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Advancements in sequencing and laboratory technologies have enabled forensic genetic analysis on increasingly low quality and degraded DNA samples. However, existing computational methods applied to genotyping and imputation for generating DNA profiles from degraded DNA have not been tested for forensic applications. Here we simulated sequencing data of varying qualities-coverage, fragment lengths, and deamination patterns-from forty individuals of diverse genetic ancestries. We used this dataset to test the performance of commonly used genotype and imputation methods (SAMtools, GATK, ATLAS, Beagle, and GLIMPSE) on five different SNP panels (MPS-plex, FORCE, two extended kinship panels, and the Human Origins array) that are used for forensic and population genetics applications. For genome mapping and variant calling with degraded DNA, we find use of parameters and methods (such as ATLAS) developed for ancient DNA analysis provides a marked improvement over conventional standards used for next generation sequencing analysis. We find that ATLAS outperforms GATK and SAMtools, achieving over 90 % genotyping accuracy for the four largest SNP panels with coverages greater than 10X. For lower coverages, decreased concordance rates are correlated with increased rates of heterozygosity. Genotype refinement and imputation improve the accuracy at lower coverages by leveraging population reference data. For all five SNP panels, we find that using a population reference panel representative of worldwide populations (e.g., the 1000 Genomes Project) results in increased genotype accuracies across genetic ancestries, compared to ancestry-matched population reference panels. Importantly, we find that the low SNP density of commonly used forensics SNP panels can impact the reliability and performance of genotype refinement and imputation. This highlights a critical trade-off between enhancing privacy by using panels with fewer SNPs and maintaining the effectiveness of genomic tools. We provide benchmarks and recommendations for analyzing degraded DNA from diverse populations with widely used genomic methods in forensic casework., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Copy Number Variations (CNVs) are crucial in various diseases, especially cancer, but detecting them accurately from SNP genotyping arrays remains challenging. Therefore, this study benchmarked five CNV detection tools-PennCNV, QuantiSNP, iPattern, EnsembleCNV, and R-GADA-using SNP array and WGS data from 2002 individuals of the DRAGEN re-analysis of the 1000 Genomes project. Results showed significant variability in tool performance. R-GADA had the highest recall but low precision, while PennCNV was the most reliable in terms of precision and F1 score. EnsembleCNV improved recall by combining multiple callers but increased false positives. Overall, current tools, including new methods, do not outperform PennCNV in precise CNV detection. Improved reference data and consensus on true positive CNV calls are necessary. This study provides valuable insights and scalable workflows for researchers selecting CNV detection methods in future studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The MiniMUGA genotyping array is a popular tool for genetic quality control of laboratory mice and genotyping samples from most experimental crosses involving laboratory strains, particularly for reduced complexity crosses. The content of the production version of the MiniMUGA array is fixed; however, there is the opportunity to improve the array's performance and the associated report's usefulness by leveraging thousands of samples genotyped since the initial description of MiniMUGA. Here, we report our efforts to update and improve marker annotation, increase the number and the reliability of the consensus genotypes for classical inbred strains and substrains, and increase the number of constructs reliably detected with MiniMUGA. In addition, we have implemented key changes in the informatics pipeline to identify and quantify the contribution of specific genetic backgrounds to the makeup of a given sample, remove arbitrary thresholds, include the Y Chromosome and mitochondrial genome in the ideogram, and improve robust detection of the presence of commercially available substrains based on diagnostic alleles. Finally, we have updated the layout of the report to simplify the interpretation and completeness of the analysis and added a section summarizing the ideogram in table format. These changes will be of general interest to the mouse research community and will be instrumental in our goal of improving the rigor and reproducibility of mouse-based biomedical research., Competing Interests: Conflicts of interest The authors have no conflict of interest to declare. None of the authors have a financial relationship with Neogen Inc. apart from the service contracts listed above., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

The International Human Papillomavirus (HPV) Reference Center launches annual global HPV genotyping proficiency panels to enhance the precision and international standardization of HPV genotyping services. This study aims to assess the proficiency levels achieved in the global HPV genotyping proficiency panels conducted in 2022 and 2023, and to evaluate trends in performance over time. The proficiency panels comprised 44 blinded samples each, including 40 samples containing various purified plasmids corresponding to HPV types combined with human DNA, plus four control samples (one negative control and three extraction controls). Proficiency required a sensitivity of 50 International Units (IU)/5 µL for HPV 16 and HPV 18 500 IU/5 µL for HPVs 6, 11, 31, 33, 45, 52, and 58 and 500 genome equivalents (GE)/5 µL for other HPV types in both single and multiple infections, while avoiding false positivity. In 2022, 78 laboratories submitted a total of 154 data sets, and in 2023, 81 laboratories contributed 141 data sets. Most data sets (87%, 258/295) utilized commercially available HPV assays. Proficiency was common, with 77% of data sets meeting the proficiency criteria in 2022 and 79% in 2023. False positive results significantly decreased from 22% in 2022 to 13% in 2023. The high proficiency and increasing specificity in HPV genotyping services indicates progress toward more reliable HPV testing. High accuracy is crucial for supporting global efforts in HPV and cervical cancer elimination., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Patients who carry Rhesus (RH) blood group variants may develop Rh alloantibodies requiring matched red blood cell transfusions. Serologic reagents for Rh variants often fail to specifically identify variant Rh antigens and are in limited supply. Therefore, red blood cell genotyping assays are essential for managing transfusions in patients with clinically relevant Rh variants. Well-characterized DNA reference reagents are needed to ensure quality and accuracy of the molecular tests. Eight lyophilized DNA reference reagents, representing 21 polymorphisms in RHD and RHCE, were produced from an existing repository of immortalized B-lymphoblastoid cell lines at the Center for Biologics Evaluation and Research/US Food and Drug Administration. The material was validated through an international collaborative study involving 17 laboratories that evaluated each DNA candidate using molecular assays to characterize RHD and RHCE alleles, including commercial platforms and laboratory-developed testing, such as Sanger sequencing, next-generation sequencing, and third-generation sequencing. The genotyping results showed 99.4% agreement with the expected results for the target RH polymorphisms and 87.9% for RH allele agreement. Most of the discordant RH alleles results were explained by a limited polymorphism coverage in some genotyping methods. Results of stability and accelerated degradation studies support the suitability of these reagents for use as reference standards. The collaborative study results demonstrate the qualification of these eight DNA reagents for use as reference standards for RH blood group genotyping assay development and analytical validation., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. All rights reserved.)

