CaptureSeq

CaptureSeq

CaptureSeq is a targeted RNA sequencing method that is able to provide higher sequencing coverage than standard RNA sequencing for novel genes and transcripts discovery using custom capture probes. This method follows the TruSeq RNA sample preparation protocol where 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 ligated with p5 and p7 adapters. These cDNA library fragments are then amplified by 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 leaves the targeted fragments that are ready to be sequenced.

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

1. Mercer T. R., Clark M. B., Crawford J., Brunck M. E., Gerhardt D. J., et al. (2014) Targeted sequencing for gene discovery and quantification using RNA CaptureSeq. Nat Protoc 9: 989-1009