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Aim: The aimof this study is to investigate if Preimplantation Genetic Testing (PGT) can effectively identify unreported variants according to American College of Medical Genetics and Genomics (ACMG)to prevent citrullinemia type 1 affection. Design: This study involves a detailed case analysis of a family with history of citrullinemia type 1, focusing on the use of PGT for monogenic diseases (PGT-M). The genetic variants were identified using ACMG guidelines, and PGT was employed to prevent the inheritance of these variants. The study included haplotype analysis and Sanger sequencing to confirm the results. Results: The study identified previously unreported variations in the ASS1 gene causing citrullinemia type 1. PGT successfully prevented the transmission of these variants, resulting in the birth of a healthy fetus. However, challenges such as allele dropout (ADO) and gene recombination were encountered during haplotype analysis, which could potentially defeat the diagnosis. The study demonstrated that combining haplotype analysis with Sanger sequencing can enhance the accuracy of PGT. Conclusion: Preimplantation Genetic Testing (PGT) targeting likely pathogenic and pathogenic variants in the ASS1 gene, as rated by ACMG, allows the birth of healthy infants free from citrullinemia type 1. Additionally, the establishment of single haplotypes and Sanger sequencing can reduce the misdiagnosis rate caused by allele dropout (ADO) and genetic recombination. [ABSTRACT FROM AUTHOR]

The black‐footed cat (Felis nigripes) is endemic to the arid regions of southern Africa. One of the world's smallest wild felids, the species occurs at low densities and is secretive and elusive, which makes ecological studies difficult. Genetic data could provide key information such as estimates on population size, sex ratios, and genetic diversity. In this study, we test if microsatellite loci can be successfully amplified from scat samples that could be noninvasively collected from the field. Using 21 blood and scat samples collected from the same individuals, we statistically tested whether nine microsatellites previously designed for use in domestic cats can be used to identify individual black‐footed cats. Genotypes recovered from blood and scat samples were compared to assess loss of heterozygosity, allele dropout, and false alleles resulting from DNA degradation or PCR inhibitors present in scat samples. The microsatellite markers were also used to identify individuals from scats collected in the field that were not linked to any blood samples. All nine microsatellites used in this study were amplified successfully and were polymorphic. Microsatellite loci were found to have sufficient discriminatory power to distinguish individuals and identify clones. In conclusion, these molecular markers can be used to monitor populations of wild black‐footed cats noninvasively. The genetic data will be able to contribute important information that may be used to guide future conservation initiatives. [ABSTRACT FROM AUTHOR]

Abstract The black‐footed cat (Felis nigripes) is endemic to the arid regions of southern Africa. One of the world's smallest wild felids, the species occurs at low densities and is secretive and elusive, which makes ecological studies difficult. Genetic data could provide key information such as estimates on population size, sex ratios, and genetic diversity. In this study, we test if microsatellite loci can be successfully amplified from scat samples that could be noninvasively collected from the field. Using 21 blood and scat samples collected from the same individuals, we statistically tested whether nine microsatellites previously designed for use in domestic cats can be used to identify individual black‐footed cats. Genotypes recovered from blood and scat samples were compared to assess loss of heterozygosity, allele dropout, and false alleles resulting from DNA degradation or PCR inhibitors present in scat samples. The microsatellite markers were also used to identify individuals from scats collected in the field that were not linked to any blood samples. All nine microsatellites used in this study were amplified successfully and were polymorphic. Microsatellite loci were found to have sufficient discriminatory power to distinguish individuals and identify clones. In conclusion, these molecular markers can be used to monitor populations of wild black‐footed cats noninvasively. The genetic data will be able to contribute important information that may be used to guide future conservation initiatives.

Moreover, 6 and 25 genes encoding DNA methyltransferase and demethylation-related factors, respectively, were identified in the peach genome (Table S12), but they all had similar expression in fruits at 31 DAFB between "ZY4" and "LXH". A large-scale behavior of allelic dropout and imbalance caused by DNA methylation changes in an early-ripening bud sport of peach Since DNA methylation is one of the main reasons for ASE (Satyaki & Gehring, [17]; da Rocha & Gendrel, [16]), DNA methylation patterns in fruits of "ZY4" and "LXH" were investigated using Whole Genome Bisulfite Sequencing (WGBS). Keywords: allelic dropout; allelic imbalance; bud sport; DNA methylation; Prunus persica EN allelic dropout allelic imbalance bud sport DNA methylation Prunus persica 13 18 6 06/05/23 20230701 NES 230701 Data availability The sequencing data generated in this study have been deposited in NCBI SRA database with the Bio-Project accession number: PRJNA939397. [Extracted from the article]

Context Genetic testing is useful not only for the diagnosis of the MEN1 proband but also for determining the putative asymptomatic variant carriers to improve the prognosis or to avoid unnecessary medical intervention. However, we must be aware of the putative pitfalls of polymerase chain reaction (PCR)-based genetic testing in specific conditions that lead to medical mismanagement. Objective To warn of the putative pitfalls of PCR-based genetic testing, we report an overlooked case of MEN1 due to PCR allelic dropout. Methods A 69-year-old man was clinically diagnosed with MEN1, and genetic testing revealed that he had a pathogenic variant in the MEN1 gene. His 36-year-old son was completely asymptomatic. As the son was 50% at risk of MEN1, he was willing to undergo genetic testing himself after genetic counseling. Results Genetic testing was carried out in 2 independent laboratories. Although laboratory A showed that he carried a pathogenic variant, laboratory B showed that he had the wild-type genotype of MEN1. The discrepancy in these results was due to PCR allelic dropout by single-nucleotide variations of the MEN1 gene in the 5′ region. The surveillance revealed that he had asymptomatic primary hyperparathyroidism and a neuroendocrine tumor of the pancreas. Conclusion PCR-dependent genetic analysis may be susceptible to PCR allelic dropout in an SNV-specific manner. We must be careful when genetically testing individuals of relatives with clinical MEN1 disease. [ABSTRACT FROM AUTHOR]