Next-generation sequencing offers highly multiplexed and accurate detection of nucleic acid sequences but at the expense of complex workflows and high input requirements. The ease of use of CRISPR-Cas12 assays is attractive and may enable highly accurate detection of sequences implicated in, for example, cancer pathogenic variants. CRISPR assays often employ end-point measurements of Cas12 trans-cleavage activity after Cas12 activation by the target; however, end point-based methods can be limited in accuracy and robustness by arbitrary experimental choices. To overcome such limitations, we develop and demonstrate here an accurate assay targeting a mutation of the epidermal growth factor gene implicated in lung cancer (exon 19 deletion). The assay is based on characterizing the kinetics of Cas12 trans-cleavage to discriminate the mutant from wild-type targets. We performed extensive experiments (780 reactions) to calibrate key assay design parameters, including the guide RNA sequence, reporter sequence, reporter concentration, enzyme concentration, and DNA target type. Interestingly, we observed a competitive reaction between the target and reporter molecules that has important consequences for the design of CRISPR assays, which use preamplification to improve sensitivity. Finally, we demonstrate the assay on 18 tumor-extracted amplicons and 100 training iterations with 99% accuracy and discuss discrimination parameters and models to improve wild type versus mutant classification.

5-Fluorouracil (5-FU) and oral fluoropyrimidines, such as capecitabine, are widely used in the treatment of cancer, especially gastrointestinal tumors and breast cancer, but their administration can produce serious and even lethal toxicity. This toxicity is often related to the partial or complete deficiency of the dihydropyrimidine dehydrogenase (DPD) enzyme, which causes a reduction in clearance and a longer half-life of 5-FU. It is advisable to determine if a DPD deficiency exists before administering these drugs by genotyping DPYD gene polymorphisms. The objective of this consensus of experts, in which representatives from the Spanish Pharmacogenetics and Pharmacogenomics Society and the Spanish Society of Medical Oncology participated, is to establish clear recommendations for the implementation of genotype and/or phenotype testing for DPD deficiency in patients who are candidates to receive fluoropyrimidines. The genotyping of DPYD previous to treatment classifies individuals as normal, intermediate, or poor metabolizers. Normal metabolizers do not require changes in the initial dose, intermediate metabolizers should start treatment with fluoropyrimidines at doses reduced to 50%, and poor metabolizers are contraindicated for fluoropyrimidines., (© 2021. The Author(s).)

Based on molecular markers, genomic prediction enables us to speed up breeding schemes and increase the response to selection. There are several high-throughput genotyping platforms able to deliver thousands of molecular markers for genomic study purposes. However, even though its widely applied in plant breeding, species without a reference genome cannot fully benefit from genomic tools and modern breeding schemes. We used a method to assemble a population-tailored mock genome to call single-nucleotide polymorphism (SNP) markers without an available reference genome, and for the first time, we compared the results with standard genotyping platforms (array and genotyping-by-sequencing (GBS) using a reference genome) for performance in genomic prediction models. Our results indicate that using a population-tailored mock genome to call SNP delivers reliable estimates for the genomic relationship between genotypes. Furthermore, genomic prediction estimates were comparable to standard approaches, especially when considering only additive effects. However, mock genomes were slightly worse than arrays at predicting traits influenced by dominance effects, but still performed as well as standard GBS methods that use a reference genome. Nevertheless, the array-based SNP markers methods achieved the best predictive ability and reliability to estimate variance components. Overall, the mock genomes can be a worthy alternative for genomic selection studies, especially for those species where the reference genome is not available., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Human papillomavirus (HPV) is the main cause of cervical cancer. Polymerase chain reaction (PCR)-based techniques are associated with accurate results with respect to HPV detection and genotyping, being able to identify viral DNA at low levels. However, differences in primer design influence their sensibility and specificity, depending on the HPV type assessed., Objective: The aim of the study was to comparatively evaluate the effectiveness of three different PCR-based strategies for HPV detection and genotyping from cervical samples., Study Design: The procedures were based on different primer design strategies, using MY09/MY11, EntroA, and type specific multiplex PCR primers., Results: Out of 411 samples of cervical scrapings, 45 (10.9%), 50 (12.2%), and 117 (28.5%) were positive for MY09/MY11, EntroA, and multiplex PCR, respectively. For MY09/MY11 positive samples, 36 were negative for EntroA and 23 for multiplex PCR. For EntroA positive samples, 40 were negative for MY09/MY11 and 26 for multiplex PCR. For multiplex PCR positive samples, 96 were negative for MY09/MY11 and 94 for EntroA. MY09/MY11 identified 12 different HPV types, EntroA detected eight types and multiplex PCR detected 11 HPV types. EntroA primers were able to detect HPV in more samples than MY09/MY11, while multiplex PCR, despite the limited targeted HPV types, presented higher sensibility than the other methods., Conclusions: The three methods presented different advantages and disadvantages, and the present study reinforces the need to use more than one molecular strategy for HPV detection and genotyping, and the development of novel methods which could overcome the limitations of the existing tests., (© 2021 Wiley Periodicals LLC.)

Next generation sequencing (NGS) is being applied for HLA typing in research and clinical settings. NGS HLA typing has made it feasible to sequence exons, introns and untranslated regions simultaneously, with significantly reduced labor and reagent cost per sample, rapid turnaround time, and improved HLA genotype accuracy. NGS technologies bring challenges for cost-effective computation, data processing and exchange of NGS-based HLA data. To address these challenges, guidelines and specifications such as Genotype List (GL) String, Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING), and Histoimmunogenetics Markup Language (HML) were proposed to streamline and standardize reporting of HLA genotypes. As part of the 17th International HLA and Immunogenetics Workshop (IHIW), we implemented standards and systems for HLA genotype reporting that included GL String, MIRING and HML, and found that misunderstanding or misinterpretations of these standards led to inconsistencies in the reporting of NGS HLA genotyping results. This may be due in part to a historical lack of centralized data reporting standards in the histocompatibility and immunogenetics community. We have worked with software and database developers, clinicians and scientists to address these issues in a collaborative fashion as part of the Data Standard Hackathons (DaSH) for NGS. Here we report several categories of challenges to the consistent exchange of NGS HLA genotyping data we have observed. We hope to address these challenges in future DaSH for NGS efforts., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Array technology to genotype single-nucleotide variants (SNVs) is widely used in genome-wide association studies (GWAS), clinical diagnostics, and linkage studies. Arrays have undergone a tremendous growth in both number and content over recent years making a comprehensive comparison all the more important. We have compared 28 genotyping arrays on their overall content, genome-wide coverage, imputation quality, presence of known GWAS loci, mtDNA variants and clinically relevant genes (i.e., American College of Medical Genetics (ACMG) actionable genes, pharmacogenetic genes, human leukocyte antigen (HLA) genes and SNV density). Our comparison shows that genome-wide coverage is highly correlated with the number of SNVs on the array but does not correlate with imputation quality, which is the main determinant of GWAS usability. Average imputation quality for all tested arrays was similar for European and African populations, indicating that this is not a good criterion for choosing a genotyping array. Rather, the additional content on the array, such as pharmacogenetics or HLA variants, should be the deciding factor. As the research question of a study will in large part determine which class of genes are of interest, there is not just one perfect array for all different research questions. This study can thus help as a guideline to determine which array best suits a study's requirements., (© 2021. The Author(s).)

