CaptureSeq

CaptureSeq

CaptureSeq is a targeted RNA sequencing method that is able to provide higher sequencing coverage for selected regions of the genome. 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.

Pros:

• Highly suitable for discovering novel exons, genes, and splice isoforms

Cons:

• 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

• CaptureSeq: Mercer T. R., Gerhardt D. J., Dinger M. E., Crawford J., Trapnell C., et al. (2012) Targeted RNA sequencing reveals the deep complexity of the human transcriptome. Nat Biotechnol 30: 99-104

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3. Luciano D. J. and Belasco J. G. NAD in RNA: unconventional headgear. Trends Biochem Sci. 2015;40:245-247

1. 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
2. Clark M. B., Mercer T. R., Bussotti G., et al. Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing. Nat Methods. 2015;
3. 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