title: Haplotype counting by next-generation sequencing for ultrasensitive human DNA detection.
abstract: 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.
authors.affiliation: Department of Pathology, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Marija
authors.initials: M
authors.lastname: Debeljak
authors.affiliation: Department of Neuroscience, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Donald N
authors.initials: DN
authors.lastname: Freed
authors.affiliation: Department of Genetics, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Jane A
authors.initials: JA
authors.lastname: Welch
authors.affiliation: Department of Pathology, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Lisa
authors.initials: L
authors.lastname: Haley
authors.affiliation: Department of Pathology, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Katie
authors.initials: K
authors.lastname: Beierl
authors.affiliation: Department of Medicine, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Brian S
authors.initials: BS
authors.lastname: Iglehart
authors.affiliation: Department of Pathology, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Aparna
authors.initials: A
authors.lastname: Pallavajjala
authors.affiliation: Department of Pathology, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Gocke
authors.affiliation: Department of Medicine, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Mary S
authors.initials: MS
authors.lastname: Leffell
authors.affiliation: Department of Pathology, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Ming-Tseh
authors.initials: MT
authors.lastname: Lin
authors.affiliation: Department of Neuroscience, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Pevsner
authors.affiliation: Department of Oncology, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Wheelan
authors.affiliation: Department of Pathology, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland; Department of Oncology, Johns Hopkins University, Johns Hopkins Medical Institutions, Baltimore, Maryland. Electronic address: jeshlem@jhmi.edu.
authors.forename: James R
authors.initials: JR
authors.lastname: Eshleman
mesh_terms: D000483:Alleles; D001854:Bone Marrow Cells; D045744:Cell Line, Tumor; D019295:Computational Biology; D004247:DNA; D005820:Genetic Testing / Q000379:methods / Q000592:standards; D015894:Genome, Human; D015234:HLA-A Antigens / Q000235:genetics; D006239:Haplotypes; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods* / Q000592:standards; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D015234:HLA-A Antigens; D004247:DNA