A Snakemake Toolkit for the Batch Assembly, Annotation and Phylogenetic Analysis of Mitochondrial Genomes and Ribosomal Genes From Genome Skims of Museum Collections.

Low coverage 'genome-skims' are often used to assemble organelle genomes and ribosomal gene sequences for cost-effective phylogenetic and barcoding studies. Natural history collections hold invaluable biological information, yet poor preservation resulting in degraded DNA often hinders polymerase chain reaction-based analyses. However, it is possible to generate libraries and sequence the short fragments typical of degraded DNA to generate genome-skims from museum collections. Here we introduce a snakemake toolkit comprised of three pipelines skim2mito, skim2rrna and gene2phylo, designed to unlock the genomic potential of historical museum specimens using genome skimming. Specifically, skim2mito and skim2rrna perform the batch assembly, annotation and phylogenetic analysis of mitochondrial genomes and nuclear ribosomal genes, respectively, from low-coverage genome skims. The third pipeline gene2phylo takes a set of gene alignments and performs phylogenetic analysis of individual genes, partitioned analysis of concatenated alignments and a phylogenetic analysis based on gene trees. We benchmark our pipelines with simulated data, followed by testing with a novel genome skimming dataset from both recent and historical solariellid gastropod samples. We show that the toolkit can recover mitochondrial and ribosomal genes from poorly preserved museum specimens of the gastropod family Solariellidae, and the phylogenetic analysis is consistent with our current understanding of taxonomic relationships. The generation of bioinformatic pipelines that facilitate processing large quantities of sequence data from the vast repository of specimens held in natural history museum collections will greatly aid species discovery and exploration of biodiversity over time, ultimately aiding conservation efforts in the face of a changing planet.

Exome capture of Antarctic krill (Euphausia superba) for cost effective genotyping and population genetics with historical collections.

Antarctic krill (Euphausia superba Dana) is a keystone species in the Southern Ocean ecosystem, with ecological and commercial significance. However, its vulnerability to climate change requires an urgent investigation of its adaptive potential to future environmental conditions. Historical museum collections of krill from the early 20th century represent an ideal opportunity to investigate how krill have changed over time due to predation, fishing and climate change. However, there is currently no cost-effective method for implementing population scale collection genomics for krill given its genome size (48 Gbp). Here, we assessed the utility of two inexpensive methods for population genetics using historical krill samples, specifically low-coverage shotgun sequencing (i.e. 'genome-skimming') and exome capture. Two full-length transcriptomes were generated and used to identify 166 putative gene targets for exome capture bait design. A total of 20 historical krill samples were sequenced using shotgun and exome capture. Mitochondrial and nuclear ribosomal sequences were assembled from both low-coverage shotgun and off-target of exome capture data demonstrating that endogenous DNA sequences could be assembled from historical collections. Although, mitochondrial and ribosomal sequences are variable across individuals from different populations, phylogenetic analysis does not identify any population structure. We find exome capture provides approximately 4500-fold enrichment of sequencing targeted genes, suggesting this approach can generate the sequencing depth required to call identify a significant number of variants. Unlocking historical collections for genomic analyses using exome capture, will provide valuable insights into past and present biodiversity, resilience and adaptability of krill populations to climate change.

Measuring air metagenomic diversity in an agricultural ecosystem.

