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

BMC Genomics

Methodology Article

Richard A. Moore¹  Philip Tsao¹  Jacquie Schein¹  Duane Smailus¹  Rob A. Holt¹  Tina MacLeod¹  Pawan Pandoh¹  Steven J. Jones¹  Steve Bilobram¹  Heather Kirk¹  Helen McDonald¹  Yongjun Zhao¹  Robin J. Coope¹  Diane Miller¹  Miruna Bala¹  Richard D. Corbett¹  Yussanne Ma¹  Andrew J. Mungall¹  Simon Haile¹  Marco A. Marra²  Martin Hirst³ 

[1] Canada’s Michael Smith Genome Sciences Centre, BC Cancer Agency, 675 West 10th Avenue, V5Z 1L3, Vancouver, BC, Canada;Canada’s Michael Smith Genome Sciences Centre, BC Cancer Agency, 675 West 10th Avenue, V5Z 1L3, Vancouver, BC, Canada;Department of Medical Genetics, University of British Columbia, V6T 1Z3, Vancouver, BC, Canada;Canada’s Michael Smith Genome Sciences Centre, BC Cancer Agency, 675 West 10th Avenue, V5Z 1L3, Vancouver, BC, Canada;Department of Microbiology and Immunology, Michael Smith Laboratories Centre for High-Throughput Biology, University of British Columbia, V6T 1Z4, Vancouver, BC, Canada;

关键词: Ampure XP magnetic beads;    Next-generation sequencing;    Library construction;    Strand-specific;    dUTP;    Uracil DNA N-Glycosylase;    Ligation;    mRNA;    RNA-seq;    Illumina;    Ligation;    Reverse transcriptase;   

DOI  :  10.1186/s12864-017-3900-6

 received in 2016-10-25, accepted in 2017-06-22,  发布年份 2017

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© The Author(s). 2017