Biology subjects

Mitchell, A.

Publications and source records attributed to Mitchell, A..

35 records · Page 2Linked to original sources

bioRxiv2022

Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-Seq

Phage Immunoprecipitation-Sequencing (PhIP-Seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-Seq for autoantigen discovery, including our previous work (Vazquez et al. 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki Disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), and finally, mild and severe forms of COVID19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as PDYN in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in 2 patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-Seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID19, including the endosomal protein EEA1. Together, scaled PhIP-Seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.

immunology↗

bioRxiv2022

A primary patient-derived model for investigating functional heterogeneity within the human Leukemic Stem Cell Compartment

The ability of leukemic stem cells (LSC) to evade therapy and fuel leukemic progression causing relapse impedes therapeutic success in acute myeloid leukemia (AML). The LSC pool within a patient sample is not homogenous but comprises distinct LSC subsets that vary in self-renewal and propagation properties. The stemness programs that underlie LSC types are poorly understood since human LSC studies require primary patient samples where LSC numbers are low and isolation methods impure. To overcome these challenges, we developed a patient-derived AML model system (OCI-AML22) displaying a functionally, transcriptionally and epigenetically defined cellular hierarchy driven by functional LSCs that can be immunophenotypically identified and isolated. Through single cell and functional approaches, the OCI-AML22 LSC fraction was found to contain distinct LSCs that vary in proliferative and differentiation properties. OCI-AML22 represents a valuable resource to decipher mechanisms driving stemness and the multiple layers of heterogeneity within LSCs.

cancer biology↗

bioRxiv2022

A cellular hierarchy framework for understanding heterogeneity and predicting drug response in AML

The treatment landscape of AML is evolving with promising therapies entering clinical translation, yet patient responses remain heterogeneous and biomarkers for tailoring treatment are lacking. To understand how disease heterogeneity links with therapy response, we determined the leukemia cell hierarchy make-up from bulk transcriptomes of over 1000 patients through deconvolution using single-cell reference profiles of leukemia stem, progenitor, and mature cell types. Leukemia hierarchy composition was associated with functional, genomic, and clinical properties and converged into four overall classes, spanning Primitive, Mature, GMP, and Intermediate. Critically, variation in hierarchy composition along the Primitive vs GMP or Primitive vs Mature axes were associated with response to chemotherapy or drug sensitivity profiles of targeted therapies, respectively. A 7-gene biomarker derived from the Primitive vs Mature axis was predictive of patient response to 105 investigational drugs. Thus, hierarchy composition constitutes a novel framework for understanding disease biology and advancing precision medicine in AML.

cancer biology↗

bioRxiv2021

Vertex-wise characterization of Non-Human Primate cortical development with prenatal insights.

Characterization of the interwoven complexities of early cortical thickness development has been an ongoing undertaking in neuroscience research. Longitudinal studies of Non-Human Primates (NHP) offer unique advantages to categorizing the diverse patterns of cortical growth trajectories. Here, we used latent growth models to characterize the trajectories of typical cortical thickness development in Japanese macaques at each cortical surface vertex (i.e. grayordinate). Cortical thickness from 4 to 36 months showed regional specific linear and non-linear trajectories and distinct maturation timing across the cortex. Intriguingly, we revealed a "accumulation/ablation phenomenon" of cortical maturation where the most profound development changes in cortical thickness occur in the accumulation or ablation zones surrounding the focal points (i.e., a center of a delineated regions where cortical thickness is thickest or thinnest) throughout the brain. We further examined maternal diet and inflammation in the context of these typical brain trajectories and known network architecture. A well-controlled NHP model of a maternal "Western-style" diet was used alongside measures of inflammatory cytokine interleukin-6 (IL-6) in the mothers during gestation. We observed that these accumulation and ablation zones of variable change might be most susceptible to environmental effects. The maternal factors, diet and inflammation during pregnancy were distinctively associated with different aspects of offspring cortical development reflected in regions related to distinctive functional networks. Our findings characterize the versatile intricacies of typical cortical thickness development and highlight how the maternal environment plays a role in offspring cortical development.

