Biology subjects

Larsen, P. A.

Publications and source records attributed to Larsen, P. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Ultrasensitive Detection of Alpha-Synuclein Oligomers in Human Plasma Using Optimized Nano-QuIC

Early diagnosis of Parkinsons disease (PD) is critical, as clinical symptoms typically emerge only after substantial neuronal loss. While -synuclein (-Syn) oligomers in blood are promising biomarkers for early detection, their clinical utility is limited by their low abundance and the presence of inhibitory components in the plasma matrix. To address these limitations, we tailored the Nanoparticle-enhanced Quaking-Induced Conversion (Nano-QuIC) platform specifically for the ultrasensitive detection of -Syn oligomers in human plasma. We identified critical reaction determinants by investigating buffer pH, ionic strength, detergent types, and shaking conditions. Furthermore, the integration of silica nanoparticles (siNPs) proved essential in mitigating plasma matrix interference, ensuring robust and reproducible protein aggregation. Under these optimized conditions, the assay achieved a detection limit of 100 pg/mL for -Syn oligomers spiked into human plasma. These results demonstrate that our adapted Nano-QuIC platform provides a highly sensitive and minimally invasive method for detecting pathological -Syn species, offering a significant advancement toward the development of early-stage PD diagnostics.

bioengineering↗

bioRxiv2025

Prion Seeding Activity in DNA Extractions: Implications for Laboratory Biosafety

Infectious prions (PrPSc) are largely resistant to proteolytic digestion, including proteinase K digestion. While nucleic acid extracts are generally considered non-infectious, we investigated whether standard DNA purification methods can co-purify PrPSc, posing an unrecognized biosafety risk. Two laboratories, the University of Minnesota Center for Prion Research and Outreach (MNPRO) and the Canadian Food Inspection Agency (CFIA), independently tested filter-based and magnetic bead-based DNA extraction kits using tissues from chronic wasting disease (CWD)-positive and -negative white-tailed deer (WTD; Odocoileus virginianus), as well as prion-infected and control Syrian hamster (Mesocricetus auratus) brains. CFIA used two filter-based kits (one automated and one manual), while MNPRO tested two manual kits (one filter-based and one magnetic bead-based). PrPSc seeding activity was measured in extracted DNA and source tissues using real-time quaking-induced conversion (RT-QuIC). MNPRO found substantial to almost perfect agreement (kappa ({kappa}) = 0.789 - 0.816) between RT-QuIC seeding activity of DNA eluates from both extraction methods and that of the source WTD tissue homogenate. CFIA optimized RT-QuIC to a 30-hour runtime, achieving 74% sensitivity and 94% specificity in 88 archived WTD DNA samples. Both laboratories concluded that commercial DNA extraction kits do not eliminate PrPSc, enabling its carry-over into DNA eluates. Until infectivity is resolved by animal bioassay, DNA from PrPSc-positive tissues should be managed under biosafety protocols appropriate for the originating prion disease, with appropriate decontamination and containment procedures.

molecular biology↗

bioRxiv2025

Evaluating the diagnostic capabilities of nanopore sequencing for Borrelia burgdorferi detection in blacklegged ticks

