•Inter-laboratory evaluation of two early access Globalfiler MPS panels with respect to STR performance.•We assessed concordance, sensitivity and casework samples (including mixtures).•Our study indicates that the early access Globalfiler MPS panels are powerful tools with benefits for practical forensic casework. The current gold standard for analysing forensic genetic variation focuses on human identity testing using multiplex autosomal short tandem repeat (STR) genotyping using polymerase chain reaction (PCR)- and capillary electrophoresis (CE)-based approaches [[1]Jobling M.A. Gill P. Encoded evidence: DNA in forensic analysis.Nat. Rev. Genet. 2004; 5: 739-751Crossref PubMed Scopus (408) Google Scholar,[2]Butler J.M. Forensic DNA Typing: Biology, Technology, and Genetics of STR Markers.second ed. Elsevier Academic Press, New York2005Google Scholar]. In recent years, massively parallel sequencing (MPS) has been under continuous development for forensic genetics and, while CE-based STR genotyping is generally sufficiently discriminatory for routine forensic applications, shown some benefits over this technology [[3]Churchill J.D. Schmedes S.E. King J.L. Budowle B. Evaluation of the illumina® Beta version ForenSeq DNA signature prep kit for use in genetic profiling.Forensic Sci. Int. Genet. 2016; 20: 20-29Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar]. For instance, in CE-based PCR kits (containing up to 30 STR markers) amplicons need to be designed to avoid overlapping fragment lengths. Consequently, some PCR products are longer than would be necessary to reflect the information of the polymorphic repeat block only. Target amplicons between 80 and 500 bp are common [4ThermoFisherScientific GlobalFiler PCR Amplification Kit, User Guide.2016Google Scholar, 5Life-Technologies-Corporation AmpFlSTR® NGM SElect™ PCR Amplification Kit User Guide.2012Google Scholar, 6Westen A.A. Kraaijenbrink T. Robles de Medina E.A. Harteveld J. Willemse P. Zuniga S.B. van der Gaag K.J. Weiler N.E. Warnaar J. Kayser M. Sijen T. de Knijff P. Comparing six commercial autosomal STR kits in a large Dutch population sample.Forensic Sci. Int. Genet. 2014; 10: 55-63Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 7Butler J.M. Hill C.R. Biology and genetics of new autosomal STR loci useful for forensic DNA analysis.Forensic Sci. Rev. 2012; 24: 15-26PubMed Google Scholar, 8Oostdik K. Lenz K. Nye J. Schelling K. Yet D. Bruski S. Strong J. Buchanan C. Sutton J. Linner J. Frazier N. Young H. Matthies L. Sage A. Hahn J. Wells R. Williams N. Price M. Koehler J. Staples M. Swango K.L. Hill C. Oyerly K. Duke W. Katzilierakis L. Ensenberger M.G. Bourdeau J.M. Sprecher C.J. Krenke B. Storts D.R. Developmental validation of the PowerPlex® fusion system for analysis of casework and reference samples: a 24-locus multiplex for new database standards.Forensic Sci. Int. Genet. 2014; 12: 69-76Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar]. In contrast, MPS offers the ability to target dozens to hundreds of amplicons regardless of their size in one assay. Short amplicons are beneficial for the analysis of degraded DNA [[9]Calafell F. Anglada R. Bonet N. Gonzalez-Ruiz M. Prats-Munoz G. Rasal R. Lalueza-Fox C. Bertranpetit J. Malgosa A. Casals F. An assessment of a massively parallel sequencing approach for the identification of individuals from mass graves of the Spanish Civil War (1936-1939).Electrophoresis. 2016; 37: 2841-2847Crossref PubMed Scopus (17) Google Scholar,[10]Fattorini P. Previdere C. Carboni I. Marrubini G. Sorcaburu-Cigliero S. Grignani P. Bertoglio B. Vatta P. Ricci U. Performance of the ForenSeq™ DNA signature prep kit on highly degraded samples.Electrophoresis. 2017; 38: 1163-1174Crossref PubMed Scopus (44) Google Scholar]. Due to the nature of degraded DNA, samples analysed using conventional DNA typing methods often result either in partial profiles with lower discriminatory power or in the total lack of retrievable genetic information. Further, many studies demonstrated that sequencing of STRs provides new insights into sequence variation of established STR markers, elucidating the structure of micro-variants and improving the detection of mixtures [11Scheible M. Loreille O. Just R. Irwin J. Short tandem repeat typing on the 454 platform: strategies and considerations for targeted sequencing of common forensic markers.Forensic Sci. Int. Genet. 