Aim: The aim of this study is to investigate if Preimplantation Genetic Testing (PGT) can effectively identify unreported variants according to American College of Medical Genetics and Genomics (ACMG)to prevent citrullinemia type 1 affection., Design: This study involves a detailed case analysis of a family with history of citrullinemia type 1, focusing on the use of PGT for monogenic diseases (PGT-M). The genetic variants were identified using ACMG guidelines, and PGT was employed to prevent the inheritance of these variants. The study included haplotype analysis and Sanger sequencing to confirm the results., Results: The study identified previously unreported variations in the ASS1 gene causing citrullinemia type 1. PGT successfully prevented the transmission of these variants, resulting in the birth of a healthy fetus. However, challenges such as allele dropout (ADO) and gene recombination were encountered during haplotype analysis, which could potentially defeat the diagnosis. The study demonstrated that combining haplotype analysis with Sanger sequencing can enhance the accuracy of PGT., Conclusion: Preimplantation Genetic Testing (PGT) targeting likely pathogenic and pathogenic variants in the ASS1 gene, as rated by ACMG, allows the birth of healthy infants free from citrullinemia type 1. Additionally, the establishment of single haplotypes and Sanger sequencing can reduce the misdiagnosis rate caused by allele dropout (ADO) and genetic recombination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wu, Liang, Liu, Ding and Shu.)

Comprehensive and accurate human leukocyte antigen (HLA) typing within a short turnaround time is a crucial initial step for allocating deceased donor organs for transplantation. Erroneous HLA typing of deceased donors can be catastrophic and result in recipient death, failed transplant, and organ wastage due to inappropriately matched donors. The real-time polymerase chain reaction method is widely used as the sole method for HLA typing of deceased donors because of its simplified workflow. Herein, we have reported cases of four deceased donors showing discrepant HLA typing discovered using two independent methods concurrently. The HLA typing of these donors could have been erroneously reported if a single method had been used, which would have profound patient safety implications. In one case, the drop out of HLA-DR7 using a single method could have resulted in harmful organ allocation if the organ was transplanted after a virtual crossmatch to a sensitized candidate showing strong donor-specific HLA-DR7 antibodies. In conclusion, this case series suggests that concurrent dual typing is essential for accurate HLA typing of deceased donors. This strategy is vital because precise HLA typing is critical for accurate virtual crossmatching, which facilitates continuous distribution and broader geographic sharing of the deceased donor organ. [ABSTRACT FROM AUTHOR]

Next generation sequencing (NGS) is a widespread molecular biology method integrated into clinical practice to detect genetic variants, for diagnostic and prognostic purposes. The scheduled external quality assessments (EQA) is integral part of clinical molecular laboratory quality assurance. The EQA provides an efficient system to compare analytic test performances among different laboratories, which is essential to evaluate consistency of molecular test. EQA failures demands targeted corrective action plans. In this context, the complexity of the NGS techniques requires careful and continuous quality control procedures. We report a tumor BRCA1/2 (tBRCA) testing benchmark discrepancy provided by the European Molecular Genetics Quality Network in our laboratory during a round of EQA for somatic mutation testing of BRCA genes in relation to ovarian cancer. The critical analysis emerging from the tBRCA EQA is presented. We underline that harmonization processes are still required for the EQA in the molecular biology field, especially if applied to the evaluation of methods characterized by high complexity. [ABSTRACT FROM AUTHOR]

Hematopoietic stem cell transplantation (HSCT) from HLA-matched donors significantly decreases the risks of graft-rejection and graft-versus-host disease. Long-range PCR- amplicon-based next-generation sequencing (NGS) is increasingly used as a standalone method in clinical laboratories to determine HLA compatibility for HSCT and solid-organ transplantation. We hypothesized that an allelic dropout is a frequent event in the long-range PCR amplicon-based NGS HLA typing method. To test the hypothesis, we typed 4,006 samples concurrently using a commercially available long-range PCR amplicon-based NGS-typing and short exon-specific amplicon-based reverse sequence-specific oligonucleotide (rSSO) methods. The concordance between the NGS and rSSO typing results was 100% at HLA-A, -B, -C, -DRB1, -DRB3, -DRB5, -DQA1, DPA1 loci. However, 4.5% of the samples (179/4006) showed allelic-dropouts at one of the other three loci: HLA-DRB4 (3.9%), HLA-DPB1 (0.4%), and HLA-DQB1* (0.15%). The allelic-dropouts are not associated with specific haplotypes, and some dropouts can be reagent lot-specific. Although DRB1-DRB3/4/5-DQB1 linkages help to diagnose these allelic-dropouts in some cases, the rSSO typing was crucial to identify the dropouts in DQB1 and DPB1 loci. These results uncover the critical limitations of using long-range PCR amplicon-based NGS as a standalone method in clinical histocompatibility laboratories and advocate the need for strategies to diagnose and resolve allelic-dropouts. [ABSTRACT FROM AUTHOR]

Genotyping‐by‐sequencing methods such as RADseq are popular for generating genomic and population‐scale data sets from a diverse range of organisms. These often lack a usable reference genome, restricting users to RADseq specific software for processing. However, these come with limitations compared to generic next generation sequencing (NGS) toolkits. Here, we describe and test a simple pipeline for reference‐free RADseq data processing that blends de novo elements from STACKS with the full suite of state‐of‐the art NGS tools. Specifically, we use the de novo RADseq assembly employed by STACKS to create a catalogue of RAD loci that serves as a reference for read mapping, variant calling and site filters. Using RADseq data from 28 zebra sequenced to ~8x depth‐of‐coverage we evaluate our approach by comparing the site frequency spectra (SFS) to those from alternative pipelines. Most pipelines yielded similar SFS at 8x depth, but only a genotype likelihood based pipeline performed similarly at low sequencing depth (2–4x). We compared the RADseq SFS with medium‐depth (~13x) shotgun sequencing of eight overlapping samples, revealing that the RADseq SFS was persistently slightly skewed towards rare and invariant alleles. Using simulations and human data we confirm that this is expected when there is allelic dropout (AD) in the RADseq data. AD in the RADseq data caused a heterozygosity deficit of ~16%, which dropped to ~5% after filtering AD. Hence, AD was the most important source of bias in our RADseq data. [ABSTRACT FROM AUTHOR]

