Sequencing-induced artefacts in NGS STR data.

Significant progress has been made in recent years in the development of techniques for Next Generation Sequencing (NGS), or Massively Parallel Sequencing (MPS), of forensically relevant short tandem repeat (STR) loci. However, as these technologies are investigated and adopted by forensic laboratories, new challenges unfold that require further scrutiny. In the analysis of DNA profiles generated using the MiSeq FGx sequencing system, we have observed noise sequences with relatively high readcounts that are challenging to distinguish from genuine alleles. These high read count noise sequences appear as allele sequences with one or a few substituted bases compared to a known allele sequence within the profile. An examination of ForenSeq DNA Signature Prep Kit STR noise sequences revealed that the substituted base of a parent allele can align to the same position on the sequence across noise sequences. This suggests that these substitution events occur at specific positions within the amplicon, resulting in multiple noise reads with substitutions at the same position. Mapping of the noise events onto the original raw read positions revealed a high number of events, or "noise spikes", occurring at specific positions within a given sequencing run. These noise spikes affected reads across the entire run, agnostic of locus or sample, while the position, occurrence, and amplitude of the spikes differed across runs. The majority of noise sequences with high read counts in a DNA profile were generated from base changes at these spike positions, and could be classified as "noise spike artefacts". In this paper we present evidence of the noise spike artefacts and their genesis during the sequencing process in the sequencing-by-synthesis (SBS) cycles, as well as the methods developed to detect them. The information and methods will assist laboratories with detecting noise spikes in MiSeq FGx sequencing runs, differentiating authentic allele sequences from noise spike artefacts, and developing protocols for analyst review and handling of MiSeq FGx data.

An overview of autosomal STRs and identity SNPs in a Norwegian population using massively parallel sequencing.

In recent years, probabilistic genotyping software has been adapted for the analysis of massively parallel sequencing (MPS) forensic data. Likelihood ratios (LR) are based on allele frequencies selected from populations of interest. This study provides an outline of sequence-based (SB) allele frequencies for autosomal short tandem repeats (aSTRs) and identity single nucleotide polymorphisms (iSNPs) in 371 individuals from Southern Norway. 27 aSTRs and 94 iSNPs were previously analysed with the ForenSeq™ DNA Signature Prep Kit (Verogen). The number of alleles with frequencies less than 0.05 for sequenced-based alleles was 4.6 times higher than for length-based alleles. Consistent with previous studies, it was observed that sequence-based data (both with and without flanks) exhibited higher allele diversity compared to length-based (LB) data; random match probabilities were lower for SB alleles confirming their advantage to discriminate between individuals. Two alleles in markers D22S1045 and Penta D were observed with SNPs in the 3´ flanking region, which have not been reported before. Also, a novel SNP with a minor allele frequency (MAF) of 0.001, was found in marker TH01. The impact of the sample size on minor allele frequency (MAF) values was studied in 88 iSNPs from Southern Norway (n = 371). The findings were then compared to a larger Norwegian population dataset (n = 15,769). The results showed that the smaller Southern Norway dataset provided similar results, and it was a representative sample. Population structure was analyzed for regions within Southern Norway; FST estimates for aSTR and iSNPs did not indicate any genetic structure. Finally, we investigated the genetic differences between Southern Norway and two other populations: Northern Norway and Denmark. Allele frequencies between these populations were compared, and we found no significant frequency differences (p-values > 0.0001). We also calculated the pairwise FST values per marker and comparisons between Southern and Northern Norway showed small differences. In contrast, the comparisons between Southern Norway and Denmark showed higher FST values for some markers, possibly driven by distinct alleles that were present in only one of the populations. In summary, we propose that allele frequencies from each population considered in this study could be used interchangeably to calculate genotype probabilities.

Forensic efficiency and genetic polymorphisms of 12 X-chromosomal STR loci in Northeastern Thai populations.

