Publications by authors named "Migun Shakya"

17 Publications

  • Page 1 of 1

Comparative genomic and phenotypic characterization of invasive non-typhoidal Salmonella isolates from Siaya, Kenya.

PLoS Negl Trop Dis 2021 Feb 1;15(2):e0008991. Epub 2021 Feb 1.

Los Alamos National Laboratory, Los Alamos, New Mexico, United States.

Non-typhoidal Salmonella (NTS) is a major global health concern that often causes bloodstream infections in areas of the world affected by malnutrition and comorbidities such as HIV and malaria. Developing a strategy to control the emergence and spread of highly invasive and antimicrobial resistant NTS isolates requires a comprehensive analysis of epidemiological factors and molecular pathogenesis. Here, we characterize 11 NTS isolates that caused bloodstream infections in pediatric patients in Siaya, Kenya from 2003-2010. Nine isolates were identified as S. Typhimurium sequence type 313 while the other two were S. Enteritidis. Comprehensive genotypic and phenotypic analyses were performed to compare these isolates to those previously identified in sub-Saharan Africa. We identified a S. Typhimurium isolate referred to as UGA14 that displayed novel plasmid, pseudogene and resistance features as compared to other isolates reported from Africa. Notably, UGA14 is able to ferment both lactose and sucrose due to the acquisition of insertion elements on the pKST313 plasmid. These findings show for the first time the co-evolution of plasmid-mediated lactose and sucrose metabolism along with cephalosporin resistance in NTS further elucidating the evolutionary mechanisms of invasive NTS phenotypes. These results further support the use of combined genomic and phenotypic approaches to detect and characterize atypical NTS isolates in order to advance biosurveillance efforts that inform countermeasures aimed at controlling invasive and antimicrobial resistant NTS.
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http://dx.doi.org/10.1371/journal.pntd.0008991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7877762PMC
February 2021

NCBI's Virus Discovery Codeathon: Building "FIVE" -The Federated Index of Viral Experiments API Index.

Viruses 2020 12 10;12(12). Epub 2020 Dec 10.

National Center for Biotechnology Information, U.S. National Library of Medicine, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20894, USA.

Viruses represent important test cases for data federation due to their genome size and the rapid increase in sequence data in publicly available databases. However, some consequences of previously decentralized (unfederated) data are lack of consensus or comparisons between feature annotations. Unifying or displaying alternative annotations should be a priority both for communities with robust entry representation and for nascent communities with burgeoning data sources. To this end, during this three-day continuation of the Virus Hunting Toolkit codeathon series (VHT-2), a new integrated and federated viral index was elaborated. This Federated Index of Viral Experiments (FIVE) integrates pre-existing and novel functional and taxonomy annotations and virus-host pairings. Variability in the context of viral genomic diversity is often overlooked in virus databases. As a proof-of-concept, FIVE was the first attempt to include viral genome variation for HIV, the most well-studied human pathogen, through viral genome diversity graphs. As per the publication of this manuscript, FIVE is the first implementation of a virus-specific federated index of such scope. FIVE is coded in BigQuery for optimal access of large quantities of data and is publicly accessible. Many projects of database or index federation fail to provide easier alternatives to access or query information. To this end, a Python API query system was developed to enhance the accessibility of FIVE.
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http://dx.doi.org/10.3390/v12121424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764237PMC
December 2020

A Public Website for the Automated Assessment and Validation of SARS-CoV-2 Diagnostic PCR Assays.

Bioinformatics 2020 Aug 10. Epub 2020 Aug 10.

Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico.

Summary: Polymerase chain reaction-based assays are the current gold standard for detecting and diagnosing SARS-CoV-2. However, as SARS-CoV-2 mutates, we need to constantly assess whether existing PCR-based assays will continue to detect all known viral strains. To enable the continuous monitoring of SARS-CoV-2 assays, we have developed a web-based assay validation algorithm that checks existing PCR-based assays against the ever-expanding genome databases for SARS-CoV-2 using both thermodynamic and edit-distance metrics. The assay screening results are displayed as a heatmap, showing the number of mismatches between each detection and each SARS-CoV-2 genome sequence. Using a mismatch threshold to define detection failure, assay performance is summarized with the true positive rate (recall) to simplify assay comparisons.

