Evaluation of the PowerPlex 35GY Amplification System in Combination with the Spectrum Compact Capillary Electrophoresis System on Complex Samples from Valparaíso
José Manríquez*, María Oriana Yáñez, Wilson Campos. Forensic Genetics Unit of Valparaíso, Servicio Médico Legal, Chile
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The analysis of complex forensic samples poses a constant challenge for laboratory staff. Factors such as PCR inhibitors, low DNA quantity, or significant sample degradation due to environmental conditions like temperature or decomposition often result in genetic profiles that are difficult to interpret. These issues complicate identity and familial determinations, increasing the laboratory’s response time a critical factor in emergencies and mass disasters.
When inhibitors or fragmented DNA are present, many laboratories repeat the extraction process using a different method to enhance the removal of these compounds. If the extracted DNA is insufficient in quantity or highly fragmented, a new extraction is performed with a larger sample quantity or an alternative segment to obtain higher-quality DNA. For unique or limited samples, concentration methods are often employed, although these can also concentrate inhibitors and may not resolve issues related to DNA fragmentation.
In such cases, analysts may adjust amplification parameters, such as increasing the DNA template quantity for the reaction (if possible) or the number of PCR cycles. Adjustments to electrophoresis parameters, such as extending injection times, are also implemented to detect as many alleles as possible. The use of amplification systems with small genetic markers is particularly effective for samples with highly fragmented DNA. Many laboratories adopt a combination of amplification systems that include varying distributions of miniSTRs (≤300 bp) to increase the likelihood of obtaining more alleles.
However, the primary drawback of this strategy is the need for multiple amplifications, which increases analysis time, consumes more of the sample, and does not guarantee successful results. Additionally, in catastrophic events, familial grouping is often required because direct parent-child comparisons may not always be possible. In such cases, claims are often made by relatives such as siblings, half-siblings, or grandparents, necessitating the use of lineage markers to establish these relationships. This, in turn, increases the number of analyses needed to resolve the case.
In February of this year, Chile experienced a massive wildfire, one of the largest disasters in the country’s history. The event affected 15,500 homes, burned 8,500 hectares, and resulted in 135 fatalities. The Forensic Genetics Unit (UGF-V) of the Servicio Médico Legal of Valparaíso (Chile’s Legal Medical Service) was tasked with analyzing complex samples derived from victims’ remains and assisting 117 affected family members. Due to the significant degradation of the samples, many required re-extraction and/or re-amplification, with some undergoing analysis using all available amplification systems. This extended the processing time and delayed the return of the victims’ remains.
Recently, Promega® introduced the PowerPlex 35GY® (35GY) amplification system, with eight-color detection. A key advantage of this system is the reduction of the size of 22 autosomal STR loci to less than 300 bp, which increases the likelihood of detecting more alleles in degraded samples during a single analysis. Additionally, the 35GY system includes 11 Y-STR loci amplified in the same PCR reaction, facilitating lineage studies for familial determinations and significantly reducing processing time in both amplification and detection.
The 35GY system is compatible with the Spectrum Compact Capillary Electrophoresis (SCCE) equipment, capable of detecting eight colors. This instrument was introduced to our laboratory as a demonstration unit to evaluate the performance of the 35GY system. The SCCE shares similarities with ThermoFisher® ABI devices commonly used in forensic laboratories, minimizing training time and ensuring compatibility with most amplification systems already in use at our unit.
Materials and Methods
Preliminary Analysis
Residual DNA extracts from samples collected during the wildfire in Valparaíso, previously analyzed in our laboratory, were utilized. These DNA extracts were obtained from fluid samples, coagulated blood, tissues, and cartilage fragments using the Maxwell FSC system in combination with the Maxwell FSC DNA IQ™ Casework Kit. Soft and semi-soft tissue samples were pretreated with the Casework Extraction Kit, following the manufacturer’s instructions for both systems (Promega). Liquid blood samples from family members were fixed on autolytic cards and amplified using the VeriFiler®Express system (VFE, Applied Biosystems) or the F6C system. Blood samples from deceased individuals, fixed on filter paper, were pretreated with PunchSolution® (Promega Corp.) or extracted using the Maxwell FSC system and subsequently amplified using F6C. The resulting DNA extracts were quantified via qPCR using the ABI-7300 system (ThermoFisher®) and the QuantiFiler DUO system, in accordance with the manufacturer’s instructions. DNA concentrations ranged from 3 ng to below 0.023 ng/μl, the quantification limit of our laboratory. Previously, DNA extracts were amplified using the PowerPlex® Fusion 6C (F6C), PowerPlex® Y23 (Y23) (Promega), GlobalFiler® (GF), and MiniFiler® (MF) (Applied Biosystems) systems, depending on case requirements. Fragment detection was performed via capillary electrophoresis using the POP-4® polymer in an ABI-3500 genetic analyzer (Applied Biosystems). Fragment analysis was conducted with GeneMapper ID-X v1.4 software.
