Biology subjects

Walker, M.

Publications and source records attributed to Walker, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Genetic variation at 11q23.1 confers colorectal cancer risk by dysregulation of colonic tuft cell transcriptional activator POU2AF2

Common genetic variation at 11q23.1 is associated with colorectal cancer (CRC) risk, and exerts local (cis) expression quantitative trait locus (cis-eQTL) effects on POU2AF2, COLCA1 and POU2AF3 genes. However, complex linkage disequilibrium and correlated expression at the 11q23.1 locus has thus far hindered elucidation of the mechanisms by which genetic variants impart CRC risk. Here, we establish that rs3087967 is the likely causal eQTL at this locus, co-localising with expression of POU2AF2 and CRC risk. Furthermore, we show trans-eQTL effects on 21 distant target genes, which are highly enriched for Tuft cell markers. Analysis of available scRNAseq, ChIPseq and scATACseq data implicates POU2AF2 as the primary controller of the tuft cell specific trans-genes through POU2F3-correlated genetic regulation. Immunofluorescence demonstrates that the rs3087967 risk genotype (T) is associated with lower tuft cell abundance in human colonic epithelium. CRISPR-mediated deletion of the 11q23.1 risk locus in the mouse germline exacerbated the ApcMin/+ mouse phenotype upon abrogation of Pou2af2 expression specifically. Taken together, we implicate a key protective role of tuft cells in the large bowel and the importance of mis-regulation of POU2AF2 as the prime tuft cell transcriptional activator at this locus.

genetics↗

bioRxiv2023

Neural Extracellular Matrix Remodeling Signatures in Genetic and Acquired Mouse Models of Epilepsy

Epilepsies are multifaceted neurological disorders characterized by abnormal brain activity, e.g., caused by imbalanced synaptic excitation and inhibition. The neural extracellular matrix (ECM) is dynamically modulated by physiological and pathophysiological activity and critically involved in controlling the brains excitability. We used different epilepsy models, i.e. mice lacking the presynaptic scaffolding protein Bassoon at excitatory, inhibitory or all synapse types as genetic models for rapidly generalizing early-onset epilepsy, and intra-hippocampal kainate injection, a model for acquired temporal lobe epilepsy, to study the relationship between epileptic seizures and ECM composition. Electroencephalogram recordings revealed Bassoon deletion at excitatory or inhibitory synapses having diverse effects on epilepsy-related phenotypes. While constitutive Bsn mutants and GABAergic neuron-specific knockouts (BsnDlx5/6cKO) displayed severe epilepsy with more and stronger seizures than kainate-injected animals, mutants lacking Bassoon solely in excitatory forebrain neurons (BsnEmx1cKO) showed only mild impairments. By semiquantitative immunoblotting and immunohistochemistry we show model-specific patterns of neural ECM remodeling, and we also demonstrate significant upregulation of the ECM receptor CD44 in null and BsnDlx5/6cKO mutants. ECM-associated WFA-binding chondroitin sulfates were strongly augmented in seizure models. Strikingly, Brevican, Neurocan, Aggrecan and link protein Hapln1 levels reliably predicted seizure properties across models, suggesting a link between ECM state and epileptic phenotype.

neuroscience↗

bioRxiv2023

Mind the viscous modulus: The mechanotransductive response to the viscous nature of isoelastic matrices regulates stem cell chondrogenesis

The design of hydrogels as mimetics of tissues matrices typically disregards the viscous nature of native tissues and focuses only on their elastic properties. In the case of stem cell chondrogenesis, this has led to contradictory results, likely due to unreported changes of the matrices viscous modulus. Here, by employing isoelastic matrices with a Youngs modulus of ~12 kPa, we demonstrate that variations in viscous properties alone (i.e., loss tangent between 0.1-0.25) are sufficient to drive efficient growth factor-free chondrogenesis of human mesenchymal stem cells, both in 2D and 3D cultures. The increase of the viscous component of RGD-functionalised polyacrylamide or polyethylene glycol maleimide hydrogels promotes a phenotype with reduced adhesion, alters mechanosensitive signalling, and boosts cell-cell contacts. In turn, this upregulates the chondrogenic transcription factor SOX9 and supports neocartilage formation, demonstrating that the mechanotransductive response to the viscous nature of the matrix can be harnessed to direct cell fate.

