Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing

Simon Haile,Richard A Moore,Diane Miller,Yongjun Zhao,Pawan Pandoh,Miruna Bala,Jacquie Schein,Heather Kirk,Steve Bilobram,Helen Mcdonald,Andrew J Mungall,Yussanne Ma,Martin Hirst,Steven J.m Jones ,Duane E Smailus ,Robin Coope ,Philip S Tsao ,Robert A Holt ,Richard Corbett ,Tina Macleod,Marco A Marra

doi:10.1186/s12864-017-3900-6

Abstract

BackgroundRNA-Sequencing (RNA-seq) is now commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. However, standard approaches for library construction typically require relatively high amounts of input RNA, are labor intensive, and are time consuming.MethodsHere, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction.ResultsThis work has resulted in the identification of an optimized messenger RNA isolation protocol, a potent reverse transcriptase for cDNA synthesis, and an efficient chemistry and a simplified formulation of library construction reagents. We also present an optimization of bead-based purification and size selection designed to maximize the recovery of cDNA fragments.ConclusionsThese developments have allowed us to assemble a rapid high throughput pipeline that produces high quality data from amounts of total RNA as low as 25 ng. While the focus of this study is on RNA-seq sample preparation, some of these developments are also relevant to other next-generation sequencing library types.

Highlights

RNA-Sequencing (RNA-seq) is commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts and landscapes of expressed mutations
Haile et al BMC Genomics (2017) 18:515 the double-stranded cDNA fragments are ligated to adapters, the dUTP-marked strand is selectively destroyed by the enzyme Uracil DNA N-Glycosylase (UNG)
Universal Human Reference (UHR) was spiked with External RNA Controls Consortium (ERCC) spike-in mix from Ambion (Catalog# 4456740). 0.02 μL of the spike-in mix was used per 1 μg UHR total RNA

Summary

Introduction

RNA-Sequencing (RNA-seq) is commonly used to reveal quantitative spatiotemporal snapshots of the transcriptome, the structures of transcripts (splice variants and fusions) and landscapes of expressed mutations. One of the key factors in the generation of high quality NGS data is the process of library construction. The main purpose of the multi-step library construction process is to provide priming sites and sample-specific. Library construction involves a series of enzymatic reactions, each typically followed by a purification step. A typical library sample preparation for RNA-seq, for example, involves 6–8 such purification steps. These purification steps involved laborious processes such as phenol-chloroform or column-based purifications. The replacement of these purifications with paramagnetic bead-based solid phase reversible immobilization (SPRI) is a significant advance rendering library construction simpler and more amenable to high throughput or robotic handling [7, 8]. The most widely applied and commercialized form of SPRI magnetic beads are Ampure XP beads

Methods

Results

Conclusion