2014; 12: 107-119Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 12Zeng X. King J.L. Stoljarova M. Warshauer D.H. LaRue B.L. Sajantila A. Patel J. Storts D.R. Budowle B. High sensitivity multiplex short tandem repeat loci analyses with massively parallel sequencing.Forensic Sci. Int. Genet. 2015; 16: 38-47Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 13Gettings K.B. Kiesler K.M. Faith S.A. Montano E. Baker C.H. Young B.A. Guerrieri R.A. Vallone P.M. Sequence variation of 22 autosomal STR loci detected by next generation sequencing.Forensic Sci. Int. Genet. 2016; 21: 15-21Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 14Novroski N.M.M. King J.L. Churchill J.D. Seah L.H. Budowle B. Characterization of genetic sequence variation of 58 STR loci in four major population groups.Forensic Sci. Int. Genet. 2016; 25: 214-226Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 15Gettings K.B. Aponte R.A. Vallone P.M. Butler J.M. STR allele sequence variation: current knowledge and future issues.Forensic Sci. Int. 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Butler J.M. STR sequence analysis for characterizing normal, variant, and null alleles.Forensic Sci. Int. Genet. 2011; 5: 329-332Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 20van der Heijden S. de Oliveira S.J. Kampmann M.L. Borsting C. Morling N. Comparison of manual and automated AmpliSeq workflows in the typing of a Somali population with the precision ID identity panel.Forensic Sci. Int. Genet. 2017; 31: 118-125Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 21Wang Z. Zhou D. Wang H. Jia Z. Liu J. Qian X. Li C. Hou Y. Massively parallel sequencing of 32 forensic markers using the precision ID GlobalFiler NGS STR panel and the ion PGM system.Forensic Sci. Int. Genet. 2017; 31: 126-134Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 22Kiesler K.M. Steffen C.R. Coble M.D. Vallone P.M. Initial assessment of the precision ID globalfiler Mixture ID panel on the ion Torrent S5XL DNA sequencer and converge v2.0 software.Forensic Sci. Int. Genet. 2017; 6: E94-E95Abstract Full Text Full Text PDF Scopus (2) Google Scholar]. However, before MPS can be implemented into routine forensic casework both the new technology as well as resulting data have to be rigorously validated with respect to robustness, performance and backward compatibility to the large body of CE-based STR data stored in national DNA intelligence databases. For instance, in 2017 Alonso et al. [[23]Alonso A. Mueller P. Roewer L. Willuweit S. Budowle B. Parson W. European survey on forensic applications of massively parallel sequencing.Forensic Sci. Int. Genet. 2017; 29: e23-e25Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar] reported the need for a sequencing platform independent analysis software based on the considerations of the DNA commission of the International Society for Forensic Genetics (ISFG) [[24]Parson W. Ballard D. Budowle B. Butler J.M. Gettings K.B. Gill P. Gusmao L. Hares D.R. Irwin J.A. King J.L. Knijff P. Morling N. Prinz M. Schneider P.M. Neste C.V. Willuweit S. Phillips C. Massively parallel sequencing of forensic STRs: considerations of the DNA commission of the International Society for Forensic Genetics (ISFG) on minimal nomenclature requirements.Forensic Sci. Int. Genet. 