title: A SNaPshot of next generation sequencing for forensic SNP analysis.
abstract: Forensic phenotyping can provide useful intelligence regarding the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of an evidentiary sample. Currently, single nucleotide polymorphism (SNP) based inference of BGA and EVCs is performed most commonly using SNaPshot(®), a single base extension (SBE) assay. However, a single SNaPshot multiplex PCR is limited to 30-40 SNPs. Next generation sequencing (NGS) offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. The PCR multiplexes from five SNaPshot assays (SNPforID 52plex, SNPforID 34plex, Eurasiaplex, IrisPlex and an unpublished BGA assay) were applied to three different DNA template amounts (0.1, 0.2 and 0.3 ng) in three samples (9947A and 007 control DNAs and a male donor). The pooled PCR amplicons containing 136 unique SNPs were sequenced using Life Technologies' Ion Torrent™ PGM system. Approximately 72 Mb of sequence was generated from two 10 Mb Ion 314™ v1 chips. Accurate genotypes were readily obtained from all three template amounts. Of a total of 408 genotypes, 395 (97%) were fully concordant with SNaPshot across all three template amounts. Of those genotypes discordant with SNaPshot, six Ion Torrent sequences (1.5%) were fully concordant with Sanger sequencing across the three template amounts. Seven SNPs (1.7%) were either discordant between template amounts or discordant with Sanger sequencing. Sequence coverage observed in the negative control, and, allele coverage variation for heterozygous genotypes highlights the need to establish a threshold for background levels of sequence output and heterozygous balance. This preliminary study of the Ion Torrent PGM system has demonstrated considerable potential for use in forensic DNA analyses as a low to medium throughput NGS platform using established SNaPshot assays.
authors.affiliation: Office of the Chief Forensic Scientist, Forensic Services Department, Victoria Police, Australia. Electronic address: runa.daniel@police.vic.gov.au.
authors.forename: R
authors.initials: R
authors.lastname: Daniel
authors.affiliation: Forensic Genetics Unit, Institute of Forensic Science "Luis Concheiro", University of Santiago de Compostela, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Santos
authors.affiliation: Forensic Genetics Unit, Institute of Forensic Science "Luis Concheiro", University of Santiago de Compostela, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Phillips
authors.affiliation: Forensic Genetics Unit, Institute of Forensic Science "Luis Concheiro", University of Santiago de Compostela, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Fondevila
authors.affiliation: Office of the Chief Forensic Scientist, Forensic Services Department, Victoria Police, Australia.
authors.forename: R A H
authors.initials: RA
authors.lastname: van Oorschot
authors.affiliation: Forensic Genetics Unit, Institute of Forensic Science "Luis Concheiro", University of Santiago de Compostela, Spain; CIBERER, Genomic Medicine Group, University of Santiago de Compostela, Spain; Center of Excellence in Genomic Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia.
authors.forename: A
authors.initials: A
authors.lastname: Carracedo
authors.affiliation: Forensic Genetics Unit, Institute of Forensic Science "Luis Concheiro", University of Santiago de Compostela, Spain.
authors.forename: M V
authors.initials: MV
authors.lastname: Lareu
authors.affiliation: National Centre for Forensic Studies, University of Canberra, Australia.
authors.forename: D
authors.initials: D
authors.lastname: McNevin
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D053803:Forensic Genetics; D005787:Gene Frequency; D006801:Humans; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide
chemical_list: D017931:DNA Primers
keywords: Biogeographical ancestry (BGA); Externally visible characteristics (EVCs); Molecular photofitting; Next generation sequencing (NGS); SNaPshot; Single nucleotide polymorphisms (SNPs)