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BioMed Research International
Volume 2015, Article ID 182389, 8 pages
Research Article

Evaluation and Application of the Strand-Specific Protocol for Next-Generation Sequencing

1Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan
2YourGene Biotechnology, Taipei, Taiwan
3Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Taiwan
4Department of Parasitology, National Cheng Kung University Medical College, Tainan, Taiwan
5Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan
6Bioinformatics Center, Chang Gung University, Taoyuan, Taiwan
7Genomics and Proteomics Core Laboratory, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan

Received 11 September 2014; Accepted 3 February 2015

Academic Editor: Dongchun Liang

Copyright © 2015 Kuo-Wang Tsai et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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.