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- Title
Improving the diversity of captured full-length isoforms using a normalized single-molecule RNA-sequencing method.
- Authors
Hu, Yueming; Shu, Xing-Sheng; Yu, Jiaxian; Sun, Ming-an; Chen, Zewei; Liu, Xianming; Fang, Qiongfang; Zhang, Wei; Hui, Xinjie; Ying, Ying; Fu, Li; Lu, Desheng; Kumar, Rakesh; Wang, Yejun
- Abstract
Human genes form a large variety of isoforms after transcription, encoding distinct transcripts to exert different functions. Single-molecule RNA sequencing facilitates accurate identification of the isoforms by extending nucleotide read length significantly. However, the gene or isoform diversity is lowly represented by the mRNA molecules captured by single-molecule RNA sequencing. Here, we show that a cDNA normalization procedure before the library preparation for PacBio RS II sequencing captures 3.2–6.0 fold more full-length high-quality isoform species for different human samples, as compared to the non-normalized capture procedure. Many lowly expressed, functionally important isoforms can be detected. In addition, normalized PacBio RNA sequencing also resolves more allele-specific haplotype transcripts. Finally, we apply the cDNA normalization based long-read RNA sequencing method to profile the transcriptome of human gastric signet-ring cell carcinomas, identify new cancer-specific transcriptome signatures, and thus, bring out the utility of the improved protocols in gene expression studies. Hu et al. combine cDNA normalization before library preparation with a software tool to increase the capture of RNA isoform species in single-molecule RNA sequencing. They demonstrate that this approach can detect previously unknown transcripts in gastric signet-ring cell carcinomas that are not present in non-malignant tissue.
- Subjects
RNA sequencing; NUCLEOTIDES; MESSENGER RNA; ANTISENSE DNA; TRANSCRIPTOMES
- Publication
Communications Biology, 2020, Vol 3, Issue 1, p1
- ISSN
2399-3642
- Publication type
Article
- DOI
10.1038/s42003-020-01125-7