All species shed DNA during life or in death, providing an opportunity to monitor biodiversity via environmental DNA (eDNA). In recent years, combining eDNA, high-throughput sequencing technologies, bioinformatics, and increasingly complete sequence databases has promised a non-invasive and non-destructive environmental monitoring tool. Modern agricultural systems are often large monocultures and so are highly vulnerable to disease outbreaks. Pest and pathogen monitoring in agricultural ecosystems is key for efficient and early disease prevention, lower pesticide use, and better food security. Although the air is rich in biodiversity, it has the lowest DNA concentration of all environmental media and yet is the route for windborne spread of many damaging crop pathogens. Our work suggests that ecosystems can be monitored efficiently using airborne nucleic acid information. Here, we show that the airborne DNA of microbes can be recovered, shotgun sequenced, and taxonomically classified, including down to the species level. We show that by monitoring a field growing key crops we can identify the presence of agriculturally significant pathogens and quantify their changing abundance over a period of 1.5 months, often correlating with weather variables. We add to the evidence that aerial eDNA can be used as a source for biomonitoring in terrestrial ecosystems, specifically highlighting agriculturally relevant species and how pathogen levels correlate with weather conditions. Our ability to detect dynamically changing levels of species and strains highlights the value of airborne eDNA in agriculture, monitoring biodiversity changes, and tracking taxa of interest.

Genomics-informed captive breeding can reduce inbreeding depression and the genetic load in zoo populations.

Zoo populations of threatened species are a valuable resource for the restoration of wild populations. However, their small effective population size poses a risk to long-term viability, especially in species with high genetic load. Recent bioinformatic developments can identify harmful genetic variants in genome data. Here, we advance this approach, analysing the genetic load in the threatened pink pigeon (Nesoenas mayeri). We lifted the mutation-impact scores that had been calculated for the chicken (Gallus gallus) to estimate the genetic load in six pink pigeons. Additionally, we perform in silico crossings to predict the genetic load and realized load of potential offspring. We thus identify the optimal mate pairs that are theoretically expected to produce offspring with the least inbreeding depression. We use computer simulations to show how genomics-informed conservation can reduce the genetic load whilst reducing the loss of genome-wide diversity. Genomics-informed management is likely to become instrumental in maintaining the long-term viability of zoo populations.

Deficiencies with CAGE-AID questionnaire in identifying nonmedical opioid use-a report of two cases.

BACKGROUND: The CAGE-AID questionnaire (Cut-down, Annoyed, Guilty, Eye-opener scale Adapted to Include Drugs) is used to screen patients for substance use disorder and nonmedical opioid use (NMOU). Major pain guidelines encourage using such screening tools for all patients including cancer patients before initiating opioids. We present two cases where the CAGE-AID results did not accurately identify the risk for NMOU. CASE DESCRIPTION: Patient 1 is a male in his 60s with metastatic prostate cancer was admitted for uncontrolled pain. Imaging revealed extensive spinal metastasis, needing initiation of methadone and hydromorphone. The CAGE-AID score was positive, placing him at risk for NMOU. This likely biased the providers, delaying opioid titration. Subsequently, doses were adjusted, and he was discharged with adequate pain control and no evidence of NMOU. Patient 2 is a male in his 40s with metastatic cholangiocarcinoma admitted for uncontrolled abdominal pain. The patient had multiple hospitalizations at different facilities with similar symptoms. The CAGE-AID score was negative. Despite this, the patient demonstrated behaviors such as demanding intravenous opioids, dose escalation, or interventions such as nerve blocks. The workup did not identify any etiology for the increased pain. The patient left the hospital against medical advice when his demands for intravenous opioids were not met. CONCLUSIONS: The CAGE-AID questionnaire alone does not accurately identify risks for NMOU. Screening tools must always be accompanied by a thorough clinical assessment of behaviors and pain mechanism. More research is needed to better characterize CAGE-AID false positives and negatives among patients with cancer pain.

Reliability of urine drug testing among patients on buprenorphine transdermal patch.

