Clinical Chemistry - Journal Club
The Clinical Chemistry Journal Club allows readers to discuss key articles by using focused slides as teaching tools. Each month Clinical Chemistry posts the original article and slides online, and they are then distributed to individuals and university Journal Clubs.
Original Article: https://doi.org/10.1093/clinchem/hvae076
Slides: Download ppt
The analysis of haplotypes of variants is important for pharmacogenomics analysis and noninvasive prenatal testing for monogenic diseases. However, there is a lack of robust methods for targeted haplotyping.
We developed digital PCR haplotype sequencing (dHapSeq) for targeted haplotyping of variants, which is a method that compartmentalizes long DNA molecules into droplets. Within one droplet, 2 target regions are PCR amplified from one template molecule, and their amplicons are fused together. The fused products are then sequenced to determine the phase relationship of the single nucleotide polymorphism (SNP) alleles. The entire haplotype of 10s of SNPs can be deduced after the phase relationship of individual SNPs are determined in a pairwise manner. We applied dHapSeq to noninvasive prenatal testing in 4 families at risk for thalassemia and utilized it to detect NUDT15 diplotypes for predicting drug tolerance in pediatric acute lymphoblastic leukemia (72 cases and 506 controls).
For SNPs within 40 kb, phase relation can be determined with 100% accuracy. In 7 trio families, the haplotyping results for 97 SNPs spanning 185 kb determined by dHapSeq were concordant with the results deduced from the genotypes of both parents and the fetus. In 4 thalassemia families, a 19.3-kb Southeast Asian deletion was successfully phased with 97 downstream SNPs, enabling noninvasive determination of fetal inheritance using relative haplotype dosage analysis. In the NUDT15 analysis, the variant status and phase of the variants were successfully determined in all cases and controls.
The dHapSeq represents a robust and scalable haplotyping approach with numerous clinical and research applications.