Targeted sequencing for gene discovery and quantification using RNA CaptureSeq.
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 ∼7 d.
1] Garvan Institute of Medical Research, Sydney, New South Wales, Australia. [2].
Tim R
TR
Mercer
1] Garvan Institute of Medical Research, Sydney, New South Wales, Australia. [2] Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia. [3].
Michael B
MB
Clark
1] Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia. [2].
Joanna
J
Crawford
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia.
Marion E
ME
Brunck
Mater Research Institute-The University of Queensland, Translational Research Institute, Woolloongabba, Queensland, Australia.
Daniel J
DJ
Gerhardt
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
Ryan J
RJ
Taft
Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland, Australia.
Lars K
0000000181913511
LK
Nielsen
Garvan Institute of Medical Research, Sydney, New South Wales, Australia.
Marcel E
000000034423934X
ME
Dinger
Garvan Institute of Medical Research, Sydney, New South Wales, Australia.
John S
0000000276807527
JS
Mattick
D005091:Exons / Q000235:genetics; D056726:Genetic Association Studies / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D015345:Oligonucleotide Probes / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*; D012984:Software
D015345:Oligonucleotide Probes