Ticks pose substantial threats to public health. Blacklegged ticks (Ixodes scapularis) are responsible for most tick-borne diseases in the US, transmitting seven human pathogens. Molecular surveillance for tick-borne pathogens has been outpaced by their emergence, revealing a critical need to develop agnostic strategies that characterize emerging and putative pathogens. Oxford Nanopore Technologys nanopore adaptive sampling (NAS), an approach that selectively enriches or depletes for target genomes or genetic loci, provides an opportunity to generate real-time genomic insights into tick-borne pathogens. In the current study, we performed PCR and NAS on pooled Borrelia burgdorferi-infected and -uninfected ticks to evaluate the diagnostic capability of NAS. We found that NAS generates extensive datasets on tick-borne pathogens from individual ticks that aid in distinguishing true and false positive samples. Using a pooled approach consisting of whole genomic DNA from 168 total ticks multiplexed over seven sequencing experiments, our results indicated that NAS is extremely specific (0.97 [95% CI: 0.93, 1.00]) with moderate sensitivity (0.48 [95% CI: 0.41, 0.55]), suggesting a strong capacity to confirm B. burgdorferi when present at the expense of an elevated false-negative rate. We found that quality-based filtering of sequence data has a profound influence on diagnostic metrics, emphasizing the need to optimize pooling strategy, wet-lab procedures, and bioinformatic pipelines to enhance the sensitivity of NAS for detecting tick-borne pathogens. Author summaryTicks pose substantial threats to public health. In the United States, the blacklegged tick (Ixodes scapularis) can transmit seven (known) human pathogens and is responsible for most tick-borne disease cases. The most prevalent tick-borne pathogen, Borrelia burgdorferi, causes Lyme disease, which is consequently the most common tick-borne disease in the United States. As blacklegged ticks continue to expand across the United States, it is imperative to develop rapid molecular surveillance tools that agnostically detect emerging and putative pathogens. In the current study, we employ Oxford Nanopore Technologys nanopore adaptive sampling on PCR-infected and PCR-uninfected blacklegged ticks to determine the diagnostic capability of this technology for rapid Borrelia burgdorferi detection. Our results suggest that nanopore adaptive sampling can generate extensive sequence datasets on user-specified target reference genomes. We demonstrate that nanopore adaptive sampling is extremely specific, capable of confirming B. burgdorferi presence when detected, yet lacks sensitivity, leading to a high false negative rate. We highlight the methodological limitations that undoubtedly led to this lower sensitivity and provide future research directions for enhancing nanopore adaptive sampling as a real-time, unbiased molecular tool for tick-borne pathogen surveillance.

molecular biology↗

bioRxiv2024

AI-QuIC: Machine Learning for Automated Detection of Misfolded Proteins in Seed Amplification Assays

Advancements in AI, particularly deep learning, have revolutionized protein folding modeling, offering insights into biological processes and accelerating drug discovery for protein misfolding diseases. However, detecting misfolded proteins associated with neurodegenerative disorders, such as Alzheimers, Parkinsons, ALS, and prion diseases, relies on Seed Amplification Assays (SAAs) analyzed through manual, time-consuming, and potentially inconsistent methods. We introduce AI-QuIC, an AI-driven platform that automates the analysis of Real-Time Quaking- Induced Conversion (RT-QuIC) assay data, a type of SAA crucial for detecting misfolded proteins. Utilizing a well-labeled RT-QuIC dataset of over 8,000 wells--the largest curated dataset for chronic wasting disease prion detection--we applied various AI models to classify true positive, false positive, and negative reactions. Notably, our deep-learning-based model achieved over 98% sensitivity and 97% specificity. By learning directly from raw fluorescence data, deep learning simplifies the SAA-analysis workflow. Automating and standardizing SAA data interpretation with AI-QuIC provides robust, scalable, and consistent diagnostic solutions.

bioinformatics↗

bioRxiv2024

QuICSeedR: An R package for analyzing fluorophore-assisted seed amplification assay data

SummaryFluorophore-assisted seed amplification assays (F-SAAs), such as real-time quaking-induced conversion (RT-QuIC) and fluorophore-assisted protein misfolding cyclic amplification (F-PMCA), have become indispensable tools for studying protein misfolding in neurodegenerative diseases. However, analyzing data generated by these techniques often requires complex and time-consuming manual processes. Additionally, the lack of standardization in F-SAA data analysis presents a significant challenge to the interpretation and reproducibility of F-SAA results across different laboratories and studies. Here, we present QuICSeedR (pronounced as "quick seeder"), an R package that addresses these challenges by providing a comprehensive toolkit for the automated processing, analysis, and visualization of F-SAA data. Importantly, QuICSeedR also sets up the foundation for building an F-SAA data management and analysis framework, enabling more consistent and comparable results across different research groups. Availability and implementationQuICSeedR source code is freely available at: https://github.com/mancili/QuICSeedR. Data and code used in this manuscript are provided in Supplementary Materials. Supplementary informationSupplementary Materials are available with the manuscript.

