TY - JOUR
T1 - Evaluation and application of the strand-specific protocol for next-generation sequencing
AU - Tsai, Kuo Wang
AU - Chang, Bill
AU - Pan, Cheng Tsung
AU - Lin, Wei Chen
AU - Chen, Ting Wen
AU - Li, Sung Chou
N1 - Publisher Copyright:
© 2015 Kuo-Wang Tsai et al.
PY - 2015
Y1 - 2015
N2 - 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.
AB - 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.
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U2 - 10.1155/2015/182389
DO - 10.1155/2015/182389
M3 - Article
C2 - 25893191
AN - SCOPUS:84928392606
VL - 2015
JO - BioMed Research International
JF - BioMed Research International
SN - 2314-6133
M1 - 182389
ER -