A long-read RNA-seq approach to identify novel transcripts of very large genes

© 2020 Uapinyoying et al.; Published by Cold Spring Harbor Laboratory Press..

RNA-seq is widely used for studying gene expression, but commonly used sequencing platforms produce short reads that only span up to two exon junctions per read. This makes it difficult to accurately determine the composition and phasing of exons within transcripts. Although long-read sequencing improves this issue, it is not amenable to precise quantitation, which limits its utility for differential expression studies. We used long-read isoform sequencing combined with a novel analysis approach to compare alternative splicing of large, repetitive structural genes in muscles. Analysis of muscle structural genes that produce medium (Nrap: 5 kb), large (Neb: 22 kb), and very large (Ttn: 106 kb) transcripts in cardiac muscle, and fast and slow skeletal muscles identified unannotated exons for each of these ubiquitous muscle genes. This also identified differential exon usage and phasing for these genes between the different muscle types. By mapping the in-phase transcript structures to known annotations, we also identified and quantified previously unannotated transcripts. Results were confirmed by endpoint PCR and Sanger sequencing, which revealed muscle-type-specific differential expression of these novel transcripts. The improved transcript identification and quantification shown by our approach removes previous impediments to studies aimed at quantitative differential expression of ultralong transcripts.

Medienart:

E-Artikel

Erscheinungsjahr:

2020

Erschienen:

2020

Enthalten in:

Zur Gesamtaufnahme - volume:30

Enthalten in:

Genome research - 30(2020), 6 vom: 13. Juni, Seite 885-897

Sprache:

Englisch

Beteiligte Personen:

Uapinyoying, Prech [VerfasserIn]
Goecks, Jeremy [VerfasserIn]
Knoblach, Susan M [VerfasserIn]
Panchapakesan, Karuna [VerfasserIn]
Bonnemann, Carsten G [VerfasserIn]
Partridge, Terence A [VerfasserIn]
Jaiswal, Jyoti K [VerfasserIn]
Hoffman, Eric P [VerfasserIn]

Links:

Volltext

Themen:

Journal Article
RNA, Messenger
Research Support, N.I.H., Extramural

Anmerkungen:

Date Completed 20.07.2021

Date Revised 15.11.2022

published: Print-Electronic

Citation Status MEDLINE

doi:

10.1101/gr.259903.119

funding:

Förderinstitution / Projekttitel:

PPN (Katalog-ID):

NLM312375727