Developmental validation of the PowerPlex® Fusion 6C System

Ensenberger, Martin G.; Lenz, Kristy A.; Matthies, Learden K.; Hadinoto, Gregory M.; Schienman, John E.; Przech, Angela J.; Morganti, Michael W.; Renstrom, Daniel; Baker, Victoria M.; Gawrys, Kori M.; Hoogendoorn, Marlijn; Steffen, Carolyn R.; Martı́n, Pablo; Alonso, Antonio; Olson, Hope R.; Sprecher, Cynthia J.; Storts, Douglas R.

doi:10.1016/j.fsigen.2015.12.011

Cited by 98 publications

(38 citation statements)

References 14 publications

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“…The profiles obtained from primates can be distinguished from human profiles by the presence of many off-ladder peaks and the overall imbalance. Similar findings have been reported in previous studies [16]. For Investigator 24plex QS, Macaque DNA produced an Amelogenin Xpeak, one allele call for D1S1656 allele 12 and further off-ladder peaks in the FAM, BTG and BTR panel.…”

Section: Species Specificitysupporting

confidence: 89%

Developmental validation of QIAGEN Investigator® 24plex QS Kit and Investigator® 24plex GO! Kit: Two 6-dye multiplex assays for the extended CODIS core loci

Kraemer

Prochnow

Bussmann

et al. 2017

Forensic Science International: Genetics

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The original CODIS database based on 13 core STR loci has been overwhelmingly successful for matching suspects with evidence. In order to increase the power of discrimination, reduce the possibility of adventitious matches, and expand global data sharing, the CODIS Core Loci Working Group determined the expansion of the CODIS core loci to 20 STR plus three additional "highly recommended" loci (SE33, DY391, Amelogenin) Hares, 2015Hares, , 2012. The QIAGEN Investigator 24plex QS and Investigator 24plex GO! Kits are 6-dye multiplex assays that contain all markers of the expanded 23 CODIS core loci along with a unique internal performance control that is co-amplified with the STR markers. The "Quality Sensor" generates additional information for quality control and performance checks. Investigator 24plex QS is designed for purified DNA from casework and reference samples, whereas 24plex GO! is dedicated to direct amplification of reference samples, like blood or buccal cells on FTA or swabs.A developmental validation study was performed on both assays. Here, we report the results of this study which followed the recommendations of the European Network of Forensic Science Institutes (ENFSI) [3] and the Revised Validation Guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM) [4]. Data included are for PCR-based procedures e.g. reaction conditions, effects of PCR annealing temperature variations, amplification cycles or cyclers, sensitivity (also in the context of the Quality Sensor), performance with simulated inhibition, stability and efficiency, precision, reproducibility, mixture study, concordance, stutter, species specificity, and case-type samples. The validation results demonstrate that the Investigator 24plex QS and Investigator 24plex GO! Kits are robust and reliable identification assays as required for forensic DNA typing in forensic casework analysis and databasing.

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Section: Species Specificitysupporting

confidence: 89%

Developmental validation of QIAGEN Investigator® 24plex QS Kit and Investigator® 24plex GO! Kit: Two 6-dye multiplex assays for the extended CODIS core loci

Kraemer

Prochnow

Bussmann

et al. 2017

Forensic Science International: Genetics

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“…2 and S1). In general, results for the ForenSeq DNA Signature Prep Kit aSTR sensitivity were comparable to CE-based STR kits [58][59][60][61] and in line with earlier published MPS studies [1,3,4,14,54].…”

Section: Sensitivitysupporting

confidence: 88%

Inter-laboratory study on standardized MPS libraries: evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA

et al. 2019

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We present results from an inter-laboratory massively parallel sequencing (MPS) study in the framework of the SeqForSTRs project to evaluate forensically relevant parameters, such as performance, concordance, and sensitivity, using a standardized sequencing library including reference material, mixtures, and ancient DNA samples. The standardized library was prepared using the ForenSeq DNA Signature Prep Kit (primer mix A). The library was shared between eight European laboratories located in Austria, France, Germany, The Netherlands, and Sweden to perform MPS on their particular MiSeq FGx sequencers. Despite variation in performance between sequencing runs, all laboratories obtained quality metrics that fell within the manufacturer's recommended ranges. Furthermore, differences in locus coverage did not inevitably adversely affect heterozygous balance. Inter-laboratory concordance showed 100% concordant genotypes for the included autosomal and Y-STRs, and still, X-STR concordance exceeded 83%. The exclusive reasons for X-STR discordances were drop-outs at DXS10103. Sensitivity experiments demonstrated that correct allele calling varied between sequencing instruments in particular for lower DNA amounts (≤ 125 pg). The analysis of compromised DNA samples showed the drop-out of one sample (FA10013B01A) while for the remaining three degraded DNA samples MPS was able to successfully type ≥ 87% of all aSTRs, ≥ 78% of all Y-STRs, ≥ 68% of all X-STRs, and ≥ 92% of all iSNPs demonstrating that MPS is a promising tool for human identity testing, which in return, has to undergo rigorous in-house validation before it can be implemented into forensic routine casework.

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“…Since the mixtures were used to test the performance of the software and also to determine analysis thresholds that are fit for purpose, we balanced the influence of varying DNA inputs in the PCR and increased drop-out due to low DNA input. This was achieved by the use of a minimum of the minor component of 60 pg in the 0.5%, 1% and 5% mixtures, resulting in a total DNA input of 12 ng, 6 ng and 1.2 ng, respectively (60 pg resulted in less than 20% drop-out in the validation of Powerplex 6C [14]). The same total DNA input of 1.2 ng was used for the 5%, 10%, 20% and 50% mixtures (resulting in 120 pg and 240 pg of the minor components in the 10% and 20% mixtures, respectively).…”

Section: Sample Preparationmentioning

confidence: 99%

FDSTools: A software package for analysis of massively parallel sequencing data with the ability to recognise and correct STR stutter and other PCR or sequencing noise

Hoogenboom

Gaag

Leeuw

et al. 2017

Forensic Science International: Genetics

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Massively parallel sequencing (MPS) is on the advent of a broad scale application in forensic research and casework. The improved capabilities to analyse evidentiary traces representing unbalanced mixtures is often mentioned as one of the major advantages of this technique. However, most of the available software packages that analyse forensic short tandem repeat (STR) sequencing data are not well suited for high throughput analysis of such mixed traces. The largest challenge is the presence of stutter artefacts in STR amplifications, which are not readily discerned from minor contributions. FDSTools is an open-source software solution developed for this purpose. The level of stutter formation is influenced by various aspects of the sequence, such as the length of the longest uninterrupted stretch occurring in an STR. When MPS is used, STRs are evaluated as sequence variants that each have particular stutter characteristics which can be precisely determined. FDSTools uses a database of reference samples to determine stutter and other systemic PCR or sequencing artefacts for each individual allele. In addition, stutter models are created for each repeating element in order to predict stutter artefacts for alleles that are not included in the reference set. This information is subsequently used to recognise and compensate for the noise in a sequence profile. The result is a better representation of the true composition of a sample. Using Promega Powerseq™ Auto System data from 450 reference samples and 31 two-person mixtures, we show that the FDSTools correction module decreases stutter ratios above 20% to below 3%. Consequently, much lower levels of contributions in the mixed traces are detected. FDSTools contains modules to visualise the data in an interactive format allowing users to filter data with their own preferred thresholds.

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Developmental validation of the PowerPlex® Fusion 6C System

Cited by 98 publications

References 14 publications

Developmental validation of QIAGEN Investigator® 24plex QS Kit and Investigator® 24plex GO! Kit: Two 6-dye multiplex assays for the extended CODIS core loci

Developmental validation of QIAGEN Investigator® 24plex QS Kit and Investigator® 24plex GO! Kit: Two 6-dye multiplex assays for the extended CODIS core loci

Inter-laboratory study on standardized MPS libraries: evaluation of performance, concordance, and sensitivity using mixtures and degraded DNA

FDSTools: A software package for analysis of massively parallel sequencing data with the ability to recognise and correct STR stutter and other PCR or sequencing noise

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