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Short tandem repeats (STRs) are the preferred genetic markers in forensic DNA analysis, routinely measured by capillary electrophoresis (CE) method based on the fragment length features. While, the massive parallel sequencing (MPS) technology could simultaneously target a large number of intriguing forensic STRs, bypassing the intrinsic limitations of amplicon size separation and accessible fluorophores in CE, which is efficient and promising for enabling the identification of forensic biological evidence. Here, we developed a novel MPS-based Forensic Analysis System Multiplecues SetB Kit of 133-plex forensic STR markers (52 STRs and 81 Y-STRs) and one Y-InDel (M175) based on multiplex PCR and single-end 400 bp sequencing strategy. This panel was subjected to developmental validation studies according to the SWGDAM Validation Guidelines. Approximately 2185 MPS-based reactions using 6 human DNA standards and 8 male donors were conducted for substrate studies (filter paper, gauze, cotton swab, four different types of FTA cards, peripheral venous blood, saliva, and exfoliated cells), sensitivity studies (from 2 ng down to 0.0625 ng), mixture studies (two-person DNA mixtures), PCR inhibitor studies (seven commonly encountered PCR inhibitors), species specificity studies (11 non-human species), and repeatability studies. Results of concordance studies (413 Han males and 6 human DNA standards) generated by STRait Razor and in-house Python scripts indicated 99.98% concordance rate in STR calling relative to CE for STRs between 41,900 genotypes at 100 STR markers. Moreover, the limitations of present studies, the nomenclature rules and forensic MPS applications were also described. In conclusion, the validation studies based on ~ 2200 MPS-based and ~ 2500 CE-based DNA profiles demonstrated that the novel MPS-based panel meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities, and the STR nomenclature rules should be further regulated to integrate the inconformity between MPS-based and CE-based methods.

Short tandem repeats (STRs) are the preferred genetic markers in forensic DNA analysis, routinely measured by capillary electrophoresis (CE) method based on the fragment length features. While, the massive parallel sequencing (MPS) technology could simultaneously target a large number of intriguing forensic STRs, bypassing the intrinsic limitations of amplicon size separation and accessible fluorophores in CE, which is efficient and promising for enabling the identification of forensic biological evidence. Here, we developed a novel MPS-based Forensic Analysis System Multiplecues SetB Kit of 133-plex forensic STR markers (52 STRs and 81 Y-STRs) and one Y-InDel (M175) based on multiplex PCR and single-end 400 bp sequencing strategy. This panel was subjected to developmental validation studies according to the SWGDAM Validation Guidelines. Approximately 2185 MPS-based reactions using 6 human DNA standards and 8 male donors were conducted for substrate studies (filter paper, gauze, cotton swab, four different types of FTA cards, peripheral venous blood, saliva, and exfoliated cells), sensitivity studies (from 2 ng down to 0.0625 ng), mixture studies (two-person DNA mixtures), PCR inhibitor studies (seven commonly encountered PCR inhibitors), species specificity studies (11 non-human species), and repeatability studies. Results of concordance studies (413 Han males and 6 human DNA standards) generated by STRait Razor and in-house Python scripts indicated 99.98% concordance rate in STR calling relative to CE for STRs between 41,900 genotypes at 100 STR markers. Moreover, the limitations of present studies, the nomenclature rules and forensic MPS applications were also described. In conclusion, the validation studies based on ~ 2200 MPS-based and ~ 2500 CE-based DNA profiles demonstrated that the novel MPS-based panel meets forensic DNA quality assurance guidelines with robust, reliable, and reproducible performance on samples of various quantities and qualities, and the STR nomenclature rules should be further regulated to integrate the inconformity between MPS-based and CE-based methods.

Due to the formation of the Qiongzhou Strait by climate change and marine transition, Hainan island isolated from the mainland southern China during the Last Glacial Maximum. Hainan island, located at the southernmost part of China and separated from the Leizhou Peninsula by the Qiongzhou Strait, laid on one of the modern human northward migration routes from Southeast Asia to East Asia. The Hlai-language speaking Li minority, the second largest population after Han Chinese in Hainan island, is the direct descendants of the initial migrants in Hainan island and has unique ethnic properties and derived characteristics, however, the forensic associated studies on Hainan Li population are still insufficient.Hence, 136 Hainan Li individuals were genotyped in this study using the MPS-based ForenSeq™DNA Signature Prep Kit (DNA Primer Set A) to characterize the forensic genetic polymorphism landscape, and DNA profiles were obtained from 152 different molecular genetic markers (27 autosomal STRs, 24 Y-STRs, 7 X-STRs, and 94 iiSNPs). A total of 419 distinct length variants and 586 repeat sequence sub-variants, with 31 novel alleles (at 17 loci), were identified across the 58 STR loci from the DNA profiles of Hainan Li population. We evaluated the forensic characteristics and efficiencies of DAPA, it demonstrated that the STRs and iiSNPs in DAPA were highly polymorphic in Hainan Li population and could be employed in forensic applications. In addition, we set up three Datasets, which included the genetic data of (I). iiSNPs (27 populations, 2640 individuals), (II). Y-STRs (42 populations, 8281 individuals), and (III). Y-haplogroups (123 populations, 4837 individuals) along with the population ancestries and language families, to perform population genetic analyses separately from different perspectives.In conclusion, the phylogenetic analyses indicated that Hainan Li, with a southern East Asia origin and Tai-Kadai language-speaking language, is an isolated population relatively. But the genetic pool of Hainan Li influenced by the limited gene flows from other Tai-Kadai populations and Hainan populations. Furthermore, the establishment of isolated population models will be beneficial to clarify the exquisite population structures and develop specific genetic markers for subpopulations in forensic genetic fields.