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There is increasing interest in the African spiny mouse (Acomys cahirinus) as a model organism because of its ability for regeneration of tissue after injury in skin, muscle, and internal organs such as the kidneys. A high-quality reference genome is needed to better understand these regenerative properties at the molecular level. Here, we present an improved reference genome for A. cahirinus generated from long Nanopore sequencing reads. We confirm the quality of our annotations using RNA sequencing data from 4 different tissues. Our genome is of higher contiguity and quality than previously reported genomes from this species and will facilitate ongoing efforts to better understand the regenerative properties of this organism., Competing Interests: Conflicts of interest DEM is engaged in a research agreement with Oxford Nanopore, and they have paid for him to travel to speak on their behalf. DEM is on a scientific advisory board at Oxford Nanopore. The other authors have no conflicts to declare., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Background: The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse-transcription PCR (RT-qPCR). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce. To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction., Methods: We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral inactivation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance., Results: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/µL, 100% sensitivity, and 99.4% specificity when compared side by side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time., Conclusion: SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics., (© American Association for Clinical Chemistry 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The genus Rhododendron (Ericaceae), which includes horticulturally important plants such as azaleas, is a highly diverse and widely distributed genus of >1,000 species. Here, we report the chromosome-scale de novo assembly and genome annotation of Rhododendron williamsianum as a basis for continued study of this large genus. We created multiple short fragment genomic libraries, which were assembled using ALLPATHS-LG. This was followed by contiguity preserving transposase sequencing (CPT-seq) and fragScaff scaffolding of a large fragment library, which improved the assembly by decreasing the number of scaffolds and increasing scaffold length. Chromosome-scale scaffolding was performed by proximity-guided assembly (LACHESIS) using chromatin conformation capture (Hi-C) data. Chromosome-scale scaffolding was further refined and linkage groups defined by restriction-site associated DNA (RAD) sequencing of the parents and progeny of a genetic cross. The resulting linkage map confirmed the LACHESIS clustering and ordering of scaffolds onto chromosomes and rectified large-scale inversions. Assessments of the R. williamsianum genome assembly and gene annotation estimate them to be 89% and 79% complete, respectively. Predicted coding sequences from genome annotation were used in syntenic analyses and for generating age distributions of synonymous substitutions/site between paralgous gene pairs, which identified whole-genome duplications (WGDs) in R. williamsianum. We then analyzed other publicly available Ericaceae genomes for shared WGDs. Based on our spatial and temporal analyses of paralogous gene pairs, we find evidence for two shared, ancient WGDs in Rhododendron and Vaccinium (cranberry/blueberry) members that predate the Ericaceae family and, in one case, the Ericales order., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled ∼1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: (i) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, (ii) deletions are predominantly associated with microhomology and (iii) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Combined Annotation-Dependent Depletion (CADD) is a widely used measure of variant deleteriousness that can effectively prioritize causal variants in genetic analyses, particularly highly penetrant contributors to severe Mendelian disorders. CADD is an integrative annotation built from more than 60 genomic features, and can score human single nucleotide variants and short insertion and deletions anywhere in the reference assembly. CADD uses a machine learning model trained on a binary distinction between simulated de novo variants and variants that have arisen and become fixed in human populations since the split between humans and chimpanzees; the former are free of selective pressure and may thus include both neutral and deleterious alleles, while the latter are overwhelmingly neutral (or, at most, weakly deleterious) by virtue of having survived millions of years of purifying selection. Here we review the latest updates to CADD, including the most recent version, 1.4, which supports the human genome build GRCh38. We also present updates to our website that include simplified variant lookup, extended documentation, an Application Program Interface and improved mechanisms for integrating CADD scores into other tools or applications. CADD scores, software and documentation are available at https://cadd.gs.washington.edu.

