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Forensic validation of the PowerPlex® ESI 16 STR Multiplex and comparison of performance with AmpFlSTR® SGM Plus®

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Abstract

We describe the forensic validation of Promega’s PowerPlex® European Standard Investigator 16 (ESI 16) multiplex kit and compare results generated with the AmpFlSTR® SGM Plus® (SGM+) multiplex. ESI 16 combines the loci contained within the SGM+ multiplex with five additional loci: D2S441, D10S1248, D22S1045, D1S1656, and D12S391. A relative reduction in amplicon size of the SGM+ loci facilitates an increased robustness and amplification success of these amplicons with degraded DNA samples. Tests performed herein supplement ESI 16 data published previously with sensitivity, profile quality, mock casework, inhibitor and mixture study data collected in our laboratories in alignment with our internal technical and quality guidelines and those issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), the DNA Advisory Board (DAB) and the DNA working group (DNAWG) of the European Network of Forensic Science Institutes (ENFSI). Full profiles were routinely generated from a fully heterozygous single source DNA template using 62.5 pg for ESI 16 and 500 pg for SGM+. This increase in sensitivity has a consequent effect on mixture analyses and the detection of minor mixture components. The improved PCR chemistry confers enhanced tolerance to high levels of laboratory prepared inhibitors compared with SGM+ results. In summary, our results demonstrate that the ESI 16 multiplex kit is more robust and sensitive compared with SGM+ and will be a suitable replacement system for the analysis of forensic DNA samples providing compliance with the European standard set of STR loci.

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Acknowledgements

The authors would like to acknowledge the generosity of Promega Corporation, with special thanks to Dr. Doug Storts, Dr. Bob McLaren, Cindy Sprecher and Dawn Rabbach. Thanks also to FSS staff: Gareth Stead, Kate Lomas, Marcus Adams, Emily Rowlands, Laura Pelleymounter, Rebecca Lewis, Alison Peters, Nina Fairless and Tarek Leane for technical assistance with this work.

Ethical standards

Experiments contained herein were performed in the UK and comply with current laws. All samples were collected with fully informed consent.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Research and Development, the Forensic Science Service, 2960 Trident Court, Birmingham Business Park, Solihull Parkway, Birmingham, B37 7YN, UK

    Valerie C. Tucker, Amanda J. Kirkham & Andrew J. Hopwood

Authors
  1. Valerie C. Tucker
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  2. Amanda J. Kirkham
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  3. Andrew J. Hopwood
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Corresponding author

Correspondence to Valerie C. Tucker.

Electronic supplementary materials

Below is the link to the electronic supplementary material.

Online Resource 1

SGM+ and ESI 16 sensitivity study. Ten replicates of a fully heterozygous male DNA sample were amplified for each template level on both multiplex systems and mean peak heights across all loci were calculated. Error bars represent the standard error of the mean. ESI 16 amplifications were injected at 3 kV for 5 and 10 s to assess peak heights obtained in our laboratory against manufacturer data. SGM+ amplifications were injected at 1.5 kV for 10 s (DOC 30 kb)

Online Resource 2

Inter-locus balance in ESI 16 and SGM+. Mean peak height was determined at each locus from ten amplifications of 500 pg of DNA from sensitivity data for ESI 16 (a) and 1,500 pg DNA from sensitivity data for SGM+ (b). Mean peak height (Y-axis) in rfu is plotted against each locus. Loci are arranged in order (left to right) from smallest to largest amplicons in blue, green, yellow, and red dye channels (DOC 33 kb)

Online Resource 3

Peak height ratio with respect to DNA mass. Mean peak height ratio of alleles across all loci (Y-axis) was determined from sensitivity data and plotted against mass of DNA template (X-axis). Results are shown for ESI 16 injected at 3 kV for 10 s (a), and SGM+ injected at 1.5 kV for 10 s (b) (DOC 32 kb)

Online Resource 4

Limit of detection. The baseline fluorescence of 20 PCR negative control samples was measured by applying a 1 rfu analysis threshold in GeneMapper® ID. The height of the first 50 peaks in each locus were recorded and analysed in groups according to dye channel. The mean height of baseline fluorescence in each dye channel is presented above in rfu (DOC 35 kb)

