Inter-laboratory validation study of the ForenSeq™ DNA Signature Prep Kit.

The implementation of massively parallel sequencing (MPS) in forensic science revealed the advantages of the new method compared to the forensic benchmark in DNA-STR analysis, the capillary-electrophoresis (CE): Sequence information and the possibility to multiplex hundreds of markers in one multiplex PCR increase the discrimination power of a forensic (STR-) profile. The EU funded project DNASeqEx (DNA-STR Massive Sequencing & International Information Exchange) aims to evaluate MPS-based materials in their respective developmental stages using the two established platforms MiSeq FGx (Illumina) and Ion S5™ (Thermo Fisher Scientific). As part of this project, we present here an inter-laboratory validation of the Forenseq™ DNA Signature Prep Kit, focussing on STRs included in primer mix A. Our study comprises tests of concordance, reproducibility, sensitivity (1 ng, 500 pg, 250 pg, 125 pg, 63 pg, 31 pg) and mixtures (male-male and male-female at ratios of 1:1, 1:5, 1:10, 1:15, 1:20, 1:100, 1:500, 1:1000). Sequencing results found to be virtually concordant to CE results, to reference profiles and reproducible between duplicates and between both laboratories. We observed first locus drop-outs (LDO) at a DNA input of 63 pg (20 sample pool) and 125 pg (38 sample pool). Alleles were found to be well balanced at a DNA input of 250 pg or more. We found the kit to perform well on moderate mixtures (1:1-1:20).

[1]  Peter M Vallone,et al.  Production and certification of NIST Standard Reference Material 2372 Human DNA Quantitation Standard , 2009, Analytical and bioanalytical chemistry.

[2]  Francesc Calafell,et al.  An assessment of a massively parallel sequencing approach for the identification of individuals from mass graves of the Spanish Civil War (1936–1939) , 2016, Electrophoresis.

[3]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[4]  Bruce Budowle,et al.  Comparative tolerance of two massively parallel sequencing systems to common PCR inhibitors , 2017, International Journal of Legal Medicine.

[5]  Francesc Calafell,et al.  Length and repeat-sequence variation in 58 STRs and 94 SNPs in two Spanish populations. , 2017, Forensic science international. Genetics.

[6]  Anantharaman Ramani,et al.  Ancestry prediction in Singapore population samples using the Illumina ForenSeq kit. , 2017, Forensic science international. Genetics.

[7]  Wei Zhu,et al.  Next generation sequencing: Improved resolution for paternal/maternal duos analysis. , 2016, Forensic science international. Genetics.

[8]  Niels Morling,et al.  Second-generation sequencing of forensic STRs using the Ion Torrent™ HID STR 10-plex and the Ion PGM™. , 2015, Forensic science international. Genetics.

[9]  J M Butler,et al.  Biology and Genetics of New Autosomal STR Loci Useful for Forensic DNA Analysis. , 2013, Forensic science review.

[10]  Walther Parson,et al.  Evaluation of the Illumina ForenSeq™ DNA Signature Prep Kit - MPS forensic application for the MiSeq FGx™ benchtop sequencer. , 2017, Forensic science international. Genetics.

[11]  Bruce Budowle,et al.  Genetic analysis of the Yavapai Native Americans from West-Central Arizona using the Illumina MiSeq FGx™ forensic genomics system. , 2016, Forensic science international. Genetics.

[12]  Ann Marie Gross,et al.  Internal Validation of the AmpFlSTR Yfiler™ Amplification Kit for Use in Forensic Casework , 2008, Journal of forensic sciences.

[13]  Douglas R Storts,et al.  Developmental validation of the PowerPlex(®) Fusion System for analysis of casework and reference samples: A 24-locus multiplex for new database standards. , 2014, Forensic science international. Genetics.

[14]  Hoi Yan Chow,et al.  Qualitative and quantitative assessment of Illumina’s forensic STR and SNP kits on MiSeq FGx™ , 2017, PloS one.

[15]  Dennis Y. Wang,et al.  Developmental validation of the GlobalFiler(®) Express PCR Amplification Kit: A 6-dye multiplex assay for the direct amplification of reference samples. , 2015, Forensic science international. Genetics.

[16]  Jianye Ge,et al.  Developmental validation of the Yfiler(®) Plus PCR Amplification Kit: An enhanced Y-STR multiplex for casework and database applications. , 2016, Forensic science international. Genetics.

[17]  David Ballard,et al.  Concordance of the ForenSeq™ system and characterisation of sequence-specific autosomal STR alleles across two major population groups. , 2017, Forensic science international. Genetics.

[18]  Rebecca Just,et al.  Short tandem repeat typing on the 454 platform: strategies and considerations for targeted sequencing of common forensic markers. , 2014, Forensic science international. Genetics.

[19]  Paul Roffey,et al.  Comparison between magnetic bead and qPCR library normalisation methods for forensic MPS genotyping , 2017, International Journal of Legal Medicine.