The domestic cat (Felis silvestris catus) is a valued companion animal throughout the world. Over 60 different cat breeds are accepted for competition by the cat fancy registries in different countries. Genetic markers, including short tandem repeats and SNPs, are available to evaluate and manage levels of inbreeding and genetic diversity, population and breed structure relationships, and individual identification for forensic and registration purposes. The International Society of Animal Genetics (ISAG) hosts the Applied Genetics in Companion Animals Workshop, which supports the standardization of genetic marker panels and genotyping for the identification of cats via comparison testing. SNP panels have been in development for many species, including the domestic cat. An ISAG approved core panel of SNPs for use in cat identification and parentage analyses is presented. SNPs (n = 121) were evaluated by different university-based and commercial laboratories using 20 DNA samples as part of the ISAG comparison testing procedures. Different SNP genotyping technologies were examined, including DNA arrays, genotyping-by-sequencing and mass spectroscopy, to select a robust and efficient panel of 101 SNPs as the ISAG core panel for cats. The SNPs are distributed across all chromosomes including two on the X chromosome and an XY pseudo-autosomal sexing marker (zinc-finger XY; ZFXY). A population study demonstrated that the markers have an average polymorphic information content of 0.354 and a power of exclusion greater than 0.9999. The SNP panel should keep testing affordable while also allowing for the development of additional panels to monitor health, phenotypic traits, hybrid cats and highly inbred cats., (© 2021 The Authors. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.)

Objectives: Genotyping of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in monitoring viral evolution and transmission during the pandemic. The quality of the sequence data obtained from these genotyping efforts depends on several factors, including the quantity/integrity of the input material, the technology, and laboratory-specific implementation. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in genomic epidemiology studies. We aimed to establish clear and broadly applicable recommendations for reliable virus genotyping., Methods: We established and used a sequencing data analysis workflow that reliably identifies and removes technical artefacts; such artefacts can result in miscalls when using alternative pipelines to process clinical samples and synthetic viral genomes with an amplicon-based genotyping approach. We evaluated the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination., Results: We found that at least 1000 viral genomes are necessary to confidently detect variants in the SARS-CoV-2 genome at frequencies of ≥10%. The broad applicability of our recommendations was validated in over 200 clinical samples from six independent laboratories. The genotypes we determined for clinical isolates with sufficient quality cluster by sampling location and period. Our analysis also supports the rise in frequencies of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was facilitated by travel during the summer of 2020., Conclusions: We present much-needed recommendations for the reliable determination of SARS-CoV-2 genome sequences and demonstrate their broad applicability in a large cohort of clinical samples., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Inadequate sensitivity has been the primary limitation for implementing high-throughput sequencing for studies of tick-borne agents. Here we describe the development of TBDCapSeq, a sequencing assay that uses hybridization capture probes that cover the complete genomes of the eleven most common tick-borne agents found in the United States. The probes are used for solution-based capture and enrichment of pathogen nucleic acid followed by high-throughput sequencing. We evaluated the performance of TBDCapSeq to surveil samples that included human whole blood, mouse tissues, and field-collected ticks. For Borrelia burgdorferi and Babesia microti, the sensitivity of TBDCapSeq was comparable and occasionally exceeded the performance of agent-specific quantitative PCR and resulted in 25 to > 10,000-fold increase in pathogen reads when compared to standard unbiased sequencing. TBDCapSeq also enabled genome analyses directly within vertebrate and tick hosts. The implementation of TBDCapSeq could have major impact in studies of tick-borne pathogens by improving detection and facilitating genomic research that was previously unachievable with standard sequencing approaches.

Cryptosporidiosis is ranked sixth in the list of the most important food-borne parasites globally, and it is an important contributor to mortality in infants and the immunosuppressed. Recently, the number of genome sequences available for this parasite has increased drastically. The majority of the sequences are derived from population studies of Cryptosporidium parvum and Cryptosporidium hominis , the most important species causing disease in humans. Work with this parasite is challenging since it lacks an optimal, prolonged, in vitro culture system, which accurately reproduces the in vivo life cycle. This obstacle makes the cloning of isolates nearly impossible. Thus, patient isolates that are sequenced represent a population or, at times, mixed infections. Oocysts, the lifecycle stage currently used for sequencing, must be considered a population even if the sequence is derived from single-cell sequencing of a single oocyst because each oocyst contains four haploid meiotic progeny (sporozoites). Additionally, the community does not yet have a set of universal markers for strain typing that are distributed across all chromosomes. These variables pose challenges for population studies and require careful analyses to avoid biased interpretation. This review presents an overview of existing population studies, challenges, and potential solutions to facilitate future population analyses.

Species identification of the components of various duck breeds has revealed that the lowest identifiable number of components depends on the breed. The results (shown on the agarose gel) of a species-specific PCR reaction for Rouen ducks were less intense than the results for the same amount of components from other popular duck breeds, suggesting differences in the Rouen duck genome. Therefore, the present study aimed to identify part of the Rouen duck's gene sequences and to develop two new primer pairs. The first pair enables breed-independent identification of duck DNA, and the second distinguishes Rouen ducks from Chinese and Indian Runner ducks. The sequencing reaction yielded sequences of 1386 bp in length, and the identified sequence differs by around 7% from the sequences of Chinese duck species. The detected sequence contributes to improving species identification methods for duck DNA. On its basis, two primers for the identification of duck DNA were designed. The first allows for DNA amplification with the same sensitivity regardless of duck breed. The second primer's pair is breed specific, and it distinguishes Rouen ducks from Chinese and Indian Runner ducks. Both methods are very sensitive (0.05%).

Cutaneous warts are infectious disorders caused by human papillomavirus (HPV). A recent study revealed that the HPV genotype influences the natural course and response to treatment for plantar warts, suggesting that HPV genotyping could potentially be used to optimize wart treatment schemes. For this purpose, a wart-associated HPV genotyping assay was developed. The assay was subjected to an intensive validation process including, i.a., empiric determination of the annealing temperature, primer-probe optimization, evaluation of the analytical specificity and sensitivity, viral load quantification, and qualitative as well as quantitative analysis of intra-run repeatability and inter-run reproducibility. The newly developed assay was employed in a small-scale HPV genotyping study of wart biopsies (n = 50). The assay exhibited an analytical type-specific sensitivity and specificity of 100% (95% confidence interval [CI]: 83.9%-100%). The limit of quantification of the tested sequences corresponded to less than 17 viral copies/µl, while the limit of detection was less than 5 copies/µl. Very good to excellent agreements were gained between intra- and inter-run measurements (κ = 0.85-1.00) and coefficients of variation of the quantitative agreements were less then 3%. 22.5% (95% CI: 11%-39%) of the analyzed biopsies were negative for the tested HPV types, while 35% (95% CI: 21%-52%) contained multiple infections. The wart-associated HPV quantitative polymerase chain reaction assay was proven to be highly sensitive and specific. Multiple HPV infections were detected in 35% of lesions, contradicting the current literature claiming that in immunocompetent patients only 4%-16% of warts exhibit multiple HPV infections. This assay is qualified to be implemented in development of future genotype specific wart treatment strategies., (© 2020 Wiley Periodicals LLC.)

