Rapture
Restriction-Site Associated DNA Capture
Rapture is a massively parallel, targeted DNA sequencing technique that combines RAD-seq and sequence capture to compare multiple genes of interest among large numbers of samples (Ali et al., 2016). The targeted sequencing is based on identifying restriction enzyme sites specifically near the loci of interest.
DNA samples are pooled into individual wells in plates and digested with selected restriction enzymes. Biotinylated RAD adapters with well-specific barcodes are ligated to the sticky ends before pooling all the wells from each plate. The barcoded DNA fragments are randomly sheared and bound to streptavidin beads. Again, using restriction enzymes, fragments are cleaved from the streptavidin beads and used in standard DNA library preparation kits with plate-specific barcode labels. Next, libraries from both plates are pooled and hybridized with biotinylated bait-oligos specific to each RAD tag, before a final streptavidin pull-down. The isolated DNA fragments are sequenced and arranged according to their RAD tags, plate barcodes, and well barcodes.
Advantages:
- Massively parallel, targeted DNA sequencing for SNP identification
- Improved number of mapped fragments and locus coverage compared to RAD-seq
Disadvantages:
- RAD tags needs to be designated prior to the experiment
- Additional cost to synthesize baits
- Only a small number of loci are interrogated
- Biased toward sequences closer to the restriction cut site
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
Andrews K. R., Good J. M., Miller M. R., Luikart G. and Hohenlohe P. A. Harnessing the power of RADseq for ecological and evolutionary genomics. Nat Rev Genet. 2016;
References:
Ali O. A., O’Rourke S. M., Amish S. J., et al. RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping. Genetics. 2016;202:389-400
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History: Rapture
Revision by sbrumpton on 2017-06-21 07:50:21 - Show/Hide
Restriction-Site Associated DNA Capture
Rapture is a massively parallel, targeted DNA sequencing technique that combines RAD-seq and sequence capture to compare multiple genes of interest among large numbers of samples (Ali et al., 2016). The targeted sequencing is based on identifying restriction enzyme sites specifically near the loci of interest.
DNA samples are pooled into individual wells in plates and digested with selected restriction enzymes. Biotinylated RAD adapters with well-specific barcodes are ligated to the sticky ends before pooling all the wells from each plate. The barcoded DNA fragments are randomly sheared and bound to streptavidin beads. Again, using restriction enzymes, fragments are cleaved from the streptavidin beads and used in standard DNA library preparation kits with plate-specific barcode labels. Next, libraries from both plates are pooled and hybridized with biotinylated bait-oligos specific to each RAD tag, before a final streptavidin pull-down. The isolated DNA fragments are sequenced and arranged according to their RAD tags, plate barcodes, and well barcodes.
Advantages:- Massively parallel, targeted DNA sequencing for SNP identification
- Improved number of mapped fragments and locus coverage compared to RAD-seq
Disadvantages:- RAD tags needs to be designated prior to the experiment
- Additional cost to synthesize baits
- Only a small number of loci are interrogated
- Biased toward sequences closer to the restriction cut site
Reagents:Illumina Library prep and Array Kit SelectorReviews:Andrews K. R., Good J. M., Miller M. R., Luikart G. and Hohenlohe P. A. Harnessing the power of RADseq for ecological and evolutionary genomics. Nat Rev Genet. 2016;References:Ali O. A., O'Rourke S. M., Amish S. J., et al. RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping. Genetics. 2016;202:389-400