A rapid molecular approach for chromosomal phasing.
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).
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, United States of America.
John F
JF
Regan
Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America; Program in Medical and Population Genetics and Stanley Center for Psychiatric Research, Cambridge, Massachusetts, United States of America.
Nolan
N
Kamitaki
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, United States of America.
Tina
T
Legler
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, United States of America.
Samantha
S
Cooper
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, United States of America.
Niels
N
Klitgord
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, United States of America.
George
G
Karlin-Neumann
Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America.
Catherine
C
Wong
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, United States of America.
Shawn
S
Hodges
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, United States of America.
Ryan
R
Koehler
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, California, United States of America.
Svilen
S
Tzonev
Department of Genetics, Harvard Medical School, Boston, Massachusetts, United States of America; Program in Medical and Population Genetics and Stanley Center for Psychiatric Research, Cambridge, Massachusetts, United States of America.
Steven A
SA
McCarroll
D000465:Algorithms; D002460:Cell Line; D002875:Chromosomes / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D006801:Humans; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA; D013997:Time Factors