2016; 22: 54-63Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar]. We evaluated the current state of MPS technology and chemistry to facilitate STR sequencing in the framework of the EU funded project DNASEQEX (DNA-STR Massive Sequencing & International Information Exchange (HOME/2014/ISFP/AG/LAWX/4000007135)) including commercially available products from Illumina (San Diego, USA), pre-release products from Thermo Fisher Scientific (TFS; Waltham, USA) and analysis software at their respective developmental stages. The consortium consisted of the following partners: The Biology Service of the National Institute of Toxicology and Forensic Science, Madrid (Spain) acting as coordinator, the Department of Forensic Genetics of the Institute of Legal Medicine and Forensics Science, Berlin (Germany) and the Institute of Legal Medicine, Medical University of Innsbruck (Austria) as beneficiaries and the Institute of Applied Genetics, Department of Molecular and Medical Genetics, University of North Texas Health Science Center, USA acting as consultant to the consortium. Here, we present the evaluation of two MPS-prototypes, Early Access Applied Biosystems Precision ID Globalfiler Mixture ID and Early Access Applied Biosystems Precision ID Globalfiler NGS STR Panels (both Thermo Fisher Scientific (TFS), Waltham, USA), including experiments to assess concordance, sensitivity, mock casework (single source and mixtures) as well as degraded DNA samples. We note that only the STR genotype calls were considered for this study in agreement with the goals defined in the DNASEQEX project. Inter-laboratory concordance was assessed by performing all experiments in a uniform manner at the Institute of Legal Medicine, Medical University of Innsbruck, Austria (GMI) and the Biology Service of the National Institute of Toxicology and Forensic Science, Madrid, Spain (INTCF). This inter-laboratory evaluation study combines data produced at the GMI and the INTCF. A set of forensically relevant samples was agreed upon in order to assess the inter-laboratory performance of the Early Access Applied Biosystems Precision ID Globalfiler Mixture ID (GF-Mix) and the Globalfiler NGS STR Panels (GF-STR) for the Ion S5 System (TFS, see Section 2.1). Time estimates for a typical automated workflow for both panels amounted to 24.5 h for eight samples. This includes 7.3 h (0.3 h hands-on time, 7 h instrument time) for library preparation, 1.2 h (0.5 h hands-on time, 0.7 h instrument time) for library quantitation, 13 h (0.3 h hands-on time, 12.7 h instrument time) for template preparation and 2–3 h for sequencing. The GF-Mix panel included 113 markers (29 autosomal STRs (aSTRs), one Y-STR, 42 SNPs, two Y-SNPs, 36 micro-haplotypes, Amelogenin and Y-InDel (rs2032678)) whereas with the GF-STR panel 33 targets could be analysed simultaneously (29 aSTRs, one Y-STR, Amelogenin and Y-InDel (rs2032678)). Both laboratories performed a concordance study using the same single donor standard reference material (SRM) purchased from the National Institute of Standards and Technology (NIST; component 2391c A–C [[25]National-Institute-of-Standards-&-Technology, Standard Reference Material® 2391c PCR-Based DNA Profiling Standard (Certificate of Analysis), (2011, Certificate Revision History: 03 April 2015).Google Scholar]; Gaithersburg, USA) and the forensic standard control DNA 9947A (TFS). The final DNA input for the concordance study was 1 ng according to the manufacturer’s recommendation. Concordance was determined using 29 aSTRs and one Y-STR for the GF-Mix and 28 aSTRs and one Y-STR for the GF-STR panel for 2391c A–C, known from reference [[25]National-Institute-of-Standards-&-Technology, Standard Reference Material® 2391c PCR-Based DNA Profiling Standard (Certificate of Analysis), (2011, Certificate Revision History: 03 April 2015).Google Scholar], and 20 aSTRs for 9947A and both panels known from CE results. At GMI serial dilutions for both Globalfiler trials consisted of forensic standard control DNA 9947A and NIST SRM 2372A [[26]National-Institute-of-Standards-&-Technology, Standard Reference Material® 2372 Human DNA Quantitation Standard, (2013, Certificate Revision History: 08 Jan 2013).Google Scholar]. To examine the impact of manual library preparation on sensitivity testing, additional libraries using 2372A for the GF-STR and control DNA 2800 M (Promega, Madison, WI, USA) for the GF-Mix panel (DNA input/assay: 1 ng to 31 pg, single approach) were manually prepared at GMI following [[27]ThermoFisherScientific Precision ID Panels With Ion PGM™ System, Application