Primary cardiomyopathies (CMPs) are monogenic but multi-allelic disorders with dozens of genes involved in pathogenesis. The implementation of next-generation sequencing (NGS) approaches has resulted in more time- and cost-efficient DNA diagnostics of cardiomyopathies. However, the diagnostic yield of genetic testing for each subtype of CMP fails to exceed 60%. The aim of this study was to demonstrate that allelic dropout (ADO) is a common phenomenon that reduces the diagnostic yield in primary cardiomyopathy genetic testing based on targeted gene panels assayed on the Ion Torrent platform. We performed mutational screening with three custom targeted gene panels based on sets of oligoprimers designed automatically using AmpliSeq Designer® containing 1049 primer pairs for 37 genes with a total length of 153 kb. DNA samples from 232 patients were screened with at least one of these targeted gene panels. We detected six ADO events in both IonTorrent PGM (three cases) and capillary Sanger sequencing (three cases) data, identifying ADO-causing variants in all cases. All ADO events occurred due to common or rare single nucleotide variants (SNVs) in the oligoprimer binding sites and were detected because of the presence of “marker” SNVs in the target DNA fragment. We ultimately identified that PCR-based NGS involves a risk of ADO that necessitates the use of Sanger sequencing to validate NGS results. We assume that oligoprimer design without ADO data affects the amplification efficiency of up to 0.77% of amplicons.

Primary cardiomyopathies (CMPs) are monogenic but multi-allelic disorders with dozens of genes involved in pathogenesis. The implementation of next-generation sequencing (NGS) approaches has resulted in more time- and cost-efficient DNA diagnostics of cardiomyopathies. However, the diagnostic yield of genetic testing for each subtype of CMP fails to exceed 60%. The aim of this study was to demonstrate that allelic dropout (ADO) is a common phenomenon that reduces the diagnostic yield in primary cardiomyopathy genetic testing based on targeted gene panels assayed on the Ion Torrent platform. We performed mutational screening with three custom targeted gene panels based on sets of oligoprimers designed automatically using AmpliSeq Designer® containing 1049 primer pairs for 37 genes with a total length of 153 kb. DNA samples from 232 patients were screened with at least one of these targeted gene panels. We detected six ADO events in both IonTorrent PGM (three cases) and capillary Sanger sequencing (three cases) data, identifying ADO-causing variants in all cases. All ADO events occurred due to common or rare single nucleotide variants (SNVs) in the oligoprimer binding sites and were detected because of the presence of "marker" SNVs in the target DNA fragment. We ultimately identified that PCR-based NGS involves a risk of ADO that necessitates the use of Sanger sequencing to validate NGS results. We assume that oligoprimer design without ADO data affects the amplification efficiency of up to 0.77% of amplicons. [ABSTRACT FROM AUTHOR]

The use of non-invasively collected DNA source material for genetic and genomic applications is usually characterized by low target DNA concentration and quality, genotyping errors and cost-intensive lab procedures. However, for otters (Lutrinae) as elusive species of conservation concern, genetic non-invasive sampling has become an important tool to study their ecology and demography. To increase cost-efficiency of monitoring programmes and to promote the expansion of genomic approaches to non-invasive samples, we aimed to refine sample collection and preparation. Therefore, we examined the effects of intrinsic sample characteristics (including diet), environmental conditions in the field and sample treatment in the molecular laboratory on the success of genotyping and allelic dropout (ADO) rates using microsatellite markers in 1970 fresh Eurasian otter (Lutra lutra) scats. Using fresh samples only, we probably eliminated one of the most important impediments of genotyping DNA from otter faecal samples beforehand. But, we observed higher genotyping success and lower ADO rates for anal glad secretions and faecal samples containing high proportions of mucus. Moist conditions during sample collection may promote DNA degradation and PCR inhibition, leading to decreased genotyping success rates. ADO was further affected by the type of extraction kit. However, a high proportion of variance remaining unexplained by our models implied that additional parameters were acting (amount of PCR inhibitors, non-uniform distribution of intestinal cells, efficiency of PCRs, specific microclimate at marking sites). We summarized influential factors maximizing genotyping quality of otter scats and give recommendations for sample collection, storage and DNA extraction based on our results and current literature. [ABSTRACT FROM AUTHOR]

Loss of one allele during polymerase chain reaction (PCR) amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST. We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10. These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations.

When using microsatellite loci for DNA paternity testing, genetic inconsistencies sometimes occur in true trios and duos and may be erroneously attributed to germline mutations of microsatellite alleles. Here, we reported a typical case and discussed the issue of how to find out the cause of a genetic inconsistency. In our case, a genetic inconsistency in a true trio was observed at the D8S1179 locus, where the father has only allele 10 as compared to only allele 16 of his son. A set of tests were then performed. The results showed that the inconsistency was not result from the germline mutation of allele 10 to allele 16, or from the presence of null alleles due to primer binding site mutations, but from the microdeletion at 8q24.13, about 2.99 to 49.76 kb, detected in both the father and his son, which revealed by deletion mapping using short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). In conclusion, genetic inconsistencies observed in true trios or duos cannot be rashly attributed to germline STR mutations, especially multistep mutations, in the absence of verification or specification; otherwise, the reliability of the genetic proofs established will be challenged. [ABSTRACT FROM AUTHOR]

