A rapid molecular approach for chromosomal phasing

PLoS One. 2015 Mar 4;10(3):e0118270. doi: 10.1371/journal.pone.0118270. eCollection 2015.

Abstract

Determining the chromosomal phase of pairs of sequence variants - the arrangement of specific alleles as haplotypes - is a routine challenge in molecular genetics. Here we describe Drop-Phase, a molecular method for quickly ascertaining the phase of pairs of DNA sequence variants (separated by 1-200 kb) without cloning or manual single-molecule dilution. In each Drop-Phase reaction, genomic DNA segments are isolated in tens of thousands of nanoliter-sized droplets together with allele-specific fluorescence probes, in a single reaction well. Physically linked alleles partition into the same droplets, revealing their chromosomal phase in the co-distribution of fluorophores across droplets. We demonstrated the accuracy of this method by phasing members of trios (revealing 100% concordance with inheritance information), and demonstrate a common clinical application by phasing CFTR alleles at genomic distances of 11-116 kb in the genomes of cystic fibrosis patients. Drop-Phase is rapid (requiring less than 4 hours), scalable (to hundreds of samples), and effective at long genomic distances (200 kb).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms*
  • Cell Line
  • Chromosomes / genetics*
  • Genomics / methods*
  • Humans
  • Polymorphism, Single Nucleotide
  • Sequence Analysis, DNA
  • Time Factors

Grant support

This work was supported by Broad Institute (http://www.broadinstitute.org/). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.