Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™

M Eduardoff; C Santos; M de la Puente; T E Gross; M Fondevila; C Strobl; B Sobrino; D Ballard; P M Schneider; Á Carracedo; M V Lareu; W Parson; C Phillips

doi:10.1016/j.fsigen.2015.04.007

Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™

Forensic Sci Int Genet. 2015 Jul:17:110-121. doi: 10.1016/j.fsigen.2015.04.007. Epub 2015 Apr 15.

Authors

M Eduardoff¹, C Santos², M de la Puente², T E Gross³, M Fondevila², C Strobl¹, B Sobrino⁴, D Ballard⁵, P M Schneider³, Á Carracedo⁶, M V Lareu², W Parson⁷, C Phillips⁸

Affiliations

¹ Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria.
² Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain.
³ Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Cologne, Germany.
⁴ Grupo de Medicina Xenómica (GMX), Faculty of Medicine, University of Santiago de Compostela, Spain.
⁵ Department of Forensic and Analytical Science, Faculty of Life Science, King's College, London, UK.
⁶ 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.
⁷ Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria; Forensic Science Program, The Pennsylvania State University, PA, USA.
⁸ Forensic Genetics Unit, Institute of Forensic Sciences, University of Santiago de Compostela, Santiago de Compostela, Spain. Electronic address: c.phillips@mac.com.

PMID: 25955683
DOI: 10.1016/j.fsigen.2015.04.007

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.

Keywords: Identification SNPs; Ion PGM™; Ion Torrent; Massively parallel sequencing; Next generation sequencing.

Publication types

Comparative Study
Evaluation Study
Research Support, Non-U.S. Gov't

MeSH terms

DNA / analysis
DNA / genetics
Female
Forensic Genetics / methods*
Genotyping Techniques / methods*
High-Throughput Nucleotide Sequencing / methods*
Humans
Male
Polymorphism, Single Nucleotide*
Reproducibility of Results
Sequence Analysis, DNA

Substances

DNA