According to sequences from the dbSNP database, X-InDel markers were selected to meet the following criteria: (1) the difference of allelic X-InDel fragment in length, the inserted or deleted base number in other words, ranged from 1 to 25 bp; (2) all loci were located in introns; (3) the minor allele frequency (MAF) varied from 0.4 to 0.5 in East Asians; and (4) all loci comply with the Hardy–Weinberg equilibrium in the Chinese population. Finally, we got 38 optimal X-InDel markers, among which four multi-InDel markers (rs59605609, rs79829945, rs143123845, and rs35574346) with more than two alleles were involved in order to improve the discrimination power. Besides, the amelogenin gene was added for the design of a sex-confirmation marker. The information about the 38 selected X-InDel loci and amelogenin marker are shown in Table 1.

The Polymerase Chain Reaction (PCR) primers corresponding to the InDel loci and amelogenin locus were filtered and obtained using the Oligo 6.0 software (Premier Biosoft International, Palo Alto, CA, United States). All primers were chosen based on the following requirements: (1) primer pairs had similar melting temperatures (Tm) to be amplified at a similar efficiency, (2) without self-complementarity, hairpin structure, and non-specific amplification, (3) BLAST was used as an alignment algorithm to design target-specific primer sequences, and (4) SNP locations were excluded in primer sequences for a successful amplification experiment.

After optimization, PCR amplification was performed in a 25.0-μl reaction volume comprising 10.0 μl of 2.5 × PCR buffer mixture (consisting of 125 mmol/L Tris-HCl buffer, 125 mmol/L KCl, 7.5 mmol/L dNTPs, 5 mmol/L MgCl₂, 2 mg/ml BSA, and 1% PCR intensifier), 5.0 μl of 5 × primer mix, 1.0 μl of 5 U/μl heat-activated Taq polymerase (AGCU ScienTech Incorporation) and 0.5–2 ng of template DNA. sdH₂O was adjusted to keep the total volume at 25.0 μl.

The PCR cycling conditions were as follows: initial denaturation at 95°C for 2 min, annealing elongation of 94°C for 30 s, 58.5°C for 1 min, and 72°C for 1 min for 29 cycles, final extension of 72°C for 10 min, and then hold at 4°C.

Following amplification, 1 μl of amplified product was added to 10 μl of Hi-Di^TM formamide and 0.3 μl of AGCU Marker SIZ-500 size standard, followed by being centrifuged for 3 min at 3,000 rpm. The mixture was denatured at 95°C for 3 min and then chilled on ice for 3 min. Subsequently, using the Applied Biosystems 3130XL Genetic Analyzer, fragments were separated with filter of E5 and polymer of POP-4 (Thermo Fisher Scientific, South San Francisco, CA, United States) by capillary electrophoresis, under the following parameters: injection at 2 kV for 10 s and electrophoresis at 15 kV for 1,500 s, 60°C. The raw CE data were genotyped by GeneMapper IDX 1.3 (Life Technologies).

All experiments were performed according to the recommendations for the use of nomenclature and the guidelines for quality control as well as statistical analysis proposed by the SWGDAM and the International Society of Forensic Genetics (ISFG) (Schneider, 2007).

As shown in Figure 1, complete profiles were acquired when the template DNA concentration was higher than or equal to 62.5 pg. Allele dropouts were observed when template DNA inputs were further reduced to 31.25 pg. Therefore, the sensitivity of AGCU X-InDel 38 kit is 62.5 pg/10 μl.

The results of species specificity testing showed that no specific amplification products were observed (Supplementary Figure 1). Therefore, the genotyping results of AGCU X-InDel 38 kit were not affected by other common non-human DNA.

The results indicated that consistent allele calls were obtained consistently for all studied samples. The electropherograms of allelic ladder from three independent labs are shown in Supplementary Figure 2.

Figure 2 shows that complete profiles were obtained when amounts of inhibitors were not higher than 50 μmol/L hematin, 100 μmol/L hemoglobin, 16 mmol/L indigo, 50 mg/L of humic acid, 1.2 mmol/L EDTA, or 1.6 mmol/L calcium ion. Furthermore, once the amounts of inhibitors increased to 75 μmol/L hematin, 150 μmol/L hemoglobin, 20 mmol/L indigo, 75 mg/L humic acid, 1.5 mmol/L EDTA, and 2.0 mmol/L calcium ion, allele dropouts were observed. Those results revealed that this X-InDel system could tolerate considerable concentrations of six common inhibitors.

