Blind study evaluation illustrates utility of the Ion PGM™ system for use in human identity DNA typing.

218 FORENSIC SCIENCE Croat Med J. 2015;56:218-29 doi: 10.3325/cmj.2015.56.218 Blind study evaluation illustrates utility of the Ion PGM™ system for use in human identity DNA typing Jennifer D. Churchill1, Joseph Chang2, Jianye Ge2, Narasimhan Rajagopalan2, Sharon C. Wootton2, Chien-Wei Chang2, Robert Lagacé2, Wenchi Liao2, Jonathan L. King1, Bruce Budowle1,3 Institute of Applied Genetics, Department of Molecular and Medical Genetics, University of North Texas Health Science Center, Fort Worth, TX, USA 1 Aim To perform a blind study to assess the capability of the Ion Personal Genome Machine® (PGM™) system to sequence forensically relevant genetic marker panels and to characterize unknown individuals for ancestry and possible relatedness. Methods Twelve genomic samples were provided by a third party for blinded genetic analysis. For these 12 samples, the mitochondrial genome and three PGM™ panels containing human identity single nucleotide polymorphisms (SNPs), ancestry informative SNPs, and short tandem repeats (STRs) were sequenced on the PGM™ system and analyzed. Human Identification, Thermo Fisher Scientific, South San Francisco, CA, USA 2 Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia 3 Results All four genetic systems were run and analyzed on the PGM™ system in a reasonably quick time frame. Completeness of genetic profiles, depth of coverage, strand balance, and allele balance were informative metrics that illustrated the quality and reliability of the data produced. SNP genotypes allowed for identification of sex, paternal lineage, and population ancestry. STR genotypes were shown to be in complete concordance with genotypes generated by standard capillary electrophoresis-based technologies. Variants in the mitochondrial genome data provided information on population background and maternal relationships. Conclusion All results from analysis of the 12 genomic samples were consistent with sample information provided by the sample providers at the end of the blinded study. The relatively easy identification of intra-STR allele SNPs offered the potential for increased discrimination power. The promising nature of these results warrants full validation studies of this massively parallel sequencing technology and its further development for forensic data analysis. Received: March 2, 2015 Accepted: May 25, 2015 Correspondence to: Jennifer D. Churchill 3500 Camp Bowie Blvd, CBH-250 Fort Worth, TX 76107, USA www.cmj.hr 219 Churchill et al: Evaluation of the Ion PGM™ System for human identity DNA typing The advent of massively parallel sequencing (MPS) technologies offers an alternative to current DNA typing methods. Comprehensive coverage of multiple forensically relevant genetic markers made possible by MPS technologies can provide a wealth of data for use in criminal investigations (1-6). While short tandem repeats (STRs) have been the primary marker system for human identity typing due to their polymorphic nature and high discrimination power, MPS allows for the examination of repeat and sequence variants in these STRs and for the inclusion of single nucleotide polymorphisms (SNPs) and mitochondrial DNA into the analysis pipeline. Using MPS to analyze STRs allows for the exact sequence of each allele to be obtained and for SNPs to be identified within the STR repeat structure. These intra-STR SNPs offer greater resolving power when analyzing mixtures and performing kinship analyses (1,4,7). Alternative marker types can facilitate analysis of degraded or low template samples. SNPs and mitochondrial DNA can aid in the analysis of degraded and low quantity samples. SNPs reflect a single base change, thus short amplicons can be used in their analysis (2,8). Sequencing the entire mitochondrial genome allows a greater discrimination power to be obtained and more accurate haplogroup assignments to be generated (3,5). This capability allows for better population background predictions and identification of maternal lineage relationships. The multitude of SNPs provides information on identity, ancestry, and lineage, which can help produce investigative leads that were not previously possible (6,8-11). The Ion Torrent Personal Genome Machine® (PGM™) (Thermo Fisher Scientific, Waltham, MA USA) is one available MPS benchtop platform. The PGM™ is a high-throughput sequencer that employs semiconductor-sequencing technology (12). This sequence-by-synthesis chemistry measures the release of hydrogen ions as nucleotides are incorporated into the growing DNA strand. The PGM™ measures the associated, real-time pH change of the surrounding solution on a semiconductor chip thereby allowing for direct translation of chemically-encoded information into digital information (12). This process uses customized chemistries in a laboratory workflow that enables high-throughput and fast run times at a reasonable cost. In fact, the PGM™’s read length, sequencing time, running costs, and scalability lend itself to effective incorporation into diagnostic workflow of the forensic laboratory. to evaluate the potential of MPS on the PGM™ system to reliably analyze unknown samples, based on the self-declared information of the donors of these samples. The mitochondrial genome, identity SNPs, ancestry informative SNPs, and STRs (the latter three enabled by using PGM™ panels) were sequenced on the PGM™ system and resultant data were analyzed for these 12 samples. Materials and methods Samples The study was conducted in June of 2014. Twelve blinded genomic DNA samples were kindly provided by George Duncan, Ron Fourney, and Bruce McCord for this study. The samples were obtained under informed consent from volunteers at the Broward Sheriff’s Office. Additionally, the policies and procedures approved by the Institutional Review Board for the University of North Texas Health Science Center in Fort Worth, TX were followed. These singlesource samples arrived with arbitrarily assigned Sample Identification Numbers that were used to delineate the samples throughout the study. Extraction and quantification were completed by the sample providers using a Qiagen EZ1Advanced robot with a Qiagen DNA Investigator kit (Qiagen, Valencia, CA, USA) and a Promega Plexor HY real-time kit (Promega, Madison, WI, USA) on a 7500 Real Time instrument (Thermo Fisher Scientific), respectively. The concentrations of these samples ranged from 6.1 nanograms (ng)/microliter (μL) to 40 ng/μL, and a total volume of 7 μL per sample was provided. Capillary electrophoresis concordance data Conventional STR typing was performed on the 12 genomic samples using the AmpFlSTR® Identifiler® Plus PCR Amplification Kit (Thermo Fisher Scientific) and the AmpFlSTR® Yfiler® PCR Amplification Kit (Thermo Fisher Scientific) and one nanogram of DNA for each reaction per the recommended manufacturer’s protocols (13,14). The GeneAmp® PCR System 9700 thermal cycler (Thermo Fisher S (...truncated)