Biology subjects

King, R.

Publications and source records attributed to King, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Modelling sterilisation strategies to maximise population impact and cost-efficiency in free-roaming dog populations

Free-roaming dog populations pose challenges to public health and animal welfare, and their management and protection is legally mandated in many countries to control zoonotic diseases such as rabies, reduce bites, mitigate fear of aggressive dogs, and preserve animal welfare. Although international organisations recommend holistic dog population management with surgical sterilisation as a key component, stakeholders report a lack of evidence-based guidance on effective and cost-efficient strategies. To address this, we developed a deterministic model of a closed free-roaming dog (FRD) population, including dogs dependent on and independent of humans, parameterised using data from southern India. We evaluated a wide range of sterilisation strategies varying duration, interval, frequency, intensity, targeting different population subsets, and fixed or responsive approaches. Strategies were assessed based on their effects on independent FRD population size and implementation costs. Female sterilisation coverage (FSC) strongly determined population reduction and cost-effectiveness. Repeated, short sterilisation periods with appropriately timed intervals and high early intensity best balanced population reduction and cost-efficiency. Excluding dependent dogs (cared for by humans) severely constrained population reduction, while male sterilisation enhanced impact at little extra cost, assuming direct effects on pregnancy rates. Costs and population impacts can be visualised on an interactive web application (https://field.shinyapps.io/DogPopSimApp), allowing practitioners and policymakers to explore sterilisation strategy outcomes. Overall, cost-effective population reduction required sustained commitment and coverage-driven strategies that balance welfare, population reduction and the operational needs of dog-mediated rabies control. Author SummaryFree-roaming street dogs live closely with people. While these animals are often valued members of the community, they can raise public health and safety concerns, including disease transmission, bites, and traffic accidents. To protect both human and animal health, many regions use surgical sterilisation to humanely manage street dog populations. However, running these programmes is complex and expensive, and community managers often lack clear guidance on how to maximise their limited resources. To address this, we used data from southern India to create a mathematical model of a street dog population. We simulated thousands of different sterilisation strategies to see how factors like campaign length, frequency, and targeted sub-groups impacted population size and total costs. We found that short, repeated sterilisation campaigns offer the best balance of population reduction and cost-efficiency. Crucially, these programmes must include dogs that are partially cared for by humans; otherwise, their puppies will continually replenish the street population. Although sterilisation of females drives population reduction, sterilising males alongside females enhanced the overall impact. Ultimately, our study provides practical, evidence-based guidance to help communities design effective and financially sustainable dog management programmes that improve public health and animal welfare.

ecology↗

bioRxiv2026

Automated AI image recognition tools improve the efficiency of aerial wildlife counts: A multi-species case study on breeding seabirds and pinnipeds at the sub-Antarctic Bounty Islands.

Accurate monitoring of populations is essential for conservation management, including for vulnerable seabirds. Yet traditional ground-based surveys are logistically challenging and time-consuming, especially in remote environments such as the sub-Antarctic islands. Advances in aerial imagery and artificial intelligence (AI) offer opportunities to improve the efficiency and repeatability of population surveys. In this study, we evaluate an AI-based approach for counting Salvins albatross from high-resolution aerial imagery collected using a piloted fixed-wing aircraft at the Bounty Islands, New Zealand. Imagery acquired during a single-day survey was processed to create orthomosaic images, which were previously analysed using manual counts by an experienced observer. We applied an automated detection and counting model based on a Faster R-CNN architecture with Slicing-Aided Hyper-Inference, and compared AI-derived counts with original human counts in terms of accuracy, consistency, and processing time. The AI achieved an initial F1 score of 92.8% for albatross detection and produced counts within 3% of the manual results, while reducing processing time from approximately 66 hours to just over four minutes. The model was also capable of simultaneously detecting additional species present within the mixed breeding colony, including erect-crested penguins, fulmar prions, and New Zealand fur seals, adding scalable efficiency gains for future surveys. Our results demonstrate that combining piloted aircraft surveys with AI-based image analysis provides a rapid, scalable, and accurate method for monitoring seabird populations, with substantial benefits for conservation management in remote and logistically constrained regions.