A rapid shift from traditional Sanger sequencing-based molecular methods to the phylogenomic approach with large numbers of loci is underway. Among phylogenomic methods, restriction site associated DNA (RAD) sequencing approaches have gained much attention as they enable rapid generation of up to thousands of loci randomly scattered across the genome and are suitable for nonmodel species. RAD data sets however suffer from large amounts of missing data and rapid locus dropout along with decreasing relatedness among taxa. The relationship between locus dropout and the amount of phylogenetic information retained in the data has remained largely uninvestigated. Similarly, phylogenetic hypotheses based on RAD have rarely been compared with phylogenetic hypotheses based on multilocus Sanger sequencing, even less so using exactly the same species and specimens. We compared the Sanger-based phylogenetic hypothesis (8 loci; 6172 bp) of 32 species of the diverse moth genus Eupithecia (Lepidoptera, Geometridae) to that based on double-digest RAD sequencing (3256 loci; 726,658 bp). We observed that topologies were largely congruent, with some notable exceptions that we discuss. The locus dropout effect was strong. We demonstrate that number of loci is not a precise measure of phylogenetic information since the number of single-nucleotide polymorphisms (SNPs) may remain low at very shallow phylogenetic levels despite large numbers of loci. As we hypothesize, the number of SNPs and parsimony informative SNPs (PIS) is low at shallow phylogenetic levels, peaks at intermediate levels and, thereafter, declines again at the deepest levels as a result of decay of available loci. Similarly, we demonstrate with empirical data that the locus dropout affects the type of loci retained, the loci found in many species tending to show lower interspecific distances than those shared among fewer species. We also examine the effects of the numbers of loci, SNPs, and PIS on nodal bootstrap support, but could not demonstrate with our data our expectation of a positive correlation between them. We conclude that RAD methods provide a powerful tool for phylogenomics at an intermediate phylogenetic level as indicated by its broad congruence with an eight-gene Sanger data set in a genus of moths. When assessing the quality of the data for phylogenetic inference, the focus should be on the distribution and number of SNPs and PIS rather than on loci. [ABSTRACT FROM AUTHOR]

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High-throughput sequencing methods that multiplex a large number of individuals have provided a cost-effective approach for discovering genome-wide genetic variation in large populations. These sequencing methods are increasingly being utilized in population genetic studies across a diverse range of species. Two side-effects of these methods, however, are (1) sequencing errors and (2) heterozygous genotypes called as homozygous due to only one allele at a particular locus being sequenced, which occurs when the sequencing depth is insufficient. Both of these errors have a profound effect on the estimation of linkage disequilibrium (LD) and, if not taken into account, lead to inaccurate estimates. We developed a new likelihood method, GUS-LD, to estimate pairwise linkage disequilibrium using low coverage sequencing data that accounts for undercalled heterozygous genotypes and sequencing errors. Our findings show that accurate estimates were obtained using GUS-LD, whereas underestimation of LD results if no adjustment is made for the errors. [ABSTRACT FROM AUTHOR]

The interpretation of DNA evidence may rely upon the assumption that the forensic short tandem repeat (STR) profile is composed of multiple genotypes, or partial genotypes, originating from n contributors. In cases where the number of contributors (NOC) is in dispute, it may be justifiable to compute likelihood ratios that utilize different NOC parameters in the numerator and denominator, or present different likelihoods separately. Therefore, in this work, we evaluate the impact of allele dropout on estimating the NOC for simulated mixtures with up to six contributors in the presence or absence of a major contributor. These simulations demonstrate that in the presence of dropout, or with the application of an analytical threshold (AT), estimating the NOC using counting methods was unreliable for mixtures containing one or more minor contributors present at low levels. The number of misidentifications was only slightly reduced when we expand the number of STR loci from 16 to 21. In many of the simulations tested herein, the minimum and actual NOC differed by more than two, suggesting that low-template, high-order mixtures with allele counts fewer than six may be originating from as many as four-, five-, or six-persons. Thus, there is justification for the use of differing or multiple assumptions on the NOC when computing the weight of DNA evidence for low-template mixtures, particularly when the peak heights are in the vicinity of the signal threshold or allele counting methods are the mechanism by which the NOC is assessed. [ABSTRACT FROM AUTHOR]

Loss of one allele during polymerase chain reaction (PCR) amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST. We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10. These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations. [ABSTRACT FROM AUTHOR]

Several methods exist for weight of evidence calculations on DNA mixtures. Especially if dropout is a possibility, it may be difficult to estimate mixture specific parameters needed for the evaluation. For semi-continuous models, the LR for a person to have contributed to a mixture depends on the specified number of contributors and the probability of dropout for each. We show here that, for the semi-continuous model that we consider, the weight of evidence can be accurately obtained by applying the standard statistical technique of integrating the likelihood ratio against the parameter likelihoods obtained from the mixture data. This method takes into account all likelihood ratios belonging to every choice of parameters, but LR's belonging to parameters that provide a better explanation to the mixture data put in more weight into the final result. We therefore avoid having to estimate the number of contributors or their probabilities of dropout, and let the whole evaluation depend on the mixture data and the allele frequencies, which is a practical advantage as well as a gain in objectivity. Using simulated mixtures, we compare the LR obtained in this way with the best informed LR, i.e., the LR using the parameters that were used to generate the data, and show that results obtained by integration of the LR approximate closely these ideal values. We investigate both contributors and non-contributors for mixtures with various numbers of contributors. For contributors we always obtain a result close to the best informed LR whereas non-contributors are excluded more strongly if a smaller dropout probability is imposed for them. The results therefore naturally lead us to reconsider what we mean by a contributor, or by the number of contributors. [ABSTRACT FROM AUTHOR]

Microsatellite genetic marker data are exploited in a variety of fields, including forensics, gene mapping, kinship inference and population genetics. In all of these fields, inference can be thwarted by failure to quantify and account for data errors, and kinship inference in particular can benefit from separating errors into two distinct classes: allelic dropout and false alleles. Pedant is MS Windows software for estimating locus-specific maximum likelihood rates of these two classes of error. Estimation is based on comparison of duplicate error-prone genotypes: neither reference genotypes nor pedigree data are required. Other functions include: plotting of error rate estimates and confidence intervals; simulations for performing power analysis and for testing the robustness of error rate estimates to violation of the underlying assumptions; and estimation of expected heterozygosity, which is a required input. The program, documentation and source code are available from http://www.stats.gla.ac.uk/~paulj/pedant.html.

