A Rapid Molecular Approach for Chromosomal Phasing |
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| Authors: | John F Regan Nolan Kamitaki Tina Legler Samantha Cooper Niels Klitgord George Karlin-Neumann Catherine Wong Shawn Hodges Ryan Koehler Svilen Tzonev Steven A McCarroll |
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| Institution: | 1. Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, United States of America.; 2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.; 3. Program in Medical and Population Genetics and Stanley Center for Psychiatric Research, Cambridge, Massachusetts, United States of America.; Johns Hopkins University, UNITED STATES, |
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| 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). |
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