ecology↗

bioRxiv2025

A complete diploid human genome benchmark for personalized genomics

Human genome resequencing typically involves mapping reads to a reference genome to call variants; however, this approach suffers from both technical and reference biases, leaving many duplicated and structurally polymorphic regions of the genome unmapped. Consequently, existing variant benchmarks, generated by the same methods, fail to assess these complex regions. To address this limitation, we present a telomere-to-telomere genome benchmark that achieves near-perfect accuracy (i.e. no detectable errors) across 99.4% of the complete, diploid HG002 genome. This benchmark adds 701.4 Mb of autosomal sequence and both sex chromosomes (216.8 Mb), totaling 15.3% of the genome that was absent from prior benchmarks. We also provide a diploid annotation of genes, transposable elements, segmental duplications, and satellite repeats, including 39,144 protein-coding genes across both haplotypes. To facilitate application of the benchmark, we developed tools for measuring the accuracy of sequencing reads, phased variant call sets, and genome assemblies against a diploid reference. Genome-wide analyses show that state-of-the-art de novo assembly methods resolve 2-7% more sequence and outperform variant calling accuracy by an order of magnitude, yielding just one error per 100 kb across 99.9% of the benchmark regions. Adoption of genome-based benchmarking is expected to accelerate the development of cost-effective methods for complete genome sequencing, expanding the reach of genomic medicine to the entire genome and enabling a new era of personalized genomics.

genomics↗

bioRxiv2024

Towards a global barcode reference library for subterranean fauna

Implementation of environmental DNA (eDNA) metabarcoding for biodiversity discovery and assessment offers a unique opportunity to gain new insights into subterranean communities around the world. However, for effective and meaningful identification of species from anonymous eDNA barcodes, a library of known reference sequences with associated correct taxonomic metadata -also called a barcode reference library (BRL) - is required. Here we propose an open, publicly accessible information resource for eDNA biomonitoring of subterranean fauna following findable, accessible, interoperable and reusable (FAIR) principles that can be expanded globally. While similar proposals have been made by other authors for individual taxon groups, here we have analysed a curated BRL of subterranean fauna compiled from existing GenBank and BOLD DNA sequence databases for a minimum of four genes (COI, 18S, 12S, and 16S rRNA). We demonstrate the value of such an initiative to eDNA metabarcoding where custom libraries are used to characterise entire ecosystems under examination. To examine the effectiveness of a custom BRL, we generated metabarcoding data for an exemplar system at Bungaroo Creek in the Pilbara (Australia), a globally significant location of stygofaunal diversity. We compared results of BLAST queries of eDNA Operational Taxonomic Units (OTUs) to our BRL and the GenBank nucleotide database, and observed that for all barcoding regions, the custom BRL identified subterranean ZOTUs that could not be identified using GenBank and worked better in tandem. We use the BRL presented here to propose a four-stage plan for developing data infrastructure for subterranean fauna, especially with respect to eDNA metabarcoding data. To our knowledge, this represents the first published instance of a subterranean BRL being tested against real eDNA metabarcoding data. These findings provide a step forward towards robust DNA-based bioassessments for subterranean biodiversity and further emphasise the need for the eDNA community to work together in facilitating a global BRL.

evolutionary biology↗

bioRxiv2024

The interplay of recombination landscape, a transposable element and population history in European populations of Chironomus riparius

Genome resolution is often constrained for non-model species. This can be challenging for population genomic studies as estimations are highly dependent on the quality of the reference genome. This is the case for population history inferences where accuracy relies on the correct detection of single-nucleotide polymorphisms (SNPs) and accurate recombination rates. Here, we utilize a novel long-read genome assembly of Chironomus riparius with high resolution at a chromosome-scale and reanalyse Illumina resequencing data of five European populations. With the model MSMC2, new population demographies were inferred and compared to an older study based on a fragmented genome. Assembly contiguity and completeness led to an increase in accuracy of past demography, suggesting the onset of divergence of an ancestral population between late Pleistocene and early Holocene. These estimates are additionally supported by paleotemperature data, which reveal significant climate shifts in Central Europe during these times. Recombination severely influences population history estimates. With the new reference genome, it was possible to resolve the recombination landscape on the genome-wide scale across different populations using the tool iSMC. Recombination and the dispersal of transposable elements (TEs) in the genome are suspected to influence each other. One TE, known as Cla-element, is suspected to be involved in the divergence of the different C. riparius populations. As it seems to be highly dynamic in the genome, its potential impact on the recombination landscape was explored. No global pattern could be detected which demonstrated a higher resistance of the recombination landscape to the impact of repetitive elements on genome integrity.