BACKGROUND: Urine drug testing (UDT) plays a significant role in monitoring patients on chronic opioid therapy (COT) for non-medical opioid use (NMOU). UDT, at times, can be inconsistent and misleading. We present a case where a patient on a buprenorphine patch had false negative results. CASE DESCRIPTION: A female in her 70s with metastatic breast cancer presented with uncontrolled pain from a T6 compression fracture. She had no relief with tramadol 50 mg every 6 hours as needed. Due to an allergic reaction to hydromorphone, our team prescribed a buprenorphine patch of 5 µg/h. Subsequently, she expressed excellent pain control, and the clinician confirmed the patch placement on examination. She underwent a UDT during the visit. The UDT was negative for both buprenorphine and its metabolites. The literature review showed that false negative UDT results are relatively common among patients with low-dose buprenorphine patches. The combination of a thorough physical examination, a review of the Prescription Drug Monitoring Program, and reassuring scores on screening tools placed her at low risk for NMOU. DISCUSSION: Buprenorphine has a ceiling effect on respiratory depression and a lower risk for addiction. However, when used in low doses, the drug might not have enough metabolites in the urine, leading to a false negative UDT. Such results might affect patient-physician relationships. CONCLUSION: In addition to the UDT, a thorough history, screening for NMOU, physical exam, a review of PDMP, and a good understanding of opioid metabolism are necessary to help guide pain management.

Fluoroquinolone-Induced Seizure in a Patient Receiving End-of-Life Care: A Case Report.

When caring for patients nearing the end of live (EOL), healthcare providers must carefully assess the potential benefits and drawbacks of common medical interventions, such as starting antibiotic treatment. Antibiotic use during this stage can be a challenging and multifaceted situation, encompassing important clinical, social, and ethical considerations. While physicians may be motivated to prescribe antibiotics to terminally ill patients in hopes of prolonging survival and alleviating symptoms, it's crucial to recognize that these drugs can have significant implications for individuals at the EOL. Factors like advanced age, frailty, and multiple medication use make these patients more vulnerable to adverse events caused by antibiotics. For instance, fluoroquinolones, a specific type of antibiotics, have been linked to central nervous system toxicity and neurological side effects, including seizures. Geriatric patients, who often have underlying risk factors, are particularly susceptible to fluoroquinolone-induced seizures. However, there have also been reports of otherwise healthy individuals experiencing seizures as a result of fluoroquinolone use. This report sheds light on the complexities associated with initiating antibiotic therapy in patients nearing the EOL.

Grapevine trunk diseases of cold-hardy varieties grown in Northern Midwest vineyards coincide with canker fungi and winter injury.

Grapevine trunk diseases make up a disease complex associated with several vascular fungal pathogenic species. Surveys to characterize the composition of grapevine trunk diseases have been conducted for most major grape growing regions of the world. This study presents a similar survey characterizing the fungi associated with grapevine trunk diseases of cold-hardy interspecific hybrid grape varieties grown nearly exclusively in the atypical harsh winter climate of Northern Midwestern United states vineyards. From the 172 samples collected in 2019, 640 isolates obtained by culturing were identified by ITS sequencing and represent 420 sample-unique taxa. From the 420 representative taxa, opportunistic fungi of the order Diaporthales including species of Cytospora and Diaporthe were most frequently identified. Species of Phaeoacremonium, Paraconiothyrium, and Cadophora were also prevalent. In other milder Mediterranean growing climates, species of Xylariales and Botryosphaeriales are often frequently isolated but in this study they were isolated in small numbers. No Phaeomoniellales taxa were isolated. We discuss the possible compounding effects of winter injury, the pathogens isolated, and management strategies. Additionally, difficulties in researching and understanding the grapevine trunk disease complex are discussed.

Candidate resistance genes to foliar phylloxera identified at Rdv3 of hybrid grape.