neuroscience↗

bioRxiv2021

cAMP controls a trafficking mechanism that directs the neuron specificity and subcellular placement of electrical synapses

Electrical synapses are established between specific neurons and within distinct subcellular compartments, but the mechanisms that direct gap junction assembly in the nervous system are largely unknown. Here we show that a transcriptional program tunes cAMP signaling to direct the neuron-specific assembly and placement of electrical synapses in the C. elegans motor circuit. For these studies, we use live cell imaging to visualize electrical synapses in vivo and a novel optogenetic assay to confirm that they are functional. In VA motor neurons, the UNC-4 transcription factor blocks expression of cAMP antagonists that promote gap junction miswiring. In unc-4 mutants, VA electrical synapses are established with an alternative synaptic partner and are repositioned from the VA axon to soma. We show that cAMP counters these effects by driving gap junction trafficking into the VA axon for electrical synapse assembly. Thus, our experiments in an intact nervous system establish that cAMP regulates gap junction trafficking for the biogenesis of electrical synapses.

neuroscience↗

bioRxiv2021

Accessibility Over Transposable Elements Reveals Genetic Determinants of Stemness Properties in Normal and Leukemic Hematopoiesis

Despite most acute myeloid leukemia (AML) patients achieving complete remission after induction chemotherapy, two-thirds will relapse with fatal disease within five years. AML is organized as a cellular hierarchy sustained by leukemia stem cells (LSC) at the apex, with LSC properties directly linked to tumor progression, therapy failure, and disease relapse 1-5. Despite the central role of LSC in poor patient outcomes, little is known about the genetic determinants driving their stemness properties. As LSCs share many functional and molecular properties with normal hematopoietic stem cells (HSC) 6, we investigated accessible chromatin unique across normal hematopoietic and cancer cell states and identified transposable elements (TEs) as genetic determinants of both primitive populations in comparison with their downstream mature progeny. A clinically-relevant TE chromatin accessibility-based LSCTE121 signature was developed that enabled patient classification based on survival outcomes. Through functional assays, primitive cell specific-TE subfamilies were found to serve as docking sites for stem cell-associated regulators of genome topology or lineage-specific transcription factors, including LYL1 in LSCs. Finally, using chromatin editing tools, we establish that chromatin accessibility at LTR12C elements in LSCs are necessary to maintain stemness properties. Our work identifies TEs as genetic drivers of primitive versus mature cell states, where distinct TE subfamilies account for stemness properties in normal versus leukemic hematopoietic stem cells.

cancer biology↗

bioRxiv2020

Psychological Well-being, Age and Gender can Mediate Soundscapes Pleasantness and Eventfulness: A large sample study

Soundscape studies aim to consider the holistic human perception of a sound environment, including both the physical phenomena and how these are mediated by internal factors: the mechanisms underpinning the interactions between these two aspects are not well understood. This study aims to assess the influence of psychological well-being and demographic factors including age, gender, occupation status, and education levels on the dimensions of the soundscape circumplex, i.e., Pleasantness and Eventfulness. Data was collected in eleven urban locations in London through a large-scale (N=1134) soundscape survey according to the ISO 12913-2 technical specifications and incorporating the WHO-5 well-being index. Linear mixed-effects modelling applying backwards-step feature selection was used to model the interactions between internal factors including psychological well-being, age, gender, occupation status, education levels and the soundscape Pleasantness and Eventfulness, while accounting for the random effects of the survey location. The findings suggest that internal factors account for approximately 1.4% of the variance for Pleasantness and 3.9% for Eventfulness, while the influence of the locations accounted for approximately 34% and 14%, respectively. Psychological well-being is positively associated with perceived Pleasantness, while there is a negative association with Eventfulness only for males. Occupation status, in particular retirement as a proxy of age and gender, was identified as a significant factor for both dimensions. These findings offer empirical grounds for developing theories of the interaction between internal factors and soundscape formation whilst highlighting the importance of the location, namely: the context.

neuroscience↗

bioRxiv2020

Mitochondrial profiling reveals dynamic, sex- and age-specific mitochondrial phenotypes in human immune cell subtypes

Using a high-throughput mitochondrial phenotyping platform to quantify multiple mitochondrial features among molecularly-defined immune cell subtypes, we quantify the natural variation in citrate synthase, mitochondrial DNA copy number (mtDNAcn), and respiratory chain enzymatic activities in human neutrophils, monocytes, B cells, and naive and memory T lymphocyte subtypes. In mixed peripheral blood mononuclear cells (PBMCs) from the same individuals, we show to what extent mitochondrial measures are confounded by both cell type distributions and contaminating platelets. Cell subtype-specific measures among women and men spanning 4 decades of life indicate potential age- and sex-related differences, including an age-related elevation in mtDNAcn, which are masked or blunted in mixed PBMCs. Finally, a proof-of-concept, repeated-measures study in a single individual validates cell type differences and also reveals week-to-week changes in mitochondrial activities. Larger studies are required to validate and mechanistically extend these findings. These mitochondrial phenotyping data build upon established immunometabolic differences among leukocyte sub-populations, and provide foundational quantitative knowledge to develop interpretable blood-based assays of mitochondrial health.

cell biology↗

bioRxiv2020

Light into the darkness: Unifying the known and unknown coding sequence space in microbiome analyses

Genes of unknown function are among the biggest challenges in molecular biology, especially in microbial systems, where 40%-60% of the predicted genes are unknown. Despite previous attempts, systematic approaches to include the unknown fraction into analytical workflows are still lacking. Here, we propose a conceptual framework and a computational workflow that bridge the known-unknown gap in genomes and metagenomes. We showcase our approach by exploring 415,971,742 genes predicted from 1,749 metagenomes and 28,941 bacterial and archaeal genomes. We quantify the extent of the unknown fraction, its diversity, and its relevance across multiple biomes. Furthermore, we provide a collection of 283,874 lineage-specific genes of unknown function for Cand. Patescibacteria, being a significant resource to expand our understanding of their unusual biology. Finally, by identifying a target gene of unknown function for antibiotic resistance, we demonstrate how we can enable the generation of hypotheses that can be used to augment experimental data.

microbiology↗

bioRxiv2020

An updated global COI barcode reference data set for Fall Armyworm (Spodoptera frugiperda) and first record of this species in Bhutan

We report the first detection of Fall Armyworm (FAW), Spodoptera frugiperda (Smith, 1797), in Bhutan. FAW feeds on more than 300 plant species and is a serious pest of many. It has been spreading through Africa since 2016 and Asia since 2018. In Bhutan, this species was first detected in maize fields in the western part of the country in September 2019 and subsequently found infesting maize crop in southern parts of the country in December 2019 and April 2020. Using morphological and molecular techniques the presence of the first invading populations of S. frugiperda in Bhutan is confirmed through this study. We present an updated reference DNA barcode data set for FAW comprising 374 sequences, which can be used to reliably identify this serious pest species, and discuss some of the reasons why such compiled reference data sets are necessary, despite the publicly availability of the underlying data. We also report on a second armyworm species, the Northern Armyworm, Mythimna separata (Walker, 1865), in rice, maize and other crops in eighteen districts of Bhutan.

evolutionary biology↗

bioRxiv2020

Structural insights into loss of function of a pore forming toxin and its role in pneumococcal adaptation to an intracellular lifestyle

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-{pi} interaction, governing formation of the transmembrane {beta}-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with loss of pore forming ability of Ply facilitating a benign association of SPN in an alternative, intracellular host niche. AUTHOR SUMMARYStreptococcus pneumoniae, the main causative agent of pneumonia, triggers inflammation and tissue damage by expressing a pore-forming toxin, pneumolysin (Ply). Ply-induced inflammation drives pneumococcal transmission from nasopharynx (its primary reservoir), but also contributes to host mortality, limiting its occupiable habitats. Here, we uncovered the structural basis for loss of pore-forming activity of a Ply variant, present in Serotype 1 ST306, and observed that this enabled adoption of an intracellular lifestyle, attenuating inflammatory responses and prolonging host tolerance of pneumococcus in the lower airways. This commensal-like lifestyle, resembling that of members of the mitis group of Streptococci, might have evolved within ST306 by loss of function ply mutations, compensating for limited nasopharyngeal carriage capacity by facilitating adaptation to an alternate niche.

