title: Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™.
abstract: Next generation sequencing (NGS) offers the opportunity to analyse forensic DNA samples and obtain massively parallel coverage of targeted short sequences with the variants they carry. We evaluated the levels of sequence coverage, genotyping precision, sensitivity and mixed DNA patterns of a prototype version of the first commercial forensic NGS kit: the HID-Ion AmpliSeq™ Identity Panel with 169-markers designed for the Ion PGM™ system. Evaluations were made between three laboratories following closely matched Ion PGM™ protocols and a simple validation framework of shared DNA controls. The sequence coverage obtained was extensive for the bulk of SNPs targeted by the HID-Ion AmpliSeq™ Identity Panel. Sensitivity studies showed 90-95% of SNP genotypes could be obtained from 25 to 100pg of input DNA. Genotyping concordance tests included Coriell cell-line control DNA analyses checked against whole-genome sequencing data from 1000 Genomes and Complete Genomics, indicating a very high concordance rate of 99.8%. Discordant genotypes detected in rs1979255, rs1004357, rs938283, rs2032597 and rs2399332 indicate these loci should be excluded from the panel. Therefore, the HID-Ion AmpliSeq™ Identity Panel and Ion PGM™ system provide a sensitive and accurate forensic SNP genotyping assay. However, low-level DNA produced much more varied sequence coverage and in forensic use the Ion PGM™ system will require careful calibration of the total samples loaded per chip to preserve the genotyping reliability seen in routine forensic DNA. Furthermore, assessments of mixed DNA indicate the user's control of sequence analysis parameter settings is necessary to ensure mixtures are detected robustly. Given the sensitivity of Ion PGM™, this aspect of forensic genotyping requires further optimisation before massively parallel sequencing is applied to routine casework.
authors.affiliation: Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: M
authors.initials: M
authors.lastname: Eduardoff
authors.affiliation: Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Santos
authors.affiliation: Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain.
authors.forename: M
authors.initials: M
authors.lastname: de la Puente
authors.affiliation: Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Cologne, Germany.
authors.forename: T E
authors.initials: TE
authors.lastname: Gross
authors.affiliation: Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Fondevila
authors.affiliation: Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: C
authors.initials: C
authors.lastname: Strobl
authors.affiliation: Grupo de Medicina Xenómica (GMX), Faculty of Medicine, University of Santiago de Compostela, Spain.
authors.forename: B
authors.initials: B
authors.lastname: Sobrino
authors.affiliation: Department of Forensic and Analytical Science, Faculty of Life Science, King's College, London, UK.
authors.forename: D
authors.initials: D
authors.lastname: Ballard
authors.affiliation: Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Cologne, Germany.
authors.forename: P M
authors.initials: PM
authors.lastname: Schneider
authors.affiliation: Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain; Grupo de Medicina Xenómica (GMX), Faculty of Medicine, University of Santiago de Compostela, Spain.
authors.forename: Á
authors.initials: Á
authors.lastname: Carracedo
authors.affiliation: Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain.
authors.forename: M V
authors.initials: MV
authors.lastname: Lareu
authors.affiliation: Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria; Forensic Science Program, The Pennsylvania State University, PA, USA.
authors.forename: W
authors.initials: W
authors.lastname: Parson
authors.affiliation: Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain. Electronic address: c.phillips@mac.com.
authors.forename: C
authors.initials: C
authors.lastname: Phillips
mesh_terms: D004247:DNA / Q000032:analysis / Q000235:genetics; D005260:Female; D053803:Forensic Genetics / Q000379:methods*; D060005:Genotyping Techniques / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
chemical_list: D004247:DNA
keywords: Identification SNPs; Ion PGM™; Ion Torrent; Massively parallel sequencing; Next generation sequencing