Errors in genotype calling can have perverse effects on genetic analyses, confounding association studies, and obscuring rare variants. Analyses now routinely incorporate error rates to control for spurious findings. However, reliable estimates of the error rate can be difficult to obtain because of their variance between studies. Most studies also report only a single estimate of the error rate even though genotypes can be miscalled in more than one way. Here, we report a method for estimating the rates at which different types of genotyping errors occur at biallelic loci using pedigree information. Our method identifies potential genotyping errors by exploiting instances where the haplotypic phase has not been faithfully transmitted. The expected frequency of inconsistent phase depends on the combination of genotypes in a pedigree and the probability of miscalling each genotype. We develop a model that uses the differences in these frequencies to estimate rates for different types of genotype error. Simulations show that our method accurately estimates these error rates in a variety of scenarios. We apply this method to a dataset from the whole-genome sequencing of owl monkeys (Aotus nancymaae) in three-generation pedigrees. We find significant differences between estimates for different types of genotyping error, with the most common being homozygous reference sites miscalled as heterozygous and vice versa. The approach we describe is applicable to any set of genotypes where haplotypic phase can reliably be called and should prove useful in helping to control for false discoveries., (© The Author(s) 2020. Published by Oxford University Press on behalf of Genetics Society of America. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of X O and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research., (Copyright © 2020 by the Genetics Society of America.)

Objectives: Accurate assays for hepatitis C virus (HCV) quantitation and genotyping are important for the management of HCV infection. In this study, we evaluated the performance of cobas HCV and cobas HCV GT assays (Roche) for HCV quantitation and genotyping on the cobas 4800 System., Methods: We compared the performance of the cobas HCV assays with another commercial system (Abbott m 2000) using a panel of well-characterized patient samples and proficiency testing samples., Results: The limit-of-detection of the cobas HCV assay in our center was higher (15 IU/mL) than the manufacturer claim (9.2 IU/mL). The assay showed high analytical specificity, accuracy, precision, and linearity. Performance was congruent with the RealTi m e HCV assay (Abbott). For genotyping, the cobas HCV GT assay only showed moderate agreement with the RealTi m e HCV Genotype II assay (kappa = 0.550). The cobas assay outperformed the RealTi m e assay for typing HCV genotypes 1b and 6 (p = 0.033)., Conclusion: Our results confirm that the cobas 4800 System is a reliable platform for HCV quantitation and genotyping. The cobas HCV GT assay is a good choice for genotype 1b/6 endemic areas in east Asia, clearly outperforming the RealTi m e HCV Genotype II assay.

Background: The SureX HPV genotyping test (SureX HPV test), which targets the human papillomavirus (HPV) E6/E7 genes was compared with the Cobas 4800 and Venus HPV tests for detecting 14 high-risk HPV (HR-HPV) types in clinical referral and follow-up patients to evaluate its value for cervical cancer screening., Methods: Two different populations were enrolled in the study. The first population comprised 185 cases and was used for comparing the SureX HPV test (Health, China) with the Cobas 4800 test (Roche, USA). The second population comprised 290 cases and was used for comparing the SureX HPV test (Health, China) with the Venus HPV test (Zhijiang, China). Polymerase chain reaction (PCR) sequencing was performed for further confirmation of discordant results., Results: In the first population, the overall agreement rate was 95.6% for 14 high-risk HPV types. Eight discordant cases were confirmed by PCR sequencing, which showed that the agreement rates were 75.0% between the SureX HPV test and PCR sequencing and 25.0% between the Cobas 4800 test and PCR sequencing (P < 0.01). In the second population, the overall agreement rate was 95.5%. Thirteen discordant cases were confirmed by PCR sequencing, which showed that the agreement rates were 76.9% between the SureX HPV test and PCR sequencing and 23.1% between the Venus HPV test and PCR sequencing (P < 0.01). With cervical intraepithelial neoplasia grade 2+ (CIN2+) as the reference standard, the sensitivity values of the SureX HPV test and the Venus HPV test were 93.5% and 92.0%, (P > 0.05), while the specificity values were 43.3% and 46.7%, respectively (P > 0.05)., Conclusion: The SureX HPV test had good consistency with both the Cobas 4800 and Venus HPV tests for 14 HR-HPV types. In addition, it avoided some false negatives and false positives. Therefore, the SureX HPV test can be used for cervical cancer screening.

Male-to-female sex reversal in horses is a developmental disorder in which phenotypic females have a male genetic constitution. Male-to-female sex reversal is the second most common genetic sex abnormality, after X chromosome monosomy. All male-to-female sex reversal cases studied to date have been found to be infertile. Therefore, a screening test is particularly useful in laboratories doing DNA genotyping in horses. Our laboratory has tested > 209,000 horses for parentage using a panel of microsatellite markers and the sex marker gene amelogenin ( AMEL ). Suspect XY sex reversal cases are reported females with a male profile by AMEL testing. After routine genotyping, 49 cases were detected and further tested using the sex-determining region Y ( SRY ) gene, confirming the XY SRY -negative genotype of suspect sex reversal cases. When some inconsistencies arose in the initial result, a molecular panel of X- and Y-linked markers was analyzed for these samples. Of the 49 cases, 33 were confirmed as XY SRY -negative. The remaining 16 cases were identified as false-positives as a result of anomalies of AMEL testing in horses.

With the widespread use of single nucleotide variants generated through mutagenesis screens and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end-point PCR in Caenorhabditis elegans Each SS primer consists of a 5' "anchor" that hybridizes to the template, followed by a noncomplementary "bridge," and a "foot" corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3' nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length and sequence composition of each SS primer segment affected selectivity and efficiency in various genetic contexts in order to develop simple rules for primer design that allow for differentiation between alleles over a broad range of annealing temperatures. Manipulating bridge length affected amplification efficiency, while modifying the foot sequence altered discriminatory power. Changing the anchor position enabled SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. After defining primer design parameters, we demonstrated the utility of SS primers for genotyping crude C. elegans lysates, suggesting that this approach could also be used for SNP mapping and screening of CRISPR mutants. Further, since SS primers reliably detect point mutations, this method has potential for broad application in all genetic systems., (Copyright © 2020 by the Genetics Society of America.)

