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 (CPEduos) and in trios (CPEtrios) 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.

On the purpose of enhancing the forensic efficiency of CODIS STR loci, new STR loci have been gradually discovered and developed into some commercial multiplex systems. Recently, 22 STR loci including 18 non-CODIS STR loci and four CODIS STR loci were investigated in 501 unrelated healthy individuals of Kazakh ethnic group. Seven to 20 alleles at the different loci were identified and altogether 276 alleles for 22 selected loci were detected with the corresponding allelic frequencies ranging from 0.0010 to 0.3623. No significant deviation was observed from the Hardy-Weinberg equilibrium test for any of the 22 STRs. The value of cumulative power of discrimination in Kazakh group was 1-1.00E-28. Analyses of population differentiations and genetic distances between Kazakh and other Chinese groups presented that the Kazakh group with the Uygur group. These 22 STR loci evenly distributed on 22 different autosomal chromosomes were characterized by high genetic diversities and therefore could be utilized in the forensic cases to further increase the discrimination performance.

Short tandem repeats (STRs) with a high level of polymorphisms and convenient detection method play an indispensable role in human population and forensic genetics. Recently, we detected the 21 autosomal non-combined DNA index system (non-CODIS) STR loci in a Kyrgyz ethnic group, calculated their forensic parameters and analysed its genetic relationships with reference populations from China. In total, 168 alleles were observed at 21 non-CODIS STRs with corresponding allelic frequencies from 0.0016 to 0.4788. No significant deviations at these STRs were observed from the Hardy-Weinberg equilibrium. The values of cumulative power of discrimination and probability of exclusion for all the 21 non-CODIS STRs were 0.99999999999999999998835 and 0.9999994002, respectively. Furthermore, the analyses of phylogenetic trees, genetic distances and interpopulation differentiations demonstrated that the Kyrgyz group had relatively close genetic relationships with the Uygur and Kazak groups. These 21 non-CODIS STRs were characterized by high genetic diversities in the Kyrgyz group and could be applied as a robust tool for individual identification and kinship testing in forensic sciences.