bioinformatics↗

bioRxiv2024

Detection and decontamination of chronic wasting disease prions during venison processing

Prion diseases, including chronic wasting disease (CWD), are caused by prions, which are misfolded aggregates of normal cellular prion protein. Prions possess many characteristics that distinguish them from conventional pathogens, in particular, an extraordinary recalcitrance to inactivation and a propensity to avidly bind to surfaces. In mid to late stages of CWD, prions begin accumulating in cervid muscle tissues. These features collectively create scenarios where occupational hazards arise for workers processing venison and pose risks to consumers through direct prion exposure via ingestion and cross-contamination of food products. In this work, we show that steel and plastic surfaces used in venison processing can be directly contaminated with CWD prions and that cross-contamination of CWD-negative venison can occur from equipment that had previously been used with CWD-positive venison. We also show that several decontaminant solutions (commercial bleach and potassium peroxymonosulfate) are efficacious for prion inactivation on these same surfaces.

microbiology↗

bioRxiv2023

Multifaceted impact of specialized neuropeptide-intensive neurons on the selective vulnerability in Alzheimer's disease

INTRODUCTIONWidespread disruption of neuropeptide (NP) networks in Alzheimers disease (AD) and disproportionate absence of neurons expressing high NP-producing, coined as HNP neurons, have been reported for the entorhinal cortex (EC) of AD brains. Hypothesizing that functional features of HNP neurons are involved in the early pathogenesis of AD, we aim to understand the molecular mechanisms underlying these observations. METHODSMultiscale and spatiotemporal transcriptomic analysis was used to investigate AD-afflicted and healthy brains. Our focus encompassed NP expression dynamics in AD, AD-associated NPs (ADNPs) trajectories with aging, and the neuroanatomical distribution of HNP neuron. RESULTSFindings include that 1) HNP neurons exhibited heightened metabolic needs and an upregulation of gene expressions linked to protein misfolding; 2) dysfunctions of ADNP production occurred in aging and mild cognitive decline; 3) HNP neurons co-expressing ADNPs were preferentially distributed in brain regions susceptible to AD. DISCUSSIONWe identified potential mechanisms that contribute to the selective vulnerability of HNP neurons to AD. Our results indicate that the functions of HNP neurons predispose them to oxidative stress and protein misfolding, potentially serving as inception sites for misfolded proteins in AD.

neuroscience↗

bioRxiv2023

Blood-based Nano-QuIC: Accelerated and Inhibitor-resistant Detection of Misfolded Alpha-synuclein

A hallmark of -synucleinopathies (e.g. Parkinsons disease) is the misfolding and aggregation of -synuclein in tissues and biological fluids. Protein amplification assays like real-time quaking-induced conversion (RT-QuIC) are sensitive yet currently limited to semi-invasive sample types such as cerebrospinal fluid because more accessible samples, such as blood, contain inhibitors. Here, we show that Nanoparticle-enhanced Quaking-induced Conversion (Nano-QuIC) can double the speed of reactions spiked with misfolded -synuclein while increasing sensitivity 100-fold in human plasma. Nano-QuIC detected spike concentrations down to 90 pg/ml in lysed whole blood, while reactions without nanoparticles (RT-QuIC) failed to have any detection due to the presence of strong inhibitors. Moreover, Nano-QuIC showed increased seeding activity in plasma samples from Parkinsons patients (n=4) versus healthy controls (n=4). This sets the groundwork for the noninvasive diagnostic use of Nano-QuIC, potentially enabling early disease detection and management through blood-based testing. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/552630v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1141573org.highwire.dtl.DTLVardef@18c63d6org.highwire.dtl.DTLVardef@1ee9524org.highwire.dtl.DTLVardef@81af13_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Microfluidic Quaking-Induced Conversion (Micro-QuIC) for Rapid On-Site Amplification and Detection of Misfolded Proteins