Northeastern Thailand comprises one-third of the country and is home to various populations, with Lao Isan constituting the majority, while others are considered minority groups. Previous studies on forensic short tandem repeats (STRs) in Thailand predominantly focused on autosomal STRs but there was a paucity of X-STRs, exclusively reported from the North and Central regions of the country. In this study, we have newly established a 12 X-STRs from a total of 896 samples from Northeastern Thailand, encompassing Lao Isan as the major group in the region, alongside nine minor populations (Khmer, Mon, Nyahkur, Bru, Kuy, Phutai, Kalueang, Nyaw, and Saek). Across all ten populations, the combined powers of discrimination in both genders were high and the combined mean exclusion chance (MEC) indices calculated for deficiency, normal trio and duo cases were also high (> 0.99999). DXS10148 emerged as the most informative marker, while DXS7423 was identified as the least informative. Genetic comparison based on X-STRs frequency supported genetic distinction of cerain minor groups such as Kuy, Saek and Nyahkur from other northeastern Thai groups as well as genetic differences according to the geographic region of Thai groups (Northeast, North and Central). In sum, the overall results on population genetics are in agreement with earlier reports on other genetic systems, indicating the informativeness of X-STRs for use in anthropological genetics studies. From a forensic perspective, despite the limitations of small sample sizes for minority groups, the present results contribute to filling the gap in the reference X-STRs database of the major group Lao Isan, providing valuable frequency data for forensic applications in Thailand and neighboring countries.

Evaluation of the usefulness of insertion-null markers in critical skeletal remains.

Forensic DNA analysis in compromised skeletal remains may pose challenges due to DNA degradation, often resulting in partial or negative autosomal STRs profiles. To address this issue, alternative approaches such as mitochondrial DNA or SNPs typing may be employed; however, they are labour-intensive and costly. Insertion-null alleles (INNULs), short interspersed nuclear elements, have been suggested as a valuable tool for human identification in challenging samples due to their small amplicon size. A commercial kit including 20 INNULs markers along with amelogenin (InnoTyper® 21) has been developed. This study assesses its utility using degraded skeletal remains, comparing the results obtained (the number of detected alleles, RFU values, PHR, and the number of reportable markers) to those obtained using GlobalFiler™. Subsequently, the random match probability of the two profiles for each sample was determined using Familias version 3 to evaluate the power of discrimination of the results obtained from each kit. In every sample, InnoTyper® 21 yielded more alleles, higher RFU values, and a greater number of reportable loci. However, in most cases, both profiles were similarly informative. In conclusion, InnoTyper® 21 serves as a valuable complement to the analysis of challenging samples in cases where a poor or negative profile was obtained.

Development and validation of YARN: A novel SE-400 MPS kit for East Asian paternal lineage analysis.

Y-chromosomal short tandem repeat polymorphisms (Y-STRs) and Y-chromosomal single nucleotide polymorphisms (Y-SNPs) are valuable genetic markers used in paternal lineage identification and population genetics. Currently, there is a lack of an effective panel that integrates Y-STRs and Y-SNPs for studying paternal lineages, particularly in East Asian populations. Hence, we developed a novel Y-chromosomal targeted panel called YARN (Y-chromosome Ancestry and Region Network) based on multiplex PCR and a single-end 400 massive parallel sequencing (MPS) strategy, consisting of 44 patrilineage Y-STRs and 260 evolutionary Y-SNPs. A total of 386 reactions were validated for the effectiveness and applicability of YARN according to SWGDAM validation guidelines, including sensitivity (with a minimum input gDNA of 0.125 ng), mixture identification (ranging from 1:1-1:10), PCR inhibitor testing (using substances such as 50 µM hematin, 100 µM hemoglobin, 100 µM humic acid, and 2.5 mM indigo dye), species specificity (successfully distinguishing humans from other animals), repeatability study (achieved 100% accuracy), and concordance study (with 99.91% accuracy for 1121 Y-STR alleles). Furthermore, we conducted a pilot study using YARN in a cohort of 484 Han Chinese males from Huaiji County, Zhaoqing City, Guangdong, China (GDZQHJ cohort). In this cohort, we identified 52 different Y-haplogroups and 73 different surnames. We found weak to moderate correlations between the Y-haplogroups, Chinese surnames, and geographical locations of the GDZQHJ cohort (with λ values ranging from 0.050 to 0.340). However, when we combined two different categories into a new independent variable, we observed stronger correlations (with λ values ranging from 0.617 to 0.754). Overall, the YARN panel, which combines Y-STR and Y-SNP genetic markers, meets forensic DNA quality assurance guidelines and holds potential for East Asian geographical origin inference and paternal lineage analysis.

Forensic autosomal and gonosomal short tandem repeat marker reference database for populations in Burkina Faso.

