Publications by authors named "Matthew D Clark"

34 Publications

Evaluation of Antibiotic Initiation Tools in End-of-Life Care.

Am J Hosp Palliat Care 2021 Jun 25:10499091211027806. Epub 2021 Jun 25.

173678Hospice of Southern Illinois, Inc, Belleville, IL, USA.

Background: Hospice patients are frequently confronted with potentially infectious complications necessitating antibiotic consideration. Information regarding the appropriate use of antibiotics and their impact on symptom management in hospice patients are unknown.

Objectives: This study aimed to evaluate and describe the use of an antibiotic initiation tool in patients admitted to outpatient hospice services. The primary outcome assessed the percentage of antibiotics that were appropriately initiated based on Loeb's Minimum Criteria (LMC) for Antibiotic Initiation Tool. Secondary outcomes included the number of patients with documented symptom resolution following antibiotic completion, the number of antibiotic courses that were successfully completed, and treatment-related adverse events.

Methods: This was a retrospective, multisite, descriptive analysis of hospice patients treated with antibiotics between April 2019 and September 2020.

Results: Two hundred and thirty patients were assessed for inclusion, with 172 meeting eligibility criteria and receiving a total of 201 antibiotic courses. Based on LMC, 84 of the 201 (42%) antibiotics ordered were appropriate, with 60% of these LMC-approved courses resulting in symptom resolution. Out of 201 total courses, 99 (49%) resulted in symptom resolution. Overall, 160 (80%) antibiotic courses were successfully completed.

Conclusion: In this study, antibiotic initiation in hospice patients frequently did not meet LMC. Less than half of the antibiotics prescribed led to symptom resolution despite antibiotic course completion in most patients. There is no consensus or guidelines directing appropriate antibiotic decision-making in hospice patients. The appropriate use of antibiotics in terminally ill patients warrants additional research.
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http://dx.doi.org/10.1177/10499091211027806DOI Listing
June 2021

Pharmacogenomics in Pain Management: A Review of Relevant Gene-Drug Associations and Clinical Considerations.

Ann Pharmacother 2021 12 26;55(12):1486-1501. Epub 2021 Mar 26.

Southern Illinois University Edwardsville School of Pharmacy, Edwardsville, IL, USA.

Objective: To provide an overview of clinical recommendations regarding genomic medicine relating to pain management and opioid use disorder.

Data Sources: A literature review was conducted using the search terms , , , , , and on PubMed (inception to February 1, 2021), CINAHL (2016 through February 1, 2021), and EMBASE (inception through February 1, 2021).

Study Selection And Data Extraction: All relevant clinical trials, review articles, package inserts, and guidelines evaluating applicable pharmacogenotypes were considered for inclusion.

Data Synthesis: More than 300 Food and Drug Administration-approved medications contain pharmacogenomic information in their labeling. Genetic variability may alter the therapeutic effects of commonly prescribed pain medications. Pharmacogenomic-guided therapy continues to gain traction in clinical practice, but a multitude of barriers to widespread pharmacogenomic implementation exist.

Relevance To Patient Care And Clinical Practice: Pain is notoriously difficult to treat given the need to balance safety and efficacy when selecting pharmacotherapy. Pharmacogenomic data can help optimize outcomes for patients with pain. With improved technological advances, more affordable testing, and a better understanding of genomic variants resulting in treatment disparities, pharmacogenomics continues to gain popularity. Unfortunately, despite these and other advancements, pharmacogenomic testing and implementation remain underutilized and misunderstood in clinical care, in part because of a lack of health care professionals trained in assessing and implementing test results.

Conclusions: A one-size-fits-all approach to pain management is inadequate and outdated. With increasing genomic data and pharmacogenomic understanding, patient-specific genomic testing offers a comprehensive and personalized treatment alternative worthy of additional research and consideration.
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http://dx.doi.org/10.1177/10600280211003875DOI Listing
December 2021

A low-cost pipeline for soil microbiome profiling.

Microbiologyopen 2020 12 22;9(12):e1133. Epub 2020 Nov 22.

Natural History Museum (NHM, London, UK.