microbiology↗

bioRxiv2020

Cooperative foraging during larval stage affects fitness in Drosophila

Cooperative behavior can confer advantages to animals. This is especially true for cooperative foraging which provides fitness benefits through more efficient acquisition and consumption of food. While examples of group foraging have been widely described, the principles governing formation of such aggregations and rules that determine group membership remain poorly understood. Here we take advantage of an experimental model system featuring cooperative foraging behavior in Drosophila. Under crowded conditions, fly larvae form coordinated digging groups (clusters), where individuals are linked together by sensory cues and group membership requires prior experience. However, fitness benefits of Drosophila larval clustering remain unknown. We demonstrate that animals raised in crowded conditions on food partially processed by other larvae experience a developmental delay presumably due to the decreased nutritional value of the substrate. Intriguingly, same conditions promote formation of cooperative foraging clusters which further extends larval stage compared to non-clustering animals. Remarkably, this developmental retardation also results in a relative increase in wing size, serving an indicator of adult fitness. Thus, we find that the clustering-induced developmental delay is accompanied by fitness benefits. Therefore, cooperative foraging, while delaying development, may have evolved to give Drosophila larvae benefits when presented with competition for limited food resources.

animal behavior and cognition↗

bioRxiv2020

Recurrent pre-leukemic deletions in myeloid malignancies are the result of DNA double-strand breaks followed by microhomology-mediated end joining

The mechanisms underlying myeloid malignancies deletions are not well understood, nor is it clear why specific genomic hotspots are predisposed to particular deletions. In the current study we inspected the genomic regions around recurrent deletions in myeloid malignancies, and identified microhomology-mediated end-joining (MMEJ) signatures in recurrent deletions in CALR, ASXL1 and SRSF2 loci. Since MMEJ deletions are the result of DNA double-strand breaks (DSBs), we introduced CRISPR Cas9 DSBs into exon 12 of ASXL1, successfully generating recurrent ASXL1 deletion in human hematopoietic stem and progenitor cells (HSPCs). A systematic search of COSMIC dataset for MMEJ deletions in all cancers revealed that recurrent deletions enrich myeloid malignancies. Despite this myeloid predominance, we provide evidence that MMEJ deletions occur in multipotent HSCs. An analysis of DNA repair pathway gene expression in single human adult bone marrow HSPCs could not identify a subpopulation of multipotent HSPCs with increased MMEJ expression, however exposed differences between myeloid and lymphoid biased progenitors. Our data indicate an association between MMEJ-repaired DSBs and recurrent MMEJ deletions in human HSCs and in myeloid leukemia. A better understanding of the source of these DSBs and the regulation of the HSC MMEJ repair pathway might aid with preventing recurrent deletions in human pre-leukemia.

genomics↗

bioRxiv2019

Characterizing upper extremity motor behavior in the first week after stroke

BackgroundAnimal models of brain recovery identify the first days after lesioning as a time of great flux in sensorimotor function and physiology; these findings have implications for human stroke recovery. After rodent motor system lesioning, daily skill training in the less affected forelimb reduces skill acquisition in the more affected forelimb. We asked whether spontaneous human motor behaviors of the less affected upper extremity (UE) early after stroke resemble the animal training model, with the potential to suppress clinical recovery.\n\nMethodsThis prospective observational study used a convenience sample of patients <7 days after stroke (n=25) with a wide severity range; Controls were hospitalized for non-neurological conditions (n=12). Outcome measures were Accelerometry, Upper-Extremity Fugl-Meyer (UEFM), Action Research Arm Test (ARAT), Shoulder Abduction/ Finger Extension Test (SAFE), NIH Stroke Scale (NIHSS).\n\nResultsAccelerometry indicated total paretic UE movement was reduced compared to controls, primarily due to a 44% reduction of bilateral UE use. Unilateral paretic movement was unchanged. Movement shifted to unilateral use of the nonparetic UE, which increased by 77%. Low correlations between movement time and motor performance prompted an exploratory factor analysis (EFA) revealing a 2-component solution; motor performance tests load on one component (motor performance) whereas accelerometry-derived variables load on a second non-orthogonal component (quantity of movement).\n\nConclusionsEarly after stroke, spontaneous overall UE movement is reduced, and movement shifts to unilateral use of the nonparetic UE. Thus, spontaneously-arising UE motor behaviors early after stroke are potential substrates for two mechanisms associated with poorer motor outcomes in animal models: learned non-use and inhibition of motor recovery through training of the nonparetic side. Accurate UE motor assessment requires two independent constructs: motor performance and quantity of movement. These findings provide opportunities and measurement methods for studies to develop new behaviorally-based stroke recovery treatments that begin early after onset.