Analysis with 35GY
Our laboratory analyzed DNA extracts from nine samples collected during the wildfire, which had shown highly degraded profiles in preliminary analyses conducted during the emergency. These extracts, with a concentration of 0.2 [ng/μl], were stored at -20°C. Additionally, frozen tissue remnants from one of the emergency cases were preserved, and DNA was extracted from these using the Maxwell FSC system with the Casework Extraction Kit and DNA IQ™ Casework Kit cartridge. The extract was quantified using the QuantiFiler DUO kit and QuantStudio 5 equipment. The extracts were amplified using the F6C and 35GY systems, both at 30 PCR cycles. Fragment detection was performed with the SCCE equipment, adhering to the manufacturer’s instructions and the predefined run module settings for the F6C and 35GY systems: injection voltage of 1.5 kV, injection time of 9 seconds, running voltage of 13 kV, running temperature of 60°C, and a run duration of 1930 seconds. Allelic determination was carried out using a trial version of the GeneMarker HID v3.2.0 software, with a detection threshold set at 50 RFU.
Concordance Analysis
A small concordance test between the F6C and 35GY systems was conducted using SCCE equipment to analyze 10 known-profile samples from laboratory staff, fixed on autolytic cards. Amplification and electrophoresis were performed following the manufacturer’s recommended conditions, with 26 PCR cycles for the 35GY system. F6C amplification was carried out under the system’s internal validation conditions, using the same number of PCR cycles as 35GY.
Figure 1: (Top) Amplification of the STR markers Penta D and SE33 for sample No. 93 using the F6C system. A significant imbalance is observed in the Penta D marker (34%), along with allelic loss in the SE33 marker. (Bottom) Amplification of the STR markers Penta D and SE33 for sample No. 93 using the 35GY system. Balanced signals are observed in the Penta D marker (80%). For the SE33 marker, a heterozygous genotype is observed, recovering allele 21.2, which was lost in the amplification with the F6C kit.
Sample 118: Allelic loss was observed in the TPOX marker, and amplification failure occurred in the D22S1045 marker with the F6C system, with signals below the detection threshold (<175 RFU). For the TPOX marker, genotype 11 was detected, although a faint signal below the threshold suggested the sibling allele. No alleles were detected for D22S1045. Recovery of both markers was achieved using the GF system, yielding genotypes 10–11 for TPOX and 15–16 for D22S1045. These results were corroborated with the 35GY system (Figure 2).
Figure 2: Amplification of the STR markers TPOX and D22S1045 for sample No. 118 using the F6C system (top left) and the GF system (top right). Allelic losses are observed in both markers with the F6C system, whereas amplification is observed for both STRs with the GF system, due to its generation of smaller amplicons. (Bottom) Amplification of the STR markers TPOX and D22S1045 for sample No. 118 using the 35GY system. Balanced allelic amplification is observed for both markers. Red arrows indicate the fragment sizes obtained.
Sample 128: Significant amplification imbalance was observed in the CSF1PO, TPOX, and SE33 markers with the GF system, resulting in partial allelic loss. Concentrating the sample and using the maximum allowable input volume partially resolved this issue. In contrast, the 35GY system successfully corroborated genotypes for all markers without any allelic losses or prior sample concentration (Figure 3).
Figure 3: (Top) Amplification of the STR markers CSF1PO, TPOX, and SE33 for sample No. 128 using the GF system with the total sample volume available for the reaction (15 μl). Allelic signals in the SE33 marker appear poorly defined, and a signal imbalance (60%) is observed in the TPOX marker. (Bottom) Amplification of the STR markers CSF1PO, TPOX, and SE33 for sample No. 128 using the 35GY system with 1 ng of DNA for the reaction. Allelic signals appear balanced for all markers, with signal heights exceeding those achieved with other systems.
Sample 134: A genotypic inconsistency was observed between the GF and VFE systems compared to the F6C system for the TPOX marker. AB systems detected an off-ladder microvariant, designated as 8.2 following Butler’s recommendation (Forensic DNA Typing, Second Edition, Elsevier, 2005), resulting in the genotype 8.2–12. This microvariant was transmitted to the deceased’s descendant and observed in the son’s sample with genotype 8.2–10. The F6C system failed to detect the microvariant, resulting in a null allele with genotype 10–10. The 35GY system resolved the discrepancy by detecting the null allele as allele 8, yielding the genotype 8–12 in the deceased’s sample (Figure 4).
