A Rapid Molecular Approach for Chromosomal Phasing

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).

Determining the impact of uncharacterized inversions in the human genome by droplet digital PCR

“…Recently, it was shown that this technique could also be used to phase variants separated by up to 200 Kb (Regan et al 2015), which has already been applied to fusion transcript detection (Hoff et al 2016) or to phase deletions into haplotypes (Boettger et al 2016). Here, we have developed new ddPCR assays to genotype quickly and reliably human polymorphic inversions flanked by large IRs, and thanks to the genotype data we demonstrate that most of these inversions are recurrent and that inversion alleles are associated to gene expression changes.…”

“…AFR, Africans; EAS, East-Asians; EUR, Europeans. ddPCR technology allows us to quantify how close two independent sequences are within a DNA molecule based on their simultaneous amplification in a higher number of droplet partitions than expected by chance (Regan et al 2015). Thus, for each inversion we designed three amplicons in the unique sequence outside the IRs (A or D) and at both ends of the inverted segment (B and C) ( Fig.…”

“…1B). The linkage between the two targeted amplicons (percentage of DNA molecules containing both amplicons) was obtained as the excess of double-positive droplets over what is statistically expected (Regan et al 2015). From these values we could calculate the total linkage and the O1 and O2 linkage ratios that allow us to genotype inversions.…”

Fundamentals of multiplexing with digital PCR

“…In the case of two different targets in cis and nearby, there will be an excess of double positive partitions. We can measure the concentration of each target using standard approaches but may miss the fact the targets are indeed linked [48]. Current standard analysis tools can report the concentration of the linked molecules using linkage-based assays to measure cis versus trans configuration of targets and potential structural rearrangements [49].…”

Biallelic COLGALT1 variants are associated with cerebral small vessel disease

“…Reactions contained 20 ng of genomic DNA from Patient 2, a 5′ fluorescein (FAM)‐labeled locked nucleic acid (LNA) probe targeting the c.460G > C variant, a hexachlorofluorescein (HEX)‐labeled LNA probe targeting the c.1129G > C variant, 2 primer sets to amplify the respective target regions, and ddPCR Supermix for Probes (no dUTP; Bio‐Rad Laboratories). We phased 2 variants as previously described …”