Population genetic analyses of Eastern Chinese Han nationality using ForenSeq™ DNA Signature Prep Kit.

Currently, the most commonly used method for human identification and kinship analysis in forensic genetics is the detection of length polymorphism in short tandem repeats (STRs) using polymerase chain reaction (PCR) and capillary electrophoresis (CE). However, numerous studies have shown that considerable sequence variations exist in the repeat and flanking regions of the STR loci, which cannot be identified by CE detection. Comparatively, massively parallel sequencing (MPS) technology can capture these sequence differences, thereby enhancing the identification capability of certain STRs. In this study, we used the ForenSeq™ DNA Signature Prep Kit to sequence 58 STRs and 94 individual identification SNPs (iiSNPs) in a sample of 220 unrelated individuals from the Eastern Chinese Han population. Our aim is to obtain MPS-based STR and SNP data, providing further evidence for the study of population genetics and forensic applications. The results showed that the MPS method, utilizing sequence information, identified a total of 486 alleles on autosomal STRs (A-STRs), 97 alleles on X-chromosome STRs (X-STRs), and 218 alleles on Y-chromosome STRs (Y-STRs). Compared with length polymorphism, we observed an increase of 260 alleles (157, 31, and 72 alleles on A-STRs, X-STRs, and Y-STRs, respectively) across 36 STRs. The most substantial increments were observed in DYF387S1 and DYS389II, with increases of 287.5% and 250%, respectively. The most increment in the number of alleles was found at DYF387S1 and DYS389II (287.5% and 250%, respectively). The length-based (LB) and sequence-based (SB) combined random match probability (RMP) of 27 A-STRs were 6.05E-31 and 1.53E-34, respectively. Furthermore, other forensic parameters such as total discrimination power (TDP), cumulative probability of exclusion of trios (CPEtrio), and duos (CPEduo) were significantly improved when using the SB data, and informative data were obtained for the 94 iiSNPs. Collectively, these findings highlight the advantages of MPS technology in forensic genetics, and the Eastern Chinese Han genetic data generated in this study could be used as a valuable reference for future research in this field.

Application of SNaPshot Technology in Semen-Specific cSNP Genetic Marker.

OBJECTIVES: To explore the feasibility of genetic marker detection of semen-specific coding region single nucleotide polymorphism (cSNP) based on SNaPshot technology in semen stains and mixed body fluid identification. METHODS: Genomic DNA (gDNA) and total RNA were extracted from 16 semen stains and 11 mixtures composed of semen and venous blood, and the total RNA was reverse transcribed into complementary DNA (cDNA). The cSNP genetic markers were screened on the validated semen-specific mRNA coding genes. The cSNP multiplex detection system based on SNaPshot technology was established, and samples were genotyped by capillary electrophoresis (CE). RESULTS: A multiplex detection system containing 5 semen-specific cSNPs was successfully established. In 16 semen samples, except the cSNP located in the TGM4 gene showed allele loss in cDNA detection results, the gDNA and cDNA typing results of other cSNPs were highly consistent. When detecting semen-venous blood mixtures, the results of cSNP typing detected were consistent with the genotype of semen donor and were not interfered by the genotype of venous blood donor. CONCLUSIONS: The method of semen-specific cSNPs detection by SNaPshot technology method can be applied to the genotyping of semen (stains) and provide information for determining the origin of semen in mixed body fluids (stains).

Internal validation of an improved system for forensic application: a 41-plex Y-STR panel.