bioengineering↗

bioRxiv2022

Variation in histone configurations correlates with gene expression across nine inbred strains of mice

It is well established that epigenetic features, such as histone modifications and DNA methylation, are associated with variation in gene expression across cell types. Less well known is the extent to which epigenetic states vary across genetically diverse individuals, and whether such variation corresponds to inter-individual variation in gene expression. To investigate genetically driven variation in epigenetics, we conducted a survey of epigenetic modifications and gene expression in hepatocytes of nine inbred mouse strains. We surveyed four histone modifications (H3K4me1, H3K4me3, H3K27me3, and H3K27ac), and DNA methylation. We used ChromHMM to identify 14 chromatin states, each of which represented a distinct combination of the four histone modifications. We found that chromatin states varied widely across the nine strains and that epigenetic state was strongly correlated with local gene expression. We replicated this correspondence between chromatin state and gene expression in an independent population of Diversity Outbred mice in which we imputed local chromatin state. In contrast, we found that DNA methylation did not vary across the inbred strains and was not correlated with variation in gene expression in DO mice. This work suggests that chromatin state is highly influenced by local genotype and may be a primary mode through which expression quantitative trait loci (eQTLs) are mediated. Through examples, we demonstrate that naturally occurring chromatin state variation, in conjunction with gene expression, can aid in functional annotation of the mouse genome. Finally, we provide a data resource that documents variation in chromatin state in hepatocytes across genetically distinct mice.

genomics↗

bioRxiv2022

GATK-gCNV: A Rare Copy Number Variant Discovery Algorithm and Its Application to Exome Sequencing in the UK Biobank

Copy number variants (CNVs) are major contributors to genetic diversity and disease. To date, exome sequencing (ES) has been generated for millions of individuals in international biobanks, human disease studies, and clinical diagnostic screening. While standardized methods exist for detecting short variants (single nucleotide and insertion/deletion variants) using tools such as the Genome Analysis ToolKit (GATK), technical challenges have confounded similarly uniform large-scale CNV analyses from ES data. Given the profound impact of rare and de novo coding CNVs on genome organization and human disease, the lack of widely-adopted and robustly benchmarked rare CNV discovery tools has presented a barrier to routine exome-wide assessment of this critical class of variation. Here, we introduce GATK-gCNV, a flexible algorithm to discover rare CNVs from genome sequencing read-depth information, which we distribute as an open-source tool packaged in GATK. GATK-gCNV uses a probabilistic model and inference framework that accounts for technical biases while simultaneously predicting CNVs, which enables self-consistency between technical read-depth normalization and variant calling. We benchmarked GATK-gCNV in 7,962 exomes from individuals in quartet families with matched genome sequencing and microarray data. These analyses demonstrated 97% recall of rare ([≤]1% site frequency) coding CNVs detected by microarrays and 95% recall of rare coding CNVs discovered by genome sequencing at a resolution of more than two exons. We applied GATK-gCNV to generate a reference catalog of rare coding CNVs in 197,306 individuals with ES from the UK Biobank. We observed strong correlations between CNV rates per gene and measures of mutational constraint, as well as rare CNV associations with multiple traits. In summary, GATK-gCNV is a tunable approach for sensitive and specific CNV discovery in ES, which can easily be applied across trait association and clinical screening.

genomics↗

bioRxiv2022

Genome-wide analysis of Structural Variants in Parkinson's Disease using Short-Read Sequencing data