Common bottlenecks in environmental and crop microbiome studies are the consumable and personnel costs necessary for genomic DNA extraction and sequencing library construction. This is harder for challenging environmental samples such as soil, which is rich in Polymerase Chain Reaction (PCR) inhibitors. To address this, we have established a low-cost genomic DNA extraction method for soil samples. We also present an Illumina-compatible 16S and ITS rRNA gene amplicon library preparation workflow that uses common laboratory equipment. We evaluated the performance of our genomic DNA extraction method against two leading commercial soil genomic DNA kits (MoBio PowerSoil® and MP Biomedicals™ FastDNA™ SPIN) and a recently published non-commercial extraction method by Zou et al. (PLoS Biology, 15, e2003916, 2017). Our benchmarking experiment used four different soil types (coniferous, broad-leafed, and mixed forest plus a standardized cereal crop compost mix) assessing the quality and quantity of the extracted genomic DNA by analyzing sequence variants of 16S V4 and ITS rRNA amplicons. We found that our genomic DNA extraction method compares well to both commercially available genomic DNA extraction kits in DNA quality and quantity. The MoBio PowerSoil® kit, which relies on silica column-based DNA extraction with extensive washing, delivered the cleanest genomic DNA, for example, best A260:A280 and A260:A230 absorbance ratios. The MP Biomedicals™ FastDNA™ SPIN kit, which uses a large amount of binding material, yielded the most genomic DNA. Our method fits between the two commercial kits, producing both good yields and clean genomic DNA with fragment sizes of approximately 10 kb. Comparative analysis of detected amplicon sequence variants shows that our method correlates well with the two commercial kits. Here, we present a low-cost genomic DNA extraction method for soil samples that can be coupled to an Illumina-compatible simple two-step amplicon library construction workflow for 16S V4 and ITS marker genes. Our method delivers high-quality genomic DNA at a fraction of the cost of commercial kits and enables cost-effective, large-scale amplicon sequencing projects. Notably, our extracted gDNA molecules are long enough to be suitable for downstream techniques such as full gene sequencing or even metagenomics shotgun approaches using long reads (PacBio or Nanopore), 10x Genomics linked reads, and Dovetail genomics.
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http://dx.doi.org/10.1002/mbo3.1133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7755778PMC
December 2020

Enfortumab Vedotin-ejfv: A First-in-Class Anti-Nectin-4 Antibody-Drug Conjugate for the Management of Urothelial Carcinoma.

Ann Pharmacother 2021 06 18;55(6):772-782. Epub 2020 Sep 18.

The University of Texas MD Anderson Cancer Center, Houston, TX, USA.

Objective: To evaluate the pharmacology, pharmacokinetics, clinical efficacy, safety, dosing, cost, and clinical implications of enfortumab vedotin-ejfv (EV) in the treatment of locally advanced or metastatic urothelial carcinoma (UC).

Data Sources: A literature search of PubMed (inception to August 2020) was conducted using the terms , and . Data were also obtained from package inserts, meeting abstracts, and ongoing studies from ClinicalTrials.gov.

Study Selection And Data Extraction: All relevant published articles, package inserts, and meeting abstracts evaluating EV for the treatment of UC were analyzed.

Data Synthesis: Antibody-drug conjugates (ADCs) deliver potent cytotoxic agents using highly selective monoclonal antibodies. Targeting the near-universal expression of Nectin-4 on UC cells is a viable therapeutic strategy. In a pivotal phase II trial, EV demonstrated an overall response rate of 44%, and a median duration of response of 7.6 months. Estimated overall survival was 11.7 months with a median estimated progression-free survival of 5.6 months. Results were similar among difficult-to-treat patients, including those with liver metastases. Unique toxicity concerns with EV require careful consideration and monitoring.

Relevance To Patient Care And Clinical Practice: EV, a first-in-class anti-Nectin-4 ADC, provides impressive response rates with manageable toxicities, making it a promising treatment option for patients with multiply relapsed or refractory UC.

Conclusion: The US Food and Drug Administration-approved EV demonstrates antitumor activity in heavily pretreated patients with UC but harbors important adverse effects and financial concerns. Additional studies are required to identify the optimal sequencing, patient population, and place in therapy for EV.
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http://dx.doi.org/10.1177/1060028020960402DOI Listing
June 2021

Rapid MinION profiling of preterm microbiota and antimicrobial-resistant pathogens.

Nat Microbiol 2020 03 16;5(3):430-442. Epub 2019 Dec 16.

Earlham Institute, Norwich Research Park, Norwich, UK.

The MinION sequencing platform offers near real-time analysis of DNA sequence; this makes the tool attractive for deployment in fieldwork or clinical settings. We used the MinION platform coupled to the NanoOK RT software package to perform shotgun metagenomic sequencing and profile mock communities and faecal samples from healthy and ill preterm infants. Using Nanopore data, we reliably classified a 20-species mock community and captured the diversity of the immature gut microbiota over time and in response to interventions such as probiotic supplementation, antibiotic treatment or episodes of suspected sepsis. We also performed rapid real-time runs to assess gut-associated microbial communities in critically ill and healthy infants, facilitated by NanoOK RT software package, which analysed sequences as they were generated. Our pipeline reliably identified pathogenic bacteria (that is, Klebsiella pneumoniae and Enterobacter cloacae) and their corresponding antimicrobial resistance gene profiles within as little as 1 h of sequencing. Results were confirmed using pathogen isolation, whole-genome sequencing and antibiotic susceptibility testing, as well as mock communities and clinical samples with known antimicrobial resistance genes. Our results demonstrate that MinION (including cost-effective Flongle flow cells) with NanoOK RT can process metagenomic samples to a rich dataset in < 5 h, which creates a platform for future studies aimed at developing these tools and approaches in clinical settings with a focus on providing tailored patient antimicrobial treatment options.
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http://dx.doi.org/10.1038/s41564-019-0626-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7044117PMC
March 2020

Plant gene editing through de novo induction of meristems.