Y-chromosome short tandem repeats (Y-STRs) have a unique role in forensic investigation. However, low-medium mutating Y-STRs cannot meet the requirements for male lineage differentiation in inbred populations, whereas rapidly mutating (RM) high-resolution Y-STRs might cause unexpected exclusion of paternal lineages. Thus, combining Y-STRs with low and high mutation rates helps to distinguish male individuals and lineages in family screening and analysis of genetic relationships. In this study, a novel 6-dye, 41-plex Y-STR panel was developed and validated, which included 17 loci from the Yfiler kit, nine RM Y-STR loci, 15 low-medium mutating Y-STR loci, and three Y-InDels. Developmental validation was performed for this panel, including size precision testing, stutter analysis, species specificity analysis, male specificity testing, sensitivity testing, concordance evaluation, polymerase chain reaction inhibitors analysis, and DNA mixture examination. The results demonstrated that the novel 41-plex Y-STR panel, developed in-house, was time efficient, accurate, and reliable. It showed good adaptability to directly amplify a variety of case-type samples. Furthermore, adding multiple Y-STR loci significantly improved the system's ability to distinguish related males, making it highly informative for forensic applications. In addition, the data obtained were compatible with the widely used Y-STR kits, facilitating the search and construction of population databases. Moreover, the addition of Y-Indels with short amplicons improves the analyses of degraded samples. Key Points: A novel multiplex comprising 41 Y-STR and 3 Y-InDel was developed for forensic application.The multiplex included rapidly mutating Y-STRs and low-medium mutating Y-STRs, which is compatible with many commonly used Y-STR kits.The multiplex is a powerful tool for distinguishing related males, familial searching, and constructing DNA databases.

Developmental validation of a 381 Y-chromosome SNP panel for haplogroup analysis in the Chinese populations.

Y-chromosome single nucleotide polymorphism (Y-SNP) shows great variation in geographical distribution and population heterogeneity and can be used to map population genetics around the world. Massive parallel sequencing (MPS) methodology enables high-resolution Y-SNP haplogrouping for a certain male and is widely used in forensic genetics and evolutionary studies. In this present study, we used MPS to develop a customized 381 Y-SNP panel (SifaMPS 381 Y-SNP panel) to investigate the basic structure and subbranches of the haplogroup tree of the Chinese populations. The SifaMPS 381 Y-SNP panel covers all the Y-SNPs from our previously designed 183 Y-SNP panel and additional SNPs under the predominant haplogroups in the Chinese populations based on certain criteria. We also evaluated the sequencing matrix, concordance, sensitivity, repeatability of this panel and the ability to analyze mixed and case-type samples based on the Illumina MiSeq System. The results demonstrated that the novel MPS Y-SNP panel possessed good sequencing performance and generated accurate Y-SNP genotyping results. Although the recommended DNA input was greater than 1.25 ng, we observed that a lower DNA amount could still be used to analyze haplogroups correctly. In addition, this panel could handle mixed samples and common case-type samples and had higher resolution among Chinese Han males than previously reported. In conclusion, the SifaMPS 381 Y-SNP panel showed an overall good performance and offers a better choice for Y-SNP haplogrouping of the Chinese population, thereby facilitating paternal lineage classification, familial searching and other forensic applications.

Multi-locus identification of Psilocybe cubensis by high-resolution melting (HRM).

Hallucinogenic mushroom is a kind of toxic strain containing psychoactive tryptamine substances such as psilocybin, psilocin and ibotenic acid, etc. The mushrooms containing hallucinogenic components are various, widely distributed and lack of standard to define, which made a great challenge to identification. Traditional identification methods, such as morphology and toxicology analysis, showed shortcomings in old or processed samples, while the DNA-based identification of hallucinogenic mushrooms would allow to identify these samples due to the stability of DNA. In this paper, four primer sets are designed to target Psilocybe cubensis DNA for increasing resolution of present identification method, and the target markers include largest subunit of RNA polymerase II (marked as PC-R1), psilocybin-related phosphotransferase gene (marked as PC-PT), glyceraldehyde 3-phosphate dehydrogenase (marked as PC-3) and translation EF1α (marked as PC-EF). Real-time PCR with high-resolution melting (HRM) assay were used for the differentiation of the fragments amplified by these primer sets, which were tested for specificity, reproducibility, sensitivity, mixture analysis and multiplex PCR. It was shown that the melting temperatures of PC-R1, PC-PT, PC-3 and PC-EF of P. cubensis were (87.93 ± 0.12) °C, (82.21 ± 0.14) °C, (79.72 ± 0.12) °C and (80.11 ± 0.19) °C in our kinds of independent experiments. Significant HRM characteristic can be shown with a low concentration of 62.5 pg/µL DNA sample, and P. cubensis could be detected in mixtures with Homo sapiens or Cannabis sativa. In summary, the method of HRM analysis can quickly and specifically distinguish P. cubensis from other species, which could be utilized for forensic science, medical diagnosis and drug trafficking cases. Supplemental data for this article are available online at https://doi.org/10.1080/20961790.2021.1875580.