Guide.2016Google Scholar]. At INTCF serial dilutions were prepared using DNA control 9947A and 2372A for the GF-Mix panel as well as 2372A for the GF-STR panel. Dilutions were prepared enabling DNA input of 250 pg and 125 pg, following the manufacturer´s workbooks. In addition DNA input (2372A) of 500 pg, 250 pg, 125 pg, 62 pg were examined in duplicate at INTCF according to the manufacturer’s recommendations. Moreover, to improve the total number of sequencing reads INTCF tested adapted PCR cycling conditions (28 instead of 24 PCR cycles) using 2372A at the following genomic DNA input amounts in duplicate: 125 pg, 62 pg, 31 pg and 15 pg. Sensitivity was determined using 20 aSTRs and one Y-STR known from CE experiments. The casework study sample set at GMI consisted of 16 single source and six mixture stains (2–3 persons) from past GEDNAP (German DNA Profiling; http://www.gednap.org) proficiency tests listed in Table S1. The final DNA input for each sample was 1 ng according to the manufacturer´s recommendation. The casework study sample set at INTCF consisted of 12 single source and 13 mixture stains (2–3 persons) from past GEDNAP proficiency tests listed in Table S1. The final DNA input was adjusted to 1 ng for all single source samples, while mixtures were analysed using 2–3 ng final DNA input (see Tables S1–S6_INTCF) according to the manufacturer´s recommendation. The casework study sample set utilized at GMI consisted of 17 single source and six mixture stains (2–3 persons) from past GEDNAP proficiency tests plus SRM 2391c E [[28]NIST Certificate of Analysis - Standard Reference Material® 2391c.2015Google Scholar] (Table S1). The final DNA input for each sample was 1 ng according to the manufacturer’s recommendation. The casework study sample set at INTCF consisted of 10 single source and 13 mixture stains (2–3 persons) from past GEDNAP proficiency tests, three single source samples with known profile from volunteers, one single source and two mixture samples (2–3 persons) from past GHEP-ISFG (Spanish and Portuguese-Speaking Group of the ISFG; https://ghep-isfg.org/) proficiency tests and four challenging DNA samples (one tooth, one neonatal femur, one neonatal pars petrosa bone and one FFPE (formalin fixed and paraffin embedded) sample) (Table S1). At both laboratories the amount of genomic DNA was determined using the Quantifiler Trio DNA Quantification Kit (TFS) [[29]ThermoFisherScientific Quantifiler Trio DNA Quantification Kit, User Guide.2015Google Scholar]. For calibration the Quantifiler THP DNA Standard was diluted in Quantifiler THP DNA Dilution Buffer to 50 ng μL−1, 5 ng μL−1, 0.500 ng μL−1, 0.050 ng μL−1 and 0.005 ng μL−1. The final volume of each reaction was 20 μL consisting of 10 μL Quantifiler THP PCR Reaction Mix, 8 μL Quantifiler Trio Primer Mix and 2 μL sample of unknown quantity. Samples were run in duplicate using an Applied Biosystems 7500 Fast Real-Time PCR Instrument (TFS) using the HID Real-Time PCR Software v 2.3. Thermal cycler protocol was performed according to [[29]ThermoFisherScientific Quantifiler Trio DNA Quantification Kit, User Guide.2015Google Scholar]. GMI: STR typing was conducted using the Globalfiler PCR Amplification Kit (GF-PCR) (TFS). DNA samples were diluted in UV-irradiated Molecular Biology Grade water according to the manufacturer´s protocol [[4]ThermoFisherScientific GlobalFiler PCR Amplification Kit, User Guide.2016Google Scholar] to the final DNA input amount of 1 ng. All DNA extracts were amplified with 29 PCR cycles except of GEDNAP-47_S1 and GEDNAP-43_S3, where 30 PCR cycles were applied. Amplification was performed on an Applied Biosystems GeneAmp 9700 thermal cycler (TFS) following the manufacturer´s protocol [[4]ThermoFisherScientific GlobalFiler PCR Amplification Kit, User Guide.2016Google Scholar]. CE analysis of PCR products was performed on an Applied Biosystems Prism 3500XL Genetic Analyzer (TFS). The analysis of STR fragments utilized the GeneMapper ID-X software, version 1.2 (TFS) by applying the in-house validated colour channel specific analytical thresholds: blue – 50 relative fluorescence units (RFU), green – 80 RFU, yellow – 100 RFU, red – 100 RFU, purple – 175 RFU. INTCF: Standardized STR typing was conducted using the GF-PCR kit according to Martin et al. [[30]Martin P. de Simon L.F. Luque G. Farfan M.J. Alonso A. Improving DNA data exchange: validation studies on a single 6 dye STR kit with 24 loci.Forensic Sci. Int. Genet. 