genomics↗

bioRxiv2024

Extreme temperatures impede the release success of captive-bred avian scavengers

Conservation translocations (reinforcements and reintroductions) are central for managing various endangered species, yet, their implementation is logistically and financially challenging. Because many translocations fail due to the mortality of released individuals, identifying and preventing these factors is crucial. Here we examine risk factors affecting the post-release survival of the Griffon vulture (Gyps fulvus). The Israeli population is facing extinction, and the recovery efforts by the local Nature and Parks Authority include supplemental releases of individuals from captive-breeding or rehabilitated birds (mostly imported from Spain). We use GPS tracking and thermometers to compare movement, behavior, and weather conditions experienced by released and wild-born individuals active in the same area and time. Our results show that the Judean Desert release site had a significantly lower survival rate than other release sites included in the program. After excluding several possible factors (e.g. known pathogens) with pathological examinations, we found that released individuals at this area were exposed to higher temperatures preceding their death (compared to wild-born griffons nearby), suggesting heat stress as the cause of death. Arguably, they failed to avoid the harsh environmental conditions, resulting in overheating due to their inexperience and undeveloped flight ability, as reflected by their lower probabilities of flying and shorter travel distances per day compared to wild-born vultures. These findings have led to adjustments of the local release protocol, (namely winter releases) resulting in a significant improvement in the early survival of translocated Griffons in the Judean Desert. Accounting for the harmful effects of extreme weather conditions is particularly important in a world facing climate change. More broadly, this sequence of scientific investigation with data integration from ecological, clinical, and biotelemetry sources, leading to improvement in translocation success demonstrates how conservation practices can be optimized by supporting studies to ensure the survival of endangered species in the wild.

ecology↗

bioRxiv2023

An experimental target-based platform in yeast for screening Plasmodium vivax deoxyhypusine synthase inhibitors

The enzyme deoxyhypusine synthase (DHS) catalyzes the first step in the post-translational modification of the eukaryotic translation factor 5A (eIF5A). This is the only protein known to contain the amino acid hypusine, which results from this modification. Both eIF5A and DHS are essential for cell viability in eukaryotes, and inhibiting DHS can be a promising strategy for the development of new therapeutic alternatives. The human and parasitic orthologous proteins are different enough to render selective targeting against infectious diseases; however, no DHS inhibitor selective for the parasite ortholog has previously been reported. Here, we established a yeast surrogate genetics platform to identify inhibitors of DHS from Plasmodium vivax, one of the major causative agents of malaria. We constructed genetically modified Saccharomyces cerevisiae strains expressing DHS genes from Homo sapiens (HsDHS) or P. vivax (PvDHS) in place of the endogenous DHS gene from S. cerevisiae. This new strain background was [~]60-fold more sensitive to an inhibitor of human DHS than the one previously used. Initially, a virtual screen using datasets from the ChEMBL-NTD database was performed. Candidate ligands were tested in growth assays using the newly generated yeast strains expressing heterologous DHS genes. Among these, two showed promise by preferentially reducing the growth of the PvDHS-expressing strain. Further, in a robotized assay, we screened 400 compounds from the Pathogen Box library using the same S. cerevisiae strains, and one compound preferentially reduced the growth of the PvDHS-expressing yeast strain. Western blot revealed that these compounds significantly reduced eIF5A hypusination in yeast. Our study demonstrates that this yeast-based platform is suitable for identifying and verifying candidate small molecule DHS inhibitors, selective for the parasite over the human ortholog.

synthetic biology↗

bioRxiv2023

Improved gene annotation of the fungal wheat pathogen Zymoseptoria tritici based on combined Iso-Seq and RNA-Seq evidence

Despite large omics datasets, the establishment of a reliable gene annotation is still challenging for eukaryotic genomes. Here, we used the reference genome of the major fungal wheat pathogen Zymoseptoria tritici (isolate IPO323) as a case study to develop methods to improve eukaryotic gene prediction. Four previous IPO323 annotations identified 10,933 to 13,260 gene models, but only one third of these coding sequences (CDS) have identical structures. To resolve these discrepancies and improve gene models, we generated full-length transcripts using long-read sequencing. This dataset was used together with other evidence (RNA-Seq transcripts and protein sequences) to generate novel ab initio gene models. The selection of the best structure among novel and existing gene models was performed according to transcript and protein evidence using InGenAnnot, a novel bioinformatics suite. Overall, 13,414 re-annotated gene models (RGMs) were predicted, including 671 new genes among which 53 encoded effector candidates. This process corrected many of the errors (15%) observed in previous gene models (coding sequence fusions, false introns, missing exons). While fungal genomes have poor annotations of untranslated regions (UTRs), our Iso-Seq long-read sequences outlined 5 and 3UTRs for 73% of the RGMs. Alternative transcripts were identified for 13% of RGMs, mostly due to intron retention (75%), likely corresponding to unprocessed pre-mRNAs. A total of 353 genes displayed alternative transcripts with combinations of previously predicted or novel exons. Long non-coding transcripts (lncRNAs) and double-stranded RNAs from two fungal viruses were also identified. Most lncRNAs corresponded to antisense transcripts of genes (52%). lncRNAs that were up or down regulated during infection were enriched in antisense transcripts (70%), suggesting their involvement in the control of gene expression. Our results showed that combining different ab initio gene predictions and evidence-driven curation using InGenAnnot improved the quality of gene annotations of a compact eukaryotic genome. Our analysis also provided new insights into the transcriptional landscape of Z. tritici, helping develop an increasingly complex picture of its biology.

