Bats are capable of asymptomatically carrying a diverse number of microorganisms, including human... more Bats are capable of asymptomatically carrying a diverse number of microorganisms, including human pathogens, due to their unique immune system. Because of the close contact between bats and humans, there is a possibility for interspecies transmission and consequential disease outbreaks. Herein, high-throughput sequencing was used to determine the kidney-associated microbiome of a bat species abundant in Grenada, West Indies, Artibeus spp. Results indicate that the kidney of these bats can carry potential human pathogens. An endogenous retrovirus, Desmodus rotundus endogenous retrovirus isolate 824, phylogenetically related to betaretroviruses from rodents and New World primates, was also identified.
As the 2nd year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ... more As the 2nd year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to control the pandemic and to prepare for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and to prepare for future outbreaks. The review lays out the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium-an international research collective that has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytical sample processing, target amplification and amplicon detection, then the hardware and software required for deployment, and finally a summary of the current regulatory landscape. Included as well are a series of first-person accounts of LAMP method development and deployment. The final conclusions and recommendations section provides the reader with a distillation of the most validated testing methods and their paths to implementation. The review aims to provide practical information and insight for a range of audiences: for a research audience to help accelerate research through sharing of best practices, for an implementation audience to help get testing up and running quickly, and for public health, clinical, and policy audience to help convey the breadth of impact that LAMP methods have to offer.
The read counts and relative abundances for Bacillus anthracis identified by various tools after ... more The read counts and relative abundances for Bacillus anthracis identified by various tools after the whole genome sequencing of two samples from the New York City subway system. (XLSX 13 kb)
Tool accuracy per taxon. Each file is categorized by taxonomic level. Inside each file, the first... more Tool accuracy per taxon. Each file is categorized by taxonomic level. Inside each file, the first sheet shows the accuracy (with additional columns at the species level for mean GC content, genome length, and class(es) for associated strains based on the number of repeats). The second sheet details the number of false positives and the third sheet details the number of false negatives of each classifier for each taxon in each taxonomic level. The three ensemble classifiers (Community, Blast Ensemble, Diamond Ensemble) are included in this analysis for comparison. (ZIP 1590 kb)
Features of datasets included in the analysis. Mean AUPR across tools provides an indication of t... more Features of datasets included in the analysis. Mean AUPR across tools provides an indication of the difficulty of a dataset. (XLSX 18 kb)
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus d... more Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is a respiratory virus primarily transmitted from person to person through inhalation of droplets or aerosols, laden with viral particles. However, as some studies have shown, virions can remain infectious for up to 72 hours on surfaces, which can lead to transmission through contact. For this reason, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end (E2E) study showed that the effective combination of monitoring SARS-CoV-2 on surfaces include using an Isohelix swab as a collection tool, DNA/RNA Shield as a preservative, an automated system for RNA extraction, and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) as the detection assay. Using this E2E approach, this study showed that, in some cases, SARS-CoV-2 viral standards were still recovered from su...
Since the introduction of nanopore sequencing in 2013, countless new methods and techniques have ... more Since the introduction of nanopore sequencing in 2013, countless new methods and techniques have been developed that has enabled researcher to study nucleic acids like never before. Nanopore sequencing has many characteristics including ultra-long DNA sequencing of single read lengths of up to a megabase, direct RNA sequencing with modified base detection, and amplicon sequencing, to name a few. These sequencing systems come in many formats for both small and large core laboratories as well a multitude of library synthesis methods and barcoding options to address many techniques required for many aspects of both genomic and transcriptional studies. In this session we will discuss some of the popular techniques as well as an open panel discussion for audience interaction. Speaker 1: Scott Tighe: Introduction to Nanopore Sequencing 5 min Speaker 2: High Molecular Weight DNA Extractions for Long Read Sequencing and Other Genomic Applications Kelvin Liu Circulomics 20 min Speaker 3: Det...
As microbiome and metagenomic studies span nearly all aspects of science, the need to characteriz... more As microbiome and metagenomic studies span nearly all aspects of science, the need to characterize the limitations and strengths of the full laboratory process is critical. This includes DNA and RNA extraction efficiencies, type of sequencing, and bioinformatics, to name a few. Presently, a commercial accurately enumerated whole cell microbial standard does not exist, except for the MGRG WCM Standard that is near completion. The minimum design characteristics included 1) accuracy cellular enumeration of each species, 2) intact cellular integrity and morphology that came be confirmed by microscopy and staining, 3) preserved, 4) fully sequenced genomes, 5) various Gram reaction and GC content, and 6) able to maintain cell wall digestion with lytic enzymes. In this study, we have performed initial experiments to characterize this standard using various kits and sequencing approaches. We will present both RNA and DNA extractions and sequence results as well as open up the discussion to ...
