Kit Information

The PowerPlex® Fusion 6C System (Fusion 6C) (Promega Corporation) amplify 27 markers in total, including 23 autosomal STRs, 3 Y-STRs and Amelogenin.

The SureID® 27comp Human DNA Identification Kit (SureID27) (Ningbo Health Gene Technologies Co., Ltd. (HGT)) amplify 27 markers in total, including 26 autosomal STR, but no Y-STRs.

There are 7 overlapping markers between the two kits: AMEL, D1S1656, D2S441, D10S1248, D16S539, D12S391, D22S1045. Marker configuration for the two kits is shown in the figure below.

Experimental Design

The analytical threshold (AT) was determined from negative (non-template) PCR controls amplified with the respective kit according to recommendations. The results from the capillary electrophoresis were analysed in GeneMapper using a peak amplitude threshold (PAT) of 1 RFU. No stutter filter or global cut-off was applied. Pull-up or bleed-through from the internal lane standard (ILS) were excluded from the analysis.

Data from 4 allelic ladders was used to determine the allele sizing precision.

Data from 85 authentic FTA reference samples, direct PCR amplified using Fusion 6C and SureID27. A reference data set with known profiles is available. The DNA profiles have been anonymized by scrambling the alleles. The results from the capillary electrophoresis were analysed in GeneMapper using the determined analytical threshold, without applied stutter filter and with no global cut-off.

The data for this workshop was kindly provided by Siv Gilfillan (Forensic Department, Oslo University Hospital).

Source Data Files

Fusion 6C

Sample Data: fusion_data.txt (2022-08-10 09:42:33)

Known profiles: fusion_ref.txt (2022-08-10 09:44:40)

Negative amplification controls: fusion_neg_SamplePlotSizingTable.txt (2022-08-20 10:22:29)

SureID27

Sample Data: sureid_data.txt (2022-08-12 14:23:19)

Known profiles: sureid_ref.txt (2022-08-12 14:43:08)

Negative amplification controls: sureid_neg_SamplePlotSizingTable.txt (2022-08-10 13:27:34)

R Packages and Operating System

strvalidator version 2.4.0

R version 4.2.1 (2022-06-23 ucrt)

Platform: x86_64-w64-mingw32/x64 (64-bit)

Running under: Windows 10 x64 (build 19043)

Analytical Threshold

The analytical threshold should be based on signal-to-noise analyses of internally derived empirical data. An analytical threshold defines the minimum height requirement at and above which detected peaks can be reliably distinguished from background noise. Because the analytical threshold is based upon a distribution of noise values, it is expected that occasional, non-reproducible noise peaks may be detected above the analytical threshold. Usage of an exceedingly high analytical threshold increases the risk of allelic data loss. SWGDAM Interpretation Guidelines for Autosomal STR Typing by Forensic DNA Testing Laboratories (APPROVED 01/12/2017).

Estimated analytical thresholds (k=3) in RFU based on amplification negative control samples for Fusion 6C (top) and SureID27 (bottom). Global across-dyes estimates in the leftmost column followed by dye-specific estimates.

Fusion 6C had the least noise with a global across-dyes estimated analytical threshold AT=25 RFU compared to AT=39 RFU for SureID27.

The mean number of noise signals in negative amplification controls from Fusion 6C was 3308 and from SureID27 1138. All data shown in tables below:

Allele Sizing Precision

For verification of electrophoretic equipment it is recommended that “the precision of the instrument should be such that all measured alleles fall within a +/- 0.5 bp window around the measured size for the corresponding allele in the allelic ladder”. ENFSI Recommended Minimum Criteria for the Validation of Various Aspects of the DNA Profiling Process (ISSUE DATE: November 2010).

The maximum size difference for Fusion 6C was 0.12 bp and from SureID27 was 0.23 bp. A size difference of 1 corresponds to +/- 0.5 bp. All data shown in the table below:

Peak Balance

Lower template DNA may cause extreme heterozygote imbalance; as such, empirical heterozygote peak-height ratio data could be used to formulate mixture interpretation guidelines and determine the appropriate ratio by which two peaks are determined to be heterozygotes. SWGDAM Validation Guidelines for DNA Analysis Methods (Approved 12/05/2016).

The peak balance ratios of heterozygote alleles within a locus and of alleles between all loci should be >60% for good quality samples. ENFSI Recommended Minimum Criteria for the Validation of Various Aspects of the DNA Profiling Process (ISSUE DATE: November 2010).

Peak height imbalances may be seen in the typing results from, for example, a primer binding site variant that results in attenuated amplification of one allele of a heterozygous pair. Likewise, degraded, inhibited, and/or low level single-source DNA samples may exhibit poor peak height balance with heterozygous alleles. SWGDAM Interpretation Guidelines for Autosomal STR Typing by Forensic DNA Testing Laboratories (APPROVED 01/12/2017).

Heterozygote Peak Balance

The interactive box plot below show heterozygote peak balance ratios for both kits. For all markers and both kits, >75% of the heterozygote peak balance ratio was >0.70 with the exceptions of D21S2055 (0.65) and D22S1045 (0.69) in SureID27. For all markers in Fusion 6C, >75% of the heterozygote peak balance ratio was >0.80.

The interactive table below show summary statistics for heterozygote peak balance for Fusion 6C and SureID27.

Profile Balance

The interactive box plot below show profile balance normalized to the highest marker sum of peak heights within each dye. For direct PCR of FTA reference samples, the criteria of >60% between all loci in a dye is not fulfilled for either Fusion 6C (top) or SureID27 (bottom). Interestingly, the shorter markers tend to overamplify using Fusion 6C, while the longer markers tend to overamplify using SureID27. Note that the Y markers in Fusion 6C has not been compensated for being single locus markers.

Stutter Ratio

The stutter ratio is the ratio of the stutter peak height compared to the corresponding allele peak height. In general, stutter peaks have to be lower than the % of the allele peak height indicated by the manufacturer of the kit to be ignored as a biological artefact of the sample. ENFSI Recommended Minimum Criteria for the Validation of Various Aspects of the DNA Profiling Process (ISSUE DATE: November 2010). Based on internal verification of the kit, it may be necessary to adjust stutter ratios provided by the manufacturer.

Summary statistics by stutter type and marker for Fusion 6C and SureID27is shown in the interactive plot below. The largest stutter ratio observed in Fusion 6C was 0.496 and in SureID27 0.386. They have been confirmed as true stutters.

Allele Calling Concordance

Fusion 6C and SureID27 have 7 overlapping markers: AMEL, D1S1656, D2S441, D10S1248, D16S539, D12S391, D22S1045. The overall concordance was 99.5%.

Observed discordances are shown in the table below: