As the forensic utility of STRs gained prominence, global efforts toward standardization were initiated. The FBI introduced a set of 13 core loci in 1998 for the Combined DNA Index System (CODIS), later expanded to 20 loci in 2017, incorporating markers with higher polymorphism and reduced stutter. Likewise, the European Standard Set (ESS) initially comprised 7 loci and was later extended to 12, including highly discriminative markers like SE33 and D1S1656.

However, the CODIS loci are primarily optimized for North American populations, while the ESS targets European subpopulations. These standardized sets may not account for the genetic diversity found in other populations with complex structures, such as endogamous groups. This lack of representation can lead to reduced discriminatory power or inaccurate interpretations (Majumder, 2010). It remains surprising that, despite India's effective use of DNA technology since its inception in 1985, there has been minimal progress in identifying a standardized set of genetic markers tailored to its diverse and intricately structured population—particularly across the many endogamous groups.

Loci Selection: The effectiveness of a multiplex system depends on the number, polymorphism, and mutual compatibility of selected loci. Loci should exhibit high heterozygosity, low stutter rates, and minimal mutation. Modern multiplexes expand beyond traditional loci, including additional autosomal STRs, mini-STRs for degraded DNA, and Y-STRs for analyzing low-level male DNA (Butler and Hill, 2012).

Primer Design: Primers should have similar GC content and annealing temperatures to ensure specific amplification. They must avoid non-specific binding, secondary structures, or dimer formation (Vallone and Butler, 2004). Optimal primer concentration is essential for peak balance, and primers are often labeled with distinct fluorescent dyes for capillary electrophoresis (Butler, 2005; Bustin et al., 2020).

PCR Optimization: Efficient amplification requires fine-tuning parameters such as annealing temperature, denaturation time, primer and Mg2+ concentrations, and extension times (Roux, 2009; Lorenz, 2012). Additives like glycerol, DMSO, or formamide enhance polymerase stability and primer binding (Markoulatos et al., 2002; Mandaliya and Thaker, 2024). Techniques like Hot Start and Touchdown PCR improve specificity by preventing non-specific amplifications (Lebedev et al., 2008; Don et al., 1991).

Amplicon Size Reduction and Mini-STRs: Degraded DNA often inhibits the amplification of longer STR regions. Mini-STRs minimize the flanking region, producing shorter amplicons (70–150 bp) that are more likely to amplify successfully (Coble and Butler, 2005). This approach improves peak balance and reduces allele dropout, especially in challenging forensic samples (Bogas et al., 2016; Thakar et al., 2020). Most of the new generation multiplex systems are equipped with more markers in this range.

Modern commercial kits capable of amplifying more than 20 STR loci are widely used worldwide, enhancing cross-border forensic cooperation. Fifteen loci are commonly shared across most systems, including D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, and vWA, facilitating international compatibility and improved data exchange.

The global adoption of commercial short tandem repeat (STR) amplification kits capable of targeting over 20 loci has significantly enhanced the interoperability of national DNA databases. These advanced kits improve cross-border compatibility, facilitating international collaboration in forensic investigations. Currently, a set of 15 STR markers is commonly found across most kits employed worldwide, promoting standardization in forensic DNA profiling. These loci include: D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D10S1248, D12S391, D16S539, D18S51, D19S433, D21S11, D22S1045, FGA, TH01, and vWA.

False Tri-Allelic Pattern at D7S820 ( Kaitholia et al,2020)

In this reported instance, analysis with the GlobalFiler™ kit produced what appeared to be a tri-allelic D7S820 genotype (10, 11, 14.1). Subsequent investigation revealed that the extra peak (14.1) actually originated from a short SE33 allele whose amplicon size fell within the D7S820 allelic bin window, creating a false tri-allelic pattern.

Initial Observation

• Using GlobalFiler™ PCR Amplification Kit, capillary electrophoresis showed three peaks at D7S820: 10, 11, and 14.1.
• A single SE33 peak (allele 8) was also present, plus an additional off-marker peak just before the SE33 range.
• The mysterious 14.1 peak co-migrated within D7S820’s bin range, mimicking a third D7S820 allele.

Confirmation by Alternative Kits

The same DNA sample was re-typed with two other widely used multiplex systems. The alternative multiplex systems used were AmpFLSTR™ Identifiler™ Plus kit (Thermo Fisher Scientific) and PowerPlex® Fusion 6C (Promega). The obtained allele calls on locus D7S820 and SE33 were as follows:

OMR denotes an off-marker region peak later attributed to SE33. Only GlobalFiler™ misassigned the SE33-derived peak to D7S820.

• Bin Overlap: In six-dye multiplex designs, SE33 and D7S820 amplicons lie adjacent in the same dye channel.
• Virtual Bins: SE33 microvariant (1.2 repeat) fell within the predefined fragment-size window (“allelic bin”) for D7S820 in GlobalFiler™, but not in the other kits.
• Allelic Ladder Limitations: The allelic ladder lacked representation of that SE33 microvariant, so GeneMapper™ auto-binned the peak to D7S820.

1. Compromise kinship and paternity inferences.
2. Lead to erroneous database entries or match exclusions.

1. Use multiple STR kits with different dye-set layouts to confirm unexpected peaks.
2. Manually inspect off-marker peaks and compare fragment sizes against neighboring loci.
3. Adjust allelic bin ranges or update virtual-bin configurations when new microvariants are discovered.
4. Sequence ambiguous peaks to verify locus assignment, especially for rare microvariants.
5. Keep an up-to-date STRBase variant table, noting any overlapping fragment-size ranges.

• Labs should review recent reports of other locus overlaps (e.g., D1S1656 into D2S441) and incorporate those insights into internal SOPs.
• When designing new multiplexes, strategically space amplicons and dye channels to minimize adjacent-locus size conflicts.
• Emerging software tools allow in silico bin optimization, which can preemptively catch potential overlaps before validation.