The glycoprotein encoded by the ACKR1 gene expresses the Duffy blood group antigens and is a receptor for malaria parasites. We recently described 18 long-range ACKR1 alleles in an autochthonous population of a malaria endemic region. Extending this work, we sequenced the gene in a 53-sample repository established by the US Food and Drug Administration (FDA) as reference reagents for blood group genotyping. The FDA samples have been characterized for 19 genes; however, long-range haplotype information for these genes, including ACKR1, was lacking. We used a hybrid approach, novel for this type of gene, to characterize ACKR1 by combining two next-generation sequencing technologies, the short-read massively parallel sequencing and the long-read nanopore sequencing. The expedient integration of data from both next-generation sequencing systems were necessary and sufficient to allow determination of all 25 long-range ACKR1 alleles found in the 53 samples accurately. All 25 alleles identified in our current FDA cohort were novel and, unexpectedly, none had been observed among the 18 alleles in our previous study. The alleles will be useful for validation, calibration, and proficiency testing of red cell genotyping. The lack of any overlap between the ACKR1 alleles in the two studies documents differences in mutation rate and recombination frequency among populations. The exact haplotype and their interethnic or interpopulation dissimilarities can influence disease susceptibility and therapy., (Published by Elsevier Inc.)

Background: Homologous sex chromosomes can differentiate over time because recombination is suppressed in the region of the sex determining locus, leading to the accumulation of repeats, progressive loss of genes that lack differential influence on the sexes and sequence divergence on the hemizygous homolog. Divergence in the non-recombining regions leads to the accumulation of Y or W specific sequence useful for developing sex-linked markers. Here we use in silico whole-genome subtraction to identify putative sex-linked sequences in the scincid lizard Bassiana duperreyi which has heteromorphic XY sex chromosomes., Results: We generated 96.7 × 10 9 150 bp paired-end genomic sequence reads from a XY male and 81.4 × 10 9 paired-end reads from an XX female for in silico whole genome subtraction to yield Y enriched contigs. We identified 7 reliable markers which were validated as Y chromosome specific by polymerase chain reaction (PCR) against a panel of 20 males and 20 females., Conclusions: The sex of B. duperreyi can be reversed by low temperatures (XX genotype reversed to a male phenotype). We have developed sex-specific markers to identify the underlying genotypic sex and its concordance or discordance with phenotypic sex in wild populations of B. duperreyi. Our pipeline can be applied to isolate Y or W chromosome-specific sequences of any organism and is not restricted to sequence residing within single-copy genes. This study greatly improves our knowledge of the Y chromosome in B. duperreyi and will enhance future studies of reptile sex determination and sex chromosome evolution.

STRidER, the STRs for Identity ENFSI Reference Database, is a curated, freely publicly available online allele frequency database, quality control (QC) and software platform for autosomal Short Tandem Repeats (STRs) developed under the endorsement of the International Society for Forensic Genetics. Continuous updates comprise additional STR loci and populations in the frequency database and many further STR-related aspects. One significant innovation is the autosomal STR data QC provided prior to publication of datasets. Such scrutiny was lacking previously, leaving QC to authors, reviewers and editors, which led to an unacceptably high error rate in scientific papers. The results from scrutinizing 184 STR datasets containing >177,000 individual genotypes submitted in the first two years of STRidER QC since 2017 revealed that about two-thirds of the STR datasets were either being withdrawn by the authors after initial feedback or rejected based on a conservative error rate. Almost no error-free submissions were received, which clearly shows that centralized QC and data curation are essential to maintain the high-quality standard required in forensic genetics. While many errors had minor impact on the resulting allele frequencies, multiple error categories were commonly found within single datasets. Several datasets contained serious flaws. We discuss the factors that caused the errors to draw the attention to redundant pitfalls and thus contribute to better quality of autosomal STR datasets and allele frequency reports.

Giardia intestinalis is an intestinal protozoan most commonly found in humans. It has been grouped into 8 assemblages (A-H). Markers such as the glutamate dehydrogenase gene, triose phosphate isomerase and beta-giardin (β-giardin) have been widely used for genotyping. In addition, different genetic targets have been proposed as a valuable alternative to assess diversity and genetics of this microorganism. Thus, our objective was to evaluate new markers for the study of the diversity and intra-taxa genetic structure of G. intestinalis in silico and in DNA obtained from stool samples. We analysed nine constitutive genes in 80 complete genome sequences and in a group of 24 stool samples from Colombia. Allelic diversity was evaluated by locus and for the concatenated sequence of nine loci that could discriminate up to 53 alleles. Phylogenetic reconstructions allowed us to identify AI, AII and B assemblages. We found evidence of intra- and inter-assemblage recombination events. Population structure analysis showed genetic differentiation among the assemblages analysed.

Background: Individuals whose copies of the survival motor neuron 1 (SMN1) gene exist on the same chromosome are considered silent carriers for spinal muscular atrophy (SMA). Conventional screening for SMA only determines SMN1 copy number without any information regarding how those copies are arranged. A single nucleotide variant (SNV) rs143838139 is highly linked with the silent carrier genotype, so testing for this SNV can more accurately assess risk to a patient of having an affected child., Methods: Using a custom-designed SNV-specific Taqman genotyping assay, we determined and validated a model for silent-carrier detection in the laboratory., Results: An initial cohort of 21 pilot specimens demonstrated results that were 100% concordant with a reference laboratory method; this cohort was utilized to define the reportable range. An additional 177 specimens were utilized for a broader evaluation of clinical validity and reproducibility. Allelic-discrimination analysis demonstrated tight clustering of genotype groupings and excellent reproducibility, with a coefficient of variation for all genotypes ranging from 1% to 4%., Conclusion: The custom-developed Taqman SNV genotyping assay we tested provides a rapid, accurate, and cost-effective method for routine SMA silent-carrier screening and considerably improves detection rates of residual risk for SMA carriers., (© American Society for Clinical Pathology 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background and Objectives: Fetal RHD genotyping of cell-free maternal plasma DNA from RhD negative pregnant women can be used to guide targeted antenatal and postnatal anti-D prophylaxis for the prevention of RhD immunization. To assure the quality of clinical testing, we conducted an external quality assessment workshop with the participation of 31 laboratories., Materials and Methods: Aliquots of pooled maternal plasma from gestational week 25 were sent to each laboratory. One sample was fetal RHD positive, and a second sample was fetal RHD negative. A reporting scheme was supplied for data collection, including questions regarding the methodological setup, results and clinical recommendations. The samples were tested blindly., Results: Different methodological approaches were used; 29 laboratories used qPCR and two laboratories used ddPCR, employing a total of eight different combinations of RHD exon targets. Fetal RHD genotyping was performed with no false-negative and no false-positive results. One inconclusive result was reported for the RHD positive sample. All clinical conclusions were satisfactory., Conclusion: This external quality assessment workshop demonstrates that despite the different approaches taken to perform the clinical assays, fetal RHD genotyping is a reliable laboratory assay to guide targeted use of Rh prophylaxis in a clinical setting., (© 2020 International Society of Blood Transfusion.)