Nat Biotechnol 2020 01 16;38(1):84-89. Epub 2019 Dec 16.

Department of Plant and Microbial Biology, University of Minnesota, St. Paul, MN, USA.

Plant gene editing is typically performed by delivering reagents such as Cas9 and single guide RNAs to explants in culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones. The creation of edited plants through tissue culture is often inefficient, time-consuming, works for only limited species and genotypes, and causes unintended changes to the genome and epigenome. Here we report two methods to generate gene-edited dicotyledonous plants through de novo meristem induction. Developmental regulators and gene-editing reagents are delivered to somatic cells of whole plants. This induces meristems that produce shoots with targeted DNA modifications, and gene edits are transmitted to the next generation. The de novo induction of gene-edited meristems sidesteps the need for tissue culture and promises to overcome a bottleneck in plant gene editing.
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http://dx.doi.org/10.1038/s41587-019-0337-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6954279PMC
January 2020

Spatially resolved transcriptomics reveals plant host responses to pathogens.

Plant Methods 2019 10;15:114. Epub 2019 Oct 10.

Earlham Institute, Norwich Research Park, Norwich, UK.

Background: Thorough understanding of complex model systems requires the characterisation of processes in different cell types of an organism. This can be achieved with high-throughput spatial transcriptomics at a large scale. However, for plant model systems this is still challenging as suitable transcriptomics methods are sparsely available. Here we present GaST-seq (rid-ssisted, patial ranscriptome uencing), an easy to adopt, micro-scale spatial-transcriptomics workflow that allows to study expression profiles across small areas of plant tissue at a fraction of the cost of existing sequencing-based methods.

Results: We compare the GaST-seq method with widely used library preparation methods (Illumina TruSeq). In spatial experiments we show that the GaST-seq method is sensitive enough to identify expression differences across a plant organ. We further assess the spatial transcriptome response of leaves exposed to the bacterial molecule flagellin-22, and show that with eukaryotic () infection both host and pathogen spatial transcriptomes are obtained.

Conclusion: We show that our method can be used to identify known, rapidly flagellin-22 elicited genes, plant immune response pathways to bacterial attack and spatial expression patterns of genes associated with these pathways.
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http://dx.doi.org/10.1186/s13007-019-0498-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785889PMC
October 2019

New approaches for metagenome assembly with short reads.

Brief Bioinform 2020 03;21(2):584-594

Earlham Institute, Norwich Research Park, Norwich, UK.

In recent years, the use of longer range read data combined with advances in assembly algorithms has stimulated big improvements in the contiguity and quality of genome assemblies. However, these advances have not directly transferred to metagenomic data sets, as assumptions made by the single genome assembly algorithms do not apply when assembling multiple genomes at varying levels of abundance. The development of dedicated assemblers for metagenomic data was a relatively late innovation and for many years, researchers had to make do using tools designed for single genomes. This has changed in the last few years and we have seen the emergence of a new type of tool built using different principles. In this review, we describe the challenges inherent in metagenomic assemblies and compare the different approaches taken by these novel assembly tools.
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http://dx.doi.org/10.1093/bib/bbz020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299287PMC
March 2020

A critical comparison of technologies for a plant genome sequencing project.

Gigascience 2019 03;8(3)

Technology Development, Earlham Institute, Norwich Research Park, Norwich NR4 7UZ, UK.

Background: A high-quality genome sequence of any model organism is an essential starting point for genetic and other studies. Older clone-based methods are slow and expensive, whereas faster, cheaper short-read-only assemblies can be incomplete and highly fragmented, which minimizes their usefulness. The last few years have seen the introduction of many new technologies for genome assembly. These new technologies and associated new algorithms are typically benchmarked on microbial genomes or, if they scale appropriately, on larger (e.g., human) genomes. However, plant genomes can be much more repetitive and larger than the human genome, and plant biochemistry often makes obtaining high-quality DNA that is free from contaminants difficult. Reflecting their challenging nature, we observe that plant genome assembly statistics are typically poorer than for vertebrates.

Results: Here, we compare Illumina short read, Pacific Biosciences long read, 10x Genomics linked reads, Dovetail Hi-C, and BioNano Genomics optical maps, singly and combined, in producing high-quality long-range genome assemblies of the potato species Solanum verrucosum. We benchmark the assemblies for completeness and accuracy, as well as DNA compute requirements and sequencing costs.