Genetic Polymorphism of 16 X-STR Loci in Xinjiang Uygur Population.

OBJECTIVES: To study the genetic polymorphism and population genetic parameters of 16 X-STR loci in Xinjiang Uygur population. METHODS: The Goldeneye® DNA identification system 17X was used to amplify 16 X-STR loci in 502 unrelated individuals (251 females and 251 males). The amplified products were detected by 3130xl genetic analyzer. Allele frequencies and population genetic parameters were analyzed statistically. The genetic distances between Uygur and other 8 populations were calculated. Multidimensional scaling and phylogenetic tree were constructed based on genetic distance. RESULTS: In the 16 X-STR loci, a total of 67 alleles were detected in 502 Xinjiang Uygur unrelated individuals. The allele frequencies ranged from 0.001 3 to 0.572 4. PIC ranged from 0.568 8 to 0.855 3. The cumulative discrimination power in females and males were 0.999 999 999 999 999 and 0.999 999 999 743 071, respectively. The cumulative mean paternity exclusion chance in trios and in duos were 0.999 999 997 791 859 and 0.999 998 989 000 730, respectively. The genetic distance between Uygur population and Kazakh population was closer, and the genetic distance between Uygur and Han population was farther. CONCLUSIONS: The 16 X-STR loci are highly polymorphic and suitable for identification in Uygur population, which can provide a powerful supplement for the study of individual identification, paternity identification and population genetics.

A sequence-based 163plex microhaplotype assay for forensic DNA analysis.

Novel genetic marker microhaplotype has led to an upsurge in forensic genetic research. This study established a 163 microhaplotype (MH) multiplex assay based on next-generation sequencing (NGS) and evaluated the assay's performance and applicability. Our results showed that the 163 MH assay was accurate, repeatable and reliable, and could distinguish between African, European-American, Southern Asia and Eastern Asia populations. Among the 163 MH makers, 48 MHs with Ae > 3.0 in China Eastern Han were selected and confirmed to be highly polymorphic, with a combined power of discrimination of 1-8.26 × 10-44 and the combined power of exclusion in duos and trios of 1-1.26 × 10-8 and 1-8.27 × 10-16, respectively. Moreover, the mixture study demonstrated the realizability of the MHs in deconvoluting mixtures with different proportions of two to five-person. In conclusion, our findings support the use of this MH assay for ancestry inference, human identification, paternity testing and mixture deconvolution in forensic research.

Validation of the Investigator 24plex QS Kit: a 6-dye multiplex PCR assay for forensic application in the Chinese Han population.

The Investigator 24plex QS Kit (QIAGEN, Hilden, Germany) is a 6-dye fluorescent chemistry short tandem repeat (STR) polymerase chain reaction (PCR) amplification system that simultaneously amplifies 20 of the expanded Combined DNA Index System (CODIS) core STR loci, SE33, DYS391, and the standard sex-determining locus, amelogenin, as well as two special internal performance quality sensor controls (QS1 and QS2), which are included in the primer mix to check the PCR performance. This study was designed to be a pilot evaluation of this STR-PCR kit in a Chinese Han population regarding the PCR conditions, sensitivity, precision, accuracy, repeatability, reproducibility, and concordance; tolerance to PCR inhibitors; applicability to real "forensic-type" samples; species specificity; mixture, balance and stutter analyses, and utility in a population investigation. The exhaustive validation studies demonstrated that the Investigator 24plex QS system is accurate, sensitive and robust for STR genotyping. In addition, these genetic markers in the population data in our study indicated that they can also be useful for forensic identification and paternity testing in the Chinese Han population.

Establishment of Multiplex Amplification System of STR Loci in Felis Catus and Its Forensic Application.

OBJECTIVES: To construct a Felis catus STR loci multiplex amplification system and to evaluate its application value by testing the technical performance. METHODS: The published Felis catus STR loci data were reviewed and analyzed to select the STR loci and sex identification loci that could be used for Felis catus individual identification and genetic identification. The fluorescent labeling primers were designed to construct the multiplex amplification system. The system was validated for sensitivity, accuracy, balance, stability, species specificity, tissue identity and mixture analysis, and investigated the genetic polymorphisms in 145 unrelated Felis catus samples. RESULTS: Sixteen Felis catus autosomal STR loci and one sex determining region of Y (SRY) were successfully selected, and constructed a multiplex amplification system containing the above loci. The complete profile of all alleles could still be obtained when the amount of DNA template was as low as 0.25 ng. There was no specific amplification peak in other common animal samples. Population genetic surveys showed that total discrimination power (TDP) of the 16 STR loci was 1-3.57×10-20, the cumulative probability of exclusion (CPE) was 1-6.35×10-5 and the cumulative probability of matching was 3.61×10-20. CONCLUSIONS: The Felis catus STR multiplex amplification system constructed in this study is highly sensitive, species-specific, and accurate in typing results, which can provide an effective solution for Felis catus species identification, individual identification and kinship identification in the field of forensic science.

Development and Validation of a Novel and Fast Detection Method for Cannabis sativa: A 19-Plex Short Tandem Repeat Typing System.

In recent years, influenced by the legalization of Cannabis sativa in some countries and regions, the number of people who smoke or abuse C. sativa has continuously grown, cases of transnational C. sativa trafficking have also been increasing. Therefore, fast and accurate identification and source tracking of C. sativa have become urgent social needs. In this study, we developed a new 19-plex short tandem repeats (STRs) typing system for C. sativa, which includes 15 autosomal STRs (D02-CANN1, C11-CANN1, 4910, B01-CANN1, E07-CANN1, 9269, B05-CANN1, H06-CANN2, 5159, nH09, CS1, ANUCS 305, 3735, and ANUCS 302 and 9043), two X-chromosome STRs (ANUCS 501 and 1528), one sex-determining marker (DM016, on Y-chromosome), and a quality control marker (DM029, on autosome). The whole polymerase chain reaction (PCR) process could finish within 1 h, making the system suitable for fast detection. The PCR products were detected and separated with an Applied Biosystems 3500XL Genetic Analyser. Developmental validation studies indicated that the 19-plex typing system was accurate, reliable and sensitive, which could also deconvolute mixed C. sativa samples. Specifically, the sensitivity study showed that a full genotyping profile was obtainable with as low as 125 pg of C. sativa DNA. The species specificity study demonstrated that this multiplex has no cross-reactivity with common non C. sativa DNA. In the population study, a total of 162 alleles at 15 autosomal STRs and 14 alleles at two X-chromosome STRs were detected among 85 samples. The efficiency parameters, including the total discrimination power (TDP) and the combined power of exclusion (CPE) of the system, were calculated to exceed 0.999 999 999 999 988 and 0.998 455 889 684 078, respectively, further proving that the system could meet the needs of individual identification. To the extent of the known studies, this is the first study that included the C. sativa sex-determining marker. In conclusion, the developed new 19-plex STR typing system can successfully achieve the purposes of species identification, gender determination, and individual identification, which could be a powerful tool in tracing trade routes of particular drug syndicates or dealers or in linking certain C. sativa to a crime scene.

Establishing an integrated pipeline for automatic and efficient detection of trace DNA encountered in forensic applications.

The analysis of trace DNA is a crucial component in forensic applications. Biological materials containing low-level DNA collected at crime scenes, such as fingerprints, can be valuable as evidence. Automatic detection of biological samples has been largely embraced in forensic applications to meet the increasing throughput requirements. However, the amount of DNA automatically retrieved from trace evidence often tends to be small and unstable, ultimately resulting in poor detection of DNA profiles. Thus, in this work, we introduced a robust DNA extraction and purification platform named Bionewtech® BN3200 (Bionewtech®, Shanghai, China) with the goal of constructing a rapid automatic detection system for trace DNA. The establishment of automatic detection system for trace DNA mainly encompassed two parts: assessing the sensitivity of automatic extraction platform and screening the optimal short tandem repeat (STR) typing kit. The sensitivity of Bionewtech® BN3200 platform based on Ultra-sensitive DNA Extraction kit was initially estimated, demonstrating that this extraction platform might contain large potential in the trace DNA extraction. For the amplification part, three sets of commercial multiplex STR typing kits were selected as candidates, and the amplified products were further genotyped on the Applied Biosystems 3500xl Genetic Analyzer. After comparation, SiFa™ 23 Plex Kit was determined as the most suitable amplification system for trace DNA. Eventually, the newly exploited trace DNA detection system was successfully implemented in the detection of fingerprints derived from glass surfaces with the five-seconds contact time. As a result, the DNA recovered from the fingerprints fluctuated approximately from 57.60 pg to 18.05 ng, in addition, over 70% of the total STR loci were detected in 75% of the fingerprint samples.

Application of SifaInDel 45plex System in the Han and Mongolian Populations.

OBJECTIVES: To investigate the genetic polymorphism of InDel loci in SifalnDel 45plex system in the Han population in Jiangsu Province and the Mongolian population in Inner Mongolia, and to evaluate the effectiveness of the system in forensic medicine. METHODS: SifaInDel 45plex system was used for genotyping in blood samples of 398 unrelated individuals from the above two populations, and allele frequencies and population genetic parameters of the two populations were calculated respectively. Eight intercontinental populations in the gnomAD database were used as reference populations. The genetic distances between the two studied populations and eight reference populations were calculated based on the allele frequencies of 27 autosomal-InDels (A-InDels). The phylogenetic trees and multidimensional scaling (MDS) analysis diagrams were constructed accordingly. RESULTS: Among two studied populations, the 27 A-InDels and 16 X-InDels showed no linkage disequilibrium between each other and the allele frequency distributions were in Hardy-Weinberg equilibrium. The CDP of the 27 A-InDels in two studied populations were all higher than 0.999 999 999 9, and the CPEtrio were all less than 0.999 9. The CDP of the 16 X-InDels in Han in Jiangsu and Mongolian in Inner Mongolia female and male samples were 0.999 997 962, 0.999 998 389, and 0.999 818 940, 0.999 856 063, respectively. The CMECtrio were all less than 0.999 9. The results of population genetics showed that the Jiangsu Han nationality, Inner Mongolia Mongolian nationality and East Asian population clustered into one branch, showing closer genetic relationship. The other 7 intercontinental populations clustered into another group. And the above 3 populations displayed distant genetic relationships with the other 7 intercontinental populations. CONCLUSIONS: The InDels in the SifaInDel 45plex system have good genetic polymorphism in the two studied populations, which can be used for forensic individual identification or as an effective complement for paternity identification, and to distinguish different intercontinental populations.

Developmental validation of the novel six-dye GoldeneyeTM DNA ID System 35InDel kit for forensic application.

Insertion/deletion polymorphisms (InDels) have been treated as a prospective and helpful genetic marker in the fields of forensic human identification, anthropology and population genetics for the past few years. In this study, we developed a six-dye multiplex typing system consisting of 34 autosomal InDels and Amelogenin for forensic application. The contained InDels were specifically selected for Chinese population with the MAF ≥ 0.25 in East Asia, which do not overlap with the markers of Investigator® DIPplex kit. The typing system was named as GoldeneyeTM DNA ID System 35InDel Kit, and a series of developmental validation studies including repeatability/reproducibility, concordance, accuracy, sensitivity, stability, species specificity and population genetics were conducted on this kit. We confirmed that the 35InDel kit is precise, sensitive, species specific and robust for forensic practice. Moreover, the 35InDel kit is capable of typing DNA extracted from forensic routine case-type samples as well as degraded samples and mixture samples. All markers are proved to be highly polymorphic with an average observed heterozygosity (He) of 0.4582. The combined power of discrimination (CPD) is 0.999 999 999 999 978 and the combined power of exclusion in duos (CPED) and trios (CPET) are 0.978 837 and 0.999573, respectively, which are higher than those of the Investigator® DIPplex kit. Thus, the GoldeneyeTM DNA ID System 35InDel kit is suitable for forensic human identification and could serve as a supplementary typing system for paternity testing. Supplemental data for this article is available online at https://doi.org/10.1080/20961790.2021.1945723 .

Pairwise kinship analysis of 17 pedigrees using massively parallel sequencing.

With the tremendous development of massively parallel sequencing (MPS) in the last decade, it has been widely applied in basic science, clinical diagnostics, microbial genomics, as well as forensic genetics. MPS has lots of advantages that may facilitate the kinship analysis. In this study, 243 Chinese Han individuals from 17 families were involved and sequenced using the ForenSeq™ DNA Signature Prep Kit (Verogen, Inc., San Diego, USA), which provided the sequence information of 27 autosomal STRs (A-STRs), 7 X chromosomal STRs (X-STRs), 24 Y chromosomal STRs (Y-STRs) and 94 identity-informative SNPs (iSNPs). A total of 275 pairs of parent-child, 123 pairs of full siblings, 1 pair of twins, 1 pair of half siblings, 158 pairs of grandparent-grandchild, 222 pairs of uncle/aunt-nephew/niece and 121 pairs of first cousins, as well as 701 pairs of unrelated individuals were identified. Using both likelihood ratio (LR) and identical by state (IBS) methods, the kinship analysis was conducted among these relative and non-relative pairs based on the A-STRs and SNPs. As a result, the ForenSeq Signature Kit could solve the analysis of parent-child (t1 = -4, t2 = 4), full siblings (t1 = -2, t2 = 2) and most second-degree kinships (t1 = -1, t2 = 1) using the LR method. When the IBS method was applied, 123 full sibling pairs had a higher average IBS value than other kinship groups in this study. And the IBS method could play a role in the testing of parent-child and full siblings.

Development and validation of a forensic six-dye multiplex assay with 29 STR loci.

This paper describes the development and validation of a novel 31-locus, six-dye STR multiplex system, which is designed to meet the needs of the rapidly growing Chinese forensic database. This new assay combines 20 extended-CODIS core loci (D3S1358, D5S818, TPOX, CSF1PO, TH01, vWA, D7S820, D21S11, D8S1179, D18S51, D16S539, D13S317, FGA, D1S1656, D2S441, D2S1338, D10S1248, D12S391, D19S433, and D22S1045), nine highly polymorphic loci in Chinese Han population (D3S3045, D6S1043, D6S477, D8S1132, D10S1435, D15S659, D19S253, Penta D, and Penta E), and two gender determining markers, amelogenin and Y-Indel, which could amplify DNA from extracts, as well as direct amplification from substrates. To demonstrate the suitability for forensic applications, this system was validated by precision and accuracy evaluation, concordance tests, case sample tests, sensitivity, species specificity, stability, stutter calculation, and DNA mixtures, according to the guidelines described by the Scientific Working Group on DNA Analysis Methods (SWGDAM) and regulations published by the China Ministry of Public Security. The validation results indicate the robustness and reliability of this new system, and it could be a potentially helpful tool for human identification and paternity testing in the Chinese population, as well as facilitating global forensic DNA data sharing.

Parallel sequencing of 87 STR and 294 SNP markers using the prototype of the SifaMPS panel on the MiSeq FGx™ system.

Massively parallel sequencing (MPS), or next generation sequencing (NGS), is a promising methodology for the detection of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) in forensic genetics. Here, the prototype SifaMPS Panel is designed to simultaneously target 87 STRs and 294 SNPs with forensic interest in a single multiplex in conjunction with the TruSeq™ Custom Amplicon workflow and MiSeq FGx™ System. Two in-house python scripts are adopted for the fastq-to-genotype interpretation of MPS data concerning STR and SNP, respectively. In the present study, by sequencing 50 Chinese Hans and many other DNA samples involved in validation studies, system parameters including the depth of coverage (DoC), heterozygote balance (Hb) and sequence coverage ratios (SCRs), as well as different forensic parameters of STRs and SNPs in a population study, were calculated to evaluate the overall performance of this new panel and its practicality in forensic application. In general, except for two STRs (DYS505 and DYS449) and one SNP (rs4288409) that performed poorly, the other 85 STRs and 293 SNPs in our panel had good performance that could strengthen efficiency for human identification and paternity testing. In addition, discordant STR genotype results between those generated from capillary electrophoresis (CE) and from the MPS platform were clearly illustrated, and these results could be a useful reference for applying these particular non-CODIS STRs in forensic practice.

A Forensic Detection Method for Hallucinogenic Mushrooms via High-Resolution Melting (HRM) Analysis.

