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Nucleic Acids Res. 2015 Jul 27;43(13):e86. doi: 10.1093/nar/gkv297. Epub 2015 Apr 14.

Microfluidic droplet enrichment for targeted sequencing.

Author information

1
Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158, USA Mission Bio, Inc., San Francisco, CA 94107, USA.
2
Department of Neurology and Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA.
3
Mission Bio, Inc., San Francisco, CA 94107, USA.
4
Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158, USA adam.abate@ucsf.edu.

Abstract

Targeted sequence enrichment enables better identification of genetic variation by providing increased sequencing coverage for genomic regions of interest. 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 microfluidic droplets and performs TaqMan PCR reactions to identify droplets containing a desired target sequence. The TaqMan positive droplets are subsequently 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 sequence enrichment across 250 kb of targeted genomic DNA from five unique genomic loci. Significantly, this enrichment enabled a more comprehensive identification of genetic polymorphisms within the targeted loci. MESA requires low amounts of input 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.

PMID:
25873629
PMCID:
PMC4513844
DOI:
10.1093/nar/gkv297
[Indexed for MEDLINE]
Free PMC Article

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