While being well known that the diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing, not rarely, however, genetic testing needs much perseverance and cunning strategies to identify the causative mutation(s). Here we present a case of a thorny molecular diagnosis of mucolipidosis type III alpha/beta, which is an autosomal recessive lysosomal storage disorder, caused by a defect in the GNPTAB gene that codes for the a/β-subunits of the GlcNAc-1-phosphotransferase. We used both cDNA and gDNA analyses to characterize a mucolipidosis type III alpha/beta patient whose clinical diagnosis was already confirmed biochemically. In a first stage only one causal mutation was identified in heterozygosity, the already described missense mutation c.1196C>T(p.S399F), both at cDNA and gDNA levels. Only after conducting inhibition of nonsense-mediated mRNA decay (NMD) assays and after the utilization of another pair of primers the second mutation, the c.3503_3504delTC deletion, was identified. Our findings illustrate that allelic dropout due to the presence of polymorphisms and/or of mutations that trigger the NMD pathway can cause difficulties in current molecular diagnosis tests. [ABSTRACT FROM AUTHOR]

Non-invasive genetic sampling using materials such as faeces or hair can be used to monitor wildlife populations, although DNA quality is often poor. Improving sampling efficiency and minimising factors that reduce DNA quality are therefore critical. After a severe decline, the European pine marten, Martes martes, has reclaimed much of its former range in Scotland, UK. Recording this rapid range expansion requires developing techniques for accurate monitoring, but this is hampered by the species' elusive behaviour. We tested two sampling methods, hair collected from hair tubes and faeces (scat) collected along tracks, to assess the effects of key environmental and sampling variables on DNA quality and sampling efficiency. For hair, we tested the influence of hair tube location (distance from forest tracks) on collection rate and sex ratio of animals successfully sampled. For scats, we assessed the effect of time since defecation (1 to 16 days) on genotyping error rates and success under two contrasting environmental conditions (exposed to rainfall or sheltered). We found no bias in the collection rate or sex ratio of animals detected by hair samples with differing proximity to forest tracks. DNA amplification failure for scats exposed to rainfall increased from 28 to 65 % over the 16-day experimental period. During periods of low rainfall, the length of collection sessions could therefore be extended to increase sample number without risk of DNA degradation. Lack of bias in hair collection rates with proximity to forest tracks provides justification for tube placement close to tracks, as this reduces survey effort. These findings provide guidance for the development of efficient and cost-effective non-invasive sampling of Scottish pine martens. [ABSTRACT FROM AUTHOR]

While modern high-throughput sequence-based HLA genotyping methods generally provide highly accurate typing results, artefacts may nonetheless arise for numerous reasons, such as sample contamination, sequencing errors, read misalignments, or PCR amplification biases. To help detecting spurious typing results, we tested the performance of two probabilistic classifiers (binary logistic regression and random forest models) based on population-specific genotype frequencies. We trained the model using high-resolution typing results for HLA-DRB1, DQB1, and DPB1 from large samples of German, Polish and UK-based donors. The high predictive capacity of the best models replicated both in 10-fold cross-validation for each gene and in using independent evaluation data (AUC 0.820–0.893). While genotype frequencies alone provide enough predictive power to render the model generally useful for highlighting potentially spurious typing results, the inclusion of workflow-specific predictors substantially increases prediction specificity. Low initial DNA concentrations in combination with low-volume PCR reactions form a major source of stochastic error specific to the Fluidigm chip-based workflow at DKMS Life Science Lab. The addition of DNA concentrations as a predictor variable thus substantially increased AUC (0.947–0.959) over purely frequency-based models. [ABSTRACT FROM AUTHOR]

Allelic dropout in relationship problems may commonly appear in areas such as disaster victim identification and the identification of missing persons. If dropout is not accounted for, the results may be incorrect interpretation of profiles, loss of valuable information and biased results. In this paper, we explore different models for dropout in kinship cases and present an efficient implementation for one of the models. The implementation allows for dropout to be handled simultaneously with phenomena like silent alleles and mutations that may also cause discordances in relationship data, in addition to subpopulation correction. The implemented dropout model is freely available in the new version of the Familias software. The concepts and methods are illustrated on real and simulated data. [ABSTRACT FROM AUTHOR]

DNA is now routinely used in criminal investigations and court cases, although DNA samples taken at crime scenes are of varying quality and therefore present challenging problems for their interpretation. We present a statistical model for the quantitative peak information obtained from an electropherogram of a forensic DNA sample and illustrate its potential use for the analysis of criminal cases. In contrast with most previously used methods, we directly model the peak height information and incorporate important artefacts that are associated with the production of the electropherogram. Our model has a number of unknown parameters, and we show that these can be estimated by the method of maximum likelihood in the presence of multiple unknown individuals contributing to the sample, and their approximate standard errors calculated; the computations exploit a Bayesian network representation of the model. A case example from a UK trial, as reported in the literature, is used to illustrate the efficacy and use of the model, both in finding likelihood ratios to quantify the strength of evidence, and in the deconvolution of mixtures for finding likely profiles of the individuals contributing to the sample. Our model is readily extended to simultaneous analysis of more than one mixture as illustrated in a case example. We show that the combination of evidence from several samples may give an evidential strength which is close to that of a single-source trace and thus modelling of peak height information provides a potentially very efficient mixture analysis. [ABSTRACT FROM AUTHOR]

Non-invasive genetics is a powerful tool in wildlife research and monitoring, especially when dealing with elusive and rare species such as the Eurasian otter ( Lutra lutra). Nevertheless, otter DNA obtained from scats and anal secretions appears to be exposed to very quick degradation processes, and the success rate in DNA amplification is lower than in other carnivores. We collected 191 samples from April to September 2011 along the river Sangro basin (Italy) which was recently re-colonized by the Eurasian otter. Using two sets of microsatellite loci (six Lut and seven OT loci), we investigated the influence of sample type and age, collection time, storage time, temperature and humidity on genotyping success and amplification success. We also tested the efficacy of different DNA extraction kits and storage buffer mediums. Finally, we compared amplification success rate, allelic dropout and false allele rates for each locus. We obtained a mean amplification success rate of 79.0 % and a genotyping success rate of 35.1 %. Fresh pure jellies yielded the highest amplification success and genotyping rate. Six microsatellite loci should be theoretically sufficient to distinguish the individual unrelated otters (PID = 0.001), while 13 loci were needed to distinguish sibling otters (PID = 0.002) in our population. We identified 11 otters, and molecular sexing ascertained the presence of five males, four females and two uncertain individuals. Generalized linear models highlighted a significant influence of sample type and age, temperature and humidity both on genotyping and amplification success. [ABSTRACT FROM AUTHOR]