Conclusions: The field of genome sequencing and assembly is reaching maturity, and the differences we observe between assemblies are surprisingly small. We expect that our results will be helpful to other genome projects, and that these datasets will be used in benchmarking by assembly algorithm developers.
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http://dx.doi.org/10.1093/gigascience/giy163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6423373PMC
March 2019

Shifting the limits in wheat research and breeding using a fully annotated reference genome.

Science 2018 08 16;361(6403). Epub 2018 Aug 16.

GDEC (Genetics, Diversity and Ecophysiology of Cereals), INRA, Université Clermont Auvergne (UCA), 5 chemin de Beaulieu, 63039 Clermont-Ferrand, France.

An annotated reference sequence representing the hexaploid bread wheat genome in 21 pseudomolecules has been analyzed to identify the distribution and genomic context of coding and noncoding elements across the A, B, and D subgenomes. With an estimated coverage of 94% of the genome and containing 107,891 high-confidence gene models, this assembly enabled the discovery of tissue- and developmental stage-related coexpression networks by providing a transcriptome atlas representing major stages of wheat development. Dynamics of complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. This community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding.
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http://dx.doi.org/10.1126/science.aar7191DOI Listing
August 2018

Independent assessment and improvement of wheat genome sequence assemblies using Fosill jumping libraries.

Gigascience 2018 05;7(5)

John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.

Background: The accurate sequencing and assembly of very large, often polyploid, genomes remains a challenging task, limiting long-range sequence information and phased sequence variation for applications such as plant breeding. The 15-Gb hexaploid bread wheat (Triticum aestivum) genome has been particularly challenging to sequence, and several different approaches have recently generated long-range assemblies. Mapping and understanding the types of assembly errors are important for optimising future sequencing and assembly approaches and for comparative genomics.

Results: Here we use a Fosill 38-kb jumping library to assess medium and longer-range order of different publicly available wheat genome assemblies. Modifications to the Fosill protocol generated longer Illumina sequences and enabled comprehensive genome coverage. Analyses of two independent Bacterial Artificial Chromosome (BAC)-based chromosome-scale assemblies, two independent Illumina whole genome shotgun assemblies, and a hybrid Single Molecule Real Time (SMRT-PacBio) and short read (Illumina) assembly were carried out. We revealed a surprising scale and variety of discrepancies using Fosill mate-pair mapping and validated several of each class. In addition, Fosill mate-pairs were used to scaffold a whole genome Illumina assembly, leading to a 3-fold increase in N50 values.

Conclusions: Our analyses, using an independent means to validate different wheat genome assemblies, show that whole genome shotgun assemblies based solely on Illumina sequences are significantly more accurate by all measures compared to BAC-based chromosome-scale assemblies and hybrid SMRT-Illumina approaches. Although current whole genome assemblies are reasonably accurate and useful, additional improvements will be needed to generate complete assemblies of wheat genomes using open-source, computationally efficient, and cost-effective methods.
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http://dx.doi.org/10.1093/gigascience/giy053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967450PMC
May 2018

The ash dieback invasion of Europe was founded by two genetically divergent individuals.

Nat Ecol Evol 2018 06 23;2(6):1000-1008. Epub 2018 Apr 23.

John Innes Centre, Norwich Research Park, Norwich, UK.

Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H. fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H. fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H. fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.
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http://dx.doi.org/10.1038/s41559-018-0548-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5969572PMC
June 2018

Building a locally diploid genome and transcriptome of the diatom Fragilariopsis cylindrus.

Sci Data 2017 10 10;4:170149. Epub 2017 Oct 10.

School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.

The genome of the cold-adapted diatom Fragilariopsis cylindrus is characterized by highly diverged haplotypes that intersperse its homozygous genome. Here, we describe how a combination of PacBio DNA and Illumina RNA sequencing can be used to resolve this complex genomic landscape locally into the highly diverged haplotypes, and how to map various environmentally controlled transcripts onto individual haplotypes. We assembled PacBio sequence data with the FALCON assembler and created a haplotype resolved annotation of the assembly using annotations of a Sanger sequenced F. cylindrus genome. RNA-seq datasets from six different growth conditions were used to resolve allele-specifc gene expression in F. cylindrus. This approach enables to study differential expression of alleles in a complex genomic landscape and provides a useful tool to study how diverged haplotypes in diploid organisms are used for adaptation and evolution to highly variable environments.
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http://dx.doi.org/10.1038/sdata.2017.149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634323PMC
October 2017

A world of opportunities with nanopore sequencing.

J Exp Bot 2017 Nov;68(20):5419-5429

Earlham Institute, Norwich Research Park, Norwich NR4 7UZ, UK.