Protein misfolding diseases, such as prion diseases, Alzheimers, and Parkinsons, share a common molecular mechanism involving the misfolding and aggregation of specific proteins. There is an urgent need for point-of-care (POC) diagnostic technologies that can accurately detect these misfolded proteins, facilitating early diagnosis and intervention. Here, we introduce the Microfluidic Quaking Induced Conversion (Micro-QuIC), a novel acoustofluidic platform for the rapid and sensitive detection of protein misfolding diseases. We demonstrate the utility of our technology using chronic wasting disease (CWD) as a model system, as samples from wild white-tailed deer are readily accessible, and CWD shares similarities with human protein misfolding diseases. Acoustofluidic mixing enables homogeneous mixing of reagents in a high-Reynolds-number regime, significantly accelerating the turnaround time for CWD diagnosis. Our Micro-QuIC assay amplifies prions by an order of magnitude faster than the current gold standard, real-time quaking-induced conversion (RT-QuIC). Furthermore, we integrated Micro-QuIC with a gold nanoparticle-based, naked-eye detection method, which enables visual discrimination between CWD positive and negative samples without the need for a bulky fluorescence detection module. This integration creates a rapid, POC testing platform capable of detecting misfolded proteins associated with a variety of protein misfolding diseases. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/549283v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@885eeaorg.highwire.dtl.DTLVardef@10f820eorg.highwire.dtl.DTLVardef@12396eaorg.highwire.dtl.DTLVardef@188fe0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Rapid molecular species identification of mammalian scat samples using nanopore adaptive sampling

AO_SCPLOWBSTRACTC_SCPLOWAccurate species identification is essential to mammalogy. Despite this necessity, rapid and accurate identification of cryptic, understudied, and elusive mammals remains challenging. Traditional barcoding of mitochondrial genes is standard for molecular identification but requires time-consuming wet-lab methodologies. Recent bioinformatic advancements for nanopore sequencing data offer exciting opportunities for non-invasive and field-based identification of mammals. Nanopore adaptive sampling (NAS), a PCR-free method, selectively sequences regions of DNA according to user-specified reference databases. Here, we utilized NAS to enrich mammalian mitochondrial genome sequencing to identify species. Fecal DNA extractions were sequenced from nine mammals, several collected in collaboration with Minnesota Tribal Nations, to demonstrate utility for NAS-barcoding of non-invasive samples. By mapping to the entire National Center for Biotechnology Information (NCBI) mammalian mitochondrial reference genome database and bioinformatically analyzing highly similar matches, we successfully produced species identifications for all of our fecal samples. Eight of nine species identifications matched previous PCR or animal/fecal morphological identifications. For the ninth species, our genetic data indicate a misidentification stemming from the original study. Our approach has a range of applications, particularly field-based wildlife research, conservation, disease surveillance, and monitoring of wildlife trade. Of importance to Minnesota tribes is invasive species monitoring, detections, and confirmation as climate impacts causes changes in biodiversity and shifts in species distributions. The rapid assessment techniques described here will be useful as new introductions and range expansions of native and invasive species may first be detected by the presence of signs such as scat rather than direct observations and will be helpful for chronically understaffed tribal natural resources agencies.

genomics↗

bioRxiv2022

Standardization of Data Analysis for RT-QuIC-based Detection of Chronic Wasting Disease

Chronic wasting disease (CWD) is a disease affecting cervids and is caused by prions accumulating as pathogenic fibrils in lymphoid tissue and the central nervous system. Approaches for detecting CWD prions historically relied on antibody-based assays. However, recent advancements in protein amplification technology provided the foundation for a new class of CWD diagnostic tools. In particular, real-time quaking-induced conversion (RT-QuIC) has rapidly become a feasible option for CWD diagnosis. Despite its increased usage for CWD-focused research, there lacks consensus regarding the interpretation of RT-QuIC data for diagnostic purposes. It is imperative then to identify a standardized and replicable method for determining CWD status from RT-QuIC data. Here, we assessed variables that could impact RT-QuIC results and explored the use of maxpoint ratios (maximumRFU/backgroundRFU) to improve the consistency of RT-QuIC analysis. We examined a variety of statistical analyses to retrospectively analyze CWD status based on RT-QuIC and ELISA results from 668 white-tailed deer lymph nodes. Our results revealed an MPR threshold of 2.0 for determining the rate of amyloid formation, and MPR analysis showed excellent agreement with independent ELISA results. These findings suggest that the use of MPR is a statistically viable option for normalizing between RT-QuIC experiments and defining CWD status.