2014; 13: 68-78Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar], PowerPlex Fusion 6C System (Promega, Madison, WI, USA) according to Ensenberger et al. [[31]Ensenberger M.G. Lenz K.A. Matthies L.K. Hadinoto G.M. Schienman J.E. Przech A.J. Morganti M.W. Renstrom D.T. Baker V.M. Gawrys K.M. Hoogendoorn M. Steffen C.R. Martín P. Alonso A. Olson H.R. Sprecher C.J. Storts D.R. Developmental validation of the PowerPlex® fusion 6C system.Forensic Sci. Int.: Genet. 2016; 21: 134-144Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar], Minifiler, Identifiler Plus, NGM SElect NGM Detect (all four TFS) as well as PowerPlex ESX 17, PowerPlex ESI 17 and PowerPlex CS6 (all three Promega). Amplification was performed according to previous studies [[30]Martin P. de Simon L.F. Luque G. Farfan M.J. Alonso A. Improving DNA data exchange: validation studies on a single 6 dye STR kit with 24 loci.Forensic Sci. Int. Genet. 2014; 13: 68-78Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar,[31]Ensenberger M.G. Lenz K.A. Matthies L.K. Hadinoto G.M. Schienman J.E. Przech A.J. Morganti M.W. Renstrom D.T. Baker V.M. Gawrys K.M. Hoogendoorn M. Steffen C.R. Martín P. Alonso A. Olson H.R. Sprecher C.J. Storts D.R. Developmental validation of the PowerPlex® fusion 6C system.Forensic Sci. Int.: Genet. 2016; 21: 134-144Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar] and the manufacturer’s protocols [32Thermo-Fisher-Scientific AmpFlSTR Minifiler PCR Amplification Kit, User Guide.2012Google Scholar, 33Thermo-Fisher-Scientific AmpFlSTR Identifiler Plus PCR Amplification Kit, User Guide.2015Google Scholar, 34Thermo-Fisher-Scientific AppliedBiosystems® AmpFlSTR® NGM SElect™ PCR Amplification Kit, Manual.2014Google Scholar, 35Thermo-Fisher-Scientific NGM Detect PCR Amplification Kit, User Guide.2017Google Scholar, 36Promega PowerPlex® ESX 17 Fast System for Use on the Applied Biosystems® Genetic Analyzers, Technical Manual.2017Google Scholar, 37Promega PowerPlex Fusion System, Instructions for Use of Products.2017Google Scholar, 38Promega PowerPlex CS7 System, Instructions for Use of Products.2016Google Scholar, 39Promega PowerPlex ESI 17 System, Instructions for Use of Products.2017Google Scholar] on an Applied Biosystems GeneAmp 9700 thermal cycler (TFS). CE analysis of PCR products was performed on an Applied Biosystems Prism 3500 Genetic Analyzer (TFS). The analysis of STR fragments utilized the GeneMapper ID-X software, version 1.4 (TFS) by applying 100 RFU as an analytical threshold for all channels (blue, green, yellow, red and purple). Previous to library preparation on the Ion Chef System each DNA sample was diluted at GMI to a concentration of 0.067 ng μL−1 in UV-irradiated Molecular Biology Grade water to achieve a final DNA input amount of 1 ng, except for sensitivity study samples (final DNA input: 250 pg, 125 pg) according to the manufacturer´s recommendation. Samples performed at INTCF were diluted accordingly in Molecular Biology Grade water to achieve the required DNA input amount (final DNA input shown in Tables S1–S6_INTCF), except for sensitivity study samples (final DNA input: 500 pg to 15 pg). An aliquot of 15 μL of each DNA sample was pipetted into the according well of the first column on the IonCode 96 Well PCR Plate. Library preparation was facilitated using the Ion AmpliSeq Kit for Chef DL8 for HID and finished by combining all eight libraries into one labelled tube. Fully automated library preparation workflow, primer pools and PCR amplification cycles were performed according to the manufacturer´s user guides [[40]ThermoFisherScientific Early Evaluation Precision ID GlobalFiler® Mixture ID Panel, User Guide.2016Google Scholar,[41]ThermoFisherScientific Early