Oxford Nanopore Technologies' MinION sequencer was launched in pre-release form in 2014 and represents an exciting new sequencing paradigm. The device offers multi-kilobase reads and a streamed mode of operation that allows processing of reads as they are generated. Crucially, it is an extremely compact device that is powered from the USB port of a laptop computer, enabling it to be taken out of the lab and facilitating previously impossible in-field sequencing experiments to be undertaken. Many of the initial publications concerning the platform focused on provision of tools to access and analyse the new sequence formats and then demonstrating the assembly of microbial genomes. More recently, as throughput and accuracy have increased, it has been possible to begin work involving more complex genomes and metagenomes. With the release of the high-throughput GridION X5 and PromethION platforms, the sequencing of large genomes will become more cost efficient, and enable the leveraging of extremely long (>100 kb) reads for resolution of complex genomic structures. This review provides a brief overview of nanopore sequencing technology, describes the growing range of nanopore bioinformatics tools, and highlights some of the most influential publications that have emerged over the last 2 years. Finally, we look to the future and the potential the platform has to disrupt work in human, microbiome, and plant genomics.
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http://dx.doi.org/10.1093/jxb/erx289DOI Listing
November 2017

Comparative analysis of targeted long read sequencing approaches for characterization of a plant's immune receptor repertoire.

BMC Genomics 2017 07 26;18(1):564. Epub 2017 Jul 26.

Earlham Institute (EI), Norwich Research Park, Norwich, NR4 7UZ, UK.

Background: The Oxford Nanopore Technologies MinION™ sequencer is a small, portable, low cost device that is accessible to labs of all sizes and attractive for in-the-field sequencing experiments. Selective breeding of crops has led to a reduction in genetic diversity, and wild relatives are a key source of new genetic resistance to pathogens, usually via NLR immune receptor-encoding genes. Recent studies have demonstrated how crop NLR repertoires can be targeted for sequencing on Illumina or PacBio (RenSeq) and the specific gene conveying pathogen resistance identified.

Results: Sequence yields per MinION run are lower than Illumina, making targeted resequencing an efficient approach. While MinION generates long reads similar to PacBio it doesn't generate the highly accurate multipass consensus reads, which presents downstream bioinformatics challenges. Here we demonstrate how MinION data can be used for RenSeq achieving similar results to the PacBio and how novel NLR gene fusions can be identified via a Nanopore RenSeq pipeline.

Conclusion: The described library preparation and bioinformatics methods should be applicable to other gene families or any targeted long DNA fragment nanopore sequencing project.
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http://dx.doi.org/10.1186/s12864-017-3936-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530509PMC
July 2017

Construction of a map-based reference genome sequence for barley, Hordeum vulgare L.

Sci Data 2017 04 27;4:170044. Epub 2017 Apr 27.

College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China.

Barley (Hordeum vulgare L.) is a cereal grass mainly used as animal fodder and raw material for the malting industry. The map-based reference genome sequence of barley cv. 'Morex' was constructed by the International Barley Genome Sequencing Consortium (IBSC) using hierarchical shotgun sequencing. Here, we report the experimental and computational procedures to (i) sequence and assemble more than 80,000 bacterial artificial chromosome (BAC) clones along the minimum tiling path of a genome-wide physical map, (ii) find and validate overlaps between adjacent BACs, (iii) construct 4,265 non-redundant sequence scaffolds representing clusters of overlapping BACs, and (iv) order and orient these BAC clusters along the seven barley chromosomes using positional information provided by dense genetic maps, an optical map and chromosome conformation capture sequencing (Hi-C). Integrative access to these sequence and mapping resources is provided by the barley genome explorer (BARLEX).
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http://dx.doi.org/10.1038/sdata.2017.44DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5407242PMC
April 2017

A chromosome conformation capture ordered sequence of the barley genome.

Nature 2017 04;544(7651):427-433

European Molecular Biology Laboratory - The European Bioinformatics Institute, Hinxton CB10 1SD, UK.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.
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http://dx.doi.org/10.1038/nature22043DOI Listing
April 2017

An improved assembly and annotation of the allohexaploid wheat genome identifies complete families of agronomic genes and provides genomic evidence for chromosomal translocations.

Genome Res 2017 05;27(5):885-896

Earlham Institute, Norwich, NR4 7UZ, United Kingdom.

Advances in genome sequencing and assembly technologies are generating many high-quality genome sequences, but assemblies of large, repeat-rich polyploid genomes, such as that of bread wheat, remain fragmented and incomplete. We have generated a new wheat whole-genome shotgun sequence assembly using a combination of optimized data types and an assembly algorithm designed to deal with large and complex genomes. The new assembly represents >78% of the genome with a scaffold N50 of 88.8 kb that has a high fidelity to the input data. Our new annotation combines strand-specific Illumina RNA-seq and Pacific Biosciences (PacBio) full-length cDNAs to identify 104,091 high-confidence protein-coding genes and 10,156 noncoding RNA genes. We confirmed three known and identified one novel genome rearrangements. Our approach enables the rapid and scalable assembly of wheat genomes, the identification of structural variants, and the definition of complete gene models, all powerful resources for trait analysis and breeding of this key global crop.
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http://dx.doi.org/10.1101/gr.217117.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5411782PMC
May 2017

Genomic innovation for crop improvement.

