title: Direct sequencing of haplotypes from diploid individuals through a modified emulsion PCR-based single-molecule sequencing approach.
abstract: While standard DNA-sequencing approaches readily yield genotypic sequence data, haplotype information is often of greater utility for population genetic analyses. However, obtaining individual haplotype sequences can be costly and time-consuming and sometimes requires statistical reconstruction approaches that are subject to bias and error. Advancements have recently been made in determining individual chromosomal sequences in large-scale genomic studies, yet few options exist for obtaining this information from large numbers of highly polymorphic individuals in a cost-effective manner. As a solution, we developed a simple PCR-based method for obtaining sequence information from individual DNA strands using standard laboratory equipment. The method employs a water-in-oil emulsion to separate the PCR mixture into thousands of individual microreactors. PCR within these small vesicles results in amplification from only a single starting DNA template molecule and thus a single haplotype. We improved upon previous approaches by including SYBR Green I and a melted agarose solution in the PCR, allowing easy identification and separation of individually amplified DNA molecules. We demonstrate the use of this method on a highly polymorphic estuarine population of the copepod Eurytemora affinis for which current molecular and computational methods for haplotype determination have been inadequate.
authors.affiliation: Center of Rapid Evolution, University of Wisconsin, Madison, WI 53706, USA. bpmetz@umich.edu
authors.forename: Brian Patrick Hansen
authors.initials: BP
authors.lastname: Metzger
authors.forename: Gregory William
authors.initials: GW
authors.lastname: Gelembiuk
authors.forename: Carol Eunmi
authors.initials: CE
authors.lastname: Lee
mesh_terms: D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D033342:Copepoda / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D006239:Haplotypes / Q000235:genetics*; D008969:Molecular Sequence Data; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I