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

被引:17
作者
Harbers, Matthias [1 ,2 ,3 ]
Kato, Sachi [2 ,3 ]
de Hoon, Michiel [2 ,3 ]
Hayashizaki, Yoshihide [3 ,4 ]
Carninci, Piero [2 ,3 ]
Plessy, Charles [2 ,3 ]
机构
[1] KK DNAFORM, Tsurumi Ku, Yokohama, Kanagawa 2300046, Japan
[2] RIKEN, Ctr Life Sci Technol, Div Genom Technol, Yokohama, Kanagawa 2300045, Japan
[3] RIKEN, Om Sci Ctr, Yokohama, Kanagawa 2300045, Japan
[4] RIKEN, Prevent Med & Diag Innovat Program, Yokohama, Kanagawa 2300045, Japan
关键词
CAGE; Template-switching; LNA; Transcriptome; Quantitative sequencing; REVERSE-TRANSCRIPTASE; CDNA SYNTHESIS; DNA;
D O I
10.1186/1471-2164-14-665
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
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 templateswitching 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.
引用
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页数:6
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