Online Resource 5

Frequency of baseline fluorescence peaks with respect to height in rfu. Twenty PCR negative control samples were analysed by applying a 1 rfu analysis threshold in GeneMapper® ID. Peaks were analysed by dye and grouped into bins. The dye channels (top to bottom) are fluorescein (blue), JOE (green), TMR-ET (yellow) and CXR-ET (red) (DOC 25 kb)

Online Resource 6

Size accuracy of alleles amplified from the NIST Standard Reference Materials 2391b compared with allelic ladder. Sample allele sizes (in bases) were recorded, and their variance relative to the corresponding ladder allele from the same run was calculated. The largest deviation above (maximum) and below (minimum) the ladder allele are shown, together with mean size variance and standard deviation (SD). (DOC 84 kb)

Online Resource 7

System reproducibility. The mean peak height of 36 PCR positive control samples was measured by analysis in GeneMapper® ID. NSD indicates no significant difference between the test systems compared by Mann–Whitney test using a 95% confidence interval. Where Mann–Whitney returned a significant test score, it is noted as a ‘p’ value above (DOC 249 kb)

Online Resource 8

Descriptive statistics for −1 repeat stutter proportions by locus. Stutter incidence was analysed in 75 amplifications of 500 pg DNA template. Low molecular weight and high molecular weight alleles were analysed separately. The number of parent alleles (Alleles) analysed for stutter were recorded, followed by the number of observed stutters (N). Fewer high molecular weight allele stutters are recorded owing to the opportunity for the low molecular weight allele to sit in the stutter position of the high molecular weight allele. Q1 = bound of first inter-quartile range, Q3 = bound of third inter-quartile range, SD = standard deviation (DOC 182 kb)

Online Resource 9

Artefacts seen in addition to those published by the manufacturer. CE separation in our laboratory was performed with a 3130xl Genetic Analyser, employing a 3 kV for 10 s sample injection. OL = GeneMapper® software designation of ‘off ladder’. These observations were made within the set of 480 samples analysed for profile characterisation (DOC 444 kb)

Online Resource 10

Observed and expected Mx proportions. Box–plot graph and tables demonstrating the expected Mx proportions (E Mx) and the observed Mx data calculated from loci with full heterozygous major and full heterozygous minor components present (DOC 103 kb)

Online Resource 11

First pass success rates. Stacked graph to show the number of mock casework samples returning a full profile (FP), a partial profile that may be loaded to the UK NDNAD (PDB), a partial profile with five or more complete loci (PP Hi), a partial profile with less than five complete loci (PP Lo) or a null profile (NP) in a sample set of 51 (DOC 39 kb)

Online Resource 12

Comparison of results obtained from low template DNA samples where 10 and 17.5 μl of extract was amplified in a total reaction volume of 25 μl. In the table, column 2 lists the original results for the seven selected samples of the mock casework sample set when processed with SGM+. Columns 3–8 contain data from a subsequent back to back amplification using ESI 16 (DOC 55 kb)

Online Resource 13

Sensitivity of SGM+ at 28 and 34 cycles (LCN) compared with ESI 16. The mean percentage profile obtained from amplifications containing 11 DNA template amounts were plotted for three STR amplification systems. Four replicates of DNA templates 1,000 to 31.3 pg and two replicates of DNA templates 15.6 to 0.98 pg were amplified in 25 μl reaction volumes using the ESI 16 System (30 cycles), SGM+ (28 cycles) and SGM+ using LCN amplification settings (34 cycles). Capillary electrophoresis injection parameters for ESI 16 and LCN were 3 kV for 10 s, where as SGM+ reactions were injected at 1.5 kV for 10 s. Error bars indicate the standard deviation (DOC 26 kb)

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Tucker, V.C., Kirkham, A.J. & Hopwood, A.J. Forensic validation of the PowerPlex® ESI 16 STR Multiplex and comparison of performance with AmpFlSTR® SGM Plus® . Int J Legal Med 126, 345–356 (2012). https://doi.org/10.1007/s00414-011-0582-5

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