neuroscience↗

bioRxiv2019

A global perspective on microbial diversity in the terrestrial deep subsurface

While recent efforts to catalogue Earths microbial diversity have focused upon surface and marine habitats, 12% to 20% of Earths bacterial and archaeal biomass is suggested to inhabit the terrestrial deep subsurface, compared to [~]1.8% in the deep subseafloor 1-3. Metagenomic studies of the terrestrial deep subsurface have yielded a trove of divergent and functionally important microbiomes from a range of localities 4-6. However, a wider perspective of microbial diversity and its relationship to environmental conditions within the terrestrial deep subsurface is still required. Here, we show the diversity of bacterial communities in deep subsurface groundwater is controlled by aquifer lithology globally, by using 16S rRNA gene datasets collected across five countries on two continents and from fifteen rock types over the past decade. Furthermore, our meta-analysis reveals that terrestrial deep subsurface microbiota are dominated by Betaproteobacteria, Gammaproteobacteria and Firmicutes, likely as a function of the diverse metabolic strategies of these taxa. Despite this similarity, evidence was found not only for aquifer-specific microbial communities, but also for a common small consortium of prevalent Betaproteobacteria and Gammaproteobacterial OTUs across the localities. This finding implies a core terrestrial deep subsurface community, irrespective of aquifer lithology, that may play an important role in colonising and sustaining microbial habitats in the deep terrestrial subsurface. An in-silico contamination-aware approach to analysing this dataset underscores the importance of downstream methods for assuring that robust conclusions can be reached from deep subsurface-derived sequencing data. Understanding the global panorama of microbial diversity and ecological dynamics in the deep terrestrial subsurface provides a first step towards understanding the role of microbes in global subsurface element and nutrient cycling.

microbiology↗

bioRxiv2017

Common variant burden contributes significantly to the familial aggregation of migraine in 1,589 families

