Tsai Kuo-Wang, Chang Bill, Pan Cheng-Tsung, Lin Wei-Chen, Chen Ting-Wen, Li Sung-Chou
Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
YourGene Biotechnology, Taipei, Taiwan.
Biomed Res Int. 2015;2015:182389. doi: 10.1155/2015/182389. Epub 2015 Mar 29.
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.
新一代测序（NGS）已成为一种强大的测序工具，应用于广泛的生物学研究中。然而，传统的NGS样本制备方案是非链特异性的（NSS），导致对反义链上重叠转录本的表达估计存在偏差。链特异性（SS）方案最近已被开发出来。在本研究中，我们使用SS和NSS方案制备了相同的RNA样本，随后在Illumina HiSeq平台上进行测序。以实时定量PCR作为标准，我们首先证明，与NSS方案相比，SS方案能更精确地估计基因表达，特别是对于那些在反义链上重叠的基因。此外，我们还表明，来自SS方案的序列读数在许多方面与传统NSS方案的读数相当。最后，我们还将一部分序列读数映射回已知基因的反义链，这些基因最初没有注释。通过序列组装和PCR验证，我们成功地鉴定并表征了新的反义基因。我们的结果表明，SS方案比传统的NSS方案表现得更准确，可应用于未来的研究。
