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

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 RN...

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Autores principales: Mulroney, Logan, Wulf, Madalee G., Schildkraut, Ira, Tzertzinis, George, Buswell, John, Jain, Miten, Olsen, Hugh, Diekhans, Mark, Corrêa, Ivan R., Akeson, Mark, Ettwiller, Laurence
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8906549/
https://www.ncbi.nlm.nih.gov/pubmed/34728536
http://dx.doi.org/10.1261/rna.078703.121
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author Mulroney, Logan
Wulf, Madalee G.
Schildkraut, Ira
Tzertzinis, George
Buswell, John
Jain, Miten
Olsen, Hugh
Diekhans, Mark
Corrêa, Ivan R.
Akeson, Mark
Ettwiller, Laurence
author_facet Mulroney, Logan
Wulf, Madalee G.
Schildkraut, Ira
Tzertzinis, George
Buswell, John
Jain, Miten
Olsen, Hugh
Diekhans, Mark
Corrêa, Ivan R.
Akeson, Mark
Ettwiller, Laurence
author_sort Mulroney, Logan
collection PubMed
description 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(7)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(7)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(7)G caps and open chromatin, such as CAGE and DNase-HS seq, confirmed the validity of these high-confidence RNA scaffolds.
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spelling pubmed-89065492022-03-23 Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing Mulroney, Logan Wulf, Madalee G. Schildkraut, Ira Tzertzinis, George Buswell, John Jain, Miten Olsen, Hugh Diekhans, Mark Corrêa, Ivan R. Akeson, Mark Ettwiller, Laurence RNA Article 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(7)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(7)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(7)G caps and open chromatin, such as CAGE and DNase-HS seq, confirmed the validity of these high-confidence RNA scaffolds. Cold Spring Harbor Laboratory Press 2022-02 /pmc/articles/PMC8906549/ /pubmed/34728536 http://dx.doi.org/10.1261/rna.078703.121 Text en © 2022 Mulroney et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society https://creativecommons.org/licenses/by/4.0/This article, published in RNA, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Mulroney, Logan
Wulf, Madalee G.
Schildkraut, Ira
Tzertzinis, George
Buswell, John
Jain, Miten
Olsen, Hugh
Diekhans, Mark
Corrêa, Ivan R.
Akeson, Mark
Ettwiller, Laurence
Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing
title Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing
title_full Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing
title_fullStr Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing
title_full_unstemmed Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing
title_short Identification of high-confidence human poly(A) RNA isoform scaffolds using nanopore sequencing
title_sort identification of high-confidence human poly(a) rna isoform scaffolds using nanopore sequencing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8906549/
https://www.ncbi.nlm.nih.gov/pubmed/34728536
http://dx.doi.org/10.1261/rna.078703.121
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