Targeted sequencing for gene discovery and quantification using RNA CaptureSeq

Mercer, Tim R., Clark, Michael B., Crawford, Joanna, Brunck, Marion E., Gerhardt, Daniel J., Taft, Ryan J., Nielsen, Lars K., Dinger, Marcel E. and Mattick, John S. (2014) Targeted sequencing for gene discovery and quantification using RNA CaptureSeq. Nature Protocols, 9 5: 989-1009. doi:10.1038/nprot.2014.058


Author Mercer, Tim R.
Clark, Michael B.
Crawford, Joanna
Brunck, Marion E.
Gerhardt, Daniel J.
Taft, Ryan J.
Nielsen, Lars K.
Dinger, Marcel E.
Mattick, John S.
Title Targeted sequencing for gene discovery and quantification using RNA CaptureSeq
Journal name Nature Protocols   Check publisher's open access policy
ISSN 1754-2189
1750-2799
Publication date 2014-01-01
Year available 2014
Sub-type Article (original research)
DOI 10.1038/nprot.2014.058
Open Access Status DOI
Volume 9
Issue 5
Start page 989
End page 1009
Total pages 21
Place of publication London, United Kingdom
Publisher Nature Publishing Group
Language eng
Subject 1300 Biochemistry, Genetics and Molecular Biology
Abstract RNA sequencing (RNAseq) samples the majority of expressed genes infrequently, owing to the large size, complex splicing and wide dynamic range of eukaryotic transcriptomes. This results in sparse sequencing coverage that can hinder robust isoform assembly and quantification. RNA capture sequencing (CaptureSeq) addresses this challenge by using oligonucleotide probes to capture selected genes or regions of interest for targeted sequencing. Targeted RNAseq provides enhanced coverage for sensitive gene discovery, robust transcript assembly and accurate gene quantification. Here we describe a detailed protocol for all stages of RNA CaptureSeq, from initial probe design considerations and capture of targeted genes to final assembly and quantification of captured transcripts. Initial probe design and final analysis can take less than 1 d, whereas the central experimental capture stage requires ∼ 1/47 d.
Keyword Biochemical Research Methods
Biochemistry & Molecular Biology
BIOCHEMICAL RESEARCH METHODS
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID 631668
Institutional Status UQ

 
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