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Targeted sequencing for gene discovery and quantification using RNA CaptureSeq

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 approximately 7 d.

Type Journal
ISBN 1750-2799 (Electronic) 1750-2799 (Linking)
Authors Mercer, T. R.; Clark, M. B.; Crawford, J.; Brunck, M. E.; Gerhardt, D. J.; Taft, R. J.; Nielsen, L. K.; Dinger, M. E.; Mattick, J. S.;
Garvan Authors Prof John Mattick , A/Prof Marcel Dinger , Dr Michael Clark , Dr Timothy Mercer
Publisher Name NAT PROTOC
Published Date 2014-01-01 00:00:00
Published Volume 9
Published Issue 5
Published Pages 989-1009
URL http://www.ncbi.nlm.nih.gov/pubmed/24705597
Status Published In-print
OpenAccess Link https://publications.gimr.garvan.org.au/download.php?12394_12762/nprot.2014.058.pdf