genomics↗

bioRxiv2021

Self-organization and information transfer in Antarctic krill swarms

Antarctic krill swarms are one of the largest known animal aggregations. However, despite being the keystone species of the Southern Ocean, little is known about how swarms are formed and maintained, and we lack a detailed understanding of the local interactions between individuals that provide the basis for these swarms. Here we analyzed the trajectories of captive, wild-caught krill in 3D to determine individual level interaction rules and quantify patterns of information flow. Our results suggest krill operate a novel form of collective organization, with measures of information flow and individual movement adjustments expressed most strongly in the vertical dimension, a finding not seen in other swarming species. In addition, local directional alignment with near neighbors, and strong regulation of both direction and speed relative to the positions of groupmates suggest social factors are vital to the formation and maintenance of swarms. This research represents a first step in understanding the fundamentally important swarming behavior of krill.

animal behavior and cognition↗

bioRxiv2020

NERO: A Biomedical Named-entity (Recognition) Ontology with a Large, Annotated Corpus Reveals Meaningful Associations Through Text Embedding

Machine reading is essential for unlocking valuable knowledge contained in the millions of existing biomedical documents. Over the last two decades 1,2, the most dramatic advances in machine-reading have followed in the wake of critical corpus development3. Large, well-annotated corpora have been associated with punctuated advances in machine reading methodology and automated knowledge extraction systems in the same way that ImageNet 4 was fundamental for developing machine vision techniques. This study contributes six components to an advanced, named-entity analysis tool for biomedicine: (a) a new, Named-Entity Recognition Ontology (NERO) developed specifically for describing entities in biomedical texts, which accounts for diverse levels of ambiguity, bridging the scientific sublanguages of molecular biology, genetics, biochemistry, and medicine; (b) detailed guidelines for human experts annotating hundreds of named-entity classes; (c) pictographs for all named entities, to simplify the burden of annotation for curators; (d) an original, annotated corpus comprising 35,865 sentences, which encapsulate 190,679 named entities and 43,438 events connecting two or more entities; (e) validated, off-the-shelf, named-entity recognition automated extraction, and; (f) embedding models that demonstrate the promise of biomedical associations embedded within this corpus.

systems biology↗

bioRxiv2020

IDseq - An Open Source Cloud-based Pipeline and Analysis Service for Metagenomic Pathogen Detection and Monitoring

BackgroundMetagenomic next generation sequencing (mNGS) has enabled the rapid, unbiased detection and identification of microbes without pathogen-specific reagents, culturing, or a priori knowledge of the microbial landscape. mNGS data analysis requires a series of computationally intensive processing steps to accurately determine the microbial composition of a sample. Existing mNGS data analysis tools typically require bioinformatics expertise and access to local server-class hardware resources. For many research laboratories, this presents an obstacle, especially in resource limited environments. FindingsWe present IDseq, an open source cloud-based metagenomics pipeline and service for global pathogen detection and monitoring (https://idseq.net). The IDseq Portal accepts raw mNGS data, performs host and quality filtration steps, then executes an assembly-based alignment pipeline which results in the assignment of reads and contigs to taxonomic categories. The taxonomic relative abundances are reported and visualized in an easy-to-use web application to facilitate data interpretation and hypothesis generation. Furthermore, IDseq supports environmental background model generation and automatic internal spike-in control recognition, providing statistics which are critical for data interpretation. IDseq was designed with the specific intent of detecting novel pathogens. Here, we benchmark novel virus detection capability using both synthetically evolved viral sequences, and real-world samples, including IDseq analysis of a nasopharyngeal swab sample acquired and processed locally in Cambodia from a tourist from Wuhan, China, infected with the recently emergent SARS-CoV-2. ConclusionThe IDseq Portal reduces the barrier to entry for mNGS data analysis and enables bench scientists, clinicians, and bioinformaticians to gain insight from mNGS datasets for both known and novel pathogens.

bioinformatics↗

bioRxiv2020

POU6F2 Positive Retinal Ganglion Cells a Novel Group of ON-OFF Directionally Selective Subtypes in the Mouse Retina.

