CaptureSeq
RNA Capture Sequencing
CaptureSeq is a targeted RNA sequencing method that is able to provide higher sequencing coverage for selected regions of the genome(Mercer et al., 2014).This method follows the TruSeq RNA sample preparation protocol, in which mRNA is first isolated from total RNA by poly(A) selection and then fragmented. Double-stranded cDNA copies of the fragments are generated using reverse transcriptase and then ligated to p5 and p7 adapters. Next, these cDNA library fragments are amplified by the polymerase chain reaction (PCR). To increase specificity, custom capture probes are hybridized to the cDNA and bound to an array while other transcripts are washed away prior to PCR amplification. This process leaves the targeted fragments that are ready for sequencing.
Advantages:
- Highly suitable for discovering novel exons, genes, and splice isoforms
Disadvantages:
- Requires large amount of total RNA (5 µg) to yield 250 ng of amplified cDNA for capture
- Coverage accuracy drops for transcripts with repetitive sequences
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Marbaniang C. N. and Vogel J. Emerging roles of RNA modifications in bacteria. Curr Opin Microbiol. 2016;30:50-57
Gloss B. S. and Dinger M. E. The specificity of long noncoding RNA expression. Biochim Biophys Acta. 2015;
Luciano D. J. and Belasco J. G. NAD in RNA: unconventional headgear. Trends Biochem Sci. 2015;40:245-247
References:
Bussotti G., Leonardi T., Clark M. B., et al. Improved definition of the mouse transcriptome via targeted RNA sequencing. Genome Res. 2016;26:705-716
Clark M. B., Mercer T. R., Bussotti G., et al. Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing. Nat Methods. 2015;
Mercer T. R., Gerhardt D. J., Dinger M. E., et al. Targeted RNA sequencing reveals the deep complexity of the human transcriptome. Nat Biotechnol. 2012;30:99-104
Related
History: CaptureSeq
Revision by sbrumpton on 2017-06-21 09:06:28 - Show/Hide
RNA Capture Sequencing
CaptureSeq is a targeted RNA sequencing method that is able to provide higher sequencing coverage for selected regions of the genome(Mercer et al., 2014).This method follows the TruSeq RNA sample preparation protocol, in which mRNA is first isolated from total RNA by poly(A) selection and then fragmented. Double-stranded cDNA copies of the fragments are generated using reverse transcriptase and then ligated to p5 and p7 adapters. Next, these cDNA library fragments are amplified by the polymerase chain reaction (PCR). To increase specificity, custom capture probes are hybridized to the cDNA and bound to an array while other transcripts are washed away prior to PCR amplification. This process leaves the targeted fragments that are ready for sequencing.
Advantages:- Highly suitable for discovering novel exons, genes, and splice isoforms
Disadvantages:- Requires large amount of total RNA (5 µg) to yield 250 ng of amplified cDNA for capture
- Coverage accuracy drops for transcripts with repetitive sequences
Reagents:Illumina Library prep and Array Kit SelectorReviews:Marbaniang C. N. and Vogel J. Emerging roles of RNA modifications in bacteria. Curr Opin Microbiol. 2016;30:50-57Gloss B. S. and Dinger M. E. The specificity of long noncoding RNA expression. Biochim Biophys Acta. 2015;Luciano D. J. and Belasco J. G. NAD in RNA: unconventional headgear. Trends Biochem Sci. 2015;40:245-247References:Bussotti G., Leonardi T., Clark M. B., et al. Improved definition of the mouse transcriptome via targeted RNA sequencing. Genome Res. 2016;26:705-716Clark M. B., Mercer T. R., Bussotti G., et al. Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing. Nat Methods. 2015;Mercer T. R., Gerhardt D. J., Dinger M. E., et al. Targeted RNA sequencing reveals the deep complexity of the human transcriptome. Nat Biotechnol. 2012;30:99-104