Background: The Major Histocompatibility Complex (MHC) is the most important genetic marker to study patterns of adaptive genetic variation determining pathogen resistance and associated life history decisions. It is used in many different research fields ranging from human medical, molecular evolutionary to functional biodiversity studies. Correct assessment of the individual allelic diversity pattern and the underlying structural sequence variation is the basic requirement to address the functional importance of MHC variability. Next-generation sequencing (NGS) technologies are likely to replace traditional genotyping methods to a great extent in the near future but first empirical studies strongly indicate the need for a rigorous quality control pipeline. Strict approaches for data validation and allele calling to distinguish true alleles from artefacts are required. Results: We developed the analytical methodology and validated a data processing procedure which can be applied to any organism. It allows the separation of true alleles from artefacts and the evaluation of genotyping reliability, which in addition to artefacts considers for the first time the possibility of allelic dropout due to unbalanced amplification efficiencies across alleles. Finally, we developed a method to assess the confidence level per genotype a-posteriori, which helps to decide which alleles and individuals should be included in any further downstream analyses. The latter method could also be used for optimizing experiment designs in the future. Conclusions: Combining our workflow with the study of amplification efficiency offers the chance for researchers to evaluate enormous amounts of NGS-generated data in great detail, improving confidence over the downstream analyses and subsequent applications. [ABSTRACT FROM AUTHOR]

Abstract: Increases in the sensitivity of DNA profiling technology now allow profiles to be obtained from smaller and more degraded DNA samples than was previously possible. The resulting profiles can be highly informative, but the subjective elements in the interpretation make it problematic to achieve the valid and efficient evaluation of evidential strength required in criminal cases. The problems arise from stochastic phenomena such as “dropout” (absence of an allele in the profile that is present in the underlying DNA) and experimental artefacts such as “stutter” that can generate peaks of ambiguous allelic status. Currently in the UK, evidential strength evaluation uses an approach in which the complex signals in the DNA profiles are interpreted in a semi-manual fashion by trained experts aided by a set of guidelines, but also relying substantially on professional judgment. We introduce a statistical model to calculate likelihood ratios for evaluating DNA evidence arising from multiple known and unknown contributors that allows for such stochastic phenomena by incorporating peak heights. Efficient use of peak heights allows for more crime scene profiles to be reported to courts than is currently possible. The model parameters are estimated from experimental data incorporating multiple sources of variability in the profiling system. We report and analyse experimental results from the SGMPlus system, run at 28 amplification cycles with no enhancements, currently used in the UK. Our methods are readily adapted to other DNA profiling systems provided that the experimental data for the parameter estimation is available. [Copyright &y& Elsevier]

Several methods have been applied to calculate genotyping error rates (GER) for non-invasive population size estimations. However, there is a lack of comparability between these methods. Here we focused on the comparison of methods for determination of GER within one study using faeces samples of wild boars ( Sus scrofa). Error rates were calculated by (1) comparison of reference tissue samples and rectum faeces samples (2) the number of deviations between replicates and the assumed consensus genotypes, (3) re-analysis of a subsample interpreted by allelic and genotype comparisons, and (4) a blind-test of anonymously subdivided faecal samples. The error rates differed widely between these four methods (0-57.5 %) and underline the need of a consensus approach. The blind-test resulted in a GER of 4.3 %. We recommend conducting such a blind-test for estimating realistic GER when starting a pilot study in wildlife forensics. [ABSTRACT FROM AUTHOR]

Context: Genetic testing is useful not only for the diagnosis of the MEN1 proband but also for determining the putative asymptomatic variant carriers to improve the prognosis or to avoid unnecessary medical intervention. However, we must be aware of the putative pitfalls of polymerase chain reaction (PCR)-based genetic testing in specific conditions that lead to medical mismanagement., Objective: To warn of the putative pitfalls of PCR-based genetic testing, we report an overlooked case of MEN1 due to PCR allelic dropout., Methods: A 69-year-old man was clinically diagnosed with MEN1, and genetic testing revealed that he had a pathogenic variant in the MEN1 gene. His 36-year-old son was completely asymptomatic. As the son was 50% at risk of MEN1, he was willing to undergo genetic testing himself after genetic counseling., Results: Genetic testing was carried out in 2 independent laboratories. Although laboratory A showed that he carried a pathogenic variant, laboratory B showed that he had the wild-type genotype of MEN1 . The discrepancy in these results was due to PCR allelic dropout by single-nucleotide variations of the MEN1 gene in the 5' region. The surveillance revealed that he had asymptomatic primary hyperparathyroidism and a neuroendocrine tumor of the pancreas., Conclusion: PCR-dependent genetic analysis may be susceptible to PCR allelic dropout in an SNV-specific manner. We must be careful when genetically testing individuals of relatives with clinical MEN1 disease., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

Non-invasive DNA genotyping using hair samples has become a common method in population surveys of Asiatic black bears ( Ursus thibetanus) in Japan; however, the accuracy of the genotyping data has rarely been discussed in empirical studies. Therefore, we conducted a large-scale pilot study to examine genotyping accuracy and sought an efficient way of error-checking hair-trapping data. We collected 2,067 hair samples, successfully determined the genotypes of 1,245 samples, and identified 295 individuals. The genotyping data were further divided into 3 subsets of data according to the number of hairs used for DNA extraction in each sample (1-4, 5-9, and ≥10 hairs), and the error rates of allelic dropout and false alleles were estimated for each subset using a maximum likelihood method. The genotyping error rates in the samples with ≥10 hairs were found to be lower than those in the samples with 1-4 and 5-9 hairs. The presence of erroneous genotypes among the identified individuals was further checked using a post hoc goodness-of-fit test that determined the match between the expected and observed frequencies of individual homozygotes at 0-6 loci. The results indicated the presence of erroneous genotypes, possibly as a result of allelic dropout, in the samples. Therefore, for improved accuracy, it is recommended that samples containing ≥10 hairs should be used for genotyping and a post hoc goodness-of-fit test should be performed to exclude erroneous genotypes before proceeding with downstream analysis such as capture-mark-recapture estimation. [ABSTRACT FROM AUTHOR]