Availability And Implementation: The assay evaluation website and supporting software are Open Source and freely available at https://covid19.edgebioinformatics.org/#/assayValidation, https://github.com/jgans/thermonucleotideBLAST, and https://github.com/LANL-Bioinformatics/assay_validation.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa710DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7559084PMC
August 2020

A Gene Cluster That Encodes Histone Deacetylase Inhibitors Contributes to Bacterial Persistence and Antibiotic Tolerance in Burkholderia thailandensis.

mSystems 2020 Feb 11;5(1). Epub 2020 Feb 11.

Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico, USA

Persister cells are genetically identical variants in a bacterial population that have phenotypically modified their physiology to survive environmental stress. In bacterial pathogens, persisters are able to survive antibiotic treatment and reinfect patients in a frustrating cycle of chronic infection. To better define core persistence mechanisms for therapeutics development, we performed transcriptomics analyses of populations enriched for persisters via three methods: flow sorting for low proton motive force, meropenem treatment, and culture aging. Although the three persister-enriched populations generally displayed divergent gene expression profiles that reflect the multimechanistic nature of stress adaptations, there were several common gene pathways activated in two or all three populations. These include polyketide and nonribosomal peptide synthesis, Clp proteases, mobile elements, enzymes involved in lipid metabolism, and ATP-binding cassette (ABC) transporter systems. In particular, identification of genes that encode polyketide synthases (PKSs) and fatty acid catabolism factors indicates that generation of secondary metabolites, natural products, and complex lipids could be part of the metabolic program that governs the persistence state. We also found that loss-of-function mutations in the PKS-encoding gene locus BTH_I2366, which plays a role in biosynthesis of histone deacetylase (HDAC) inhibitors, resulted in increased sensitivity to antibiotics targeting DNA replication. Furthermore, treatment of multiple bacterial pathogens with a fatty acid synthesis inhibitor, CP-640186, potentiated the efficacy of meropenem against the persister populations. Altogether, our results suggest that bacterial persisters may exhibit an outwardly dormant physiology but maintain active metabolic processes that are required to maintain persistence. The discovery of antibiotics such as penicillin and streptomycin marked a historic milestone in the 1940s and heralded a new era of antimicrobial therapy as the modern standard for medical treatment. Yet, even in those early days of discovery, it was noted that a small subset of cells (∼1 in 10) survived antibiotic treatment and continued to persist, leading to recurrence of chronic infection. These persisters are phenotypic variants that have modified their physiology to survive environmental stress. In this study, we have performed three transcriptomic screens to identify persistence genes that are common between three different stressor conditions. In particular, we identified genes that function in the synthesis of secondary metabolites, small molecules, and complex lipids, which are likely required to maintain the persistence state. Targeting universal persistence genes can lead to the development of clinically relevant antipersistence therapeutics for infectious disease management.
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http://dx.doi.org/10.1128/mSystems.00609-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018527PMC
February 2020

Standardized phylogenetic and molecular evolutionary analysis applied to species across the microbial tree of life.

Sci Rep 2020 02 3;10(1):1723. Epub 2020 Feb 3.

Bioscience Division, Los Alamos National Laboratory, MS-M888, Los Alamos, NM, 87545, USA.

There is growing interest in reconstructing phylogenies from the copious amounts of genome sequencing projects that target related viral, bacterial or eukaryotic organisms. To facilitate the construction of standardized and robust phylogenies for disparate types of projects, we have developed a complete bioinformatic workflow, with a web-based component to perform phylogenetic and molecular evolutionary (PhaME) analysis from sequencing reads, draft assemblies or completed genomes of closely related organisms. Furthermore, the ability to incorporate raw data, including some metagenomic samples containing a target organism (e.g. from clinical samples with suspected infectious agents), shows promise for the rapid phylogenetic characterization of organisms within complex samples without the need for prior assembly.
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http://dx.doi.org/10.1038/s41598-020-58356-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6997174PMC
February 2020

Advances and Challenges in Metatranscriptomic Analysis.

Front Genet 2019 25;10:904. Epub 2019 Sep 25.

Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, United States.