It has long been observed that complex traits, including migraine, often aggregate in families, but the underlying genetic architecture behind this is not well understood. Two competing hypotheses exist, emphasizing either rare or common genetic variation. More specifically, familial aggregation could be predominantly explained by rare, penetrant variants that segregate according to Mendelian inheritance or rather by the sufficient polygenic accumulation of many common variants, each with an individually small effect. Some combination of both common and rare variation could also contribute towards a spectrum of disease risk.\n\nWe investigated this in a collection of 8,319 individuals across 1,589 migraine families from Finland. Family members were individually diagnosed by a migraine-specific questionnaire with either migraine without aura (MO, ICHD-3 code 1.1, n=2,357), migraine with typical aura (ICHD- 3 code 1.2.1, n=2,420), hemiplegic migraine (HM, ICHD-3 code 1.2.3, n=540), or no migraine (n=3,002). For comparison, we used population-based migraine cases (n=1,101) and controls (n=13,369) from the FINRISK study. The disease status of FINRISK individuals was assigned based on health registry data from outpatient clinics and/or prescription medication. All individuals were genotyped on the Illumina(R) CoreExome or PsychArray chip platforms and imputed to a Finnish reference panel of 6,962 haplotypes. Polygenic risk scores (PRS), representing the common variant burden in each individual, were calculated using weights from the most recent large-scale genome-wide association study of migraine. To account for family structure in our analyses, we used a mixed-model approach, adjusting for the genetic relationship matrix as a random effect.\n\nWe found a significantly higher common variant burden in familial cases of migraine (for all subtypes, measured by the odds ratio [OR] per standard deviation [SD] increase in PRS; OR = 1.76, 95% CI = 1.71-1.81, P = 1.7x10-109) compared to cases from a population cohort (OR = 1.32, 95% CI = 1.25-1.38, P = 7.2x10-17) when using the population controls as a reference group. The highest enrichment was observed for HM (OR = 1.96, 95% CI = 1.86-2.07, P = 8.7x10-36) and migraine with typical aura (OR = 1.85, 95% CI = 1.79-1.91, P = 1.4x10-86) but enrichment was also present for MO (OR = 1.57, 95% CI = 1.51-1.63, P = 1.1x10-48). Comparing within cases, there was no significant difference in common variant burden between the migraine with aura subtypes, HM and migraine with typical aura (OR = 1.09, 95% CI = 0.99-1.19, P = 0.09), but both showed significantly higher enrichment compared to MO (OR = 1.28, 95% CI = 1.17-1.38, P = 7.3x10-7, and OR = 1.17, 95% CI = 1.11-1.23, P = 4.62x10-5, respectively). Additionally, we found that higher common variant burden corresponded to earlier age of headache onset (OR per SD increase in PRS for 3,631 cases with onset before 20 years old compared to 1,686 cases with onset later than 20 years old; OR = 1.11, 95% CI = 1.05-1.18, P = 8.3x10-4). FINRISK population cases identified from national health registry data were found to have lower common variant burden in comparison to the familial migraine cases (OR = 1.32, 95% CI = 1.25-1.38, P = 6.8x10-17), unless the individuals had attended both a specialist clinic and also received prophylactic migraine treatment (OR = 1.70, 95% CI = 1.53-1.88, P = 3.9x10-9). Finally, although rare variants have been suggested as the primary cause for familial hemiplegic migraine (FHM), we found only four out of 45 sequenced FHM families (8.9%) with a pathogenic mutation in one of the known risk genes.\n\nIn summary, our results demonstrate a substantial contribution of common polygenic variation to familial aggregation in migraine, comparable to both controls and that observed in migraine cases from a population cohort. The findings also suggest that individuals with migraine aura symptoms (either typical aura, which is mostly visual, or rare motor aura) tend to have higher common variant burden on average supporting the polygenic model also in these migraine subtypes.

genetics↗

bioRxiv2017

Deep Sequencing: Intra-Terrestrial Metagenomics Illustrates The Potential Of Off-Grid Nanopore DNA Sequencing

Genetic and genomic analysis of nucleic acids from environmental samples has helped transform our perception of the Earths subsurface as a major reservoir of microbial novelty. Many of the microbial taxa living in the subsurface are under-represented in culture-dependent investigations. In this regard, metagenomic analyses of subsurface environments exemplify both the utility of metagenomics and its power to explore microbial life in some of the most extreme and inaccessible environments on Earth. Hitherto, the transfer of microbial samples to home laboratories for DNA sequencing and bioinformatics is the standard operating procedure for exploring microbial diversity. This approach incurs logistical challenges and delays the characterization of microbial biodiversity. For selected applications, increased portability and agility in metagenomic analysis is therefore desirable. Here, we describe the implementation of sample extraction, metagenomic library preparation, nanopore DNA sequencing and taxonomic classification using a portable, battery-powered, suite of off-the-shelf tools (the \"MetageNomad\") to sequence ochreous sediment microbiota while within the South Wales Coalfield. While our analyses were frustrated by short read lengths and a limited yield of DNA, within the assignable reads, Proteobacterial (-, {beta}-, {gamma}-Proteobacteria) taxa dominated, followed by members of Actinobacteria, Firmicutes and Bacteroidetes, all of which have previously been identified in coals. Further to this, the fungal genus Candida was detected, as well as a methanogenic archaeal taxon. To the best of our knowledge, this application of the MetageNomad represents an initial effort to conduct metagenomics within the subsurface, and stimulates further developments to take metagenomics off the beaten track.

microbiology↗