Comparison of RNA- or LNA-hybrid oligonucleotides in template-switching reactions for high-speed sequencing library preparationReport as inadecuate




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BMC Genomics

, 14:665

Transcriptomics

Abstract

BackgroundAnalyzing the RNA pool or transcription start sites requires effective means to convert RNA into cDNA libraries for digital expression counting. With current high-speed sequencers, it is necessary to flank the cDNAs with specific adapters. Adding template-switching oligonucleotides to reverse transcription reactions is the most commonly used approach when working with very small quantities of RNA even from single cells.

ResultsHere we compared the performance of DNA-RNA, DNA-LNA and DNA oligonucleotides in template-switching during nanoCAGE library preparation. Test libraries from rat muscle and HeLa cell RNA were prepared in technical triplicates and sequenced for comparison of the gene coverage and distribution of the reads within transcripts. The DNA-RNA oligonucleotide showed the highest specificity for capped 5′ ends of mRNA, whereas the DNA-LNA provided similar gene coverage with more reads falling within exons.

ConclusionsWhile confirming the cap-specific preference of DNA-RNA oligonucleotides in template-switching reactions, our data indicate that DNA-LNA hybrid oligonucleotides could potentially find other applications in random RNA sequencing.

KeywordsCAGE Template-switching LNA Transcriptome Quantitative sequencing AbbreviationsCAGECap analysis gene expression

LNALocked nucleic acid.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-14-665 contains supplementary material, which is available to authorized users.

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Author: Matthias Harbers - Sachi Kato - Michiel de Hoon - Yoshihide Hayashizaki - Piero Carninci - Charles Plessy

Source: https://link.springer.com/







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