Abstract: DNA samples found at a scene of crime or obtained from the debris of a mass disaster accident are often subject to degradation. When using the STR DNA technology, the DNA profile is observed via a so-called electropherogram (EPG), where the alleles are identified as signal peaks above a certain level or above a signal to noise threshold. Degradation implies that these peak intensities decrease in strength for longer short tandem repeat (STR) sequences. Consequently, long STR loci may fail to produce peak heights above the limit of detection resulting in allelic or locus drop-outs. In this paper, we present a method for measuring the degree of degradation of a sample and demonstrate how to incorporate this in estimating the probability of allelic drop-out. This is done by extending an existing method derived for non-degraded samples. The performance of the methodology is evaluated using data from degraded DNA, where cases with varying amounts of DNA and levels of degradation are investigated. [Copyright &y& Elsevier]

Optimal collection and preservation protocols for fecal DNA genotyping are not firmly established. We evaluated 3 factors that influence microsatellite genotyping success of fecal DNA extracted from coyote (Canis latrans) scats: 1) age of scat, 2) preservative, and 3) diet content. We quantified genotyping success by comparing rates of allelic dropout, false alleles, and failed amplifications among consensus genotypes. We used a panel of 6 microsatellite loci to genotype 20 scat samples, each of which was subjected to 3 age (1 day, 5 days, and 10 days post-deposition) and 3 preservation (DET buffer, 95% ethanol [EtOH], and lysis buffer) treatments. Both sample age and storage buffer had a significant effect on success and reliability. Ethanol and DET buffer preserved fecal samples with similar efficiency, and both were superior to lysis buffer. Our analysis of DNA degradation rates revealed that samples collected as early as 5 days of age yielded DNA that was highly degraded relative to samples collected on day 1. We tested the influence of dietary remains on microsatellite genotyping by using scat samples consisting predominantly of insect prey (n = 5), mammalian prey (n = 9), or the remains of juniper (Juniperus spp.) berries (n = 6) and compared EtOH and DET buffer preservation efficacy. We observed a significant interaction effect between storage buffer and diet for the probability of a false allele in a polymerase chain reaction (PCR), suggesting that the optimal preservation technique depended on the food remains comprising the scat. Scats comprised of juniper berry remains were more reliably genotyped when preserved in DET than EtOH. Mammalian prey-based scats were more reliable when stored in EtOH than DET buffer. Insect-predominant scats were preserved in EtOH and DET buffer with similar efficiency. Although accurate and reliable results can be obtained from scats collected at =5 days of age, we suggest sampling design to include collection of scats <5 days of age to minimize field and laboratory expenses. We suggest EtOH preservation for scats of obligate carnivores and of facultative carnivores with a diet consisting primarily of mammals. We suggest DET buffer preservation for animals with a diet consisting of plant-derived foods. Lysis buffer protocols that we employed should not be used for fecal DNA preservation. [ABSTRACT FROM AUTHOR]

Abstract: This paper presents a coherent probabilistic framework for taking account of allelic dropout, stutter bands and silent alleles when interpreting STR DNA profiles from a mixture sample using peak size information arising from a PCR analysis. This information can be exploited for evaluating the evidential strength for a hypothesis that DNA from a particular person is present in the mixture. It extends an earlier Bayesian network approach that ignored such artifacts. We illustrate the use of the extended network on a published casework example. [Copyright &y& Elsevier]

A year after the introduction of Identifiler™ into the forensic DNA laboratories of the Institute of Environmental Science and Research Limited (ESR), increasing occurrences of dropout of the three loci, D7S820, D18S51, and FGA, were observed in samples where the DNA was not degraded and sufficient DNA was present that full DNA profiles were to be expected. The dropout was either partial or complete at these loci. Full profiles could sometimes be obtained by reamplification of samples using the same input amount of DNA. After a thorough investigation of the methods and procedures used in the laboratory, the cause of this inhibition was identified as the cleaning agent TriGene™ ADVANCE. This was determined after the deliberate addition of varying amounts of different cleaning reagents into the DNA amplification reactions. At concentrations of 0.004% TriGene™ ADVANCE caused inhibition resulting in tri-loci dropout. At concentrations of 0.04% and higher, complete inhibition was observed. An effect was also seen on the amplification of samples using the Y STR profiling system PowerPlexY. This work highlights the importance of checking all reagents and chemicals prior to use, even those with no apparent direct influence on the DNA profiling process. [ABSTRACT FROM AUTHOR]

The article discusses research on the non-invasive DNA sampling of hair and faecal material from the Pteropus poliocephalus or grey-headed flying-fox. It references a study by Heather J. Baldwin at the Macquarie University Department of Biological Sciences in Sydney, New South Wales, and her colleagues, published in an issue of "Australian Mammalogy." The researchers concluded that the hair and faeces of P. poliocephalus serve as a good source of high-quality mitochondrial DNA. They also found that the low quality of the extracted nuclear DNA was influenced by a number of factors including spurious microsatellite profiles, false alleles and reduced amplification success.

Sampling of feces for genetic studies of wild populations can be problematic because of the low quality and quantity of template DNA obtained. We used cotton swabs in the field to isolate the mucous layer on the surface of fresh wolf (Canis lupus, C. lycaon, and their hybrids) scats followed by immediate preservation, and compared microsatellite genotyping of DNA from these fresh field swabs (FS) to that of previously frozen laboratory swabs (LS). In single polymerase chain reactions (PCRs) of 2 multiplexes, amplification at 8 loci was higher in the FS samples (FS = 50%, LS = 15%; P 5 0.02) because proportion, quantity, and quality of large fragment wolf nuclear DNA from these samples was greater (2.5-25%, 6.25-62.5 ng/swab, 35% amplified at 1,000 base pairs [bp]) than from the LS samples (1.9%-10%, 4.7-25 ng/ swab, 10% amplified at 1,000 bp). Paired blood and fresh field-swabbed samples had identical genotypes. In 84 multiplex PCRs we found no evidence of allelic dropout associated with low template quality or quantity. We conclude that field swabbing of fresh wolf scat facilitates field storage and reduces the need for multiple amplifications at single microsatellite loci, thereby reducing the genotyping costs for wildlife projects that use noninvasive samples. [ABSTRACT FROM AUTHOR]