[20]  Lu Zhang,et al.  Massively parallel sequencing of forensic STRs and SNPs using the Illumina® ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx™ Forensic Genomics System. , 2017, Forensic science international. Genetics.

[21]  Jocelyne Bruand,et al.  Developmental validation of the MiSeq FGx Forensic Genomics System for Targeted Next Generation Sequencing in Forensic DNA Casework and Database Laboratories. , 2017, Forensic science international. Genetics.

[22]  Niels Morling,et al.  Recommendations of the DNA Commission of the International Society for Forensic Genetics (ISFG) on quality control of autosomal Short Tandem Repeat allele frequency databasing (STRidER). , 2016, Forensic science international. Genetics.

[23]  Hoi Yan Chow,et al.  Systematic assessment of the performance of Illumina's MiSeq FGx™ forensic genomics system , 2017, Electrophoresis.

[24]  Peter M Vallone,et al.  STR allele sequence variation: Current knowledge and future issues. , 2015, Forensic science international. Genetics.

[25]  Walther Parson,et al.  A modular real-time PCR concept for determining the quantity and quality of human nuclear and mitochondrial DNA. , 2007, Forensic science international. Genetics.

[26]  P Gill,et al.  Report of the European Network of Forensic Science Institutes (ENSFI): formulation and testing of principles to evaluate STR multiplexes. , 2000, Forensic science international.

[27]  Magdalena Konarzewska,et al.  Application of massively parallel sequencing (MPS) in paternity testing - case report. , 2017, Archiwum medycyny sadowej i kryminologii.

[28]  Paolo Fattorini,et al.  Performance of the ForenSeqTM DNA Signature Prep kit on highly degraded samples , 2017, Electrophoresis.

[29]  Douglas R Storts,et al.  Developmental validation of the PowerPlex® Y23 System: a single multiplex Y-STR analysis system for casework and database samples. , 2013, Forensic science international. Genetics.

[30]  Haiying Jin,et al.  Developmental Validation of a novel 5 dye Y-STR System comprising the 27 YfilerPlus loci , 2016, Scientific Reports.

[31]  Peter M Vallone,et al.  Sequence variation of 22 autosomal STR loci detected by next generation sequencing. , 2016, Forensic science international. Genetics.

[32]  Ashley L. Silvia,et al.  A preliminary assessment of the ForenSeq™ FGx System: next generation sequencing of an STR and SNP multiplex , 2016, International Journal of Legal Medicine.

[33]  Bruce Budowle,et al.  Characterization of genetic sequence variation of 58 STR loci in four major population groups. , 2016, Forensic science international. Genetics.

[34]  Ugo Ricci,et al.  Forensic genetics in NGS era: New frontiers for massively parallel typing , 2015 .

[35]  María José Farfán,et al.  Improving DNA data exchange: validation studies on a single 6 dye STR kit with 24 loci. , 2014, Forensic science international. Genetics.

[36]  Bruce Budowle,et al.  Evaluation of the Illumina(®) Beta Version ForenSeq™ DNA Signature Prep Kit for use in genetic profiling. , 2016, Forensic science international. Genetics.

[37]  Bruce Budowle,et al.  Blind study evaluation illustrates utility of the Ion PGM™ system for use in human identity DNA typing , 2015, Croatian medical journal.

[38]  Bruce Budowle,et al.  Massively parallel sequencing of forensic STRs: Considerations of the DNA commission of the International Society for Forensic Genetics (ISFG) on minimal nomenclature requirements. , 2016, Forensic science international. Genetics.

[39]  Bruce Budowle,et al.  High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing. , 2015, Forensic science international. Genetics.

[40]  A. Dufour,et al.  One year variability of peak heights, heterozygous balance and inter-locus balance for the DNA positive control of AmpFℓSTR© Identifiler© STR kit. , 2011, Forensic science international. Genetics.

[41]  W Parson,et al.  "The devil's in the detail": Release of an expanded, enhanced and dynamically revised forensic STR Sequence Guide. , 2018, Forensic science international. Genetics.

[42]  Niels Morling,et al.  High-throughput sequencing of core STR loci for forensic genetic investigations using the Roche Genome Sequencer FLX platform. , 2011, BioTechniques.

[43]  Lilliana I Moreno,et al.  Performance and concordance of the ForenSeq™ system for autosomal and Y chromosome short tandem repeat sequencing of reference-type specimens. , 2017, Forensic science international. Genetics.

[44]  Yu Cao,et al.  Multiplex Y-STRs analysis using the ion torrent personal genome machine (PGM). , 2015, Forensic science international. Genetics.

[45]  Bruce Budowle,et al.  Population and performance analyses of four major populations with Illumina's FGx Forensic Genomics System. , 2017, Forensic science international. Genetics.

[46]  Bruce Budowle,et al.  Flanking region variation of ForenSeq™ DNA Signature Prep Kit STR and SNP loci in Yavapai Native Americans. , 2017, Forensic science international. Genetics.