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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Vydáno v:Frontiers in genetics Ročník 13; s. 1031355
Hlavní autoři: Bayega, Anthony, Oikonomopoulos, Spyros, Wang, Yu Chang, Ragoussis, Jiannis
Médium: Journal Article
Jazyk:angličtina
Vydáno: Switzerland Frontiers Media S.A 17.10.2022
Témata:
cDNA
Genetics
long-read sequencing
nanopore
panhandle
PCR suppression effect
RNAseq
cDNA
nanopore
RNAseq
long-read sequencing
PCR suppression effect
panhandle
ISSN:1664-8021, 1664-8021
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Shrnutí: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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content type line 23
Edited by: Eugenia Poliakov, National Eye Institute (NIH), United States
Reviewed by: Panagiotis Ioannidis, Foundation for Research and Technology Hellas (FORTH), Greece
These authors have contributed equally to this work and share first authorship
Pablo Smircich, Instituto de Investigaciones Biológicas Clemente Estable (IIBCE), Uruguay
This article was submitted to Genomic Assay Technology, a section of the journal Frontiers in Genetics
ISSN:1664-8021
1664-8021
DOI:10.3389/fgene.2022.1031355