Sequencing-based analyses of microbiomes have traditionally focused on addressing the question of community membership and profiling taxonomic abundance through amplicon sequencing of 16 rRNA genes. More recently, shotgun metagenomics, which involves the random sequencing of all genomic content of a microbiome, has dominated this arena due to advancements in sequencing technology throughput and capability to profile genes as well as microbiome membership. While these methods have revealed a great number of insights into a wide variety of microbiomes, both of these approaches only describe the presence of organisms or genes, and not whether they are active members of the microbiome. To obtain deeper insights into how a microbial community responds over time to their changing environmental conditions, microbiome scientists are beginning to employ large-scale metatranscriptomics approaches. Here, we present a comprehensive review on computational metatranscriptomics approaches to study microbial community transcriptomes. We review the major advancements in this burgeoning field, compare strengths and weaknesses to other microbiome analysis methods, list available tools and workflows, and describe use cases and limitations of this method. We envision that this field will continue to grow exponentially, as will the scope of projects (e.g. longitudinal studies of community transcriptional responses to perturbations over time) and the resulting data. This review will provide a list of options for computational analysis of these data and will highlight areas in need of development.
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http://dx.doi.org/10.3389/fgene.2019.00904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774269PMC
September 2019

Machine-Learning Classification Suggests That Many Alphaproteobacterial Prophages May Instead Be Gene Transfer Agents.

Genome Biol Evol 2019 10;11(10):2941-2953

Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire.

Many of the sequenced bacterial and archaeal genomes encode regions of viral provenance. Yet, not all of these regions encode bona fide viruses. Gene transfer agents (GTAs) are thought to be former viruses that are now maintained in genomes of some bacteria and archaea and are hypothesized to enable exchange of DNA within bacterial populations. In Alphaproteobacteria, genes homologous to the "head-tail" gene cluster that encodes structural components of the Rhodobacter capsulatus GTA (RcGTA) are found in many taxa, even if they are only distantly related to Rhodobacter capsulatus. Yet, in most genomes available in GenBank RcGTA-like genes have annotations of typical viral proteins, and therefore are not easily distinguished from their viral homologs without additional analyses. Here, we report a "support vector machine" classifier that quickly and accurately distinguishes RcGTA-like genes from their viral homologs by capturing the differences in the amino acid composition of the encoded proteins. Our open-source classifier is implemented in Python and can be used to scan homologs of the RcGTA genes in newly sequenced genomes. The classifier can also be trained to identify other types of GTAs, or even to detect other elements of viral ancestry. Using the classifier trained on a manually curated set of homologous viruses and GTAs, we detected RcGTA-like "head-tail" gene clusters in 57.5% of the 1,423 examined alphaproteobacterial genomes. We also demonstrated that more than half of the in silico prophage predictions are instead likely to be GTAs, suggesting that in many alphaproteobacterial genomes the RcGTA-like elements remain unrecognized.
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http://dx.doi.org/10.1093/gbe/evz206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6821227PMC
October 2019

Novel Insights Into the Spread of Enteric Pathogens Using Genomics.

J Infect Dis 2020 03;221(Suppl 3):S319-S330

Bioscience Division, Los Alamos National Laboratory, New Mexico.

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http://dx.doi.org/10.1093/infdis/jiz220DOI Listing
March 2020

NCBI's Virus Discovery Hackathon: Engaging Research Communities to Identify Cloud Infrastructure Requirements.

Genes (Basel) 2019 09 16;10(9). Epub 2019 Sep 16.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda MD 20894, USA.

