Evaluation and application of the strand-specific protocol for next-generation sequencing

Kuo Wang Tsai, Bill Chang, Cheng Tsung Pan, Wei Chen Lin, Ting Wen Chen, Sung Chou Li

研究成果: Article同行評審

5 引文 斯高帕斯(Scopus)

摘要

Next-generation sequencing (NGS) has become a powerful sequencing tool, applied in a wide range of biological studies. However, the traditional sample preparation protocol for NGS is non-strand-specific (NSS), leading to biased estimates of expression for transcripts overlapped at the antisense strand. Strand-specific (SS) protocols have recently been developed. In this study, we prepared the same RNA sample by using the SS and NSS protocols, followed by sequencing with Illumina HiSeq platform. Using real-time quantitative PCR as a standard, we first proved that the SS protocol more precisely estimates gene expressions compared with the NSS protocol, particularly for those overlapped at the antisense strand. In addition, we also showed that the sequence reads from the SS protocol are comparable with those from conventional NSS protocols in many aspects. Finally, we also mapped a fraction of sequence reads back to the antisense strand of the known genes, originally without annotated genes located. Using sequence assembly and PCR validation, we succeeded in identifying and characterizing the novel antisense genes. Our results show that the SS protocol performs more accurately than the traditional NSS protocol and can be applied in future studies.

原文English
文章編號182389
期刊BioMed research international
2015
DOIs
出版狀態Published - 2015

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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