One of the major challenges of the microbiome studies is unavailability of a reliable preservativ... more One of the major challenges of the microbiome studies is unavailability of a reliable preservative media for long term storage of microbes that maintains the cellular structure and integrity without causing partial lysis or cellular leakage. Such a preservative would allow for many downstream analyses without compromising DNA and RNA quality. To alleviate these technical challenges, we designed a proof of principle study which included ABRF MMRG (Metagenomics and Microbiome Research Group) Members, representing multi-institutional sites. We formulated a preservative media that was suitable for long term storage of samples, maintained cellular structure of microorganisms, exhibited no detectable DNA or RNA leakage in the solution, preserved DNA and RNA integrity, preserved Gram stain and other stainability (SYBR Green, DAPI, etc) features, and maintained enzymatic cell wall digestion by Metapolyzyme. In this proof of principle study, we preserved three Gram positive (Bacillus subtili...
Systemic lupus erythematosus (SLE) is characterized by increased DNA demethylation in T cells, al... more Systemic lupus erythematosus (SLE) is characterized by increased DNA demethylation in T cells, although it is unclear whether this occurs primarily in a subset of SLE T cells. The process driving the DNA demethylation and the consequences on overall gene expression are also poorly understood and whether this represents a secondary consequence of SLE or a primary contributing factor. Lupus-prone lpr mice accumulate large numbers of T cells with age because of a mutation in Fas (CD95). The accumulating T cells include an unusual population of CD4 2 CD8 2 TCR-ab + (DN) T cells that arise from CD8 + precursors and are also found in human SLE. We have previously observed that T cell accumulation in lpr mice is due to dysregulation of T cell homeostatic proliferation, which parallels an increased expression of numerous genes in the DN subset, including several proinflammatory molecules and checkpoint blockers. We thus determined the DNA methylome in lpr DN T cells compared with their CD8 + precursors. Our findings show that DN T cells manifest discrete sites of extensive demethylation throughout the genome, and these sites correspond to the location of a large proportion of the upregulated genes. Thus, dysregulated homeostatic proliferation in lpr mice and consequent epigenetic alterations may be a contributing factor to lupus pathogenesis. ImmunoHorizons, 2020, 4: 679-687.
The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The... more The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.
The mosquitoes Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) and Culex quinquefasciatus Say... more The mosquitoes Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) and Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) are two major vectors of arthropod-borne pathogens in Grenada, West Indies. As conventional vector control methods present many challenges, alternatives are urgently needed. Manipulation of mosquito microbiota is emerging as a field for the development of vector control strategies. Critical to this vector control approach is knowledge of the microbiota of these mosquitoes and finding candidate microorganisms that are common to the vectors with properties that could be used in microbiota modification studies. Results showed that bacteria genera including Asaia, Escherichia, Pantoea, Pseudomonas, and Serratia are common to both major arboviral vectors in Grenada and have previously been shown to be good candidates for transgenetic studies. Also, for the first time, the presence of Grenada mosquito rhabdovirus 1 is reported in C. quinquefasciatus.
Arboviruses cause diseases of significant global health concerns. Interactions between mosquitoes... more Arboviruses cause diseases of significant global health concerns. Interactions between mosquitoes and their microbiota as well as the important role of this interaction in the mosquito's capacity to harbor and transmit pathogens have emerged as important fields of research. Aedes aegypti is one of the most abundant mosquitoes in many geographic locations, a vector capable of transmitting a number of arboviruses such as dengue and Zika. Currently, there are few studies on the metavirome of this mosquito particularly in the Americas. This study analyzes the metavirome of A. aegypti from Grenada, a Caribbean nation with tropical weather, abundant A. aegypti, and both endemic and arboviral pathogens transmitted by this mosquito. Between January and December 2018, 1152 mosquitoes were collected from six semi-rural locations near houses in St. George Parish, Grenada, by weekly trapping using BG-Sentinel traps. From these, 300 A. aegypti were selected for analysis. The metavirome was analyzed using the Illumina HiSeq 1500 for deep sequencing. The generation sequencing library construction protocol used was NuGEN Universal RNA with an average read length of 125 bp. Reads were mapped to the A. aegypti assembly. Non-mosquito reads were analyzed using the tools FastViromeExplorer. The NCBI total virus, RNA virus, and eukaryotic virus databases were used as references. The metagenomic comparison analysis showed that the most abundant virus-related reads among all databases and assemblies was Phasi Charoen-like virus. The Phasi Charoen-like virus results are in agreement to other studies in America, Asia and Australia. Further studies using wild-caught mosquitoes is needed to assess the impact of this insect-specific virus on the A. aegypti lifecycle and vector capacity.