Nature 2017 03;543(7645):346-354

Earlham Institute, Norwich Research Park, Norwich NR4 7UH, UK.

Crop production needs to increase to secure future food supplies, while reducing its impact on ecosystems. Detailed characterization of plant genomes and genetic diversity is crucial for meeting these challenges. Advances in genome sequencing and assembly are being used to access the large and complex genomes of crops and their wild relatives. These have helped to identify a wide spectrum of genetic variation and permitted the association of genetic diversity with diverse agronomic phenotypes. In combination with improved and automated phenotyping assays and functional genomic studies, genomics is providing new foundations for crop-breeding systems.
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http://dx.doi.org/10.1038/nature22011DOI Listing
March 2017

Elucidation of the 'Honeycrisp' pedigree through haplotype analysis with a multi-family integrated SNP linkage map and a large apple (×) pedigree-connected SNP data set.

Hortic Res 2017 22;4:17003. Epub 2017 Feb 22.

Department of Horticultural Science, University of Minnesota , St Paul, MN 55104, USA.

The apple () cultivar Honeycrisp has become important economically and as a breeding parent. An earlier study with SSR markers indicated the original recorded pedigree of 'Honeycrisp' was incorrect and 'Keepsake' was identified as one putative parent, the other being unknown. The objective of this study was to verify 'Keepsake' as a parent and identify and genetically describe the unknown parent and its grandparents. A multi-family based dense and high-quality integrated SNP map was created using the apple 8 K Illumina Infinium SNP array. This map was used alongside a large pedigree-connected data set from the RosBREED project to build extended SNP haplotypes and to identify pedigree relationships. 'Keepsake' was verified as one parent of 'Honeycrisp' and 'Duchess of Oldenburg' and 'Golden Delicious' were identified as grandparents through the unknown parent. Following this finding, siblings of 'Honeycrisp' were identified using the SNP data. Breeding records from several of these siblings suggested that the previously unreported parent is a University of Minnesota selection, MN1627. This selection is no longer available, but now is genetically described through imputed SNP haplotypes. We also present the mosaic grandparental composition of 'Honeycrisp' for each of its 17 chromosome pairs. This new pedigree and genetic information will be useful in future pedigree-based genetic studies to connect 'Honeycrisp' with other cultivars used widely in apple breeding programs. The created SNP linkage map will benefit future research using the data from the Illumina apple 8 and 20 K and Affymetrix 480 K SNP arrays.
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http://dx.doi.org/10.1038/hortres.2017.3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321071PMC
February 2017

Genetic dissection of powdery mildew resistance in interspecific half-sib grapevine families using SNP-based maps.

Mol Breed 2017 21;37(1). Epub 2016 Dec 21.

Department of Horticultural Science, University of Minnesota, Saint Paul, MN 55108 USA.

Quantitative trait locus (QTL) identification in perennial fruit crops is impeded largely by their lengthy generation time, resulting in costly and labor-intensive maintenance of breeding programs. In a grapevine (genus ) breeding program, although experimental families are typically unreplicated, the genetic backgrounds may contain similar progenitors previously selected due to their contribution of favorable alleles. In this study, we investigated the utility of joint QTL identification provided by analyzing half-sib families. The genetic control of powdery mildew was studied using two half-sib F families, namely GE0711/1009 (MN1264 × MN1214;  = 147) and GE1025 (MN1264 × MN1246;  = 125) with multiple species in their ancestry. Maternal genetic maps consisting of 1077 and 1641 single nucleotide polymorphism (SNP) markers, respectively, were constructed using a pseudo-testcross strategy. Ratings of field resistance to powdery mildew were obtained based on whole-plant evaluation of disease severity. This 2-year analysis uncovered two QTLs that were validated on a consensus map in these half-sib families with improved precision relative to the parental maps. Examination of haplotype combinations based on the two QTL regions identified strong association of haplotypes inherited from 'Seyval blanc', through MN1264, with powdery mildew resistance. This investigation also encompassed the use of microsatellite markers to establish a correlation between 206-bp (UDV-015b) and 357-bp (VViv67) fragment sizes with resistance-carrying haplotypes. Our work is one of the first reports in grapevine demonstrating the use of SNP-based maps and haplotypes for QTL identification and tagging of powdery mildew resistance in half-sib families.
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http://dx.doi.org/10.1007/s11032-016-0586-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226326PMC
December 2016

Evolutionary genomics of the cold-adapted diatom Fragilariopsis cylindrus.

Nature 2017 01 16;541(7638):536-540. Epub 2017 Jan 16.

Zoologisches Forschungsmuseum Alexander Koenig, Leibniz Institut für Biodiversität der Tiere, Adenauerallee 160, 53113 Bonn, Germany.

