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

Roger B Brown,Scott A Ness,Hideaki Suzuki,Nathaniel J Madrid,Peng Xu

doi:10.1371/journal.pone.0176675

Roger B Brown, Scott A Ness + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0176675

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Journal: PloS one	Publication Date: May 1, 2017
Citations: 17	License type: CC BY 4.0

Affiliation: University of New Mexico, New Mexico Cancer Center

Abstract

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.

Highlights

High-throughput next-generation sequencing has become a standard method to study the complexities of the genome and transcriptome
RNA-sequencing has the ability to provide multiple types of information, including gene expression levels, alternative RNA splicing, sequence variants such as inherited single nucleotide polymorphisms (SNPs), and post-transcriptional variants introduced by RNA editing as well as gene fusions resulting from chromosomal translocations
We explored whether the Ion Proton/S5 platform could be utilized for rapid, small-scale RNA-seq assays that would still provide the advanced features of transcriptome analysis, such as identification of isoforms formed through alternative RNA splicing or variants introduced through RNA editing

Summary

Introduction

High-throughput next-generation sequencing has become a standard method to study the complexities of the genome and transcriptome. RNA-sequencing has the ability to provide multiple types of information, including gene expression levels, alternative RNA splicing, sequence variants such as inherited single nucleotide polymorphisms (SNPs), and post-transcriptional variants introduced by RNA editing as well as gene fusions resulting from chromosomal translocations. Recent studies have demonstrated the importance of changes in RNA editing [1] and alternative RNA splicing in cancer [2, 3], focusing interest on the detection of these events in clinical samples. Ion Proton transcriptome sequencing reveals RNA splicing and editing features. RNA-Sequencing can produce results that fail to adequately detect all the complexities of the transcriptome. Methods that rely on oligo-dT primed cDNA synthesis may produce 3’-end bias, and random priming methods may introduce sequence specificities [4]

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Results

Conclusion