The foliage of the native grape species Vitis riparia and certain cold-hardy hybrid grapes are particularly susceptible to the insect pest phylloxera, Daktulosphaira vitifoliae Fitch. A previous study using a cold-hardy hybrid grape biparental F1 population (N~125) detected the first quantitative trait locus (QTL) for foliar resistance on chromosome 14, designated as resistance to Daktulosphaira vitifoliae 3 (Rdv3). This locus spans a ~7-Mbp (10-20 cM) region and is too wide for effective marker-assisted selection or identification of candidate genes. Therefore, we fine mapped the QTL using a larger F1 population, GE1783 (N~1023), and genome-wide rhAmpSeq haplotype markers. Through three selective phenotyping experiments replicated in the greenhouse, we screened 184 potential recombinants of GE1783 using a 0 to 7 severity rating scale among other phylloxera severity traits. A 500-kb fine mapped region at 4.8 Mbp on chromosome 14 was identified. The tightly linked rhAmpSeq marker 14_4805213 and flanking markers can be used for future marker-assisted breeding. This region contains 36 candidate genes with predicted functions in disease resistance (R genes and Bonzai genes) and gall formation (bifunctional 3-dehydroquinate dehydratase/shikimate dehydrogenase). Disease resistance genes suggest a traditional R-gene-mediated resistance mechanism often accompanied by a hypersensitive response, which has been widely studied in the plant pathology field. A novel resistance mechanism, non-responsiveness to phylloxera gall formation is proposed as a function of the bifunctional dehydratase gene, which plays a role in gallic acid biosynthesis and is important in gall formation. This study has implications for improvement of foliar phylloxera resistance in cold-hardy hybrid germplasm and is a starting place to understand the mechanism of resistance in crops to gall-forming insects.

Nanopore adaptive sampling: a tool for enrichment of low abundance species in metagenomic samples.

Adaptive sampling is a method of software-controlled enrichment unique to nanopore sequencing platforms. To test its potential for enrichment of rarer species within metagenomic samples, we create a synthetic mock community and construct sequencing libraries with a range of mean read lengths. Enrichment is up to 13.87-fold for the least abundant species in the longest read length library; factoring in reduced yields from rejecting molecules the calculated efficiency raises this to 4.93-fold. Finally, we introduce a mathematical model of enrichment based on molecule length and relative abundance, whose predictions correlate strongly with mock and complex real-world microbial communities.

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

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.

Impact of Adult Popillia japonica (Coleoptera: Scarabaeidae) Foliar Feeding Injury on Fruit Yield and Quality of a Temperate, Cold-Hardy Wine Grape, 'Frontenac'.

Popillia japonica (Newman), is a highly polyphagous, invasive species, first recorded in the U.S. in 1916, and detected in Minnesota in the late 1960s. Historically, research on this pest in the Midwest U.S. has focused primarily on ornamental and turf crops, with little attention placed on adult feeding damage to fruit crops. Recently, wine grape producers in the region noted substantial increases in defoliation from P. japonica feeding, confirming concerns for this perennial high value crop. To address these concerns, studies were conducted during the summers of 2020-2021 to understand the impact of P. japonica foliar feeding on the quality and yield of wine grapes. Trials utilized vines of the wine grape variety, 'Frontenac.' In addition to open plots, whole vines were caged within fine mesh netting and infested with P. japonica at 0, 25, 50, and 100 beetles per meter-row of vine. Beetles used for infestations were collected from natural field populations of P. japonica and left to feed until grapes were ready for harvest. During harvest, data collection included leaf samples for obtaining average percent defoliation, cluster weights, and berry subsamples for soluble solid content, pH, titratable acidity, and phenolic compound measurements. Results from these studies demonstrated that as beetle population density and defoliation per m-row increases, at-harvest measurements of quality parameters are significantly and negatively affected (P < 0.05) when compared with uninfested vines. The negative impacts to fruit quality exhibited in these studies will be important in the development of future management strategies for P. japonica in 'Frontenac.'

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

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 pain management, pharmacogenomics, pharmacogenetics, pharmacokinetics, pharmacodynamics, and opioids 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.

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

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 enfortumab, vedotin, Padcev, and Nectin. 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.

A low-cost pipeline for soil microbiome profiling.

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.

New approaches for metagenome assembly with short reads.

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.

Plant gene editing through de novo induction of meristems.

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.

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

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.