molecular biology↗

bioRxiv2022

Single-cell sequencing of Entorhinal Cortex Reveals Wide-Spread Disruption of Neuropeptide Networks in Alzheimer's Disease

Alzheimers disease (AD) is a fatal neurodegenerative disease that involves early and significant neuropathological changes within the entorhinal cortex (EC). Many have reported on neuronal loss and synaptic dysfunction in the brains of AD patients and AD models. In parallel, abnormalities of neuropeptides (NPs) that play important roles in modulating neuronal activities are commonly observed in AD and other neurodegenerative diseases. However, the involvement of NPs has mostly been studied in the context of neurons; a cell type-specific examination of NP expression in AD brains is needed. Here, we aim to examine the NP networks in the EC of AD brains using single-nuclei and bulk transcriptomic data from other regions in the temporal cortex, focusing on the gene expression of NP and their cognate G-protein coupled receptors. We find that NP genes were expressed by all major cell types in the brain and there was a significant decrease in the quantity and the proportion of cells that express NPs in AD EC cells. On the contrary, the overall expression of GPCR genes showed an increase in AD cells, likely reflecting ongoing compensatory mechanisms in AD brains. In addition, we report that there was a disproportionate absence of cells expressing higher levels and greater diversity of NPs in AD brains. Finally, we established a negative correlation between age and the abundance of AD-associated NPs in the hippocampus, supporting that the disruption of the NP signaling network in the EC may contribute to the early pathogenesis of AD. In short, we report widespread disruption of the NP networks in AD brains at the single-cell level. In light of our results, we hypothesize that brain cells, especially neurons, that express high levels of NPs may exhibit selective vulnerability to AD. Moreover, it is likely AD brains undergo specific adaptive changes to fluctuating NP signaling, a process that can likely be targeted with therapeutic approaches aimed at stabilizing NP expression landscapes. Given that GPCRs are one of the most druggable targets for neurological diseases and disorders, we believe NP signaling pathways can be harnessed for future biomarkers and treatment strategies for AD.

neuroscience↗

bioRxiv2021

A Field-Deployable Diagnostic Assay for the Visual Detection of Misfolded Prions

Chronic Wasting Disease (CWD), a prion disease of cervids, has been identified across North America, Northern Europe and Korea. Diagnostic tools for the rapid and reliable detection of prion diseases are limited. Here, we combine gold nanoparticles (AuNPs) and quaking induced conversion (QuIC) technologies for the visual detection of amplified misfolded prion proteins from tissues of wild white-tailed deer infected with Chronic Wasting Disease (CWD). Our newly developed diagnostic test, MN-QuIC, enables both naked-eye and light-absorbance measurements for the detection of misfolded prions. The MN-QuIC assay leverages basic laboratory equipment that is cost-effective and portable, thus facilitating real-time prion diagnostics across a variety of settings. To test the portability of our assay, we deployed to a rural field station in southeastern Minnesota and tested for CWD on site. We successfully demonstrated that MN-QuIC is functional in a non-traditional laboratory setting by performing a blinded analysis in the field and correctly identifying all CWD positive and CWD not detected (independently confirmed with ELISA and/or IHC tests) animals at the field site, thus documenting the portability of the assay. Additionally, we show that electrostatic forces and concentration effects help govern the AuNP/prion interactions and contribute to the differentiation of CWD-prion positive and negative samples. We examined 17 CWD-positive and 24 CWD-not-detected white-tailed deer tissues that were independently tested using ELISA, IHC, and RT-QuIC technologies, and results secured with MN-QuIC were 100% consistent with these tests. We conclude that hybrid AuNP and QuIC assays, such as MN-QuIC, have great potential for sensitive, field-deployable diagnostics for a variety of protein misfolding diseases.