Tandem repeat genetic profiles used in forensic applications varies between populations. Despite the diversity and security issues in the Sahel that require the identification of victims (soldiers and civilians), Burkina Faso (BF) remains understudied. To fill this information gap, 396 unrelated individuals from BF were genotyped using a MICROREADER 21 ID System kit. All 20 short tandem repeat (STR) loci tested passed the Hardy-Weinberg equilibrium (HWE) test. The combined powers of exclusion for duos (CPE duos) and trios (CPE trios) for the 20 tested loci were 0.9999998 and 0.9999307, respectively. The probability that two individuals would share the same DNA profiles among the BF population was 9.80898 × 10-26. For the X-chromosome STR analysis, 292 individuals were included in this study using a MICROREADER 19X Direct ID System kit. Among the 19 loci, no significant deviations from HWE test were observed in female samples after Bonferroni correction (p < 0.05/19 = 0.0026), except for loci GATA165B12 and DXS7423. The results showed that the combined power of exclusion (CPE) and the combined power of discrimination in females (CPDF) and males (CPDM) were 0.999999760893, 0.999999999992, and 1, respectively. Comparison with other African sub-populations showed that geographical proximity is a reliable indicator of genetic relatedness.

Technical Note: A simple FTA® based method for the direct STR amplification of human foetal tissues.

Short tandem repeats (STRs) or microsatellites are short, tandemly repeated DNA sequences that involve a repetitive unit of 1-6 bp. DNA isolation and purification from a large number and often compromised samples gives problems to forensic labs for STR typing. Many of the conventional methods used in the isolation and purification of DNA from forensic samples are time consuming, expensive, hazardous for health and are often associated with greater risks of cross contamination. FTA® technology is a method designed to simplify the collection, shipment, archiving and purification of nucleic acid from a wide variety of biological samples. We report a new method for the direct STR amplification which can amplify STR loci from human foetal tissues spotted on FTA cards, bye-passing the need of DNA purification. The STR loci amplified by this method was compared with conventional method of STR profiling and was found absolutely matching. Therefore, this new method is demonstrated to be very useful for fast, less expensive and non- hazardous forensic DNA analysis.

Analysis of 26 STR loci (PowerPlex® Fusion 6C System) in a mestizo population from Mexico city.

BACKGROUND: Short tandem repeats (STRs) are the most widely used genetic markers in forensic genetics. Therefore, it is essential to document genetic population data of new kits designed for human identification purposes to enable laboratories to use these genetic systems to interpret and solve forensic casework. However, in Mexico, there are no studies with the PowerPlex Fusion 6C System, which includes 26 STRs (23 autosomal STRs and 3 Y-STRs). METHODS AND RESULTS: 600 DNA samples from Mexico City were subjected to genotyping using the PowerPlex Fusion 6C System. For autosomal STRs, 312 different alleles were observed. Combined PE and PD were 99.999999809866% and 99.99999999999999999999999818795%, respectively. Genetic distances and AMOVA test showed low but significant differentiation between Mexican populations. CONCLUSIONS: The results reported in this work demonstrate the efficacy of this system for human identification purposes in the population studied and justify its possible application in other Mexican Mestizo populations.

Male Pedigree Toolbox: A Versatile Software for Y-STR Data Analyses.

Y-chromosomal short tandem repeats (Y-STRs) are widely used in forensic, genealogical, and population genetics. With the recent increase in the number of rapidly mutating (RM) Y-STRs, an unprecedented level of male differentiation can be achieved, widening and improving the applications of Y-STRs in various fields, including forensics. The growing complexity of Y-STR data increases the need for automated data analyses, but dedicated software tools are scarce. To address this, we present the Male Pedigree Toolbox (MPT), a software tool for the automated analysis of Y-STR data in the context of patrilineal genealogical relationships. The MPT can estimate mutation rates and male relative differentiation rates from input Y-STR pedigree data. It can aid in determining ancestral haplotypes within a pedigree and visualize the genetic variation within pedigrees in all branches of family trees. Additionally, it can provide probabilistic classifications using machine learning, helping to establish or prove the structure of the pedigree and the level of relatedness between males, even for closely related individuals with highly similar haplotypes. The tool is flexible and easy to use and can be adjusted to any set of Y-STR markers by modifying the intuitive input file formats. We introduce the MPT software tool v1.0 and make it publicly available with the goal of encouraging and supporting forensic, genealogical, and other geneticists in utilizing the full potential of Y-STRs for both research purposes and practical applications, including criminal casework.