In the version of this article initially published, in Table 2, first column, "m.13095T > C" shou... more In the version of this article initially published, in Table 2, first column, "m.13095T > C" should have been "m.130 b T > C, " where " b " refers to the footnote "Characters hidden to respect confidentiality, " as with the other three from the Newcastle Group. In addition, the footnote " a " for Table 2 should have read "http://hfeaarchive.uksouth.cloudapp.azure.com/www.hfea.gov.uk/docs/Fourth_scientific_review_mitochondria_2016.pdf " rather than "Personal communication. " The following acknowledgment was omitted: "The authors thank
Background: One of the main challenges in metagenomics is the identification of microorganisms in... more Background: One of the main challenges in metagenomics is the identification of microorganisms in clinical and environmental samples. While an extensive and heterogeneous set of computational tools is available to classify microorganisms using whole-genome shotgun sequencing data, comprehensive comparisons of these methods are limited. Results: In this study, we use the largest-to-date set of laboratory-generated and simulated controls across 846 species to evaluate the performance of 11 metagenomic classifiers. Tools were characterized on the basis of their ability to identify taxa at the genus, species, and strain levels, quantify relative abundances of taxa, and classify individual reads to the species level. Strikingly, the number of species identified by the 11 tools can differ by over three orders of magnitude on the same datasets. Various strategies can ameliorate taxonomic misclassification, including abundance filtering, ensemble approaches, and tool intersection. Nevertheless, these strategies were often insufficient to completely eliminate false positives from environmental samples, which are especially important where they concern medically relevant species. Overall, pairing tools with different classification strategies (k-mer, alignment, marker) can combine their respective advantages. Conclusions: This study provides positive and negative controls, titrated standards, and a guide for selecting tools for metagenomic analyses by comparing ranges of precision, accuracy, and recall. We show that proper experimental design and analysis parameters can reduce false positives, provide greater resolution of species in complex metagenomic samples, and improve the interpretation of results.
We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacter... more We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.
Journal of biomolecular techniques : JBT, Apr 10, 2017
The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Res... more The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the wo...
Bats are capable of asymptomatically carrying a diverse number of microorganisms, including human... more Bats are capable of asymptomatically carrying a diverse number of microorganisms, including human pathogens, due to their unique immune system. Because of the close contact between bats and humans, there is a possibility for interspecies transmission and consequential disease outbreaks. Herein, high-throughput sequencing was used to determine the kidney-associated microbiome of a bat species abundant in Grenada, West Indies, Artibeus spp. Results indicate that the kidney of these bats can carry potential human pathogens. An endogenous retrovirus, Desmodus rotundus endogenous retrovirus isolate 824, phylogenetically related to betaretroviruses from rodents and New World primates, was also identified.
As the 2nd year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ... more As the 2nd year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to control the pandemic and to prepare for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and to prepare for future outbreaks. The review lays out the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium-an international research collective that has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytical sample processing, target amplification and amplicon detection, then the hardware and software required for deployment, and finally a summary of the current regulatory landscape. Included as well are a series of first-person accounts of LAMP method development and deployment. The final conclusions and recommendations section provides the reader with a distillation of the most validated testing methods and their paths to implementation. The review aims to provide practical information and insight for a range of audiences: for a research audience to help accelerate research through sharing of best practices, for an implementation audience to help get testing up and running quickly, and for public health, clinical, and policy audience to help convey the breadth of impact that LAMP methods have to offer.