A wealth of viral data sits untapped in publicly available metagenomic data sets when it might be extracted to create a usable index for the virological research community. We hypothesized that work of this complexity and scale could be done in a hackathon setting. Ten teams comprised of over 40 participants from six countries, assembled to create a crowd-sourced set of analysis and processing pipelines for a complex biological data set in a three-day event on the San Diego State University campus starting 9 January 2019. Prior to the hackathon, 141,676 metagenomic data sets from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) were pre-assembled into contiguous assemblies (contigs) by NCBI staff. During the hackathon, a subset consisting of 2953 SRA data sets (approximately 55 million contigs) was selected, which were further filtered for a minimal length of 1 kb. This resulted in 4.2 million (Mio) contigs, which were aligned using BLAST against all known virus genomes, phylogenetically clustered and assigned metadata. Out of the 4.2 Mio contigs, 360,000 contigs were labeled with domains and an additional subset containing 4400 contigs was screened for virus or virus-like genes. The work yielded valuable insights into both SRA data and the cloud infrastructure required to support such efforts, revealing analysis bottlenecks and possible workarounds thereof. Mainly: (i) Conservative assemblies of SRA data improves initial analysis steps; (ii) existing bioinformatic software with weak multithreading/multicore support can be elevated by wrapper scripts to use all cores within a computing node; (iii) redesigning existing bioinformatic algorithms for a cloud infrastructure to facilitate its use for a wider audience; and (iv) a cloud infrastructure allows a diverse group of researchers to collaborate effectively. The scientific findings will be extended during a follow-up event. Here, we present the applied workflows, initial results, and lessons learned from the hackathon.
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http://dx.doi.org/10.3390/genes10090714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6771016PMC
September 2019

Remedial Treatment of Corroded Iron Objects by Environmental Isolates.

Appl Environ Microbiol 2019 02 23;85(3). Epub 2019 Jan 23.

Laboratory of Microbiology, Institute of Biology, University of Neuchâtel, Neuchâtel, Switzerland

Using bacteria to transform reactive corrosion products into stable compounds represents an alternative to traditional methods employed in iron conservation. Two environmental strains (CA23 and CU5) were used to transform ferric iron corrosion products (goethite and lepidocrocite) into stable ferrous iron-bearing minerals (vivianite and siderite). A genomic and transcriptomic approach was used to analyze the metabolic traits of these strains and to evaluate their pathogenic potential. Although genes involved in solid-phase iron reduction were identified, key genes present in other environmental iron-reducing species are missing from the genome of CU5. Several pathogenicity factors were identified in the genomes of both strains, but none of these was expressed under iron reduction conditions. Additional tests showed hemolytic and cytotoxic activities for strain CA23 but not for strain CU5. Both strains were easily inactivated using ethanol and heat. Nonetheless, given a lesser potential for a pathogenic lifestyle, CU5 is the most promising candidate for the development of a bio-based iron conservation method stabilizing iron corrosion. Based on all the results, a prototype treatment was established using archaeological items. On those, the conversion of reactive corrosion products and the formation of a homogenous layer of biogenic iron minerals were achieved. This study shows how naturally occurring microorganisms and their metabolic capabilities can be used to develop bio-inspired solutions to the problem of metal corrosion. Microbiology can greatly help in the quest for a sustainable solution to the problem of iron corrosion, which causes important economic losses in a wide range of fields, including the protection of cultural heritage and building materials. Using bacteria to transform reactive and unstable corrosion products into more-stable compounds represents a promising approach. The overall aim of this study was to develop a method for the conservation and restoration of corroded iron items, starting from the isolation of iron-reducing bacteria from natural environments. This resulted in the identification of a suitable candidate ( sp. strain CU5) that mediates the formation of desirable minerals at the surfaces of the objects. This led to the proof of concept of an application method on real objects.
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http://dx.doi.org/10.1128/AEM.02042-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6344634PMC
February 2019

Insights into origin and evolution of α-proteobacterial gene transfer agents.

Virus Evol 2017 Jul 7;3(2):vex036. Epub 2017 Dec 7.

Department of Biological Sciences, Dartmouth College, 78 College Street, Hanover, NH 03755, USA.