As shown in Supplementary Table 1, the odds of obtaining a more complete DNA profile were found to be greater using AGCU X-InDel 38 kit than that using AGCU X19 STR kit. For example, complete profiles were obtained using AGCU X-InDel 38 kit from two samples of a 13-year-old bloodstain, while AGCU X19 STR kit only detected 10, 14 of 19 loci, respectively. Additionally, for those specimens that had been fixed in formalin for a long time, the AGCU X-InDel 38 kit was capable of getting complete profiles successfully, but the AGCU X19 STR kit could not. However, as for muscular tissue fixed in formalin, ratios of detected loci for both AGCU X-InDel 38 kit and AGCU X19 STR kit decreased significantly until immersion time ≥12 days. Supplementary Figure 3 demonstrates the electropherograms obtained from the tissues fixed for a different time by using the two kits. The results have shown the superiority of the novel X-InDel kit to genotype severely degraded DNA in forensic casework.

Ratios of the peak height of the alleles were all greater than 80% for each heterozygous locus. In addition, the peak height ratios of different loci within the same dye channel were 26, 45, 34, and 27% for blue, green, yellow, and red dye, respectively. Average RFU value for the blue dye was 5,379, 4,444 for green dye, 3,990 for yellow dye, and 3,303 for red dye.

The results showed that the largest standard deviation observed was 0.061283 bases for rs3859989, and the lowest was 0.004714 bases for rs2308033 (Figure 3), which were well below the threshold size of 0.15 bases. Overall, the AGCU X-InDel 38 kit showed high allele size accuracy in allele detection.

The allele frequencies and forensic statistical parameters for AGCU X-InDel 38 kit are provided in Figure 5 and Supplementary Tables 2, 3. The PIC ranged from 0.03843168 at rs16368 locus to 0.546322699 at rs59605609 locus. The rs16368 locus had a minimum GD value of 0.039396985, and the rs59605609 locus had a maximum value of 0.626683417. Additionally, the pairwise LD analysis was conducted to reveal allelic association between 38 loci, and a total of 703 pairs were tested. After Bonferroni correction for multiple testing was applied to adjust threshold p-value (p ≤ 0.05/703), significant LD was observed at the following 12 pairs: rs11277082–rs16368, rs16637–rs2308033, rs1160845–rs25581, rs2307707–rs3048996, rs10671504–rs3077884, rs3215490–rs36094418, rs17394–rs4030406, rs2308280–rs57843641, rs45449991–rs58595330, rs143123845–rs59605609, rs363794–rs60283667, and rs35954471–rs71671860. The existence of LD may be the results of close physical location, since all markers are located on the same chromosome. In addition, population events like drift, selection, non-random mating, stratification, or admixture may also lead to LD (Hedrick, 1987; Medina-Acosta, 2011). The LD indicated the existence of non-random association of alleles of those pairwise loci existing at population-level. As recommended by the DNA commission of the ISFG, the pairwise loci must be analyzed together, and haplotype frequencies should be used for likelihood calculations when significant association is found among markers (Tillmar et al., 2017).

The values of discrimination power in males (PD_Male) were in the range of 0.0392 (rs16368)–0.62355 (rs59605609), while those in females (PD_Female) were in the range of 0.07609504 (rs16368)–0.78105809625 (rs59605609). The combined PD_Male and PD_Female were both more than 0.999999999. The MEC_Krüger varied between 0.01921584 and 0.3336460035 for the deficiency cases, MEC_Kishida between 0.03843168 and 0.54632269875 for the normal trios, and MEC_{Desmarais  Duo} between 0.0196000000000002 and 0.39966 for the duos. Meanwhile, the combined MECs were 0.999369481, 0.999999917, and 0.999941556, for the deficiency cases, normal trios, and duos, respectively. In this study, the combined PDs and MECs, calculated in the Guangdong Han group using AGCU X-InDel 38 kit, showed higher values than those in other X-InDel studies (Freitas et al., 2010; Pereira et al., 2012; Edelmann et al., 2016). The high forensic statistical parameters for AGCU X-InDel 38 kit demonstrated its powerful performance in human identification and kinship tests, even in some deficiency cases.