molecular biology↗

bioRxiv2021

Nanopore adaptive sampling for mitogenome sequencing and bloodmeal identification in hematophagous insects

Blood-feeding insects are important vectors for an array of zoonotic pathogens. Despite significant research focused on well-documented insect vectors of One Health importance, resources for molecular species identification of a large number of hematophagous arthropods are limited. Advancements in next-generation sequencing technologies provide opportunities for targeting mitochondrial genomes of blood-feeding insects, as well as their bloodmeal hosts. This dual approach holds great promise for elucidating complex disease transmission pathways and enhancing the molecular resources for the identification of cryptic insect species. To this end, we leveraged the newly developed Oxford Nanopore Adaptive Sampling (NAS) pipeline to dually sequence the mitogenomes of hematophagous insects and their bloodmeals. Using NAS, we sequenced the entire mitogenomes of Aedes vexans, Culex restuans, Culex territans, and Chrysops niger and successfully identified bloodmeal hosts of Chrysops niger, Culex restuans, and Aedes trivittatus. We show that NAS has the utility to simultaneously molecularly identify blood-feeding insects and characterize disease transmission pathways through bloodmeal host identification. Moreover, our data indicate NAS can facilitate a wide array of molecular systematic studies through novel phylogenetic capture methods. We conclude the NAS approach has great potential for informing global One Health initiatives centered on the mitigation of vector-borne disease through dual vector and bloodmeal identification.

genomics↗

bioRxiv2021

Self-organized emergence of hyaline cartilage in hiPSC-derived multi-tissue organoids

Despite holding great therapeutic potential, existing protocols for in vitro chondrogenesis and hyaline cartilage production from human induced pluripotent stem cells (hiPSC) are laborious and complex with unclear long-term consequences. Here, we developed a simple xeno- and feeder-free protocol for human hyaline cartilage production in vitro using hydrogel-cultured multi-tissue organoids (MTOs). We investigate gene regulatory networks during spontaneous hiPSC-MTO differentiation using RNA sequencing and bioinformatic analyses. We find the interplays between BMPs and neural FGF pathways are associated with the phenotype transition of MTOs. We recognize TGF-beta/BMP and Wnt signaling likely contribute to the long-term maintenance of MTO cartilage growth and further adoption of articular cartilage development. By comparing the MTO transcriptome with human lower limb chondrocytes, we observe that the expression of chondrocyte-specific genes in MTO shows a strong correlation with fetal lower limb chondrocytes. Collectively, our findings describe the self-organized emergence of hyaline cartilage in MTO, its associated molecular pathways, and its spontaneous adoption of articular cartilage development trajectory.

cell biology↗

bioRxiv2021

Enabling metagenomic surveillance for bacterial tick-borne pathogens using nanopore sequencing with adaptive sampling

Technological and computational advancements in the fields of genomics and bioinformatics are providing exciting new opportunities for pathogen discovery and surveillance. In particular, single-molecule nucleotide sequence data originating from Oxford Nanopore Technologies (ONT) sequencing platforms can be bioinformatically leveraged, in real-time, for enhanced biosurveillance of a vast array of zoonoses. The recently released nanopore adaptive sampling (NAS) pipeline facilitates immediate mapping of individual nucleotide molecules (i.e., DNA, cDNA, and RNA) to a given reference as each molecule is sequenced. User-defined thresholds then allow for the retention or rejection of specific molecules, informed by the real-time reference mapping results, as they are physically passing through a given sequencing nanopore. Here, we show how NAS can be used to selectively sequence entire genomes of bacterial tick-borne pathogens circulating in wild populations of the blacklegged tick vector, Ixodes scapularis. The NAS method provided a two-fold increase in targeted pathogen sequences, successfully enriching for Borrelia (Borreliella) burgdorferi s.s.; Borrelia (Borrelia) miyamotoi; Anaplasma phagocytophilum; and Ehrlichia muris eauclairensis genomic DNA within our I. scapularis samples. Our results indicate that NAS has strong potential for real-time sequence-based pathogen surveillance.