The read counts and relative abundances for Bacillus anthracis identified by various tools after ... more The read counts and relative abundances for Bacillus anthracis identified by various tools after the whole genome sequencing of two samples from the New York City subway system. (XLSX 13 kb)
Tool accuracy per taxon. Each file is categorized by taxonomic level. Inside each file, the first... more Tool accuracy per taxon. Each file is categorized by taxonomic level. Inside each file, the first sheet shows the accuracy (with additional columns at the species level for mean GC content, genome length, and class(es) for associated strains based on the number of repeats). The second sheet details the number of false positives and the third sheet details the number of false negatives of each classifier for each taxon in each taxonomic level. The three ensemble classifiers (Community, Blast Ensemble, Diamond Ensemble) are included in this analysis for comparison. (ZIP 1590 kb)
Features of datasets included in the analysis. Mean AUPR across tools provides an indication of t... more Features of datasets included in the analysis. Mean AUPR across tools provides an indication of the difficulty of a dataset. (XLSX 18 kb)
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus d... more Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is a respiratory virus primarily transmitted from person to person through inhalation of droplets or aerosols, laden with viral particles. However, as some studies have shown, virions can remain infectious for up to 72 hours on surfaces, which can lead to transmission through contact. For this reason, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end (E2E) study showed that the effective combination of monitoring SARS-CoV-2 on surfaces include using an Isohelix swab as a collection tool, DNA/RNA Shield as a preservative, an automated system for RNA extraction, and reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) as the detection assay. Using this E2E approach, this study showed that, in some cases, SARS-CoV-2 viral standards were still recovered from su...
Since the introduction of nanopore sequencing in 2013, countless new methods and techniques have ... more Since the introduction of nanopore sequencing in 2013, countless new methods and techniques have been developed that has enabled researcher to study nucleic acids like never before. Nanopore sequencing has many characteristics including ultra-long DNA sequencing of single read lengths of up to a megabase, direct RNA sequencing with modified base detection, and amplicon sequencing, to name a few. These sequencing systems come in many formats for both small and large core laboratories as well a multitude of library synthesis methods and barcoding options to address many techniques required for many aspects of both genomic and transcriptional studies. In this session we will discuss some of the popular techniques as well as an open panel discussion for audience interaction. Speaker 1: Scott Tighe: Introduction to Nanopore Sequencing 5 min Speaker 2: High Molecular Weight DNA Extractions for Long Read Sequencing and Other Genomic Applications Kelvin Liu Circulomics 20 min Speaker 3: Det...
As microbiome and metagenomic studies span nearly all aspects of science, the need to characteriz... more As microbiome and metagenomic studies span nearly all aspects of science, the need to characterize the limitations and strengths of the full laboratory process is critical. This includes DNA and RNA extraction efficiencies, type of sequencing, and bioinformatics, to name a few. Presently, a commercial accurately enumerated whole cell microbial standard does not exist, except for the MGRG WCM Standard that is near completion. The minimum design characteristics included 1) accuracy cellular enumeration of each species, 2) intact cellular integrity and morphology that came be confirmed by microscopy and staining, 3) preserved, 4) fully sequenced genomes, 5) various Gram reaction and GC content, and 6) able to maintain cell wall digestion with lytic enzymes. In this study, we have performed initial experiments to characterize this standard using various kits and sequencing approaches. We will present both RNA and DNA extractions and sequence results as well as open up the discussion to ...
One of the major challenges of the microbiome studies is unavailability of a reliable preservativ... more One of the major challenges of the microbiome studies is unavailability of a reliable preservative media for long term storage of microbes that maintains the cellular structure and integrity without causing partial lysis or cellular leakage. Such a preservative would allow for many downstream analyses without compromising DNA and RNA quality. To alleviate these technical challenges, we designed a proof of principle study which included ABRF MMRG (Metagenomics and Microbiome Research Group) Members, representing multi-institutional sites. We formulated a preservative media that was suitable for long term storage of samples, maintained cellular structure of microorganisms, exhibited no detectable DNA or RNA leakage in the solution, preserved DNA and RNA integrity, preserved Gram stain and other stainability (SYBR Green, DAPI, etc) features, and maintained enzymatic cell wall digestion by Metapolyzyme. In this proof of principle study, we preserved three Gram positive (Bacillus subtili...