Parkinsons disease is a complex neurodegenerative disorder, affecting approximately one million individuals in the USA alone. A significant proportion of risk for Parkinsons disease is driven by genetics. Despite this, the majority of the common genetic variation that contributes to disease risk is unknown, in-part because previous genetic studies have focussed solely on the contribution of single nucleotide variants. Structural variants represent a significant source of genetic variation in the human genome. However, because assay of this variability is challenging, structural variants have not been cataloged on a genome-wide scale, and their contribution to the risk of Parkinsons disease remains unknown. In this study, we 1) leveraged the GATK-SV pipeline to detect and genotype structural variants in 7,772 short-read sequencing data and 2) generated a subset of matched whole-genome Oxford Nanopore Technologies long-read sequencing data from the PPMI cohort to allow for comprehensive structural variant confirmation. We detected, genotyped, and tested 3,154 "high-confidence" common structural variant loci, representing over 412 million nucleotides of non-reference genetic variation. Using the long-read sequencing data, we validated three structural variants that may drive the association signals at known Parkinsons disease risk loci, including a 2kb intronic deletion within the gene LRRN4. Further, we confirm that the majority of structural variants in the human genome cannot be detected using short-read sequencing alone, encompassing on average around 4 million nucleotides of inaccessible sequence per genome. Therefore, although these data provide the most comprehensive survey of the contribution of structural variants to the genetic risk of Parkinsons disease to date, this study highlights the need for large-scale long-read datasets to fully elucidate the role of structural variants in Parkinsons disease.

genetics↗

bioRxiv2022

Sensitivity and specificity of human point-of-care circulating cathodic antigen (POC-CCA) test in African livestock for rapid diagnosis of schistosomiasis: a Bayesian latent class analysis

Schistosomiasis is a major neglected tropical disease (NTD) affecting both humans and animals. The morbidity and mortality inflicted upon livestock in sub-Saharan Africa has been largely overlooked, in part due to a lack of validated sensitive and specific tests, which do not require specialist training or equipment to deliver and interpret. Inexpensive, non-invasive, and sensitive diagnostic tests for livestock-use would also facilitate both prevalence mapping and appropriate intervention programmes. The aim of this study was to assess the sensitivity and specificity of the currently available point-of-care circulating cathodic antigen test (POC-CCA), designed for Schistosoma mansoni detection in humans, for the detection of intestinal livestock schistosomiasis caused by Schistosoma bovis and Schistosoma curassoni. POC-CCA, together with the circulating anodic antigen (CAA) test, miracidial hatching technique (MHT) and organ and mesentery inspection (for animals from abattoirs only), were applied to samples collected from 195 animals (56 cattle and 139 small ruminants (goats and sheep) from abattoirs and living populations) from Senegal. POC-CCA sensitivity varied by ruminant group and by location/parasite species: sensitivity was greater in Barkedji (cattle: mean 81% (95% credible interval (CrI): 55%-98%); small ruminants: 49% (29%-87%), where livestock were primarily infected by S. curassoni, than in Richard Toll (cattle: 62% (41%-84%); small ruminants: 12% (1%-37%), where S. bovis was the main parasite species. Mean POC-CCA specificity across sites in small ruminants was 91% (77%-99%) with little variation between locations/parasites (Barkedji: 91% (73%-99%); Richard Toll: 88% (65% - 99%). Specificity could not be assessed in cattle owing to the low number of uninfected cattle surveyed. Overall, our results indicate that, whilst the current POC-CCA does represent a potential diagnostic tool for animal schistosomiasis, future work is needed to develop a livestock-specific affordable and field-applicable diagnostic tests to enable determination of the true extent of livestock schistosomiasis. Author summarySchistosomiasis is a debilitating neglected tropical and zoonotic disease, infecting over 230 million people and multiple millions of animals worldwide, most notably amongst the poorest regions and populations. The potential contribution of livestock schistosomiasis to disease transmission in human populations has implications for the design of effective disease management and elimination programmes. However, our understanding of the true prevalence, transmission and impact of animal schistosomiasis is severely limited, in part due to a lack of inexpensive, accessible, sensitive and specific diagnostic tools. As a point-of-care circulating cathodic antigen (POC-CCA) diagnostic test is now in widespread use to assess intestinal schistosomiasis caused by Schistosoma mansoni in humans, we hypothesised that the same test could be used to detect livestock intestinal schistosomiasis caused by Schistosoma bovis and Schistosoma curassoni. The aim of this study was thus to evaluate the sensitivity and specificity of the POC-CCA for the detection of intestinal livestock schistosomiasis in Senegal. POC-CCA sensitivity varied by ruminant group and by location/parasite species, while POC-CCA specificity in small ruminants, at least, did not vary across sites. We conclude that the currently-available POC-CCA does represent a potential diagnostic tool for animal schistosomiasis, but that the factors determining test performance warrant further investigation.