PurposePreviously we identified POU6F2 as a genetic link between central corneal thickness (CCT) and risk of open-angle glaucoma. The present study is designed to characterize the POU6F2-positive retinal ganglion cells (RGCs). MethodsThe Thy1-YFP-H mouse was used to identify the structure of POU6F2-positive RGCs in the retina. In the retina of the Thy1-YFP-H mouse approximately 3% of the RGCs were labeled with yellow fluorescent protein. These retinas were stained for POU6F2 to identify the morphology of the POU6F2 subtypes in 3D reconstructions of the labeled RGCs. Multiple retinal cell markers were also co-stained with POU6F2 to characterize the molecular signature of the POU6F2-positive RGCs. DBA/2J glaucoma models were used to test the role of POU6F2 in injury. ResultsIn the retina POU6F2 labels 32.9% of the RGCs in the DBA/2J retina (16.1% heavily and 16.8% lightly labeled). In 3D constructions of Thy1-YFP-H positive RGCs, the heavily labeled POU6F2-positive cells had dendrites in the inner plexiform layer that were bistratified and appeared to be ON-OFF directionally selective cells. The lightly labeled POU6F2 RGCs displayed 3 different dendritic distributions, with dendrites in the ON sublaminae only, OFF sublaminae only, or bistratified. The POU6F2-positive cells partially co-stained with Cdh6. The POU6F2-positive cells do not co-stain with CART and SATB2 (markers for ON-OFF directionally selective RGC), SMI32 (a marker for alpha RGCs), or ChAT and GAD67(markers for amacrine cells). The POU6F2-positive cells were sensitive to injury. In DBA/2J glaucoma model, at 8 months of age there was a 22% loss of RGCs (labeled with RBPMS) while there was 73% loss of the heavily labeled POU6F2 RGCs. ConclusionsPOU6F2 is a marker for a novel group of RGC subtypes that are ON-OFF directionally selective RGCs that are sensitive to glaucomatous injury.

genetics↗

bioRxiv2020

Designing and implementing programmable depletion in sequencing libraries with DASHit

Since Next-Generation Sequencing produces reads uniformly subsampled from an input library, highly abundant sequences may mask interesting low abundance sequences. The DASH (Depleting Abundant Sequences by Hybridization) technique takes advantage of the programmability of CRISPR/Cas9 to deplete unwanted high-abundance sequences. Because desired depletion targets vary by sample type, here we describe DASHit, software that outputs an optimal DASH target set given a sequencing dataset, an updated DASH protocol, and show depletion results with DASHit-designed targets for three different species.

microbiology↗

bioRxiv2019

The ER cargo receptor SURF4 facilitates efficient erythropoietin secretion

Erythropoietin (EPO), a glycoprotein produced by specialized peritubular fibroblasts in the kidney, is the master regulator of erythropoiesis. EPO is secreted into the plasma in response to tissue hypoxia and stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels. Using this cell line, we performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent sgRNAs resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, consistent with a role for SURF4 as an ER cargo receptor of EPO, we found that disruption of SURF4 resulted in accumulation of EPO in the ER compartment, and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO, ruling out an artifact of heterologous overexpression. This work suggests that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoeisis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.

cell biology↗

bioRxiv2019

Soft Windowing Application to Improve Analysis of High-throughput Phenotyping Data

MotivationHigh-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximises analytic power while minimising noise from unspecified environmental factors.\n\nResultsHere we introduce \"soft windowing\", a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant p-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft windowed and non-windowed approaches, respectively, from a set of 2,082 mutant mouse lines. Our method is generalisable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources.\n\nAvailability and ImplementationThe method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin.

bioinformatics↗

bioRxiv2019

A carbohydrate-binding protein, FLOURY ENDOSPERM 6 influences the initiation of A- and B-type starch granules in wheat.

