Abstract 4270: NGS based microhaplotype counting for ultrasensitive human DNA detection

Introduction: Fields of forensics, paternity, and hematopoietic stem cell transplantation (HSCT) require human identity testing. The polymorphic nature of short tandem repeats (STRs) makes them most frequently used but they are relatively insensitive. In 2000, we demonstrated proof-of-principle for the use of SNPs for bone marrow engraftment testing; however, due to the high error rate of NGS platforms, use of a single SNP for this purpose is not possible. Here, we hypothesized that using multiple SNPs on a short amplicon (microhaplotype) could overcome the inherently high error rate of NGS, and use it for ultrasensitive detection of human DNA mixes. Methods: As proof-of-principle, we identified a 119 bp region on HLA-A Exon 3 that contains a cluster of 18 SNPs. Using cell lines with known HLA-A haplotypes, we made serial dilutions from 1% to 0.001%. By sequencing each dilution at least twice we were able to determine accuracy and limit of detection. Samples collected from patients after BMT that tested as all donor were analyzed using this approach. Sample preparation and sequencing was done per manufacturer9s protocol using the Ion Torrent Personal Genome Machine and 200 base chemistry. Additionally, we analyzed the 1000 Genomes database, using 300 bp windows, and identified many other loci that contained clusters of at least 9 SNPs. Results: Alignment and analysis of common European HLA-A haplotypes of this region show mean number of differences of ∼6 SNPs. Homozygous samples with known HLA-A genotypes that varied by multiple SNPs were analyzed and demonstrated no cross-talk between them. Analyzing serially diluted samples we were able to easily achieve a limit of detection of 1 in 10,000 molecules (0.01%). In BMT samples that tested as all donor by STR analysis, we were able to detect patient DNA of up to 1.5%. From 1000 Genomes data, we identified 4,349 loci that contained at least 9 SNPs within a 300 bp window. Informativity of identified loci was determined for 3 major ethnic groups using 1000 Genomes phased haplotype data. Conclusions: Ultrasensitive detection of human DNA mixes can be achieved using multiple SNPs within a short amplicon despite the high error rates associated with NGS. This assay may be useful to detect early relapse following bone marrow transplantation and other clinical applications. Citation Format: Marija Debeljak, Donald N. Freed, Jane A. Welch, Lisa Haley, Katie Beierl, Brian S. Iglehart, Aparna Pallavajjala, Christopher D. Gocke, Mary S. Leffell, Ming-Tseh Lin, Laura D. Wood, Jonathan Pevsner, Sarah J. Wheelan, James R. Eshleman. NGS based microhaplotype counting for ultrasensitive human DNA detection. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4270. doi:10.1158/1538-7445.AM2015-4270