Comparative analysis of RNA sequencing methods for degraded or low-input samples

Xian Adiconis,Dawn Anne Thompson,Andrey Sivachenko,Michele A Busby,Joshua Z Levin,Diego Borges-Rivera,Alec Wysoker,Nathalie Pochet,David S Deluca,Aviv Regev,Timothy Fennell,Rahul Satija,Andreas Gnirke,Aaron M Berlin

doi:10.1038/nmeth.2483

Abstract

RNA-Seq is an effective method to study the transcriptome, but can be difficult to apply to scarce or degraded RNA from fixed clinical samples, rare cell populations, or cadavers. Recent studies have proposed several methods for RNA-Seq of low quality and/or low quantity samples, but their relative merits have not been systematically analyzed. Here, we compare five such methods using metrics relevant to transcriptome annotation, transcript discovery, and gene expression. Using a single human RNA sample, we constructed and sequenced ten libraries with these methods and two control libraries. We find that the RNase H method performed best for low quality RNA, and confirmed this with actual degraded samples. RNase H can even effectively replace oligo (dT) based methods for standard RNA-Seq. SMART and NuGEN had distinct strengths for low quantity RNA. Our analysis allows biologists to select the most suitable methods and provides a benchmark for future method development.

Highlights

RNA-Seq allows us to comprehensively characterize the transcripts present in a biological sample
Most standard protocols in eukaryotic cells rely on oligo to isolate polyadenylated RNA1, in order to deplete the highly abundant ribosomal RNA
For the RNase H method, we created a second library with no additional spike-in RNA (“NS” library; Online Methods) and for Duplex-Specific Nuclease (DSN)-lite, we created a second library with PCR before DSN treatment (Online Methods)

Summary

Introduction

RNA-Seq allows us to comprehensively characterize the transcripts present in a biological sample. While RNA-Seq can, in principle, be used to measure transcripts in any sample, it has been challenging to apply standard protocols to samples with either very low quantity or low quality (partially degraded) input RNA. Most standard protocols in eukaryotic cells rely on oligo (dT) to isolate polyadenylated (polyA) RNA1, in order to deplete the highly abundant ribosomal RNA (rRNA). This is a powerful technique, it excludes many non-polyadenylated transcripts other than rRNA2. For RNA that is not ACCESSION CODES.

Methods

Results

Conclusion