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Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing Haile, Simon; Corbett, Richard D.; MacLeod, Tina; Bilobram, Steve; Smailus, Duane; Tsao, Philip; Kirk, Heather; McDonald, Helen; Pandoh, Pawan; Bala, Miruna; et al.

Abstract

Background RNA-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. Methods Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. Results This 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. Conclusions These 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.

Item Metadata

Title
Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing
Creator
Haile, Simon; Corbett, Richard D.; MacLeod, Tina; Bilobram, Steve; Smailus, Duane; Tsao, Philip; Kirk, Heather; McDonald, Helen; Pandoh, Pawan; Bala, Miruna; Hirst, Martin; Miller, Diane; Moore, Richard A.; Mungall, Andrew J.; Schein, Jacquie; Coope, Robin J.; Ma, Yussanne; Zhao, Yongjun; Holt, Rob A.; Jones, Steven J. M.; Marra, Marco, 1966-
Publisher
BioMed Central
Date Issued
2017-07-05
Description
Background RNA-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. Methods Here, we report the outcome of a systematic effort to optimize and streamline steps in strand-specific RNA-seq library construction. Results This 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. Conclusions These 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.
Subject
Ampure XP magnetic beads; Next-generation sequencing; Library construction; Strand-specific; dUTP; Uracil DNA N-Glycosylase; Ligation; mRNA; RNA-seq; Illumina; Ligation; Reverse transcriptase
Genre
Article
Type
Text
Language
eng
Date Available
2018-05-18
Provider
Vancouver : University of British Columbia Library
Rights
Attribution 4.0 International (CC BY 4.0)
DOI
10.14288/1.0366953
URI
http://hdl.handle.net/2429/66007
Affiliation
Medicine, Faculty of; Science, Faculty of; Non UBC; Medical Genetics, Department of; Microbiology and Immunology, Department of
Citation
BMC Genomics. 2017 Jul 05;18(1):515
Publisher DOI
10.1186/s12864-017-3900-6
Peer Review Status
Reviewed
Scholarly Level
Faculty
Copyright Holder
The Author(s).
Rights URI
http://creativecommons.org/licenses/by/4.0/
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Attribution 4.0 International (CC BY 4.0)