Using Complementary STR Typing Systems to Genotype Problematic Samples

Publication Date: 2014

Forensic and paternity labs make great efforts to ensure that their results are accurate and objective; this includes extensive training of lab members, validation, certification and accreditation. Commercial suppliers of STR typing kits perform thorough developmental validations and concordance studies to show that their kits are robust and yield the correct DNA profile. However, despite these precautions, amplification failures and discordance can happen due to various factors such as genetic variation, mutation and insufficient resolution of microvariant alleles. There are several approaches to ensure that the correct allele calls are made in these cases, including DNA sequencing, the use of multiple STR typing kits to circumvent problems due to primer-binding-site and other mutations, and optimizing capillary electrophoresis (CE) conditions or instrumentation to improve resolution and the accuracy of allele calls. A recent paper published in Forensic Science International: Genetics ⁽¹⁾ describes the approach taken by members of the Division of Identification and Forensic Science at the Israel Police to resolve three types of problematic situations encountered during their recent adoption of the PowerPlex® ESI 16 System: failure to amplify alleles (null alleles), the presence of extreme variants and insufficient resolution of alleles during CE. The authors considered several possible approaches to resolve these anomalies and found that they could clarify ambiguous results by adopting complementary STR typing kits, which amplify the same loci but have different configurations. Specifically, they used the PowerPlex® ESI and ESX 16 Systems, which yield amplicons of different sizes (i.e., loci amplified as large amplicons with one system tend to be amplified as smaller amplicons with the other system) and use different primer pairs for most loci.

One of the challenges encountered by the lab was apparent null alleles, presumably due to mutations in the primer-binding site. One example involved casework samples from a hit-and-run accident that killed a mother and daughter. PowerPlex® ESI 16 amplifications of some samples in the case resulted in a homozygous result at D1S1656 (11,11), with a minor peak below threshold at 18.3, but amplification of other samples yielded a clear heterozygous result of 11,18.3. However, these results did not meet the lab’s balance criteria for the sample to be called a heterozygote. To resolve the ambiguity, analysts re-amplified the samples using the complementary PowerPlex® ESX 16 System. These amplifications produced an 11,18.3 heterozygous result, leading the analysts to believe that an inherited mutation at one of the D1S1656 primer-binding sites for the PowerPlex® ESI 16 System caused the failed amplification of the 18.3 allele.

The authors also described examples of extreme variants, which are allele variants that are so long or short that they do not migrate within the confines of the locus. These extreme variants are very rare and so are not included in allelic ladders of commercial STR kits. Their presence can complicate data interpretation because it can be difficult to assign the allele to the appropriate locus. Several examples of extreme variants were given, including a previously undescribed 7 allele at the D1S1656 locus, a 28 allele at D12S391 and a 29 allele at D2S1338. The D1S1656 7 allele was observed in three different samples as an off-ladder (OL) allele that migrated between the D18S51 and D1S1656 loci in three different samples amplified using the PowerPlex® ESI 16 System. Since D18S51 had two alleles in addition to the OL allele, the authors suspected that this OL allele really belonged to the D1S1656 locus, which exhibited a single allele. Using the complementary PowerPlex® ESX 16 System, where D1S1656 borders D10S1248 instead of D18S51 (Figure 1), the authors were able to show that the OL allele was associated not with D18S51 but with the D1S1656 locus. Extrapolating from the smallest allele in the D1S1656 allelic ladder, the authors identified this OL allele as a 7 allele at D1S1656. In these cases, analysts were able to flag the triallelic D18S51 pattern for additional scrutiny and determine the true genotype. In two other examples of extreme variants, the complementary kits were used to correctly assign a 28 allele at D12S391 and 29 allele at D2S1338 to the appropriate loci.

In the third and final example of situations that benefited from the use of complementary STR typing kits, observant lab members noticed that the base of some peaks were wider than normal and had a characteristic and asymmetric “ski slope” slant, where the area under the peak was skewed to one side. Upon further investigation, analysts determined that these peaks represented two alleles that differed by only one base pair and were not resolved sufficiently by their CE instrumentation. The data analysis software treated this as a single peak and, as a result, called only one of the two alleles. This phenomenon seemed to be more common at longer loci within mixture samples. Their first attempts to resolve the microvariant involved reducing the peak window size in GeneMapper® ID-X software and using a different CE platform—the Applied Biosystems® 3500xL Genetic Analyzer. When both of these approaches failed to distinguish the microvariant, lab members amplified these samples using the complementary PowerPlex® ESX 16 System, where the problematic alleles would be amplified as smaller alleles. When amplified as a smaller locus, the microvariant was separated more readily and was called correctly.

Summary

This Forensic Science International: Genetics article highlights the importance of constant vigilance in the lab to flag any anomalies or irregularities and identify samples that require additional scrutiny. For these challenging samples, the use of complementary STR typing kits often can resolve ambiguous results and confirm the true genotype. Although not discussed in the paper, the ability to re-amplify larger loci with a second, complementary kit to yield smaller amplicons also can be of value when analyzing degraded samples to maximize recovery of allelic information from as many loci as possible.

Article References

1. Oz, C. et al. (2013) Recognizing the advantage of employing the PowerPlex® ESI and PowerPlex® ESX complementary kits for purposes of clarification in routine casework. Forensic Sci. Int. Genet. 7, 461–6.

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