The XdropTM technology enables accurate single-molecule processing for targeted enrichment of long DNA fragments suitable for short read or long read sequencing, high resolution digital droplet PCR, and accurate amplifications within droplets to minimize amplification errors.
The Xdrop enrichment procedure is based on the physical separation of long DNA fragments into millions of small droplets. The droplets containing the target DNA are identified using digital droplet PCR and physically sorted using a cell sorter. The target fragments are then amplified using multiple displacement amplification. Applications include: de-novo sequencing, highly variable and partially unknown sequences, large structural variations, phasing of mutations, repeat regions, GC-rich regions, and samples in limited amounts.
Digital droplet PCR is an ultra-sensitive DNA quantification method based on droplet technology. The Xdrop droplet generator encapsulates DNA/cDNA fragments and reagents in microdroplets bounded by an oil shell that is further surrounded by an aqueous buffer. This makes it possible to use standard high throughput flow cytometers for counting droplets. In digital droplet PCR, the presence of a target DNA fragment is traced by detecting droplets in which a PCR reaction has taken place, for example droplets containing a target fragment. This ensures the target molecules can be quantified with high precision. The large number of droplets produced in the Xdrop makes it possible to accurately quantify specific molecules and rare genetic events at a wide range of target concentrations, from a few molecules to hundreds of thousands of molecules.
Minimized bias and chimeric sequences in PCR and MDA using droplet technology DNA molecules naturally tend to interact during amplification and stochastic events lead to amplification bias and errors. Single DNA molecule processing eliminates the risk of interaction between different DNA molecules. The Xdrop droplet generator encapsulates DNA/cDNA fragments into droplets. This avoids chimera formation and bias during amplification. Multiple Displacement Amplification (MDA) allows sequencing of samples in very small amounts, but is prone to significant bias and often produces chimeric fragments containing combinations of similar fragments. Droplet encapsulation prevents intermolecular recombination thereby simplifying the interpretation of results.
Droplet encapsulation also allows for unbiased PCR amplification of mixed samples, such as libraries for next generation sequencing. Each fragment is end-point amplified within a droplet and individual templates for PCR or MDA are not allowed to dominate the reaction.