Evaluation Precision ID GlobalFiler® NGS STR Panel for S5, User Guide.2016Google Scholar]. All negative controls for the sensitivity studies yielded no detectable sequences. To ensure uniform pooling, sequencing library pools were quantified in triplicate at GMI and duplicate at INTCF using a real-time PCR (qPCR) approach and the Ion Library Quantitation kit (TFS) [[42]ThermoFisherScientific Ion Library Quantitation Kit - TaqMan® Assay Quantitation of Ion Torrent Libraries, User Guide.2013Google Scholar]. Each sample library pool was diluted independently to the ratios 1:20, 1:2,000, 1:20,000 at GMI and 1:200 at INTCF in UV-irradiated Molecular Biology Grade water. For calibration, E. coli DH10B control library was diluted to a final concentration of 6.8 pM, 0.68 pM, 0.068 pM, 0.0068 pM and 0.00068 pM in Molecular Biology Grade water and tested in duplicate. According to the manufacturer´s user guide the final reaction volume was 20 μL consisting of 10 μL Ion Library TaqMan qPCR Mix, 1 μL Ion Library TaqMan Quantitation Assay and 9 μL of the diluted library sample. Amplification was performed according to [[42]ThermoFisherScientific Ion Library Quantitation Kit - TaqMan® Assay Quantitation of Ion Torrent Libraries, User Guide.2013Google Scholar]. Prior to fully automated template preparation on the Ion Chef System, all library pools were diluted to 50 pM. Both, the GF-Mix and GF-STR panel templates were prepared using the Ion 521 & 530 Kit – Chef for HID and loaded on Ion 530 sequencing chips. GMI: A total of 16 GF-Mix panel samples were loaded using the Ion Chef according to [[40]ThermoFisherScientific Early Evaluation Precision ID GlobalFiler® Mixture ID Panel, User Guide.2016Google Scholar]. For the GF-STR panel eight and 24 samples were loaded with the Ion Chef according to [[41]ThermoFisherScientific Early Evaluation Precision ID GlobalFiler® NGS STR Panel for S5, User Guide.2016Google Scholar]. At GMI the performed four sequencing runs comprised of 8, 16, 16, and 24 libraries, respectively. At INTCF five pc. of 530 sequencing chips (GF-Mix) containing 8, 16, 16, 16 and 24 samples and 12 pc. of 530 sequencing chips (GF-STR) were prepared containing 2 × 16 and 10 × 8 samples. Sequencing made use of the Ion S5 Early Access HID Sequencing Solutions, Ion S5 Early Access HID Sequencing Reagents and Ion 530 sequencing chips following the manufacturers recommendation [[40]ThermoFisherScientific Early Evaluation Precision ID GlobalFiler® Mixture ID Panel, User Guide.2016Google Scholar,[41]ThermoFisherScientific Early Evaluation Precision ID GlobalFiler® NGS STR Panel for S5, User Guide.2016Google Scholar]. Subsequently to sequencing and applying the HID Genotyper plugin, version 2.0_r1621 (TFS) generated html-files were exported from the S5 Torrent Server, version 5.2.1 (TFS) and imported into Converge software (TFS) according to the manufacturer’s workbook [[43]ThermoFisherScientific NGS on CONVERGE™ Evaluation Workbook.2016Google Scholar]. Genotype analysis was performed using Converge, version 2.0 (TFS) by applying the default relative analysis settings. The manufacturer´s default relative analytical and stochastic thresholds were 0.02 and 0.05, respectively. For example, the analytical threshold of 0.02 for an allele is defined as at least 2% coverage of the total allele coverage of the given STR marker. For stutter analysis the manufacturer´s default STR analysis parameter settings were applied to characterize stutter. Stutter settings included stutter offset and stutter ratio. Stutter offset is defined as the number of bases a stutter is offset from the mother allele. Stutter ratio is defined as the maximum expected stutter allele coverage, expressed as a percentage of the primary allele coverage at this locus. However, the design of future MPS-STR panels as well as the alignment of the sequence raw data should be performed relative to the most recent human genome reference sequence GRCh38 and follow [[24]Parson W. Ballard D. Budowle B. Butler J.M. Gettings K.B. Gill P. Gusmao L. Hares D.R. Irwin J.A. King J.L. Knijff P. Morling N. Prinz M. Schneider P.M. Neste C.V. Willuweit S. Phillips C. Massively parallel sequencing of forensic STRs: considerations of the DNA commission of the International Society for Forensic Genetics (ISFG) on minimal nomenclature requirements.Forensic Sci. Int. Genet. 