Background: Despite advances in next generation DNA sequencing (NGS), NGS-based single gene tests for diagnostic purposes require improvements in terms of completeness, quality, speed, and cost. Single-molecule molecular inversion probes (smMIPs) are a technology with unrealized potential in the area of clinical genetic testing. In this proof-of-concept study, we selected 2 frequently requested gene tests, those for the breast cancer genes BRCA1 and BRCA2, and developed an automated work flow based on smMIPs., Methods: The BRCA1 and BRCA2 smMIPs were validated using 166 human genomic DNA samples with known variant status. A generic automated work flow was built to perform smMIP-based enrichment and sequencing for BRCA1, BRCA2, and the checkpoint kinase 2 (CHEK2) c.1100del variant., Results: Pathogenic and benign variants were analyzed in a subset of 152 previously BRCA-genotyped samples, yielding an analytical sensitivity and specificity of 100%. Following automation, blind analysis of 65 in-house samples and 267 Norwegian samples correctly identified all true-positive variants (>3000), with no false positives. Consequent to process optimization, turnaround times were reduced by 60% to currently 10-15 days. Copy number variants were detected with an analytical sensitivity of 100% and an analytical specificity of 88%., Conclusions: smMIP-based genetic testing enables automated and reliable analysis of the coding sequences of BRCA1 and BRCA2. The use of single-molecule tags, double-tiled targeted enrichment, and capturing and sequencing in duplo, in combination with automated library preparation and data analysis, results in a robust process and reduces routine turnaround times. Furthermore, smMIP-based copy number variation analysis could make independent copy number variation tools like multiplex ligation-dependent probes amplification dispensable., (© 2016 American Association for Clinical Chemistry.)

Human genetics has historically depended on the identification of individuals whose natural genetic variation underlies an observable trait or disease risk. Here we argue that new technologies now augment this historical approach by allowing the use of massively parallel assays in model systems to measure the functional effects of genetic variation in many human genes. These studies will help establish the disease risk of both observed and potential genetic variants and to overcome the problem of "variants of uncertain significance.", (Copyright © 2016 by the Genetics Society of America.)

While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192-252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131-250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput 'Dial-Out PCR' protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Centromeres are essential for proper chromosome segregation. Despite extensive research, centromere locations in yeast genomes remain difficult to infer, and in most species they are still unknown. Recently, the chromatin conformation capture assay, Hi-C, has been re-purposed for diverse applications, including de novo genome assembly, deconvolution of metagenomic samples and inference of centromere locations. We describe a method, Centurion, that jointly infers the locations of all centromeres in a single genome from Hi-C data by exploiting the centromeres' tendency to cluster in three-dimensional space. We first demonstrate the accuracy of Centurion in identifying known centromere locations from high coverage Hi-C data of budding yeast and a human malaria parasite. We then use Centurion to infer centromere locations in 14 yeast species. Across all microbes that we consider, Centurion predicts 89% of centromeres within 5 kb of their known locations. We also demonstrate the robustness of the approach in datasets with low sequencing depth. Finally, we predict centromere coordinates for six yeast species that currently lack centromere annotations. These results show that Centurion can be used for centromere identification for diverse species of yeast and possibly other microorganisms., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Interpreting variants of uncertain significance (VUS) is a central challenge in medical genetics. One approach is to experimentally measure the functional consequences of VUS, but to date this approach has been post hoc and low throughput. Here we use massively parallel assays to measure the effects of nearly 2000 missense substitutions in the RING domain of BRCA1 on its E3 ubiquitin ligase activity and its binding to the BARD1 RING domain. From the resulting scores, we generate a model to predict the capacities of full-length BRCA1 variants to support homology-directed DNA repair, the essential role of BRCA1 in tumor suppression, and show that it outperforms widely used biological-effect prediction algorithms. We envision that massively parallel functional assays may facilitate the prospective interpretation of variants observed in clinical sequencing., (Copyright © 2015 by the Genetics Society of America.)

