Optimized approach for Ion Proton RNA sequencing reveals details of RNA splicing and editing features of the transcriptome

被引:13
作者
Brown, Roger B. [1 ]
Madrid, Nathaniel J. [1 ]
Suzuki, Hideaki [1 ]
Ness, Scott A. [1 ,2 ]
机构
[1] Univ New Mexico, Hlth Sci Ctr, Div Mol Med, Dept Internal Med, Albuquerque, NM 87131 USA
[2] Univ New Mexico, UNM Comprehens Canc Ctr, Albuquerque, NM 87131 USA
来源
PLOS ONE | 2017年 / 12卷 / 05期
基金
美国国家卫生研究院;
关键词
C-MYB; CELL; DATABASE; SEQ; DIFFERENTIATION; PROTEINS; LEUKEMIA;
D O I
10.1371/journal.pone.0176675
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
RNA-sequencing (RNA-seq) has become the standard method for unbiased analysis of gene expression but also provides access to more complex transcriptome features, including alternative RNA splicing, RNA editing, and even detection of fusion transcripts formed through chromosomal translocations. However, differences in library methods can adversely affect the ability to recover these different types of transcriptome data. For example, some methods have bias for one end of transcripts or rely on low-efficiency steps that limit the complexity of the resulting library, making detection of rare transcripts less likely. We tested several commonly used methods of RNA-seq library preparation and found vast differences in the detection of advanced transcriptome features, such as alternatively spliced isoforms and RNA editing sites. By comparing several different protocols available for the Ion Proton sequencer and by utilizing detailed bioinformatics analysis tools, we were able to develop an optimized random primer based RNA-seq technique that is reliable at uncovering rare transcript isoforms and RNA editing features, as well as fusion reads from oncogenic chromosome rearrangements. The combination of optimized libraries and rapid Ion Proton sequencing provides a powerful platform for the transcriptome analysis of research and clinical samples.
引用
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页数:15
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