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.

Genetic diversity and forensic statistical support for the 12 X-STR markers in the Malaysian Indian population using Qiagen Investigator® Argus X-12 QS kit.

X-chromosome short tandem repeats (X-STRs) are useful for human identification, especially in complex kinship scenarios. Since forensic statistical parameters vary among populations and the X-STRs population data for the diverse population of Peninsular Malaysia's are unavailable, this attempt for Indians (n = 201) appears forensically relevant to support the 12 X-STRs markers' evidential value for human identification in Malaysia. The Qiagen Investigator® Argus X-12 QS kit showed that DXS10135 was the most polymorphic locus with high genetic diversity, polymorphism information richness, heterozygosity, and exclusion power. Based on allele frequencies, the strength of discrimination and mean exclusion chance (MECKrüger, MECKishida, MECDesmarais, and MECDesmaraisDuo) values for the Malaysian Indians were ≥0.999997790686228. As for haplotype frequencies, the overall discrimination power and mean exclusion probability (MECKrüger, MECKishida, MECDesmarais, and MECDesmaraisDuo) were ≥0.9999984801951. The genetic distance, neighbor-joining phylogenetic tree, and principal component analysis also supported the evidential value of the 12 X-STRs markers for forensic practical caseworks in Malaysia.

Microsatellites' mutation modeling through the analysis of the Y-chromosomal transmission: Results of a GHEP-ISFG collaborative study.

The Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) organized a collaborative study on mutations of Y-chromosomal short tandem repeats (Y-STRs). New data from 2225 father-son duos and data from 44 previously published reports, corresponding to 25,729 duos, were collected and analyzed. Marker-specific mutation rates were estimated for 33 Y-STRs. Although highly dependent on the analyzed marker, mutations compatible with the gain or loss of a single repeat were 23.2 times more likely than those involving a greater number of repeats. Longer alleles (relatively to the modal one) showed to be nearly twice more mutable than the shorter ones. Within the subset of longer alleles, the loss of repeats showed to be nearly twice more likely than the gain. Conversely, shorter alleles showed a symmetrical trend, with repeat gains being twofold more frequent than reductions. A positive correlation between the paternal age and the mutation rate was observed, strengthening previous findings. The results of a machine learning approach, via logistic regression analyses, allowed the establishment of algebraic formulas for estimating the probability of mutation depending on paternal age and allele length for DYS389I, DYS393 and DYS627. Algebraic formulas could also be established considering only the allele length as predictor for DYS19, DYS389I, DYS389II-I, DYS390, DYS391, DYS393, DYS437, DYS439, DYS449, DYS456, DYS458, DYS460, DYS481, DYS518, DYS533, DYS576, DYS626 and DYS627 loci. For the remaining Y-STRs, a lack of statistical significance was observed, probably as a consequence of the small effective size of the subsets available, a common difficulty in the modeling of rare events as is the case of mutations. The amount of data used in the different analyses varied widely, depending on how the data were reported in the publications analyzed. This shows a regrettable waste of produced data, due to inadequate communication of the results, supporting an urgent need of publication guidelines for mutation studies.

Machine learning applications in forensic DNA profiling: A critical review.

Machine learning (ML) is a range of powerful computational algorithms capable of generating predictive models via intelligent autonomous analysis of relatively large and often unstructured data. ML has become an integral part of our daily lives with a plethora of applications, including web, business, automotive industry, clinical diagnostics, scientific research, and more recently, forensic science. In the field of forensic DNA, the manual analysis of complex data can be challenging, time-consuming, and error-prone. The integration of novel ML-based methods may aid in streamlining this process while maintaining the high accuracy and reproducibility required for forensic tools. Due to the relative novelty of such applications, the forensic community is largely unaware of ML capabilities and limitations. Furthermore, computer science and ML professionals are often unfamiliar with the forensic science field and its specific requirements. This manuscript offers a brief introduction to the capabilities of machine learning methods and their applications in the context of forensic DNA analysis and offers a critical review of the current literature in this rapidly developing field.

Forensic parameters and population structure based on 21 autosomal STRs with the investigator 24plex QS in mestizos from the Mexico City population.