Malformations of cortical development containing dysplastic neuronal and glial elements, including hemimegalencephaly and focal cortical dysplasia, are common causes of intractable paediatric epilepsy. In this study we performed multiplex targeted sequencing of 10 genes in the PI3K/AKT pathway on brain tissue from 33 children who underwent surgical resection of dysplastic cortex for the treatment of intractable epilepsy. Sequencing results were correlated with clinical, imaging, pathological and immunohistological phenotypes. We identified mosaic activating mutations in PIK3CA and AKT3 in this cohort, including cancer-associated hotspot PIK3CA mutations in dysplastic megalencephaly, hemimegalencephaly, and focal cortical dysplasia type IIa. In addition, a germline PTEN mutation was identified in a male with hemimegalencephaly but no peripheral manifestations of the PTEN hamartoma tumour syndrome. A spectrum of clinical, imaging and pathological abnormalities was found in this cohort. While patients with more severe brain imaging abnormalities and systemic manifestations were more likely to have detected mutations, routine histopathological studies did not predict mutation status. In addition, elevated levels of phosphorylated S6 ribosomal protein were identified in both neurons and astrocytes of all hemimegalencephaly and focal cortical dysplasia type II specimens, regardless of the presence or absence of detected PI3K/AKT pathway mutations. In contrast, expression patterns of the T308 and S473 phosphorylated forms of AKT and in vitro AKT kinase activities discriminated between mutation-positive dysplasia cortex, mutation-negative dysplasia cortex, and non-dysplasia epilepsy cortex. Our findings identify PI3K/AKT pathway mutations as an important cause of epileptogenic brain malformations and establish megalencephaly, hemimegalencephaly, and focal cortical dysplasia as part of a single pathogenic spectrum., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Unlabelled: Molecular inversion probes (MIPs) enable cost-effective multiplex targeted gene resequencing in large cohorts. However, the design of individual MIPs is a critical parameter governing the performance of this technology with respect to capture uniformity and specificity. MIPgen is a user-friendly package that simplifies the process of designing custom MIP assays to arbitrary targets. New logistic and SVM-derived models enable in silico predictions of assay success, and assay redesign exhibits improved coverage uniformity relative to previous methods, which in turn improves the utility of MIPs for cost-effective targeted sequencing for candidate gene validation and for diagnostic sequencing in a clinical setting., Availability and Implementation: MIPgen is implemented in C++. Source code and accompanying Python scripts are available at http://shendurelab.github.io/MIPGEN/., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Microbial communities consist of mixed populations of organisms, including unknown species in unknown abundances. These communities are often studied through metagenomic shotgun sequencing, but standard library construction methods remove long-range contiguity information; thus, shotgun sequencing and de novo assembly of a metagenome typically yield a collection of contigs that cannot readily be grouped by species. Methods for generating chromatin-level contact probability maps, e.g., as generated by the Hi-C method, provide a signal of contiguity that is completely intracellular and contains both intrachromosomal and interchromosomal information. Here, we demonstrate how this signal can be exploited to reconstruct the individual genomes of microbial species present within a mixed sample. We apply this approach to two synthetic metagenome samples, successfully clustering the genome content of fungal, bacterial, and archaeal species with more than 99% agreement with published reference genomes. We also show that the Hi-C signal can secondarily be used to create scaffolded genome assemblies of individual eukaryotic species present within the microbial community, with higher levels of contiguity than some of the species' published reference genomes., (Copyright © 2014 Burton et al.)

Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at 'off-target' sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate 'megaTAL', in which the DNA binding region of a transcription activator-like (TAL) effector is used to 'address' a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.

Background: The process of generating raw genome sequence data continues to become cheaper, faster, and more accurate. However, assembly of such data into high-quality, finished genome sequences remains challenging. Many genome assembly tools are available, but they differ greatly in terms of their performance (speed, scalability, hardware requirements, acceptance of newer read technologies) and in their final output (composition of assembled sequence). More importantly, it remains largely unclear how to best assess the quality of assembled genome sequences. The Assemblathon competitions are intended to assess current state-of-the-art methods in genome assembly., Results: In Assemblathon 2, we provided a variety of sequence data to be assembled for three vertebrate species (a bird, a fish, and snake). This resulted in a total of 43 submitted assemblies from 21 participating teams. We evaluated these assemblies using a combination of optical map data, Fosmid sequences, and several statistical methods. From over 100 different metrics, we chose ten key measures by which to assess the overall quality of the assemblies., Conclusions: Many current genome assemblers produced useful assemblies, containing a significant representation of their genes and overall genome structure. However, the high degree of variability between the entries suggests that there is still much room for improvement in the field of genome assembly and that approaches which work well in assembling the genome of one species may not necessarily work well for another.