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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.FS

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+Show Affiliations

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

Pub Type(s)

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

Language

eng

PubMed ID

25955683

Citation

Eduardoff, M, et al. "Inter-laboratory Evaluation of SNP-based Forensic Identification By Massively Parallel Sequencing Using the Ion PGM™." Forensic Science International. Genetics, vol. 17, 2015, pp. 110-121.
Eduardoff M, Santos C, de la Puente M, et al. Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™. Forensic Sci Int Genet. 2015;17:110-121.
Eduardoff, M., Santos, C., de la Puente, M., Gross, T. E., Fondevila, M., Strobl, C., Sobrino, B., Ballard, D., Schneider, P. M., Carracedo, Á., Lareu, M. V., Parson, W., & Phillips, C. (2015). Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™. Forensic Science International. Genetics, 17, 110-121. https://doi.org/10.1016/j.fsigen.2015.04.007
Eduardoff M, et al. Inter-laboratory Evaluation of SNP-based Forensic Identification By Massively Parallel Sequencing Using the Ion PGM™. Forensic Sci Int Genet. 2015;17:110-121. PubMed PMID: 25955683.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Inter-laboratory evaluation of SNP-based forensic identification by massively parallel sequencing using the Ion PGM™. AU - Eduardoff,M, AU - Santos,C, AU - de la Puente,M, AU - Gross,T E, AU - Fondevila,M, AU - Strobl,C, AU - Sobrino,B, AU - Ballard,D, AU - Schneider,P M, AU - Carracedo,Á, AU - Lareu,M V, AU - Parson,W, AU - Phillips,C, Y1 - 2015/04/15/ PY - 2014/11/14/received PY - 2015/03/31/revised PY - 2015/04/12/accepted PY - 2015/5/9/entrez PY - 2015/5/9/pubmed PY - 2016/4/19/medline KW - Identification SNPs KW - Ion PGM™ KW - Ion Torrent KW - Massively parallel sequencing KW - Next generation sequencing SP - 110 EP - 121 JF - Forensic science international. Genetics JO - Forensic Sci Int Genet VL - 17 N2 - 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. SN - 1878-0326 UR - https://www.unboundmedicine.com/medline/citation/25955683/Inter_laboratory_evaluation_of_SNP_based_forensic_identification_by_massively_parallel_sequencing_using_the_Ion_PGM™_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1872-4973(15)00077-0 DB - PRIME DP - Unbound Medicine ER -