Allele frequencies and forensic parameters for 21 STR autosomal markers (CSF1PO, D10S1248, D12S391, D13S317,D16S539, D18S51, D19S433, D1S1656,D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, SE33, TH01, TPOX and vWA) were reported in 289 unrelated individuals from Mexico City, Mexico. In addition, an interpopulation analysis was performed including other world populations. In brief, the established population database of 21 autosomal STR markers in the present work is adequate for human identification purposes.

Assessment of body fluid identification and DNA profiling after exposure to tropical weather conditions.

Despite current advances in body fluid identification, there are few studies evaluating the effect of environmental conditions. The present work assessed the detection of body fluids, blood, semen, and saliva, through lateral flow immunochromatographic (LFI) tests, exposed to tropical weather conditions over time, also evaluating the possibility of obtaining STR (short tandem repeat) profiles and identifying mitochondrial DNA (mtDNA) polymorphisms. Blood, semen, saliva samples, and mixtures of these fluids were deposited on polyester clothes and exposed to open-air tropical weather conditions for 1 month. The test versions from LFI (SERATEC®, Germany) Lab and crime scene (CS) used for the detection - one per each body fluid type - demonstrated that it is possible to identify body fluids and their mixtures up to 14 days after deposition. At 30 days, blood and semen were detected but not saliva. Full STR profiles were obtained from 14-day-old blood samples, and partial profiles were obtained from the remaining samples. It was possible to sequence mtDNA in the samples previously analyzed for STR profiling, and haplogroups could be assigned. In conclusion, this study demonstrated for the first time the possibility of body fluid identification and DNA profiling after exposure to tropical weather conditions for 1 month and also demonstrated the value of mtDNA analysis for compromised biological evidence.

Estimation of population specific values of theta for sequence-based STR profiles.

We describe the estimation of θ (theta) values from autosomal STR sequencing data for five metapopulations. The data were compiled from 20 publications and included 39 datasets comprising a total of 7005 samples. The estimates are suitable for use within the calculation of match probabilities in forensic casework. We also have constructed a phylogenetic tree using this data that aligns with our understanding of human evolution.

Insertion/deletion polymorphism for genetic background and forensic performance exploration of the Sui group from Guizhou.

Insertion/deletion polymorphisms (InDels) as ideal genetic markers for forensic genetics are appreciated by scholars both nationally and internationally because they integrated the favorable features of single nucleotide polymorphisms (SNPs) and short tandem repeats (STRs). Nevertheless, with the limited identification efficiency of InDels, the multiplex amplification systems of InDels might just be applied as the supplementary methods in paternity testing with respect to commonly used STRs. In the current research, we successfully genotyped 105 unrelated individuals from the Guizhou Sui population based on a six-color fluorescence multiplex panel that could simultaneously detect 64 genetic markers (59 autosomal InDels, two autosomal miniSTRs and three Y chromosomal genetic markers). In addition, frequency distributions and forensic statistical parameters of these loci in the Sui group were assessed using the STRAF software. Phylogenetic relationships among the Sui group and other reference populations were dissected by two methods (principal component analysis and phylogenetic trees) based on 59 InDels. The combined discrimination power and probability of exclusion values of 61 autosomal genetic markers in the Sui group were nearly equal to 1-1.90063 × 10-27 and 0.999998272, respectively. Furthermore, we observed that the Sui group from Guizhou had closer genetic affinities with East Asian populations with respect to other continental populations. In summary, we stated that the multiplex amplification system might be utilized as a prospective independent tool for human individual identification and parentage testing in the Sui group residing in Guizhou.

Unraveling the paternal genetic structure and forensic traits of the Hui population in Liaoning Province, China using Y-chromosome analysis.

