Haplotype counting by next-generation sequencing for ultrasensitive human DNA detection

Human identity testing is critical to the fields of forensics, paternity, and hematopoietic stem cell transplantation. Most bone marrow (BM) engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis. Single-nucleotide polymorphisms (SNPs) are theoretically a better choice among polymorphic DNA; however, ultrasensitive detection of SNPs using next-generation sequencing is currently not possible because of its inherently high error rate. We circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes. As proof-of-principle, we chose the HLA-A locus because it is highly polymorphic and is already genotyped to select proper donors for BM transplant recipients. We aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs, flanked by nonpolymorphic DNA for primer placement. Analysis of cell line mixtures shows that the assay is accurate and precise, and has a lower limit of detection of approximately 0.01%. The BM from a series of hematopoietic stem cell transplantation patients who tested as all donor by short tandem repeat analysis demonstrated 0% to 1.5% patient DNA. Comprehensive analysis of the human genome using the 1000 Genomes database identified many additional loci that could be used for this purpose. This assay may prove useful to identify hematopoietic stem cell transplantation patients destined to relapse, microchimerism associated with solid organ transplantation, forensic applications, and possibly patient identification.