Several bacterial and archaeal lineages produce nanostructures that morphologically resemble small tailed viruses, but, unlike most viruses, contain apparently random pieces of the host genome. Since these elements can deliver the packaged DNA to other cells, they were dubbed gene transfer agents (GTAs). Because many genes involved in GTA production have viral homologs, it has been hypothesized that the GTA ancestor was a virus. Whether GTAs represent an atypical virus, a defective virus, or a virus co-opted by the prokaryotes for some function, remains to be elucidated. To evaluate these possibilities, we examined the distribution and evolutionary histories of genes that encode a GTA in the α-proteobacterium (RcGTA). We report that although homologs of many individual RcGTA genes are abundant across bacteria and their viruses, RcGTA-like genomes are mainly found in one subclade of α-proteobacteria. When compared with the viral homologs, genes of the RcGTA-like genomes evolve significantly slower, and do not have higher %A+T nucleotides than their host chromosomes. Moreover, they appear to reside in stable regions of the bacterial chromosomes that are generally conserved across taxonomic orders. These findings argue against RcGTA being an atypical or a defective virus. Our phylogenetic analyses suggest that RcGTA ancestor likely originated in the lineage that gave rise to contemporary α-proteobacterial orders , , , , and , and since that time the RcGTA-like element has co-evolved with its host chromosomes. Such evolutionary history is compatible with maintenance of these elements by bacteria due to some selective advantage. As for many other prokaryotic traits, horizontal gene transfer played a substantial role in the evolution of RcGTA-like elements, not only in shaping its genome components within the orders, but also in occasional dissemination of RcGTA-like regions across the orders and even to different bacterial phyla.
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http://dx.doi.org/10.1093/ve/vex036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5721377PMC
July 2017

Functional and Evolutionary Characterization of a Gene Transfer Agent's Multilocus "Genome".

Mol Biol Evol 2016 10 24;33(10):2530-43. Epub 2016 Jun 24.

Department of Biology, Memorial University of Newfoundland, St John's, NL, Canada

Gene transfer agents (GTAs) are phage-like particles that can package and transfer a random piece of the producing cell's genome, but are unable to transfer all the genes required for their own production. As such, GTAs represent an evolutionary conundrum: are they selfish genetic elements propagating through an unknown mechanism, defective viruses, or viral structures "repurposed" by cells for gene exchange, as their name implies? In Rhodobacter capsulatus, production of the R. capsulatus GTA (RcGTA) particles is associated with a cluster of genes resembling a small prophage. Utilizing transcriptomic, genetic and biochemical approaches, we report that the RcGTA "genome" consists of at least 24 genes distributed across five distinct loci. We demonstrate that, of these additional loci, two are involved in cell recognition and binding and one in the production and maturation of RcGTA particles. The five RcGTA "genome" loci are widespread within Rhodobacterales, but not all loci have the same evolutionary histories. Specifically, two of the loci have been subject to frequent, probably virus-mediated, gene transfer events. We argue that it is unlikely that RcGTA is a selfish genetic element. Instead, our findings are compatible with the scenario that RcGTA is a virus-derived element maintained by the producing organism due to a selective advantage of within-population gene exchange. The modularity of the RcGTA "genome" is presumably a result of selection on the host organism to retain GTA functionality.
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http://dx.doi.org/10.1093/molbev/msw125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026251PMC
October 2016

A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling.

BMC Genomics 2016 Jan 14;17:55. Epub 2016 Jan 14.

Institute of Integrative Biology, University of Liverpool, Liverpool, L69 7ZB, UK.

Background: In the last 5 years, the rapid pace of innovations and improvements in sequencing technologies has completely changed the landscape of metagenomic and metagenetic experiments. Therefore, it is critical to benchmark the various methodologies for interrogating the composition of microbial communities, so that we can assess their strengths and limitations. The most common phylogenetic marker for microbial community diversity studies is the 16S ribosomal RNA gene and in the last 10 years the field has moved from sequencing a small number of amplicons and samples to more complex studies where thousands of samples and multiple different gene regions are interrogated.

Results: We assembled 2 synthetic communities with an even (EM) and uneven (UM) distribution of archaeal and bacterial strains and species, as metagenomic control material, to assess performance of different experimental strategies. The 2 synthetic communities were used in this study, to highlight the limitations and the advantages of the leading sequencing platforms: MiSeq (Illumina), The Pacific Biosciences RSII, 454 GS-FLX/+ (Roche), and IonTorrent (Life Technologies). We describe an extensive survey based on synthetic communities using 3 experimental designs (fusion primers, universal tailed tag, ligated adaptors) across the 9 hypervariable 16S rDNA regions. We demonstrate that library preparation methodology can affect data interpretation due to different error and chimera rates generated during the procedure. The observed community composition was always biased, to a degree that depended on the platform, sequenced region and primer choice. However, crucially, our analysis suggests that 16S rRNA sequencing is still quantitative, in that relative changes in abundance of taxa between samples can be recovered, despite these biases.