pathology↗

bioRxiv2022

Heavy metal pollution impacts soil bacterial community structure and antimicrobial resistance at the Birmingham 35th Avenue Superfund Site

Heavy metals (HMs) are known to modify bacterial communities both in the laboratory and in situ. As a consequence, soils in HM contaminated sites like the U.S. Environmental Protection Agency (EPA) Superfund sites are predicted to have altered ecosystem functioning, with potential ramifications for the health of organisms, including humans, that live nearby. Further, several studies have shown that metal tolerant bacteria are often also resistant to antimicrobial agents (AMR), and therefore HM contaminated soils could potentially act as reservoirs that could disseminate AMR genes into human-associated pathogenic bacteria. To explore this possibility soil samples were collected from six public locations in the zip code 35207 (the home of the North Birmingham 35th Avenue Superfund site) and in six public areas in a neighboring reference zip code (35214). Sequencing of the V4 region of the bacterial 16S rRNA gene revealed that elevated concentrations of HMs As, Mn, Pb, and Zn reduced microbial diversity and altered community structure within each zip code. While there was no difference between zip codes in the proportion of total culturable microbes that survived antimicrobial or metal exposure, bacterial isolates with HMR almost always also exhibited AMR. Metagenomes inferred using PICRUST2 also predicted significantly higher mean relative frequencies in 35207 for several AMR genes related to both specific and broad-spectrum AMR phenotypes. Together, these results support the hypothesis that chronic HM pollution alters soil bacterial community structure in ecologically meaningful ways and may also select for bacteria with increased potential to contribute to AMR in human bacterial disease.

microbiology↗

bioRxiv2022

Dynamic model of bovine brucellosis to investigate control strategies in endemic settings

Brucellosis imposes substantial impacts on livestock production and public health worldwide. A stochastic, age-structured model incorporating herd demographics was developed describing within- and between-herd transmission of Brucella abortus in dairy cattle herds. The model was fitted to data from a cross-sectional study conducted in Punjab State of India and used to evaluate the effectiveness of control strategies under consideration. Based on model results, stakeholder acceptance and constraints regarding vaccine supply, vaccination of replacement calves in large farms should be prioritised. Test and removal applied at early stages of the control programme where seroprevalence is high would not constitute an effective use of resources. Critically, under current model assumptions, significant numbers of animals removed (culled or not used for breeding) in this strategy would be removed based on false positive results. To achieve sustained reductions in brucellosis, policymakers must commit to maintaining vaccination in the long term, which may eventually reduce frequency of infection in the livestock reservoir to a low enough level for elimination to be a realistic objective. This exercise provided important insights into the control of brucellosis in India, which has the largest cattle population globally, and a general framework for evaluating control strategies in endemic settings.

microbiology↗

bioRxiv2021

A slow dynamic switch regulates processing of microRNA-21

The microRNAs are non-coding RNAs which post-transcriptionally regulate the expression of a majority of eukaryotic genes, and whose dysregulation is a driver of many human diseases. Here we report the discovery of a very slow (0.1 sec) conformational rearrangement at the Dicer cleavage site of pre-miR-21 which regulates the relative concentration of readily processed and inefficiently processed structural states. We show this dynamic switch is affected by single nucleotide mutations and can be biased by small molecule and peptide ligands, which can direct the microRNA to occupy the inefficiently processed state and reduce processing efficiency. This result reveals a new mechanism of RNA regulation and suggests a chemical approach to suppressing or activating pathogenic microRNAs by selective stabilization of the unprocessed or processed state.