Background: The androgen receptor (AR) is a nuclear receptor, encoded by the AR gene on the X chromosome. Within the first exon of the AR gene, two short tandem repeats (STR), CAG and GGC, are a source of polymorphism in the population. Therefore, high-throughput methods for screening AR, such as next-generation sequencing (NGS), are sought after; however, data generated by NGS are limited by the availability of bioinformatics tools. Here, we evaluated the accuracy of the bioinformatics tool HipSTR in detecting and quantify CAG repeats within the AR gene., Method: The AR gene of 228 infertile men was sequenced using NGSgene panel. Data generated were analyzed with HipSTR to detect CAG repeats. The accuracy was compared with the results obtained with Sanger., Results: We found that HipSTR was more accurate than Sanger in genotyping normal karyotype men (46,XY), however, it was more likely to misidentify homozygote genotypes in men with Klinefelter syndrome (47,XXY)., Conclusion: Our findings show that the bioinformatics tool HipSTR is 100% accurate in detecting and assessing AR CAG repeats in infertile men (46,XY) as well as in men with low-level mosaicism., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Recent studies have used genome-wide single-nucleotide polymorphisms (SNPs) to investigate relationships among various Jewish populations and their non-Jewish historical neighbors, often focusing on small subsets of populations from a limited geographic range or relatively small samples within populations. Here, building on the significant progress that has emerged from genomic SNP studies in the placement of Jewish populations in relation to non-Jewish populations, we focus on population structure among Jewish populations. In particular, we examine Jewish population-genetic structure in samples that span much of the historical range of Jewish populations in Europe, the Middle East, North Africa, and South Asia. Combining 429 newly genotyped samples from 29 Jewish and 3 non-Jewish populations with previously reported genotypes on Jewish and non-Jewish populations, we investigate variation in 2789 individuals from 114 populations at 486,592 genome-wide autosomal SNPs. Using multidimensional scaling analysis, unsupervised model-based clustering, and population trees, we find that, genetically, most Jewish samples fall into four major clusters that largely represent four culturally defined groupings, namely the Ashkenazi, Mizrahi, North African, and Sephardi subdivisions of the Jewish population. We detect high-resolution population structure, including separation of the Ashkenazi and Sephardi groups and distinctions among populations within the Mizrahi and North African groups. Our results refine knowledge of Jewish population-genetic structure and contribute to a growing understanding of the distinctive genetic ancestry evident in closely related but historically separate Jewish communities.

Phasing is the process of inferring haplotypes from genotype data. Efficient algorithms and associated software for accurate phasing in pedigrees are needed, especially for populations lacking reference panels of sequenced individuals. We present a novel method for phasing genotypes from whole-genome sequence data in pedigrees, called PULSAR (Phasing Using Lineage Specific Alleles/Rare variants). The method is based on the property that alleles specific to a single founding chromosome within a pedigree are highly informative for identifying haplotypes that are shared identical by descent. Simulation studies are used to assess the performance of PULSAR with various pedigree sizes and structures, and the effect of genotyping errors and the presence of nonsequenced individuals is investigated. In pedigrees with complete sequencing and realistic genotyping error rates, PULSAR correctly phases >99.9% of heterozygous genotypes, excluding sites at which all individuals are heterozygous, and does so with a switch error rate frequently below 10 -4 . PULSAR is highly accurate, capable of genotype error correction and imputation, and computationally competitive with alternative phasing software applicable to pedigrees. Our method has the significant advantage of not requiring reference panels that are essential for other population-based phasing algorithms. A software implementation of PULSAR is freely available.

In this study, we aimed to assess the value of genotyping DNA pools as a strategy to generate accurate and cost-effective genomic estimated breeding values (GEBV) of sires in multi-sire mating systems. In order to do that, we used phenotypic records of 2,436 Australian Angus cattle from 174 sires, including yearling weight (YWT; N = 1,589 records), coat score (COAT; N = 2,026 records), and Meat Standards Australia marbling score (MARB; N = 1,304 records). Phenotypes were adjusted for fixed effects and age at measurement and pools of 2, 5, 10, 15, 20, and 25 animals were explored. Pools were created either by phenotype or at random. When pools were created at random, 10 replicates were examined to provide a measure of sampling variation. The relative accuracy of each pooling strategy was measured by the Pearson correlation coefficient between the sire's GEBV with pooled progeny and the GEBV using individually genotyped progeny. Random pools allow the computation of sire GEBV that are, on average, moderately correlated (i.e., r > 0.5 at pool sizes [PS] ≤ 10) with those obtained without pooling. However, for pools assigned at random, the difference between the best and the worst relative accuracy obtained out of the 10 replicates was as high as 0.41 for YWT, 0.36 for COAT, and 0.61 for MARB. This uncertainty associated with the relative accuracy of GEBV makes randomly assigning animals to pools an unreliable approach. In contrast, pooling by phenotype allowed the estimation of sires' GEBV with a relative accuracy ≥ 0.9 at PS < 10 for all three phenotypes. Moreover, even with larger PS, the lowest relative accuracy obtained was 0.88 (YWT, PS = 20). In agreement with results using simulated data, we conclude that pooling by phenotype is a robust approach to implementing genomic evaluation using commercial herd data, and PS larger than 10 individuals can be considered., (© The Author(s) 2020. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

While genotyping studies are scavenging for suitable samples to analyze, large serum collections are currently left unused as they are assumed to provide insufficient amounts of DNA for array-based genotyping. Long-term stored serum is considered to be difficult to genotype since preanalytical treatments and storage effects on DNA yields are not well understood. Successful genotyping of such samples has the potential to activate large biobanks for future genome-wide association studies (GWAS). We aimed to evaluate genotyping of ultralow amounts of DNA from samples stored up to 45 years in the Janus Serum Bank with two commercially available platforms. 64 samples, with various preanalytical treatments, were genotyped on the Axiom Array from Thermo Fisher Scientific and a subset of 24 samples with slightly higher yield were genotyped on the HumanCoreExome array from Illumina. Our results showed that about 80% of the serum samples produced call rates with the Axiom arrays that would be satisfactory in GWAS. The mean DNA yield was 5.8 ng as measured with PicoGreen, 3-6% of recommended yield. The failed samples had on average lower input amounts of DNA. All serum samples genotyped on the HumanCoreExome with a standard and FFPE protocol produced GWAS satisfactory call rates, with mean 97.57% and 98.35% call rates, respectively. The mean yield was 10.65 ng, 6% of the recommendations. Successful array-based genotyping of ultralow DNA yields from serum samples stored up to 45 years is possible. These results demonstrate the potential to activate large serum biobank collections for future studies.