The Hui people are the second-largest ethnic minority in China, and they are distributed throughout the country. A previous study explored the paternal genetic structure of the Hui population in nine different regions of China, but it overlooked the Liaoning province. In this study, we examined the paternal genetic makeup and forensic traits of the Hui population in Liaoning province by analyzing 157 Y-chromosome single nucleotide polymorphisms (Y-SNPs) and 26 short tandem repeats (Y-STRs). We successfully genotyped 282 unrelated male individuals from the Hui population of Liaoning province using the SNaPshot® single base extension assay and Goldeneye™ Y26 system kit (PEOPLESPOT R&D, Beijing, China). The results revealed high haplotypic diversity (0.9998) and identified 46 terminal haplogroups for the Hui population. Additional analyses, such as heat maps, principal component analysis (PCA), genetic distance (FST), Multidimensional scaling (MDS) analysis, and median-joining network (MJ) analysis, showed that the Hui population could be classified into three groups: Northwest Hui populations (NWH), including Liaoning, Xinjiang, Qinghai, Gansu, Ningxia, Shaanxi, and Henan; Hui populations from Sichuan and Shandong (SSH); and Yunnan Hui populations (YNH). Pairwise genetic distance (Rst) comparisons with other Chinese populations revealed that the Hui population displayed genetic affinity with the Han population. The comprehensive understanding of the Hui population in Liaoning province, explored by Y-SNPs and Y-STRs, can be utilized to interpret their genetic structure and enhance the accuracy of forensic databases.

Robust preimplantation genetic testing of the common F8 Inv22 pathogenic variant of severe hemophilia A using a highly polymorphic multi-marker panel encompassing the paracentric inversion.

BACKGROUND: Hemophilia A (HEMA) is an X-linked bleeding disorder caused by reduced/absent coagulation factor VIII expression, as a result of pathogenic variants in the F8 gene. Preimplantation prevention of HEMA should ideally include direct pathogenic F8 variant detection, complemented by linkage analysis of flanking markers to identify the high-risk F8 allele. Linkage analysis is particularly indispensable when the pathogenic variant cannot be detected directly or identified. This study evaluated the suitability of a panel of F8 intragenic and extragenic short tandem repeat markers for standalone linkage-based preimplantation genetic testing for monogenic disorder (PGT-M) of the Inv22 pathogenic variant, an almost 600 kb paracentric inversion responsible for almost half of all severe HEMA globally, for which direct detection is challenging. METHODS: Thirteen markers spanning 1 Mb and encompassing both F8 and the Inv22 inversion interval were genotyped in 153 unrelated females of Viet Kinh ethnicity. RESULTS: All individuals were heterozygous for ≥ 1 marker, ~ 90% were heterozygous for ≥ 1 of the five F8 intragenic markers, and almost 98% were heterozygous for ≥ 1 upstream (telomeric) and ≥ 1 downstream (centromeric) markers. A prospective PGT-M couple at risk of transmitting F8 Inv22 were fully informative at four marker loci (2 intra-inversion, 1 centromeric, 1 telomeric) and partially informative at another five (2 intra-inversion, 3 centromeric), allowing robust phasing of low- and high-risk haplotypes. In vitro fertilization produced three embryos, all of which clearly inherited the low-risk maternal allele, enabling reliable unaffected diagnoses. A single embryo transfer produced a clinical pregnancy, which was confirmed as unaffected by amniocentesis and long-range PCR, and a healthy baby girl was delivered at term. CONCLUSION: Robust and reliable PGT-M of HEMA, including the common F8 Inv22 pathogenic variant, can be achieved with sufficient informative intragenic and flanking markers.

Concordance study on Y-STRs typing between SeqStudio™ genetic analyzer for HID and MiSeq™ FGx forensic genomics system.

BACKGROUND: Massively Parallel Sequencing (MPS) allowed an increased number of information to be retrieved from short tandem repeat (STR) analysis, expanding them not only to the size, as already performed in Capillary Electrophoresis (CE), but also to the sequence. MPS requires constant development and validation of the analytical parameters to ensure that the genotyping results of STRs correspond to those obtained by CE. Given the increased frequency of usage of Y-STRs as supplementary markers to the autosomal STRs analysis, it is urgent to validate the concordance of the typing results between CE and MPS analyses. METHODS AND RESULTS: DNA extracted from 125 saliva samples of unrelated males was genotyped using Yfiler™ Plus PCR Amplification Kit and ForenSeq™ DNA Signature Prep Kit, which were analyzed by SeqStudio™ Genetic Analyzer for HID and MiSeq™ FGx Forensic Genomics System, respectively. For each shared Y-STR, allele designation, number of length- and sequence-based alleles per locus, stutter percentage, and the intra-locus balance of multicopy Y-STRs were screened. CONCLUSIONS: Although the number of forensic genetics laboratories that are applying the MPS technique in routine analysis is small and does not allow a global assessment of MPS limitations, this comparative study highlights the ability of MPS to produce reliable profiles despite the generation of large amounts of raw data.