Development and validation of a next generation STR ESS-pentaplex

1. Introduction 

Two problems regularly confront forensic DNA analyses with the routine use of standard STRs: insufficient discrimination power and presence of highly degraded DNA where locus and allele drop-out can lead to complex interpretative problems. Success with highly degraded DNA is improved using short amplicon mini-STRs. We decided to develop a bolt-on STR pentaplex of five new loci, recommended as next generation markers for the European Standard Set (ESS) in order to generate allele frequency data ahead of the release of ESS kits. The ESS-pentaplex comprises two tried and tested STRs: D12S391 and D1S1656, that are highly informative but with conventional amplicon lengths, plus three mini-STRs: D2S441, D10S1248 and D22S1045 typed with amplicon size ranges 74–135bp. Space exists in this multiplex amongst the fragment sizes and green/yellow dye labels to allow additional STRs to be included in future. As part of the validation of the ESS-pentaplex we assessed its ability to amplify DNA from a range of degraded casework samples including hairs, bones, nails and washed bloodstains. In routine forensic use the ESS-pentaplex provided a valuable additional approach for the analysis of challenging DNA, even when some standard STRs in commercial kits failed or were too weak.

2. Materials and methods 

As commercial primer designs for the five new ESS STRs are not published we used our original primers for D1 and D12 [1], [2] together with those detailed in STRbase from the original developers for D2, D10 and D22 [3]. Amplicon sizes, primer sequences and dye labels are outlined in Table 1. These show that sufficient space exists for inclusion of additional informative STRs such as SE33 or D9S1120 [4] labeled with NED or VIC. For each STR reference ladders were constructed from sequenced alleles using standard procedures as previously described [4].

Table 1. PCR primer designs, dye labels and amplicon sizes of the pentaplex STRs. Observed (obs.) allele sizes obtained from an AB 3730xl and POP7.

	STR		Dye	PCR primer sequence	Obs. repeat numbers	Obs. sizes	Actual sizes
	D10S1248	F	6-FAM	TTAATGAATTGAACAAATGAGTGAG	8	79	82
		R		gCAACTCTGGTTGTATTGTCTTCAT	19	123	126
	D1S1656	F		GTGTTGCTCAAGGGTCAACT	8	131	135
		R	6-FAM	ctctctctctctctccttGAGAAATAGAATCACTAGGGA	19.3	181	182
	D12S391	F		AACAGGATCAATGGATGCAT	12	194	197
		R	6-FAM	TGGCTTTTAGACCTGGACTG	27.2	261	259
	D2S441	F	VIC	CTGTGGCTCATCTATGAAAACTT	8	74	77
		R		gAAGTGGCTGTGGTGTTATGAT	17	112	113
	D22S1045	F	NED	ATTTTCCCCGATGATAGTAGTCT	9	105	106
		R		CGGCACAGTGTGAGTGATCAC	19	135	136

The quality of results obtained from challenging forensic material was evaluated by assessing the relative performance and locus drop-out of STRs in partial profiles measured as percentage genotyping success. Detectable peaks below a prescribed minimum signal of 100 RFU were also recorded.

3. Results and discussion 

The percentage genotyping success rates observed in 49 challenging casework samples for Identifiler, MiniFiler and the ESS-pentaplex are summarized in Fig. 1. Although this study examined a wide range of degraded forensic material, the three multiplexes showed a consistent pattern of relative success. The ESS-pentaplex showed an average 97.6% success (94.7% when peaks below 100 RFU were excluded); MiniFiler an average 89.8% (88.0%) and; Identifiler 81.5% (80.8%). Clearly calculating success for the small-scale pentaplex is not completely comparable to larger multiplexes, but the limited number of PCR components benefits performance and the pentaplex is an informative supplement to either Identifiler or MiniFiler with better overall chance of success. It is interesting to note that the only ESS-pentaplex STR showing locus drop-out (8%) was D10S1248, while both normal-amplicon STRs worked almost as well as the other two mini-STRs that showed complete success with all material genotyped.

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Fig. 1. Percent genotyping success for three forensic multiplexes (Identifiler, 15 STRs; MiniFiler, 8; ESS-pentaplex, 5) in 49 challenging casework samples. Multiple overlaying points shown as grey (black if 0%). Points with dark outlines show success when excluding genotype peaks below a prescribed minimum 100RFU.

4. Conflict of interest statement 

None.