microbiology↗

bioRxiv2021

Molecular surveillance of zoonotic bacterial pathogens in farm dwelling peridomestic rodents across the upper Midwest, USA

The effective control of rodent populations on farms is a critical component of food- safety, as rodents are reservoirs and vectors for many foodborne pathogens in addition to several zoonotic pathogens. The functional role of rodents in the amplification and transmission of pathogens is likely underappreciated. Clear links have been identified between rodents and outbreaks of pathogens throughout Europe and Asia, however, comparatively little research has been devoted to studying this rodent-agricultural interface in the USA, particularly across the Midwest. Here, we address this existing knowledge gap by characterizing the metagenomic communities of rodent pests collected from Minnesota and Wisconsin food animal farms. We leveraged the Oxford Nanopore MinION sequencer to provide a rapid real-time survey of the putative zoonotic food- borne and other human pathogens. Rodents (mice and rats) were live trapped from three dairy and mixed animal farms. Tissues and fecal samples were collected from all rodents. DNA extraction was performed on 90 rodent colons along with 2 shrew colons included as outgroups in the study. Full-length 16S amplicon sequencing was performed with the MinION. Our data suggests the presence of putative foodborne pathogens including Salmonella spp., Campylobacter spp., Staphylococcus aureus, and Clostridium spp., along with many important mastitis pathogens. A critically important observation is that we discovered these pathogens within all five species of rodents (Microtus pennsylvanicus, Mus musculus, Peromyscus leucopus, Peromyscus maniculatus, and Rattus norvegicus) and shrew (Blarina brevicauda) in varying abundances. Interestingly, we observed a higher abundance of enteric pathogens (e.g. Salmonella) in shrew feces compared to the rodents analyzed in our study, however more data is required to establish that connection. Knowledge gained from our research efforts will directly inform and improve upon farm-level biosecurity efforts and public health interventions to reduce future outbreaks of foodborne and zoonotic disease.

microbiology↗

bioRxiv2021

The Mitochondrial Genome and Epigenome of the Golden Lion Tamarin from Fecal DNA using Nanopore Adaptive Sequencing

The golden lion tamarin (Leontopithecus rosalia) is an endangered Platyrrhine primate endemic to the Atlantic coastal forests of Brazil. Despite ongoing conservation efforts, genetic data on this species remains scarce. Complicating factors include limitations on sample collection and a lack of high-quality reference sequences. Here, we used nanopore adaptive sampling to resequence the L. rosalia mitogenome from feces, a sample which can be collected non-invasively. Adaptive sampling doubled the fraction of both host-derived and mitochondrial sequences compared to sequencing without enrichment. 258x coverage of the L. rosalia mitogenome was achieved in a single flow cell by targeting the unfinished genome of the distantly related emperor tamarin (Saguinus imperator) and the mitogenome of the closely related black lion tamarin (Leontopithecus chrysopygus). The L. rosalia mitogenome has a length of 16,597 bp, sharing 99.68% sequence identity with the L. chrysopygus mitogenome. A total of 38 SNPs between them were identified, with the majority being found in the non-coding D-loop region. DNA methylation and hydroxymethylation were directly detected using a neural network model applied to the raw signal from the MinION sequencer. In contrast to prior reports, DNA methylation was negligible in mitochondria in both CpG and non-CpG contexts. Surprisingly, a quarter of the 642 CpG sites exhibited DNA hydroxymethylation greater than 1% and 44 sites were above 5%, with concentration in the 3 side of several coding regions. Overall, we report a robust new mitogenome assembly for L. rosalia and direct detection of cytosine base modifications in all contexts.

genomics↗