title: Evaluation and application of the strand-specific protocol for next-generation sequencing.
abstract: 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.
authors.affiliation: Department of Medical Education and Research, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
authors.forename: Kuo-Wang
authors.initials: KW
authors.lastname: Tsai
authors.affiliation: YourGene Biotechnology, Taipei, Taiwan.
authors.forename: Bill
authors.initials: B
authors.lastname: Chang
authors.affiliation: Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Taiwan.
authors.forename: Cheng-Tsung
authors.initials: CT
authors.lastname: Pan
authors.affiliation: Department of Parasitology, National Cheng Kung University Medical College, Tainan, Taiwan.
authors.forename: Wei-Chen
authors.initials: WC
authors.lastname: Lin
authors.affiliation: Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan ; Bioinformatics Center, Chang Gung University, Taoyuan, Taiwan.
authors.forename: Ting-Wen
authors.initials: TW
authors.lastname: Chen
authors.affiliation: Genomics and Proteomics Core Laboratory, Department of Medical Research, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Sung-Chou
authors.initials: SC
authors.lastname: Li
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D019295:Computational Biology; D005091:Exons; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D023281:Genomics; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000235:genetics; D012334:RNA, Neoplasm / Q000032:analysis*; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017423:Sequence Analysis, RNA / Q000379:methods*; D013274:Stomach Neoplasms / Q000378:metabolism
chemical_list: D016376:Oligonucleotides, Antisense; D012334:RNA, Neoplasm