In recent years, trafficking and abuse of hallucinogenic mushrooms have become a serious social problem. It is therefore imperative to identify hallucinogenic mushrooms of the genus Psilocybe for national drug control legislation. An internal transcribed spacer (ITS) is a DNA barcoding tool utilized for species identification. Many methods have been used to discriminate the ITS region, but they are often limited by having a low resolution. In this study, we sought to analyze the ITS and its fragments, ITS1 and ITS2, by using high-resolution melting (HRM) analysis, which is a rapid and sensitive method for evaluating sequence variation within PCR amplicons. The ITS HRM assay was tested for specificity, reproducibility, sensitivity, and the capacity to analyze mixture samples. It was shown that the melting temperatures of the ITS, ITS1, and ITS2 of Psilocybe cubensis were 83.72 ± 0.01, 80.98 ± 0.06, and 83.46 ± 0.08 °C, and for other species, we also obtained species-specific results. Finally, we performed ITS sequencing to validate the presumptive taxonomic identity of our samples, and the sequencing output significantly supported our HRM data. Taken together, these results indicate that the HRM method can quickly distinguish the DNA barcoding of Psilocybe cubensis and other fungi, which can be utilized for drug trafficking cases and forensic science.

Forensic Application of ForenSeqTM DNA Signature Prep Kit in Zhengjiang She Ethnic Group.

OBJECTIVES: To evaluate the ability of the ForenSeqTM DNA Signature Prep kit (ForenSeq kit) in analyzing the sequence information of STRs in Zhejiang She ethnic group and its forensic application efficacy. METHODS: A total of 50 Zhejiang She ethnic group samples were sequenced with the ForenSeq kit on the MiSeq FGx platform. The data was analyzed using ForenSeqTM universal analysis software to obtain the motif structure and flank regions of the 58 STRs, then compared with PCR-CE typing results to test the consistency. At last, the allele frequency and population genetic parameters were calculated. RESULTS: A total of 448 sequence polymorphic alleles were detected in 50 samples of Zhejiang She ethnic group. Compared with fragment length polymorphism detected by PCR-CE, 82 alleles were increased by MPS detection based on ForenSeq kit, and 7 SNPs variation were detected in the flanking regions of 6 loci. The 22 male individuals were genotyped, and total 19 haplotypes were detected in 24 Y chromosome STRs of these 22 males. The cumulative discrimination power of the 27 autosomal STRs was 1-8.87×10-30, the cumulative probability of exclusion of duo-testing was 0.999 999 962 640 657, the cumulative probability of exclusion of trios-testing was 0.999 999 999 999 633. CONCLUSIONS: Based on MPS typing technology, using the ForenSeq kit greatly improves the detection efficiency. In addition, the 58 STRs have good genetic polymorphisms in Zhejiang She ethnic group, which are suitable for individual identification and paternity identification in forensic application.

Genetic investigation and phylogenetic analysis of three Chinese ethnic groups using 16 X chromosome STR loci.

Background: The value of using X-chromosomal short tandem repeats (X-STRs) as genetic markers in human genetics has been widely recognised. However, the 16 X-STRs in the Goldeneye® DNA ID System 17X kit have not been thoroughly applied.Aim: To investigate the genetic polymorphisms of 16 X-STRs in three main ethnic minorities (Tibetan, Mongolian and Kazakh) in China and to reveal the phylogenetic relationships of different populations.Subjects and methods: A total of 245 Tibetan, 168 Mongolian and 105 Kazakh individuals were genotyped using this 17X kit. The allelic frequencies and other parameters were calculated. An additional eight Chinese populations and nine global populations were included in genetic comparisons based on 16 or 8 overlapped X-STRs.Results: A total of 147 alleles were observed from 16 X-STRs with allelic frequencies ranging from 0.0024 to 0.7952 in the three studied groups. Based on 16 X-STRs, Tibetans, Kazakhs and Mongolians showed more similarity to each other and were genetically distinct from the Shanghai Han group; based on 8 X-STRs, only the genetic relationships between different nations could be clarified.Conclusions: Our study presents an extensive report on a novel X-STR assay in three Chinese ethnic groups and a comprehensive genetic comparison between different populations based on these X-STRs.