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Improved Nanopore full-length cDNA sequencing by PCR-suppression

Full-length transcript sequencing remains a main goal of RNA sequencing. However, even the application of long-read sequencing technologies such as Oxford Nanopore Technologies still fail to yield full-length transcript sequencing for a significant portion of sequenced reads. Since these technologie...

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Autores principales: Bayega, Anthony, Oikonomopoulos, Spyros, Wang, Yu Chang, Ragoussis, Jiannis
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9618600/
https://www.ncbi.nlm.nih.gov/pubmed/36324505
http://dx.doi.org/10.3389/fgene.2022.1031355
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author Bayega, Anthony
Oikonomopoulos, Spyros
Wang, Yu Chang
Ragoussis, Jiannis
author_facet Bayega, Anthony
Oikonomopoulos, Spyros
Wang, Yu Chang
Ragoussis, Jiannis
author_sort Bayega, Anthony
collection PubMed
description Full-length transcript sequencing remains a main goal of RNA sequencing. However, even the application of long-read sequencing technologies such as Oxford Nanopore Technologies still fail to yield full-length transcript sequencing for a significant portion of sequenced reads. Since these technologies can sequence reads that are far longer than the longest known processed transcripts, the lack of efficiency to obtain full-length transcripts from good quality RNAs stems from library preparation inefficiency rather than the presence of degraded RNA molecules. It has previously been shown that addition of inverted terminal repeats in cDNA during reverse transcription followed by single-primer PCR creates a PCR suppression effect that prevents amplification of short molecules thus enriching the library for longer transcripts. We adapted this method for Nanopore cDNA library preparation and show that not only is PCR efficiency increased but gene body coverage is dramatically improved. The results show that implementation of this simple strategy will result in better quality full-length RNA sequencing data and make full-length transcript sequencing possible for most of sequenced reads.
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spelling pubmed-96186002022-11-01 Improved Nanopore full-length cDNA sequencing by PCR-suppression Bayega, Anthony Oikonomopoulos, Spyros Wang, Yu Chang Ragoussis, Jiannis Front Genet Genetics Full-length transcript sequencing remains a main goal of RNA sequencing. However, even the application of long-read sequencing technologies such as Oxford Nanopore Technologies still fail to yield full-length transcript sequencing for a significant portion of sequenced reads. Since these technologies can sequence reads that are far longer than the longest known processed transcripts, the lack of efficiency to obtain full-length transcripts from good quality RNAs stems from library preparation inefficiency rather than the presence of degraded RNA molecules. It has previously been shown that addition of inverted terminal repeats in cDNA during reverse transcription followed by single-primer PCR creates a PCR suppression effect that prevents amplification of short molecules thus enriching the library for longer transcripts. We adapted this method for Nanopore cDNA library preparation and show that not only is PCR efficiency increased but gene body coverage is dramatically improved. The results show that implementation of this simple strategy will result in better quality full-length RNA sequencing data and make full-length transcript sequencing possible for most of sequenced reads. Frontiers Media S.A. 2022-10-17 /pmc/articles/PMC9618600/ /pubmed/36324505 http://dx.doi.org/10.3389/fgene.2022.1031355 Text en Copyright © 2022 Bayega, Oikonomopoulos, Wang and Ragoussis. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genetics
Bayega, Anthony
Oikonomopoulos, Spyros
Wang, Yu Chang
Ragoussis, Jiannis
Improved Nanopore full-length cDNA sequencing by PCR-suppression
title Improved Nanopore full-length cDNA sequencing by PCR-suppression
title_full Improved Nanopore full-length cDNA sequencing by PCR-suppression
title_fullStr Improved Nanopore full-length cDNA sequencing by PCR-suppression
title_full_unstemmed Improved Nanopore full-length cDNA sequencing by PCR-suppression
title_short Improved Nanopore full-length cDNA sequencing by PCR-suppression
title_sort improved nanopore full-length cdna sequencing by pcr-suppression
topic Genetics
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9618600/
https://www.ncbi.nlm.nih.gov/pubmed/36324505
http://dx.doi.org/10.3389/fgene.2022.1031355
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