2016; 22: 54-63Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar,[44]Phillips C. Gettings K.B. King J.L. Ballard D. Bodner M. Borsuk L. Parson W. The devil’s in the detail": release of an expanded, enhanced and dynamically revised forensic STR sequence guide.Forensic Sci. Int. Genet. 2018; 34: 162-169Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar]. The GF-Mix panel simultaneously targeted 29 aSTRs, one Y-STR, 42 SNPs, two Y-SNPs, 36 micro haplotypes, Amelogenin and Y-InDel (rs2032678). These loci included all European Standard-Set (ESS) [[45]EU-Monitor Exchange of DNA Analysis Results—Proposal for Amending the European Standard Set of Loci (ESS) (dl.: 13.03.2018).2009Google Scholar] and the Combined DNA Index System (CODIS) [[46]Hares D.R. Selection and implementation of expanded CODIS core loci in the United States.Forensic Sci. Int. Genet. 2015; 17: 33-34Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar,[47]Moretti T.R. Moreno L.I. Smerick J.B. Pignone M.L. Hizon R. Buckleton J.S. Bright J.A. Onorato A.J. Population data on the expanded CODIS core STR loci for eleven populations of significance for forensic DNA analyses in the United States.Forensic Sci. Int. Genet. 2016; 25: 175-181Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar] loci plus the additional STRs D1S1677, D3S4529, D4S2408, D12ATA63, D14S1434, D2S1776, D5S2800, D6S474 and D6S1043. The GF-STR panel allowed for multiplex amplification of 29 aSTRs, one Y-STR, Amelogenin and Y-InDel (rs2032678). STR markers included in the GF-STR panel were identical with those included in the GF-Mix panel core loci set except for D2S1776 that was not included in the GF-STR panel. Only STR genotype calls were considered for this study in agreement with the goals defined in the DNASEQEX project and classified as alleles when they were above the stochastic threshold (ST) and not identified as labelled stutter or noise artefacts (see Section 3.5). The study was performed in two independent laboratories to evaluate the performance of these early access prototypes using fully automated library and template preparation on the Ion Chef System (TFS) and sequencing on the Ion S5 System (TFS), except for two manually prepared sensitivity-testing libraries. To evaluate the interlocus balance of the two multiplex PCR panels the overall mean number of reads per locus for each laboratory and panel was calculated. The sample set used for this analysis was composed of: GMI (n = 22): 2391c (component A–C), DNA control 9947A, and single source GEDNAP stains; INTCF (n = 21): 2391c (component A–C), DNA controls (2800 M, 007 plus 9947A), single source GEDNAP stains as well as fresh buccal swab DNA samples from volunteers. Results for both GF panels were comparable between INTCF and GMI (Table S2). The majority of the STR markers displayed comparable results in terms of interlocus balance between GMI and INTCF, except for D3S4529, D12ATA63, D14S1434, D2S1776, D12S391, vWA, D7S820 in the GF-Mix (Fig. 1a) and D3S4529, D13S317, D7S820, D12ATA63, D5S2800, D1S1677, D1S1656 in the GF-STR panel (Fig. 1b). To evaluate potential differences in the instrumentation setup of both laboratories the theoretical number of marker-reads/sample was calculated by dividing the total number of reads at a given STR locus by the number of samples (GMI n = 22, INTCF n = 21). Table S2 shows that 21 of 32 STR (65.7%) markers included in the GF-Mix panel displayed a higher number of marker-reads/sample at GMI compared to INTCF and for the GF-STR panel 16 of 31 STRs (51.6%) displayed a higher number of marker-reads/sample at GMI compared to INTCF. The inter-laboratory variation in the total number of marker-reads/sample was reproducible for both panels (Table S2). Results for the GF-STR panel indicate that the reagent version targeting 33 markers leads to