Previously, we identified a quantitative trait locus on the group 4 chromosomes of Aegilops and bread wheat that controls B-type starch-granule content. Here, we identify a candidate gene by fine-mapping in Aegilops and confirm its function using wheat TILLING mutants. This gene is orthologous to the FLOURY ENDOSPERM 6 (FLO6) gene of rice and barley and the PTST2 gene of Arabidopsis. In Triticeae endosperm, reduction in the gene dose of functional FLO6 alleles results in reduction, or loss, of B-granules. This is due to repression of granule initiation in late-grain development, but has no deleterious impact on the synthesis of A-granules. The complete absence of functional FLO6, however, results in reduced numbers of normal A-type and B-type granules and the production of highly-abnormal granules that vary in size and shape. This polymorphous starch seen in a wheat flo6 triple mutant is similar to that observed in the barley mutant Franubet. Analysis of Franubet (fractured Nubet) starch suggests that the mutant A-granules are not fractured but compound, due to stimulation of granule initiation in plastids during early-grain development. Thus, in different situations in Triticeae, FLO6 either stimulates or represses granule initiation.

plant biology↗

bioRxiv2018

FLASH: A next-generation CRISPR diagnostic for multiplexed detection of antimicrobial resistance sequences

The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.

microbiology↗

bioRxiv2017

Genomic Locus Modulating Corneal Thickness in the Mouse Identifies POU6F2 as a Potential Risk of Developing Glaucoma

Purpose: Central corneal thickness (CCT) is one of the most heritable ocular traits and it is also a phenotypic risk factor for primary open angle glaucoma (POAG). The present study uses the BXD Recombinant Inbred (RI) strains to identify novel quantitative trait loci (QTLs) modulating CCT in the mouse with the potential of identifying a molecular link between CCT and risk of developing POAG.\n\nMethods: The BXD RI strain set was used to define mammalian genomic loci modulating CCT, with a total of 818 corneas measured from 61 BXD RI strains (between 60-100 days of age). The mice were anesthetized and the eyes were positioned in front of the lens of the Phoenix Micron IV Image-Guided OCT system or the Bioptigen OCT system. CCT data for each strain was averaged and used to identify quantitative trait loci (QTLs) modulating this phenotype using the bioinformatics tools on GeneNetwork (www.genenetwork.org). The candidate genes and genomic loci identified in the mouse were then directly compared with the summary data from a human primary open-angle glaucoma (POGA) genome wide association study (NEIGHBORHOOD) to determine if any genomic elements modulating mouse CCT are also risk factors for POAG.\n\nResults: This analysis revealed one significant QTL on Chr 13 and a suggestive QTL on Chr 7. The significant locus on Chr 13 (13 to 19 Mb) was examined further to define candidate genes modulating this eye phenotype. For the Chr 13 QTL in the mouse, only one gene in the region (Pou6f2) contained nonsynonymous SNPs. Of these five nonsynonymous SNPs in Pou6f2, two resulted in changes in the amino acid proline which could result in altered secondary structure affecting protein function. The 7 Mb region under the mouse Chr 13 peak distributes over 2 chromosomes in the human: Chr 1 and Chr 7. These genomic loci were examined in the NEIGHBORHOOD database to determine if they are potential risk factors for human glaucoma identified using meta-data from human GWAS. The top 50 hits all resided within one gene (POU6F2), with the highest significance level of p = 10-6 for SNP rs76319873. POU6F2 is found in retinal ganglion cells and in corneal limbal stem cells. To test the effect of POU6F2 on CCT we examined the corneas of a Pou6f2-null mice and the corneas were thinner than those of wild-type littermates. In addition, these POU6F2 RGCs die early in the DBA/2J model of glaucoma than most RGCs.\n\nConclusions: Using a mouse genetic reference panel, we identified a transcription factor, Pou6f2, that modulates CCT in the mouse. POU6F2 is also found in a subset of retinal ganglion cells and these RGCs are sensitive to injury.\n\nAuthors SummaryGlaucoma is a complex group of diseases with several known causal mutations and many known risk factors. One well-known risk factor for developing primary open angle glaucoma is the thickness of the central cornea. The present study leverages a unique blend of systems biology methods using BXD recombinant inbred mice and genome-wide association studies from humans to define a putative molecular link between a phenotypic risk factor (central corneal thickness) and glaucoma. We identified a transcription factor, POU6F2, that is found in the developing retinal ganglion cells and cornea. POU6F2 is also present in a subpopulation of retinal ganglion cells and in stem cells of the cornea. Functional studies reveal that POU6F2 is associated the central corneal thickness and with susceptibility of retinal ganglion cells to injury.

genetics↗