biochemistry↗

bioRxiv2021

Bacteria modulate tamoxifen-induced death via host fatty acid metabolism

Tamoxifen is a selective estrogen receptor (ER) modulator that is used to treat ER-positive breast cancer, but that at high doses kills both ER-positive and ER-negative breast cancer cells. We recapitulate this off-target effect in Caenorhabditis elegans, which does not have an ER ortholog. We find that different bacteria dramatically modulate tamoxifen toxicity in C. elegans, with a three-order of magnitude difference between animals fed Escherichia coli, Comamonas aquatica, and Bacillus subtilis. Remarkably, host fatty acid (FA) biosynthesis mitigates tamoxifen toxicity, and different bacteria provide the animal with different FAs, resulting in distinct FA profiles. Surprisingly these bacteria modulate tamoxifen toxicity by different death mechanisms, some of which are modulated by FA supplementation and others by antioxidants. Together, this work reveals a complex interplay between microbiota, FA metabolism and tamoxifen toxicity that may provide a blueprint for similar studies in more complex mammals.

systems biology↗

bioRxiv2021

Modeling the effects of Aedes aegypti's larval environment on adult body mass at emergence

Mosquitoes vector harmful pathogens that infect millions of people every year, and developing approaches to effectively control mosquitoes is a topic of great interest. However, the success of many control measures is highly dependent upon ecological, physiological, and life history traits of the mosquito species. The behavior of mosquitoes and their potential to vector pathogens can also be impacted by these traits. One trait of interest is mosquito body mass, which depends upon many factors associated with the environment in which juvenile mosquitoes develop. Our experiments examined the impact of larval density on the body mass of Aedes aegypti mosquitoes, which are important vectors of dengue, Zika, yellow fever, and other pathogens. To investigate the interactions between the larval environment and mosquito body mass, we built a discrete time mathematical model that incorporates body mass, larval density, and food availability and fit the model to our experimental data. We considered three categories of model complexity informed by data, and selected the best model within each category using Akaikes Information Criterion. We found that the larval environment is an important determinant of the body mass of mosquitoes upon emergence. Furthermore, we found that larval density has greater impact on body mass of adults at emergence than on development time, and that inclusion of density dependence in the survival of female aquatic stages in models is important. We discuss the implications of our results for the control of Aedes mosquitoes and on their potential to spread disease. Author summaryIn this work we examined how the environment in which young mosquitoes develop affects their adult body size as measured by adult body mass. Adult size has potential impacts on mosquito behavior and the ability of mosquitoes to transmit disease. We used a combination of experimental work and mathematical modeling to determine important factors affecting adult mosquito body size. In our model, we incorporated potentially interacting aspects of the mosquito life cycle and traits that affect mosquito growth as juveniles. These aspects include body mass, density of the population, and level of available resource. We compared different models to determine the one that best describes the data. As mass at emergence is linked to the success of adult mosquitoes to produce offspring and to their ability transmit pathogens, we discuss how important influences on development and survival of young mosquitoes affect mosquito control and disease spread.

ecology↗

bioRxiv2021

Connectivity and Synaptic Physiology in the Mouse and Human Cortex

To elucidate cortical microcircuit structure and synaptic properties we present a unique, extensive, and public synaptic physiology dataset and analysis platform. Through its application, we reveal principles that relate cell type to synapse properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Despite these associations, synaptic properties are heterogeneous in most subclass to subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, while the strength axis accounts for significant heterogeneity within the subclass. In human cortex, excitatory to excitatory synapse dynamics are distinct from those in mouse and short-term plasticity varies with depth across layers 2 and 3. With a novel connectivity analysis that enables fair comparisons between circuit elements, we find that intralaminar connection probability among cell subclasses exhibits a strong layer dependence.These and other findings combined with the analysis platform create new opportunities for the neuroscience community to advance our understanding of cortical microcircuits.