The Southern Ocean houses a diverse and productive community of organisms. Unicellular eukaryotic diatoms are the main primary producers in this environment, where photosynthesis is limited by low concentrations of dissolved iron and large seasonal fluctuations in light, temperature and the extent of sea ice. How diatoms have adapted to this extreme environment is largely unknown. Here we present insights into the genome evolution of a cold-adapted diatom from the Southern Ocean, Fragilariopsis cylindrus, based on a comparison with temperate diatoms. We find that approximately 24.7 per cent of the diploid F. cylindrus genome consists of genetic loci with alleles that are highly divergent (15.1 megabases of the total genome size of 61.1 megabases). These divergent alleles were differentially expressed across environmental conditions, including darkness, low iron, freezing, elevated temperature and increased CO. Alleles with the largest ratio of non-synonymous to synonymous nucleotide substitutions also show the most pronounced condition-dependent expression, suggesting a correlation between diversifying selection and allelic differentiation. Divergent alleles may be involved in adaptation to environmental fluctuations in the Southern Ocean.
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http://dx.doi.org/10.1038/nature20803DOI Listing
January 2017

Targeted capture and sequencing of gene-sized DNA molecules.

Biotechniques 2016 12 1;61(6):315-322. Epub 2016 Dec 1.

Earlham Institute (EI), Norwich Research Park, Norwich, UK.

Targeted capture provides an efficient and sensitive means for sequencing specific genomic regions in a high-throughput manner. To date, this method has mostly been used to capture exons from the genome (the exome) using short insert libraries and short-read sequencing technology, enabling the identification of genetic variants or new members of large gene families. Sequencing larger molecules results in the capture of whole genes, including intronic and intergenic sequences that are typically more polymorphic and allow the resolution of the gene structure of homologous genes, which are often clustered together on the chromosome. Here, we describe an improved method for the capture and single-molecule sequencing of DNA molecules as large as 7 kb by means of size selection and optimized PCR conditions. Our approach can be used to capture, sequence, and distinguish between similar members of the NB-LRR gene family-key genes in plant immune systems.
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http://dx.doi.org/10.2144/000114484DOI Listing
December 2016

Accelerated cloning of a potato late blight-resistance gene using RenSeq and SMRT sequencing.

Nat Biotechnol 2016 06 25;34(6):656-60. Epub 2016 Apr 25.

The Sainsbury Laboratory, Norwich Research Park, Norwich, UK.

Global yields of potato and tomato crops have fallen owing to potato late blight disease, which is caused by Phytophthora infestans. Although most commercial potato varieties are susceptible to blight, many wild potato relatives show variation for resistance and are therefore a potential source of Resistance to P. infestans (Rpi) genes. Resistance breeding has exploited Rpi genes from closely related tuber-bearing potato relatives, but is laborious and slow. Here we report that the wild, diploid non-tuber-bearing Solanum americanum harbors multiple Rpi genes. We combine resistance (R) gene sequence capture (RenSeq) with single-molecule real-time (SMRT) sequencing (SMRT RenSeq) to clone Rpi-amr3i. This technology should enable de novo assembly of complete nucleotide-binding, leucine-rich repeat receptor (NLR) genes, their regulatory elements and complex multi-NLR loci from uncharacterized germplasm. SMRT RenSeq can be applied to rapidly clone multiple R genes for engineering pathogen-resistant crops.
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http://dx.doi.org/10.1038/nbt.3540DOI Listing
June 2016

NanoOK: multi-reference alignment analysis of nanopore sequencing data, quality and error profiles.

Bioinformatics 2016 Jan 17;32(1):142-4. Epub 2015 Sep 17.

The Genome Analysis Centre (TGAC), Norwich NR4 7UH, UK.

Motivation: The Oxford Nanopore MinION sequencer, currently in pre-release testing through the MinION Access Programme (MAP), promises long reads in real-time from an inexpensive, compact, USB device. Tools have been released to extract FASTA/Q from the MinION base calling output and to provide basic yield statistics. However, no single tool yet exists to provide comprehensive alignment-based quality control and error profile analysis--something that is extremely important given the speed with which the platform is evolving.

Results: NanoOK generates detailed tabular and graphical output plus an in-depth multi-page PDF report including error profile, quality and yield data. NanoOK is multi-reference, enabling detailed analysis of metagenomic or multiplexed samples. Four popular Nanopore aligners are supported and it is easily extensible to include others.

Availability And Implementation: NanoOK is an open-source software, implemented in Java with supporting R scripts. It has been tested on Linux and Mac OS X and can be downloaded from https://github.com/TGAC/NanoOK. A VirtualBox VM containing all dependencies and the DH10B read set used in this article is available from http://opendata.tgac.ac.uk/nanook/. A Docker image is also available from Docker Hub--see program documentation https://documentation.tgac.ac.uk/display/NANOOK.

Contact: [email protected]

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btv540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681994PMC
January 2016

Zebrafish Rab5 proteins and a role for Rab5ab in nodal signalling.

