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.CME Accreditation Statement: This activity (“JMD 2014 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2014 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.Myeloablative conditioning and allogeneic stem cell transplantation have historically been limited to the treatment of lethal hematological malignancies in children or young adults. More recently, with the advent of highly immunosuppressive, nonmyeloablative regimens, the clinical use of allogeneic stem cell transplantation has expanded to include older, less fit patients with hematological malignancies and patients with nonmalignant disorders, such as sickle-cell disease.^{1, 2, 3, 4} Nonmyeloablative conditioning regimens offer the additional safeguard of recovery of autologous hematopoiesis in the event of graft rejection and may be a safer option in patients at risk for immune-mediated rejection of the donor graft.Chimerism testing at set intervals is an effective method for detecting graft rejection or recurrence of the original hematopoietic neoplasm after allogeneic hematopoietic stem cell transplantation (HSCT) with either bone marrow (BM) or peripheral blood stem cells]. Decades ago, BM engraftment (BME) monitoring was performed using Southern blot analysis and minisatellite or variable number of tandem repeats loci.⁵ Today, short tandem repeat (STR) or microsatellite loci are most commonly used for this purpose.^{6, 7, 8} STRs are composed of 10 to 60 tandemly repeated units, in which each unit is 1 to 6 bases in length. They are widely distributed throughout the human genome and highly variable between individuals; therefore, they allow for excellent differentiation between individuals, including patient and donor, even if they are closely related. Most laboratories use multiplex PCR-based kits, originally developed for forensics analysis using Combined DNA Index System loci.^{7, 9, 10} STR analysis most commonly involves PCR amplification using fluorescently labeled primers, followed by amplicon separation by capillary electrophoresis.Other polymorphic DNAs that could be used to monitor BME include single-nucleotide polymorphisms (SNPs).^{11, 12, 13} SNPs are theoretically superior to STR-based analyses because analysis of STR loci by capillary electrophoresis is relatively insensitive limit of detection (LD), 1% to 5%] and microsatellite alleles of varying length amplify with different efficiencies, thus making them inherently biased. STR amplification can also be difficult in the setting of highly degraded DNA. However, SNPs are less attractive as targets because of their inherently lower informativity (eg, only two possible bases for a bi-allelic SNP versus ≥10 alleles for some microsatellites), requiring many more SNPs to be tested to identify those that distinguish donor from recipient. For example, we previously estimated that one would need to screen >20 to 30 individual SNPs to confidently identify one SNP where the donor is homozygous for one allele and an unrelated recipient is homozygous for the other allele.¹¹ Fewer would need to be included if heterozygotes were included, but more would have to be analyzed for related individuals.Recently emerging next-generation sequencing (NGS) technologies, along with their decreasing costs, are now feasible for clinical testing. However, all NGS technologies currently have high error rates, in the range of 0.04% to 1% at each base,¹⁴ which precludes their use for ultrasensitive detection of one SNP. One solution to this problem is sequencing blocks of closely spaced SNPs (ie, haplotypes). Haplotypes are regions of the genome, where polymorphic areas are sufficiently close that they are inherited together, including either genes (eg, HLA-A and HLA-B) within a locus or multiple SNPs within a region of DNA.Herein, we first used the HLA-A locus as proof-of-principle to demonstrate that this approach permits high sensitivity, precision, and accuracy. We then studied BM samples from a cohort of patients who engrafted after HSCT and tested as all donors by STRs, and found that low-level patient DNA is commonly present. To identify additional loci that could be used for this purpose, we comprehensively analyzed the human genome and identified other regions with highly informative haplotypes. We discuss additional situations where routine haplotyping patient samples could improve patient safety.