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Open Access Highly Accessed Methodology article

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

Matthias Harbers123, Sachi Kato23, Michiel de Hoon23, Yoshihide Hayashizaki34, Piero Carninci23 and Charles Plessy23*

Author Affiliations

1 K.K. DNAFORM, Leading Venture Plaza-2, 75–1, Ono-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0046, Japan

2 RIKEN Center for Life Science Technologies, Division of Genomics Technologies, Yokohama, Kanagawa 230-0045, Japan

3 RIKEN Omics Science Center, Yokohama, Kanagawa 230-0045, Japan

4 RIKEN Preventive Medicine & Diagnosis Innovation Program, Yokohama, Kanagawa 230-0045, Japan

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BMC Genomics 2013, 14:665  doi:10.1186/1471-2164-14-665

Published: 30 September 2013

Abstract

Background

Analyzing 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.

Results

Here 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.

Conclusions

While 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.

Keywords:
CAGE; Template-switching; LNA; Transcriptome; Quantitative sequencing