a high copy number. Consequently, reads from total-RNA-seq libraries may cause ambiguous genomic alignments, demanding for flexible quantification approaches. RESULTS: Here we present Multi-Graph count (MGcount), a total-RNA-seq quantification tool combining two strategies for handling ambiguous alignments. First, MGcount assigns reads hierarchically to small-RNA and long-RNA features to account for length disparity when transcripts overlap in the same genomic position. Next, MGcount aggregates RNA products with similar sequences where reads systematically multi-map using a graph-based approach. MGcount outputs a transcriptomic count matrix compatible with RNA-sequencing downstream analysis pipelines, with both bulk and single-cell resolution, and the graphs that model repeated transcript structures for different biotypes. The software can be used as a python module or as a single-file executable program. CONCLUSIONS: MGcount is a flexible total-RNA-seq quantification tool that successfully integrates reads that align to multiple genomic locations or that overlap with multiple gene features. Its approach is suitable for the simultaneous estimation of protein-coding, long non-coding and small non-coding transcript concentration, in both precursor and processed forms. Both source code and compiled software are available at https://github.com/hitaandrea/MGcount .

Authors 5

1. Andrea Hita

   Humboldt-Universität zu Berlin · Diagenode (Belgium)

   Affiliation as printed

   Department of Biology, Humboldt-Universität zu Berlin, Berlin, Germany

   Epigenetics unit, Diagenode s.a., Liège, Belgium

2. Gilles Brocart

   Diagenode (Belgium)

   Affiliation as printed

   Epigenetics unit, Diagenode s.a., Liège, Belgium

3. Marc Rehmsmeier

   Humboldt-Universität zu Berlin

   Affiliation as printed

   Department of Biology, Humboldt-Universität zu Berlin, Berlin, Germany

4. Anna Alemany Aachen Department of Anatomy and Embryology

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Department of Anatomy and Embryology, Leiden University Medical Centre, Leiden, The Netherlands

5. Sol Schvartzman corresponding

   Diagenode (Belgium)

   Affiliation as printed

   Epigenetics unit, Diagenode s.a., Liège, Belgium. sol.schvartzman@diagenode.com

   Epigenetics unit, Diagenode s.a., Liège, Belgium