The antiplatelet agent clopidogrel, a prodrug that requires bioactivation through the cytochrome P450 2C19 (CYP2C19) enzyme, is commonly prescribed post-percutaneous coronary intervention (PCI). Genetic variation in CYP2C19 contributes to individual variability in clopidogrel response, and can lead to adverse cardiovascular events. Incorporating CYP2C19 testing during routine clinical care helps identify high-risk patients, and provides the opportunity for pharmacotherapeutic interventions in the early post-PCI period. The Spartan RX CYP2C19 System has emerged as an optimal genotyping assay for use in clinical care due to ease of use, utilization of buccal swabs, and rapid turnaround time. However, workflow constraints related to sample collection and processing, storage, time, and personnel were encountered when integrating testing into clinical care. To improve clinical workflow and successfully implement CYP2C19 genotyping at our institution, we validated the Spartan RX System to return genotype utilizing blood samples. Our Molecular Diagnostic Laboratory tested 26 known reference materials and both blood and buccal swab samples from 23 patients and volunteers using the Spartan RX Assay. Genotype results were 100% concordant between DNA from blood and buccal swabs for all patients or volunteers, and consistent with expected results for the 26 reference materials. For reproducibility, three samples were tested in at least four separate runs, with all resulting genotypes in agreement between runs. Post-validation, the laboratory began offering CYP2C19 testing during clinical care. DNA extracted from blood can serve as a genomic DNA source for the Spartan RX Assay. Alteration of the methodology allowed for clinical implementation to support genotype-guided therapy., (© 2019 The Authors. Clinical and Translational Science published by Wiley Periodicals, Inc. on behalf of the American Society for Clinical Pharmacology and Therapeutics.)

Hematopoietic stem cells (HSCs) are functionally and genetically diverse and this diversity decreases with age and disease. Numerous systems have been developed to quantify HSC diversity by genetic barcoding, but no framework has been established to empirically validate barcode sequences. Here we have developed an analytical framework, Selection of informative Amplicon Barcodes from Experimental Replicates (SABER), that identifies barcodes that are unique among a large set of experimental replicates. Amplicon barcodes were sequenced from the blood of 56 adult zebrafish divided into training and validation sets. Informative barcodes were identified and samples with a high fraction of informative barcodes were chosen by bootstrapping. There were 4.2 ± 1.8 barcoded HSC clones per sample in the training set and 3.5 ± 2.1 in the validation set (p = 0.3). SABER reproducibly quantifies functional HSCs and can accommodate a wide range of experimental group sizes. Future large-scale studies aiming to understand the mechanisms of HSC clonal evolution will benefit from this new approach to identifying informative amplicon barcodes.

Background: Short tandem repeats (STRs) are essential genetic markers for forensic applications and population estimations; thus the population genetics of STR loci have been extensively studied and discussed., Methods: In the present study, we detected 21 autosomal noncombined DNA index system (non-CODIS) STR loci in a Chinese Han population from Shanghai, calculated their forensic parameters and analyzed their genetic relationships with reported reference populations in mainland China., Results: A total of 173 alleles were observed, with corresponding allele frequencies from 0.0020 to 0.5512. The cumulative power of discrimination (CPD) and the cumulative probability of exclusion (CPE) values of the 21 STR loci were 0.999999999999999999997337058271 and 0.99999953732495, respectively. The results of interpopulation differentiation, phylogenetic, multidimensional scaling, and structure analyses indicated a closer genetic relationship of the studied population with Han populations from other regions of China than with other populations., Conclusions: The 21 STR loci exhibited high genetic polymorphism in the studied Shanghai&#95;Han population and could be used for forensic applications and population genetic studies., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Giardia duodenalis is one of the most important intestinal parasites globally, especially in children, and in Cuba is the leading cause of chronic paediatric diarrhoea in this population. G. duodenalis is composed of eight genetic groups (or assemblages), two of which (A and B) are apparently zoonotic, occurring in both humans and other animals. However, consensus on the most appropriate genotyping scheme for optimal characterization of G. duodenalis isolates is lacking. In this article we present the results of three descriptive observational studies conducted in Havana, Cuba between 2010 and 2013, with the aim of comparing the results from molecular (PCR) approaches targeting different genes in order to assign with confidence 224 isolates of G. duodenalis to the correct assemblages. In each sub-study, following DNA isolation by the phenol/chloroform/isoamyl alcohol extraction method, PCR targeting the triose phosphate isomerase (tpi) gene was used for molecular characterization, as well as one additional PCR-method targeting another gene or pair of genes. DNA amplification was obtained in 87%, 83%, and 80% in the three sub-studies. Although excellent agreement (kappa index = 1) was recorded between results from some pairs of genes, for other combinations only moderate or substantial agreement was achieved. These results highlight the importance of interpretation of genotyping data, especially when single genetic markers are used. From the results of our studies, PCR targeting a combination of the tpi gene and the intergenic spacer region of rDNA may be a useful approach for the molecular characterization of G. duodenalis isolates., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Detecting and estimating DNA sample contamination are important steps to ensure high-quality genotype calls and reliable downstream analysis. Existing methods rely on population allele frequency information for accurate estimation of contamination rates. Correctly specifying population allele frequencies for each individual in early stage of sequence analysis is impractical or even impossible for large-scale sequencing centers that simultaneously process samples from multiple studies across diverse populations. On the other hand, incorrectly specified allele frequencies may result in substantial bias in estimated contamination rates. For example, we observed that existing methods often fail to identify 10% contaminated samples at a typical 3% contamination exclusion threshold when genetic ancestry is misspecified. Such an incomplete screening of contaminated samples substantially inflates the estimated rate of genotyping errors even in deeply sequenced genomes and exomes. We propose a robust statistical method that accurately estimates DNA contamination and is agnostic to genetic ancestry of the intended or contaminating sample. Our method integrates the estimation of genetic ancestry and DNA contamination in a unified likelihood framework by leveraging individual-specific allele frequencies projected from reference genotypes onto principal component coordinates. Our method can also be used for estimating genetic ancestries, similar to LASER or TRACE , but simultaneously accounting for potential contamination. We demonstrate that our method robustly estimates contamination rates and genetic ancestries across populations and contamination scenarios. We further demonstrate that, in the presence of contamination, genetic ancestry inference can be substantially biased with existing methods that ignore contamination, while our method corrects for such biases., (© 2020 Zhang et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Individual identification is one of the most important tasks in the field of forensic genetics. Insertion/Deletion (InDel) polymorphism marker has been a promising marker for individual identification. However, a part of InDel loci in commonly used commercial kit show low polymorphisms in Chinese populations., Methods: We evaluated a panel of 35 InDel loci constructed previously for individual identifications in Hui group. Subsequently, population data of three Chinese populations from 1,000 Genomes Project database were used to evaluate individual identification performance of these 35 InDels. Forensic parameters, such as heterozygosity, power of exclusion, match probability and power of discrimination, were calculated to evaluate the forensic efficiency of these loci in Hui group. The heatmap of insertion allelic frequencies, Nei's genetic distances, pairwise fixation index values, principal component analyses and admixture analyses were used to analyze the genetic differentiations and structure between Hui group and other populations., Results: In studied Hui group, besides rs3054057, polymorphism information content values of the remaining loci were greater than 0.3. Values of expected heterozygosity of these loci were close to 0.5. The combined power of discrimination and power of exclusion values were 0.99999999999999659609 and 0.998682, respectively. Analyses of population genetics revealed that Chinese Hui group had closer genetic relationships with East Asian populations than other intercontinental populations., Conclusion: The forensic statistical analyses revealed these loci showed relatively high genetic polymorphisms in Chinese Hui group, and could be served as a useful tool for individual identifications in Hui group. Population genetic evaluations indicated that Chinese Hui group had close genetic relationships with East Asian populations., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of S. cerevisiae and C. elegans strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level., (Copyright © 2020 Morton et al.)