Sampling DNA noninvasively has advantages for identifying animals for uses such as mark–recapture modeling that require unique identification of animals in samples. Although it is possible to generate large amounts of data from noninvasive sources of DNA, a challenge is overcoming genotyping errors that can lead to incorrect identification of individuals. A major source of error is allelic dropout, which is failure of DNA amplification at one or more loci. This has the effect of heterozygous individuals being scored as homozygotes at those loci as only one allele is detected. If errors go undetected and the genotypes are naively used in mark–recapture models, significant overestimates of population size can occur. To avoid this it is common to reject low-quality samples but this may lead to the elimination of large amounts of data. It is preferable to retain these low-quality samples as they still contain usable information in the form of partial genotypes. Rather than trying to minimize error or discarding error-prone samples we model dropout in our analysis. We describe a method based on data augmentation that allows us to model data from samples that include uncertain genotypes. Application is illustrated using data from the European badger ( Meles meles). [ABSTRACT FROM AUTHOR]

ConGenR (available at ) is an R based conservation genetics script that facilitates rapid determination of consensus genotypes from replicated samples, determines overall (successful amplifications/amplification attempted) and individual sample level (proportion of samples with successful amplifications at n loci) amplification success rates, and quantifies genotyping error rates. ConGenR is intended for use with codominant, multilocus microsatellite data generated primarily through noninvasive genetic sampling and processed with a multi-tubes approach. ConGenR handles input that can be easily exported from GENEMAPPER, a program commonly used to score allele sizes. Amplification success and genotyping error rates can be evaluated by sample class (i.e., any identifiable and meaningful subdivision of samples; e.g., sex, season, region, or sample condition), offering insights into processes driving amplification success and genotyping error rates. Additionally, amplification success and genotyping error rates are calculated by locus, expediting the identification of problematic loci during pilot studies. [ABSTRACT FROM AUTHOR]

We present a new mutation in KCNH2 (c.2038delG) resulting in a frameshift and premature truncation of the IKr channel protein in a large LQTS family with several sudden death cases. This mutation was initially missed by mutation scanning with DHPLC due to allelic dropout and only retrieved after repeat genetic testing with targeted capture and massive parallel sequencing. There was full penetrance of this mutation, only if an individualized QT correction derived from 24-hour Holter data was used. This case again underscores the importance of repeat genetic testing in robust cases of LQTS that remained genotype negative with mutation scanning techniques. [ABSTRACT FROM AUTHOR]

Fecal analysis from the highly endangered North Atlantic right whale provides valuable information about health and reproductive parameters of individual animals. Genetically profiling the feces facilitates this connection when the sample originator is unknown. Although genetic analysis of feces collected in terrestrial systems has become well established, genetic studies of cetacean DNA are rare. Here, the use of free-floating feces as a source of right whale DNA and the reliability of the genotypes produced are examined. On average, fecal extracts yielded 25 ng of DNA/mg dry weight, but less than 1% was right whale DNA. Although all samples were amplified using genus-specific mitochondrial control region primers, the quantity of right whale DNA present was over estimated when compared to amplifications using nuclear primers. No correlation was found between the quantity of right whale DNA recovered and the duration the sample sat in the water. Composite microsatellite profiles from multiple amplifications of 28 fecal samples of known origin were consistent with profiles of the same individuals obtained from skin biopsies, however, the rate of allelic dropout varied depending on the amount of right whale DNA added. A screening and genotyping protocol for reliable genetic profiling based on fecal DNA quantification is presented. [ABSTRACT FROM AUTHOR]

In noninvasive genetic sampling, when genotyping error rates are high and recapture rates are low, misidentification of individuals can lead to overestimation of population size. Thus, estimating genotyping errors is imperative. Nonetheless, conducting multiple polymerase chain reactions (PCRs) at multiple loci is time-consuming and costly. To address the controversy regarding the minimum number of PCRs required for obtaining a consensus genotype, we compared consumer-style the performance of two genotyping protocols (multiple-tubes and ‘comparative method’) in respect to genotyping success and error rates. Our results from 48 faecal samples of river otters ( Lontra canadensis) collected in Wyoming in 2003, and from blood samples of five captive river otters amplified with four different primers, suggest that use of the comparative genotyping protocol can minimize the number of PCRs per locus. For all but five samples at one locus, the same consensus genotypes were reached with fewer PCRs and with reduced error rates with this protocol compared to the multiple-tubes method. This finding is reassuring because genotyping errors can occur at relatively high rates even in tissues such as blood and hair. In addition, we found that loci that amplify readily and yield consensus genotypes, may still exhibit high error rates (7–32%) and that amplification with different primers resulted in different types and rates of error. Thus, assigning a genotype based on a single PCR for several loci could result in misidentification of individuals. We recommend that programs designed to statistically assign consensus genotypes should be modified to allow the different treatment of heterozygotes and homozygotes intrinsic to the comparative method. [ABSTRACT FROM AUTHOR]

Individual genotypes determined from noninvasive DNA samples (typically extracted from shed hairs or scats) are used to estimate population size in monitoring projects of elusive species. However, polymerase chain reaction (PCR) success rates usually are lower, and genotyping errors higher than in standard population genetic surveys, due to DNA degradation or contamination in aged field samples. In this study, we evaluate the results of common garden experiments showing that DNA degradation is significant in wolf ( Canis lupus) scats older than 3 days, and it is enhanced in scats in direct contact with soil. A storage test showed that samples kept frozen in 95% ethanol performed better compared to other methods. However, variance of PCR success among samples was high, independent on sample age or storage condition. The detrimental consequences of DNA degradation can be avoided by collecting scat samples as fresh as possible, and implementing efficient multitube procedures and stringent quality control of the laboratory results. Efficient multitube procedures can produce reliable data, like in this study, which showed that the consensus genotypes obtained from excremental DNA exactly matched distinct reference genotypes obtained from wolf blood samples. [ABSTRACT FROM AUTHOR]