A newly devised multiplex assay of novel polymorphic non-CODIS STRs as a valuable tool for forensic application

DNA profiling that relies on sets of highly polymorphic autosomal STR markers is widely used in the forensic field for human identification and paternity testing. However, the number of markers that are included in the STR kits that are currently available is insufficient to conclusively prove or disprove a relationship between individuals, especially when complex family scenarios are suspected or indirect analyses are required. In these cases, it becomes necessary to increase the number of loci under analysis to reach an adequate likelihood ratio (LR). In this study, we discovered 18 new autosomal non-CODIS STR loci (D1S1616, D1S1608, D2S437, D3S2457, D4S2406, D4S3249, D5S2843, D5S2501, D6S1010, D8S1039, D12S1301, D14S586, D15S815, SHGC-145653, CHLC.GATA14D12, D1S1603, HUMUT7148, and CHLC.GATA84D07) by web scanning and experimental screening. On the basis of this discovery, we developed a novel multiplex typing system named the "SiFaSTR 21plex_NCII Typing System" comprising 1) the 18 non-CODIS autosomal STRs mentioned above, 2) a CODIS locus of D2S1338, and 3) Amelogenin and DYS391. A forensic developmental validation, including sensitivity, species specificity, concordance, reproducibility, sample suitability, testing stability, and mixture testing, was performed following SWGDAM. The results of the validation studies indicated that this system is accurate, reliable and suitable for human DNA profiling. The sensitivity study of the system demonstrated that a full profile was obtainable with DNA as low as 125 pg. Species specificity was proven by the lack of cross-reactivity with a series of common animal species. The stability study demonstrated that 1 ng of control DNA could be fully genotyped with concentrations of haematin ≤ 150 μM, indigotin ≤ 5000 ng/μl, urea ≤ 16000 ng/μl, nigrosine ≤ 100 ng/μl and humic acid ≤ 20 ng/μl. In the mixture test, all of the minor alleles could be called at mixed ratios of 1:1, 1:3 and 3:1. We also investigated the allelic frequencies and forensic parameters of the included markers in 259 Chinese Han individuals. The forensic efficiency parameters, including the total power of discrimination (TDP) and the combined exclusion power in duos (CPE_duos) and in trios (CPE_trios) of the system were calculated to be greater than 0.9999999, 0.9997347 and 0.9999997, respectively. This result proved that the system is suitable for human identification and paternity testing. The 18 newly discovered non-CODIS STRs and the developed system will be a valuable supplementary tool for the forensic community and will help solve complex paternity cases, evolutionary studies and population investigations.