Genotyping of Mycobacterium avium subsp. paratuberculosis (Map) is an indispensable tool for surveillance of this significant veterinary pathogen. For Map, multi-locus variable number tandem repeat analysis (MLVA) targeting mycobacterial interspersed repetitive units (MIRUs) and other variable number variable-number tandem repeats (VNTRs) was established using 8 markers. In the recent past this standard, portable, reproducible and discriminatory typing method has been frequently applied alone or in combinations with multi-locus short-sequence-repeat (MLSSR) sequencing. With the widespread use of these genotyping methods, standardization between laboratories needs to be managed, and knowledge of existing profiles and newly defined genotypes should be indexed and shared. To meet this need, a web application called "MAC-INMV-SSR database" was developed. This freely accessible service allows users to compare MLVA and MLSSR subtype data of their strains with those of existing reference strains analyzed with the same genotyping methods., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human papillomavirus (HPV) detection is used for screening of cervical cancer and genotype-specific persistence has shown to be mandatory for dysplasia development. Aim of this study was to evaluate the clinical performance of HPV DNA Array for cervical intraepithelial neoplasia 2+ (CIN2+)  lesion detection. HPV DNA Array is a polymerase chain reaction-based assay that targets E1 sequences of 29 HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, 85, and 97). The clinical evaluation was performed against the reference assay, BS-GP5+/6+ multiplex genotyping (MPG)-Luminex, with 600 cervical smear samples of a referral population. HPV DNA Array detected CIN2+  lesions with a sensitivity of 90.2%, identical to that of MPG-Luminex. Detection of CIN3+  lesions was with a sensitivity of 90.3%, as compared with 88.7% of MPG-Luminex. It demonstrated very good agreement for HPV detection, irrespective of type, of 91.5% (κ  =  0.832). HPV DNA Array is a simple and robust assay, with a short protocol of 4 hours hands-on time and automated readout by ELISpot AiDot software. It permits testing of up to 96 samples in one run and may be considered for use in organized screening programs and low resource settings., (© 2019 The Authors. Journal of Medical Virology Published by Wiley Periodicals, Inc.)

α1-antitrypsin deficiency (AATD) remains the only readily identified genetic cause of chronic obstructive pulmonary disease (COPD). Furthermore, there is growing evidence that even a moderate deficiency increases the risk of lung disease among smokers. Despite these facts, the uptake of testing for AATD in at-risk populations remains low for many reasons, and a lack of clarity among clinicians regarding the most appropriate diagnostic techniques presents a major deterrent. This Personal View addresses the benefits of diagnosis, the technical basis of the available diagnostic methods, and possible clinical confounders for each test. We include a series of unusual cases encountered at our National Centre of Expertise to provide context. The topics covered should equip clinicians with the core knowledge required to confidently assess patients for AATD., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This study compared imputation from lower-density commercial and customized panels to high-density panels and a combined panel (Illumina and Affymetrix) in Nelore beef cattle. Additionally, linkage disequilibrium and haplotype block conformation were estimated in individual high-density panels and compared with corresponding values in the combined panel after imputation. Overall, 814 animals were genotyped using BovineHD BeadChip (IllumHD), and 93 of these animals were also genotyped using the Axion Genome-Wide BOS 1 Array Plate (AffyHD). In general, customization considering linkage disequilibrium and minor allele frequency had the highest accuracies. The IllumHD panel had higher values of linkage disequilibrium for short distances between SNPs than AffyHD and the combined panel. The combined panel had an increased number of small haplotype blocks. The use of a combined panel is recommended due to its increased density and number of haplotype blocks, which in turn increase the probability of a marker being close to a quantitative trait locus of interest. Considering common SNPs between IllumHD and AffyHD for the customization of a low-density panel increases the imputation accuracy for IllumHD, AffyHD and the combined panel.

Short tandem repeat (STR) profiling has been routinely used in kinship testing since the introduction of commercial kits in the mid-1990s. While 15 to 23 STR loci normally give definitive results in simple kinship testing, additional loci are sometimes required to resolve complex cases. The SureID 23comp Human Identification Kit, recently released by Health Gene Technologies (China), multiplexes amelogenin and 22 autosomal STRs, 17 of which are non-CODIS STRs. This enables the profiling of 38-40 loci when used in conjunction with widely used commercial kits. In this study, the kit was evaluated for kinship applications as a supplementary STR kit following the minimum criteria for validation recommended by the European Network of Forensic Science Institutes (ENFSI) and the Scientific Working Group on DNA Analysis Methods (SWGDAM) using 500 samples. Performance was comparable with other commercial kits demonstrating: repeatability and reproducibility; precision (maximum s.d. 0.1048 nt); accuracy, all alleles were within ±0.41 nt compared to the actual sizes; heterozygous peak balances at all loci >68%; stutter ratios ranged from 3.8% to 16.15%; full profiles were generated with 125 pg DNA (95.12% of alleles at 62 pg),; and we found 100% concordance over 5 common STRs with the GlobalFiler kit.

The success of next-generation sequencing depends on the accuracy of variant calls. Few objective protocols exist for QC following variant calling from whole genome sequencing (WGS) data. After applying QC filtering based on Genome Analysis Tool Kit (GATK) best practices, we used genotype discordance of eight samples that were sequenced twice each to evaluate the proportion of potentially inaccurate variant calls. We designed a QC pipeline involving hard filters to improve replicate genotype concordance, which indicates improved accuracy of genotype calls. Our pipeline analyzes the efficacy of each filtering step. We initially applied this strategy to well-characterized variants from the ClinVar database, and subsequently to the full WGS dataset. The genome-wide biallelic pipeline removed 82.11% of discordant and 14.89% of concordant genotypes, and improved the concordance rate from 98.53% to 99.69%. The variant-level read depth filter most improved the genome-wide biallelic concordance rate. We also adapted this pipeline for triallelic sites, given the increasing proportion of multiallelic sites as sample sizes increase. For triallelic sites containing only SNVs, the concordance rate improved from 97.68% to 99.80%. Our QC pipeline removes many potentially false positive calls that pass in GATK, and may inform future WGS studies prior to variant effect analysis.

Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination Program studies and 42 additional samples that had not been characterized previously. DNA samples were distributed to volunteer testing laboratories for genotyping using a variety of commercially available and laboratory-developed tests. These publicly available samples will support the quality-assurance and quality-control programs of clinical laboratories performing CYP2D6 testing., (Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)