To Analyze Phasing
Reflex™ is an innovative method, not limited by NGS read lengths, for efficient targeted sequencing and phase determination including haplotyping of long DNA regions, such as whole genes, in large numbers of genomic DNA samples. The Reflex™ assay typically begins with the generation of long-range PCR (LRPCR) products for the target sequence on a sample-by-sample basis. Due to the design of the workflow this initial step requires only minimal starting amounts of DNA, thus making it highly attractive for use with samples where supply is limited. During the LRPCR step, samples are tagged with proprietary Reflex™ adaptors as well as unique DNA sequence tags (DNA bar-codes) to allow individual fragments of DNA to be identified throughout all subsequent steps of the process.
At this point a proprietary Reflex™ reaction is performed. This reaction produces a library of sequence-ready derivative PCR products from the original LRPCR products. Each retains the original DNA sample barcode from its starting LRPCR product, as well as a fragment linking tag that is used to re-associate fragments into the linear sequence (phase) of the original DNA molecule. The tagged, derivative PCR products comprise compositions that effectively tile across the target region with a significant degree of overlap to ensure high physical coverage and can be tailored to most NGS platforms.
“Reflex: intramolecular barcoding of long-range PCR products for sequencing multiple pooled DNAs.” Casbon et al., (2013). Nucleic Acids Res 41(10):e112
Examples of Issued US Intellectual Property
- Compositions and methods for intramolecular nucleic acid rearrangement” US issued patent 8,298,767 (Reflex method)
- “Compositions and methods for intramolecular nucleic acid rearrangement” US issued patent 8,563,274 (compositions created by the Reflex method)
- “Compositions and methods for intramolecular nucleic acid rearrangement” US issued patent 8,679,756 (methods for obtaining compositions for long reads and haplotyping).