Conclusion: We have assessed a range of experimental conditions across several next generation sequencing platforms using the most up-to-date configurations. We propose that the choice of sequencing platform and experimental design needs to be taken into consideration in the early stage of a project by running a small trial consisting of several hypervariable regions to quantify the discriminatory power of each region. We also suggest that the use of a synthetic community as a positive control would be beneficial to identify the potential biases and procedural drawbacks that may lead to data misinterpretation. The results of this study will serve as a guideline for making decisions on which experimental condition and sequencing platform to consider to achieve the best microbial profiling.
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http://dx.doi.org/10.1186/s12864-015-2194-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4712552PMC
January 2016

Improved yield of high molecular weight DNA coincides with increased microbial diversity access from iron oxide cemented sub-surface clay environments.

PLoS One 2014 17;9(7):e102826. Epub 2014 Jul 17.

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America.

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1-V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102826PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102596PMC
November 2015

A multifactor analysis of fungal and bacterial community structure in the root microbiome of mature Populus deltoides trees.

PLoS One 2013 16;8(10):e76382. Epub 2013 Oct 16.

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, United States of America ; Genome Science and Technology Program, University of Tennessee, Knoxville, Tennessee, United States of America.

Bacterial and fungal communities associated with plant roots are central to the host health, survival and growth. However, a robust understanding of the root-microbiome and the factors that drive host associated microbial community structure have remained elusive, especially in mature perennial plants from natural settings. Here, we investigated relationships of bacterial and fungal communities in the rhizosphere and root endosphere of the riparian tree species Populus deltoides, and the influence of soil parameters, environmental properties (host phenotype and aboveground environmental settings), host plant genotype (Simple Sequence Repeat (SSR) markers), season (Spring vs. Fall) and geographic setting (at scales from regional watersheds to local riparian zones) on microbial community structure. Each of the trees sampled displayed unique aspects to its associated community structure with high numbers of Operational Taxonomic Units (OTUs) specific to an individual trees (bacteria >90%, fungi >60%). Over the diverse conditions surveyed only a small number of OTUs were common to all samples within rhizosphere (35 bacterial and 4 fungal) and endosphere (1 bacterial and 1 fungal) microbiomes. As expected, Proteobacteria and Ascomycota were dominant in root communities (>50%) while other higher-level phylogenetic groups (Chytridiomycota, Acidobacteria) displayed greatly reduced abundance in endosphere compared to the rhizosphere. Variance partitioning partially explained differences in microbiome composition between all sampled roots on the basis of seasonal and soil properties (4% to 23%). While most variation remains unattributed, we observed significant differences in the microbiota between watersheds (Tennessee vs. North Carolina) and seasons (Spring vs. Fall). SSR markers clearly delineated two host populations associated with the samples taken in TN vs. NC, but overall host genotypic distances did not have a significant effect on corresponding communities that could be separated from other measured effects.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0076382PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3797799PMC
August 2014

Comparative metagenomic and rRNA microbial diversity characterization using archaeal and bacterial synthetic communities.

Environ Microbiol 2013 Jun 6;15(6):1882-99. Epub 2013 Feb 6.

Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 47841, USA.

Next-generation sequencing has dramatically changed the landscape of microbial ecology, large-scale and in-depth diversity studies being now widely accessible. However, determining the accuracy of taxonomic and quantitative inferences and comparing results obtained with different approaches are complicated by incongruence of experimental and computational data types and also by lack of knowledge of the true ecological diversity. Here we used highly diverse bacterial and archaeal synthetic communities assembled from pure genomic DNAs to compare inferences from metagenomic and SSU rRNA amplicon sequencing. Both Illumina and 454 metagenomic data outperformed amplicon sequencing in quantifying the community composition, but the outcome was dependent on analysis parameters and platform. New approaches in processing and classifying amplicons can reconstruct the taxonomic composition of the community with high reproducibility within primer sets, but all tested primers sets lead to significant taxon-specific biases. Controlled synthetic communities assembled to broadly mimic the phylogenetic richness in target environments can provide important validation for fine-tuning experimental and computational parameters used to characterize natural communities.
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http://dx.doi.org/10.1111/1462-2920.12086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3665634PMC
June 2013