Evaluation and Application of the Strand-Specific Protocol for Next-Generation Sequencing
| Autor: | Bill C.H. Chang, Sung-Chou Li, Wei Chen Lin, Kuo-Wang Tsai, Ting-Wen Chen, Cheng Tsung Pan |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Article Subject
Sequence analysis Molecular Sequence Data lcsh:Medicine Sequence assembly Genomics Biology Real-Time Polymerase Chain Reaction General Biochemistry Genetics and Molecular Biology DNA sequencing Stomach Neoplasms Cell Line Tumor Humans RNA Neoplasm Gene Genetics Base Sequence General Immunology and Microbiology Sequence Analysis RNA Oligonucleotide Gene Expression Profiling lcsh:R Intron Computational Biology High-Throughput Nucleotide Sequencing Exons Sequence Analysis DNA General Medicine Oligonucleotides Antisense Introns Gene Expression Regulation Neoplastic Gene expression profiling Research Article |
| Zdroj: | BioMed Research International, Vol 2015 (2015) BioMed Research International |
| ISSN: | 2314-6141 2314-6133 |
| DOI: | 10.1155/2015/182389 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|