Figure 4: (Top left) Amplification of the TPOX marker using the GF system for sample No. 134, and (top right) using the VFE system for the sample from the son. The allele designated as OL (Off Ladder) is observed, which was later assigned as 8.2. (Bottom left) Amplification of the TPOX marker using the F6C system for the son's sample, and (bottom right) using the 35GY system for sample No. 134. Note that the allelic loss observed with the F6C system is recovered with the 35GY system, which was also able to assign the observed microvariant in the sample as allele 8.
Sample 161: This sample exhibited significant degradation, with allelic loss above 300 bp complicating analysis of larger markers. The F6C and GF systems detected 11 of 24 and 13 of 21 markers, respectively (Figure 5). Using the MF system, all eight markers were detected, yielding a partial profile for 20 of 23 available markers. With the 35GY system, 18 of 23 markers were detected in a single amplification, recovering the Penta E marker, which failed with other systems. Markers D7S820, CSF1PO, and D5S818 failed amplification with 35GY but were successfully amplified with MF (D7S820 and CSF1PO) and GF (D5S818). No peak height imbalances or allelic losses were observed with the 35GY system (Figure 6).
Figure 5: Amplification of sample No. 161 using the F6C system. Significant DNA degradation is observed, with allelic losses in most markers larger than 300 bp.
Figure 6: Amplification of sample No. 161 using the 35GY system. Allelic signal recovery is observed, generating a profile with 18 out of 23 markers in a single reaction.
Discussion
The 35GY amplification system has proven to be a valuable tool for analyzing complex samples with high levels of degradation. Its configuration, using mini-STRs, enabled the detection of more alleles compared to previous analyses with F6C and GF systems. STRs larger than 350 bp in the F6C and GF systems frequently exhibited amplification issues, including inter- and intra-locus imbalances, allelic loss, or amplification failures, resulting in incomplete or challenging-to-interpret profiles. This observation is consistent with the performance of the MF system, which uses STRs smaller than 300 bp and achieved 100% allele detection. The 35GY system significantly reduced these issues, recovering allelic signals in some samples and improving quality parameters in most cases.
Additionally, signals obtained with the 35GY system were generally stronger than those observed with other systems, eliminating the need for duplicate analyses to confirm signal authenticity. Notably, the 35GY system avoids the need for multiple amplifications with different kits to maximize allele detection, reducing the risk of sample exhaustion. This was demonstrated with sample 161, where an incomplete profile of 18 markers was obtained in a single amplification, nearly matching the total alleles detected using three separate systems. All signals detected with 35GY exceeded 175 RFU, the internal validation threshold, although the default detection method in GeneMarker v3.2.0 uses a 50 RFU limit.
The 35GY system also demonstrated versatility by successfully amplifying degraded DNA samples and reference samples fixed on autolytic cards, a capability shared by most Promega systems. This feature reduces laboratory processing time. Another advantage is its ability to amplify autosomal and Y chromosome markers simultaneously, saving time and costs by combining both analyses into a single reaction and run. Although Y chromosome markers were not analyzed for problem samples (as all were from female victims), 100% concordance with the PowerPlex Y23 system was observed for shared markers in male laboratory staff samples.
The 35GY system also accurately detected and assigned a previously undetected microvariant that was designated as off-ladder by other systems. This suggests a potential primer-binding site mutation in the F6C system for this marker, supported by the smaller amplicon size observed with the 35GY system. Our laboratory is currently sequencing this fragment to confirm the true genotype.
At present, the 35GY system operates exclusively with Spectrum Compact equipment due to its eight-color configuration. This equipment is user-friendly, offering functionality similar to AB devices commonly used in forensic laboratories. Its intuitive interface ensures seamless operation. Results showed no artifacts from electrophoretic failures, and data generated with the SCCE was compatible with GeneMapper ID-X, ensuring excellent compatibility with existing workflows.
Data analyzed with GeneMarker v3.2.0 included all features available for Promega systems. This user-friendly software provided helpful tools such as validation assistants and kinship determination capabilities, though these were not tested. Furthermore, data from AB equipment can also be analyzed with this software, enhancing its utility across platforms.
Conclusions
The PowerPlex® 35GY amplification system, in combination with the Spectrum Compact capillary electrophoresis equipment, proved to be a versatile and highly effective system for analyzing complex samples, such as those obtained from the Valparaíso wildfire, as well as reference samples. This system demonstrated concordance with other amplification systems for both autosomal markers and Y chromosome markers. Its configuration of smaller STRs showed significant comparative advantages over the other systems used in our unit (GlobalFiler®, PowerPlex® Fusion 6C, and MiniFiler®), providing equal or greater genetic information than the other systems, whether individually or combined.