Direct sequencing of haplotypes from diploid individuals through a modified emulsion PCR-based single-molecule sequencing approach

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. ...