Dev Biol 2015 Jan 3;397(2):212-24. Epub 2014 Dec 3.

Sequencing Technology Development, The Genome Analysis Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom.

The RAB5 gene family is the best characterised of all human RAB families and is essential for in vitro homotypic fusion of early endosomes. In recent years, the disruption or activation of Rab5 family proteins has been used as a tool to understand growth factor signal transduction in whole animal systems such as Drosophila melanogaster and zebrafish. In this study we have examined the functions for four rab5 genes in zebrafish. Disruption of rab5ab expression by antisense morpholino oligonucleotide (MO) knockdown abolishes nodal signalling in early zebrafish embryos, whereas overexpression of rab5ab mRNA leads to ectopic expression of markers that are normally downstream of nodal signalling. By contrast MO disruption of other zebrafish rab5 genes shows little or no effect on expression of markers of dorsal organiser development. We conclude that rab5ab is essential for nodal signalling and organizer specification in the developing zebrafish embryo.
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http://dx.doi.org/10.1016/j.ydbio.2014.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294769PMC
January 2015

NextClip: an analysis and read preparation tool for Nextera Long Mate Pair libraries.

Bioinformatics 2014 Feb 2;30(4):566-8. Epub 2013 Dec 2.

The Genome Analysis Centre (TGAC), Norwich Research Park, Norwich NR4 7UH, UK.

Summary: Illumina's recently released Nextera Long Mate Pair (LMP) kit enables production of jumping libraries of up to 12 kb. The LMP libraries are an invaluable resource for carrying out complex assemblies and other downstream bioinformatics analyses such as the characterization of structural variants. However, LMP libraries are intrinsically noisy and to maximize their value, post-sequencing data analysis is required. Standardizing laboratory protocols and the selection of sequenced reads for downstream analysis are non-trivial tasks. NextClip is a tool for analyzing reads from LMP libraries, generating a comprehensive quality report and extracting good quality trimmed and deduplicated reads.

Availability And Implementation: Source code, user guide and example data are available from https://github.com/richardmleggett/nextclip/.
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http://dx.doi.org/10.1093/bioinformatics/btt702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928519PMC
February 2014

The zebrafish reference genome sequence and its relationship to the human genome.

Nature 2013 Apr 17;496(7446):498-503. Epub 2013 Apr 17.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
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http://dx.doi.org/10.1038/nature12111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703927PMC
April 2013

A sequence-based variation map of zebrafish.

Zebrafish 2013 Mar;10(1):15-20

CSIR- Institute of Genomics and Integrative Biology, Delhi, India.

Zebrafish (Danio rerio) is a popular vertebrate model organism largely deployed using outbred laboratory animals. The nonisogenic nature of the zebrafish as a model system offers the opportunity to understand natural variations and their effect in modulating phenotype. In an effort to better characterize the range of natural variation in this model system and to complement the zebrafish reference genome project, the whole genome sequence of a wild zebrafish at 39-fold genome coverage was determined. Comparative analysis with the zebrafish reference genome revealed approximately 5.2 million single nucleotide variations and over 1.6 million insertion-deletion variations. This dataset thus represents a new catalog of genetic variations in the zebrafish genome. Further analysis revealed selective enrichment for variations in genes involved in immune function and response to the environment, suggesting genome-level adaptations to environmental niches. We also show that human disease gene orthologs in the sequenced wild zebrafish genome show a lower ratio of nonsynonymous to synonymous single nucleotide variations.
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http://dx.doi.org/10.1089/zeb.2012.0848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3629779PMC
March 2013

Zebrafish breeding in the laboratory environment.

ILAR J 2012 ;53(2):161-8

Zebrafish International Resource Center, University of Oregon, Eugene, OR 97403, USA.

The zebrafish, Danio rerio, has become a major model organism used in biomedical studies. The widespread use of Danio rerio in research laboratories requires a comprehensive understanding of the husbandry of this species to ensure efficient propagation and maintenance of healthy and genetically diverse colonies. Breeding is a key element in zebrafish husbandry. It is a complex process influenced by a number of factors. Mate choice and mating behavior depend, for example, on olfactory cues, visual stimuli, and social interactions. Spawning is affected by the age and size of fish, interval at which fish are used for egg production, light cycle, diet, and fish health status. A number of breeding strategies, based on either single-pair matings or group crosses, are commonly employed in the laboratory to propagate lines and to identify carriers of specific mutations and/or transgenes. Propagation of zebrafish lines, in particular wild-type-derived strains, is closely monitored to ensure that genetic diversity and vigor are maintained. A robust zebrafish line typically carries a large number of polymorphic variations, which may interfere with reproducibility of experiments. To get a better insight into these variations, a wild-type hybrid Sanger AB Tübingen line has been generated from sequenced homozygous founders.
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http://dx.doi.org/10.1093/ilar.53.2.161DOI Listing
August 2013
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