Sequence investigation of 34 forensic autosomal STRs with massively parallel sequencing

Suhua Zhang,Xiling Liu,Hancheng Zheng,Chengtao Li,Yingnan Bian,Yun Bao,Rixia Dong,Chao Jin,Yong Niu

doi:10.1038/s41598-018-24495-9

Abstract

STRs vary not only in the length of the repeat units and the number of repeats but also in the region with which they conform to an incremental repeat pattern. Massively parallel sequencing (MPS) offers new possibilities in the analysis of STRs since they can simultaneously sequence multiple targets in a single reaction and capture potential internal sequence variations. Here, we sequenced 34 STRs applied in the forensic community of China with a custom-designed panel. MPS performance were evaluated from sequencing reads analysis, concordance study and sensitivity testing. High coverage sequencing data were obtained to determine the constitute ratios and heterozygous balance. No actual inconsistent genotypes were observed between capillary electrophoresis (CE) and MPS, demonstrating the reliability of the panel and the MPS technology. With the sequencing data from the 200 investigated individuals, 346 and 418 alleles were obtained via CE and MPS technologies at the 34 STRs, indicating MPS technology provides higher discrimination than CE detection. The whole study demonstrated that STR genotyping with the custom panel and MPS technology has the potential not only to reveal length and sequence variations but also to satisfy the demands of high throughput and high multiplexing with acceptable sensitivity.

Highlights

Short tandem repeats (STRs) are the most widely used polymorphism markers in forensic community[1,2]
We explored the Massively parallel sequencing (MPS) performance from sequencing reads analysis, concordance study and sensitivity testing, to document the performance capabilities and limitations of the custom panel
Libraries of them were pooled for two separate emulsion PCR (emPCR) and corresponding emPCR products which correspond to the library dilution point of 17% and 23% of template Ion Sphere Particles (ISPs) were sequenced on individual Ion 314 Chips

Summary

Results and Discussion

The custom panel allows for simultaneously detection of up to 34 polymorphic forensic autosomal STRs and the sex determination locus of Amelogenin. With the allele sequencing reads, we used the averaged values of Doc and ACR to evaluate the performance of the 34 STRs. Figure 2A illustrates the Doc information, while Fig. 2B shows the ACR values from the observed heterozygous balance at the 34 STRs. The mean DoC for the 34 loci ranged from a low value of 1144x ± 576.5 at D19S433 to a high value of 3284x ± 1163 at D14S1434. By Fisher’s exact test, no significant differences in ACR values (p = 0.1011) at the 34 STRs between each replicate sequencing were observed, indicating the variations of heterozygotes performance with different runs can be ignored. A further concordance study was performed between CE genotypes and MPS data for all 200 individual samples and all 34 STRs, resulting in the evaluation of 6800 loci. STR D1S1677 D1S1656 TPOX D2S441 D2S1776 D2S1338 D3S1358 D3S4529 D4S2408 FGA D5S2500 (AC008791) D5S818 CSF1PO D6S1043 D6S474 D7S820 D8S1179 D9S1122 D10S1248 TH01 vWA D12S391 D12ATA63 D13S317 D14S1434 Penta E D16S539 D17S1301 D18S51 D19S433 D20S482 D21S11 Penta D D22S1045

Allele Number PIC

Methods

Additional Information