neuroscience↗

bioRxiv2021

Zebrafish spinal cord oligodendrocyte formation requires boc function

The axis of the vertebrate neural tube is patterned, in part, by a ventral to dorsal gradient of Shh signaling. In the ventral spinal cord, Shh induces concentration-dependent expression of transcription factors, subdividing neural progenitors into distinct domains that subsequently produce distinct neuronal and glial subtypes. In particular, progenitors of the pMN domain express the bHLH transcription factor Olig2 and produce motor neurons followed by oligodendrocytes, the myelinating glial cell type of the central nervous system. In addition to its role in patterning ventral progenitors, Shh signaling must be maintained through development to specify pMN progenitors for oligodendrocyte fate. Using a forward genetic screen in zebrafish for mutations that disrupt development of oligodendrocytes, we identified a new mutant allele of boc, which encodes a type I transmembrane protein that functions as a coreceptor for Shh. Embryos homozygous for the bocco25 allele, which creates a missense mutation in a Fibronectin type III domain that binds Shh, have normally patterned spinal cords but fail to maintain pMN progenitors, resulting in a deficit of oligodendrocytes. Using a sensitive fluorescent detection method for in situ RNA hybridization, we found that spinal cord cells express boc in a graded fashion that is inverse to the gradient of Shh signaling activity and that boc function is necessary to maintain pMN progenitors by shaping the Shh signaling gradient.

developmental biology↗

bioRxiv2020

Creation and validation of 3D-printed head molds for stereotaxic injections of neonatal mouse brains

BackgroundAn increasing number of rodent model systems use injection of DNA or viral constructs in the neonatal brain. However, approaches for reliable positioning and stereotaxic injection at this developmental stage are limited, typically relying on handheld positioning or molds that must be re-aligned for use in a given laboratory. New methodA complete protocol and open source software pipeline for generating 3D-printed head molds derived from a CT scan of a neonatal mouse head cast, together with a universal adapter that can be placed on a standard stereotaxic stage. ResultsA series of test injections with adenovirus encoding red fluorescent protein were conducted using original clay molds and newly generated 3D printed molds. Several metrics were used to compare spread and localization of targeted injections. Comparison with existing methodsThe new method of head mold generation gave comparable results to the field standard, but also allowed the rapid generation of additional copies of each head mold with standardized positioning of the head each time. ConclusionsThis 3D printing pipeline can be used to rapidly develop a series of head molds with standardized injection coordinates across multiple laboratories. More broadly, this pipeline can easily be adapted to other perinatal ages or species.

neuroscience↗

bioRxiv2020

Flexible RSV prefusogenic fusion glycoprotein exposes multiple neutralizing epitopes that may collectively contribute to protective immunity

Human respiratory syncytial virus (RSV) is a significant cause of lower respiratory tract infection in infants, young children, and older adults. There is no licensed vaccine and prophylactic treatment options are limited and not widely available in developing countries with the greatest disease burden. The RSV fusion (F) glycoprotein is a primary target of host immunity and thus a major focus for vaccine development. The native F glycoprotein structure is flexible and undergoes significant rearrangements from the metastable prefusion to a stable postfusion structure with neutralizing epitopes on intermediate structures. We hypothesize trimeric vaccine strategies that recapitulate the breathable F quaternary structure, and provide accessibility of B-cells to epitopes on intermediate conformations, may collectively contribute to protective immunity, while ridge prefusion F structures restrict access to key protective epitopes. To test this hypothesis, we used the near full-length native prefusogenic F as a backbone to construct three prefusion F variants with substitutions in the hydrophobic head cavity: 1) disulfide bond double mutant (DS), 2) space filling hydrophobic amino acid substitutions (Cav1), and 3) DS plus Cav1 substitutions (DS-Cav1). In this study, we compared the immunogenicity of prefusogenic F to the immunogenicity of the prefusion F variants in two animal models. Native prefusogenic F was significantly more immunogenic producing high titer antibodies to prefusogenic, prefusion, and postfusion F structures compared to animals immunized with prefusion F DS or DS-Cav1. Importantly, native prefusogenic F elicited antibodies that targeted neutralizing epitopes including prefusion-specific site zero (O) and V as well as conformation-independent neutralizing sites II and IV. Immunization with prefusion F DS or DS-Cav1 elicited antibodies primarily targeting antigenic sites O and V with little or no detectable antibodies to other key neutralizing sites. Animals immunized with native prefusogenic F also had significantly higher neutralizing antibodies that cross-neutralized RSV A and B subtypes while immunization with DS or DS-Cav1 elicited neutralizing antibodies primarily to the A subtype. We conclude that breathable trimeric vaccines that closely mimic the native F-structure, and incorporate strategies for B-cell accessibility to protective epitopes, are important considerations for vaccine design. F structures locked in a single conformation restrict B-cell access to neutralizing epitopes that may collectively contribute to destabilizing F-trimers important for broad protection. These results also have implications for vaccine strategies targeting other type 1 integral membrane proteins.

