Microfluidic droplet enrichment for targeted sequencing

Targeted sequence enrichment enables better iden-tification of genetic variation by providing increased sequencing coverage for genomic regions of inter-est. Here, we report the development of a new target enrichment technology that is highly differentiated from other approaches currently in use. Our method, MESA (Microfluidic droplet Enrichment for Sequence Analysis), isolates genomic DNA fragments in mi-crofluidic droplets and performs TaqMan PCR reac-tions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subse-quently recovered via dielectrophoretic sorting, and the TaqMan amplicons are removed enzymatically prior to sequencing. We demonstrated the utility of this approach by generating an average 31.6-fold se-quence enrichment across 250 kb of targeted ge-nomic DNA from five unique genomic loci. Signifi-cantly, this enrichment enabled a more comprehen-sive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of in-put DNA, minimal prior locus sequence information and enriches the target region without PCR bias or artifacts. These features make it well suited for the study of genetic variation in a number of research and diagnostic applications