Back to Search Start Over

Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing.

Authors :
Mulroney L
Wulf MG
Schildkraut I
Tzertzinis G
Buswell J
Jain M
Olsen H
Diekhans M
Corrêa IR Jr
Akeson M
Ettwiller L
Source :
RNA (New York, N.Y.) [RNA] 2022 Feb; Vol. 28 (2), pp. 162-176. Date of Electronic Publication: 2021 Nov 02.
Publication Year :
2022

Abstract

Nanopore sequencing devices read individual RNA strands directly. This facilitates identification of exon linkages and nucleotide modifications; however, using conventional direct RNA nanopore sequencing, the 5' and 3' ends of poly(A) RNA cannot be identified unambiguously. This is due in part to RNA degradation in vivo and in vitro that can obscure transcription start and end sites. In this study, we aimed to identify individual full-length human RNA isoforms among ∼4 million nanopore poly(A)-selected RNA reads. First, to identify RNA strands bearing 5' m <superscript>7</superscript> G caps, we exchanged the biological cap for a modified cap attached to a 45-nt oligomer. This oligomer adaptation method improved 5' end sequencing and ensured correct identification of the 5' m <superscript>7</superscript> G capped ends. Second, among these 5'-capped nanopore reads, we screened for features consistent with a 3' polyadenylation site. Combining these two steps, we identified 294,107 individual high-confidence full-length RNA scaffolds from human GM12878 cells, most of which (257,721) aligned to protein-coding genes. Of these, 4876 scaffolds indicated unannotated isoforms that were often internal to longer, previously identified RNA isoforms. Orthogonal data for m <superscript>7</superscript> G caps and open chromatin, such as CAGE and DNase-HS seq, confirmed the validity of these high-confidence RNA scaffolds.<br /> (© 2022 Mulroney et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Details

Language :
English
ISSN :
1469-9001
Volume :
28
Issue :
2
Database :
MEDLINE
Journal :
RNA (New York, N.Y.)
Publication Type :
Academic Journal
Accession number :
34728536
Full Text :
https://doi.org/10.1261/rna.078703.121