immunology↗

bioRxiv2020

Expectations and blind spots for structural variation detection from short-read alignment and long-read assembly

Virtually all genome sequencing efforts in national biobanks, complex and Mendelian disease programs, and emerging clinical diagnostic approaches utilize short-reads (srWGS), which present constraints for genome-wide discovery of structural variants (SVs). Alternative long-read single molecule technologies (lrWGS) offer significant advantages for genome assembly and SV detection, while these technologies are currently cost prohibitive for large-scale disease studies and clinical diagnostics (∼5-12X higher cost than comparable coverage srWGS). Moreover, only dozens of such genomes are currently publicly accessible by comparison to millions of srWGS genomes that have been commissioned for international initiatives. Given this ubiquitous reliance on srWGS in human genetics and genomics, we sought to characterize and quantify the properties of SVs accessible to both srWGS and lrWGS to establish benchmarks and expectations in ongoing medical and population genetic studies, and to project the added value of SVs uniquely accessible to each technology. In analyses of three trios with matched srWGS and lrWGS from the Human Genome Structural Variation Consortium (HGSVC), srWGS captured ∼11,000 SVs per genome using reference-based algorithms, while haplotype-resolved assembly from lrWGS identified ∼25,000 SVs per genome. Detection power and precision for SV discovery varied dramatically by genomic context and variant class: 9.7% of the current GRCh38 reference is defined by segmental duplications (SD) and simple repeats (SR), yet 91.4% of deletions that were specifically discovered by lrWGS localized to these regions. Across the remaining 90.3% of the human reference, we observed extremely high concordance (93.8%) for deletions discovered by srWGS and lrWGS after error correction using the raw lrWGS reads. Conversely, lrWGS was superior for detection of insertions across all genomic contexts. Given that the non-SD/SR sequences span 90.3% of the GRCh38 reference, and encompass 95.9% of coding exons in currently annotated disease associated genes, improved sensitivity from lrWGS to discover novel and interpretable pathogenic deletions not already accessible to srWGS is likely to be incremental. However, these analyses highlight the added value of assembly-based lrWGS to create new catalogues of functional insertions and transposable elements, as well as disease associated repeat expansions in genomic regions previously recalcitrant to routine assessment.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genomics↗

bioRxiv2020

C. elegans methionine/S-adenosylmethionine cycle activity is sensed and adjusted by a nuclear hormone receptor

Vitamin B12 is an essential micronutrient that functions in two metabolic pathways: the canonical propionate breakdown pathway and the methionine/S-adenosylmethionine (Met/SAM) cycle. In Caenorhabditis elegans, low vitamin B12, or genetic perturbation of the canonical propionate breakdown pathway results in propionate accumulation and the transcriptional activation of a propionate shunt pathway. This propionate-dependent mechanism requires nhr-10 and is referred to as “B12-mechanism-I”. Here, we report that vitamin B12 represses the expression of Met/SAM cycle genes by a propionate-independent mechanism we refer to as “B12-mechanism-II”. This mechanism is activated by perturbations in the Met/SAM cycle, genetically or due to low dietary vitamin B12. B12-mechanism-II requires nhr-114 to activate Met/SAM cycle gene expression, the vitamin B12 transporter, pmp-5, and adjust influx and efflux of the cycle by activating msra-1 and repressing cbs-1, respectively. Taken together, Met/SAM cycle activity is sensed and transcriptionally adjusted to be in a tight metabolic regime.Competing Interest StatementThe authors have declared no competing interest.View Full Text

systems biology↗