Systemic lupus erythematosus (SLE) is characterized by increased DNA demethylation in T cells, al... more Systemic lupus erythematosus (SLE) is characterized by increased DNA demethylation in T cells, although it is unclear whether this occurs primarily in a subset of SLE T cells. The process driving the DNA demethylation and the consequences on overall gene expression are also poorly understood and whether this represents a secondary consequence of SLE or a primary contributing factor. Lupus-prone lpr mice accumulate large numbers of T cells with age because of a mutation in Fas (CD95). The accumulating T cells include an unusual population of CD4 2 CD8 2 TCR-ab + (DN) T cells that arise from CD8 + precursors and are also found in human SLE. We have previously observed that T cell accumulation in lpr mice is due to dysregulation of T cell homeostatic proliferation, which parallels an increased expression of numerous genes in the DN subset, including several proinflammatory molecules and checkpoint blockers. We thus determined the DNA methylome in lpr DN T cells compared with their CD8 + precursors. Our findings show that DN T cells manifest discrete sites of extensive demethylation throughout the genome, and these sites correspond to the location of a large proportion of the upregulated genes. Thus, dysregulated homeostatic proliferation in lpr mice and consequent epigenetic alterations may be a contributing factor to lupus pathogenesis. ImmunoHorizons, 2020, 4: 679-687.
The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The... more The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.
The mosquitoes Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) and Culex quinquefasciatus Say... more The mosquitoes Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae) and Culex quinquefasciatus Say, 1823 (Diptera: Culicidae) are two major vectors of arthropod-borne pathogens in Grenada, West Indies. As conventional vector control methods present many challenges, alternatives are urgently needed. Manipulation of mosquito microbiota is emerging as a field for the development of vector control strategies. Critical to this vector control approach is knowledge of the microbiota of these mosquitoes and finding candidate microorganisms that are common to the vectors with properties that could be used in microbiota modification studies. Results showed that bacteria genera including Asaia, Escherichia, Pantoea, Pseudomonas, and Serratia are common to both major arboviral vectors in Grenada and have previously been shown to be good candidates for transgenetic studies. Also, for the first time, the presence of Grenada mosquito rhabdovirus 1 is reported in C. quinquefasciatus.
Arboviruses cause diseases of significant global health concerns. Interactions between mosquitoes... more Arboviruses cause diseases of significant global health concerns. Interactions between mosquitoes and their microbiota as well as the important role of this interaction in the mosquito's capacity to harbor and transmit pathogens have emerged as important fields of research. Aedes aegypti is one of the most abundant mosquitoes in many geographic locations, a vector capable of transmitting a number of arboviruses such as dengue and Zika. Currently, there are few studies on the metavirome of this mosquito particularly in the Americas. This study analyzes the metavirome of A. aegypti from Grenada, a Caribbean nation with tropical weather, abundant A. aegypti, and both endemic and arboviral pathogens transmitted by this mosquito. Between January and December 2018, 1152 mosquitoes were collected from six semi-rural locations near houses in St. George Parish, Grenada, by weekly trapping using BG-Sentinel traps. From these, 300 A. aegypti were selected for analysis. The metavirome was analyzed using the Illumina HiSeq 1500 for deep sequencing. The generation sequencing library construction protocol used was NuGEN Universal RNA with an average read length of 125 bp. Reads were mapped to the A. aegypti assembly. Non-mosquito reads were analyzed using the tools FastViromeExplorer. The NCBI total virus, RNA virus, and eukaryotic virus databases were used as references. The metagenomic comparison analysis showed that the most abundant virus-related reads among all databases and assemblies was Phasi Charoen-like virus. The Phasi Charoen-like virus results are in agreement to other studies in America, Asia and Australia. Further studies using wild-caught mosquitoes is needed to assess the impact of this insect-specific virus on the A. aegypti lifecycle and vector capacity.
In the version of this article initially published, in Table 2, first column, "m.13095T > C" shou... more In the version of this article initially published, in Table 2, first column, "m.13095T > C" should have been "m.130 b T > C, " where " b " refers to the footnote "Characters hidden to respect confidentiality, " as with the other three from the Newcastle Group. In addition, the footnote " a " for Table 2 should have read "http://hfeaarchive.uksouth.cloudapp.azure.com/www.hfea.gov.uk/docs/Fourth_scientific_review_mitochondria_2016.pdf " rather than "Personal communication. " The following acknowledgment was omitted: "The authors thank
Background: One of the main challenges in metagenomics is the identification of microorganisms in... more Background: One of the main challenges in metagenomics is the identification of microorganisms in clinical and environmental samples. While an extensive and heterogeneous set of computational tools is available to classify microorganisms using whole-genome shotgun sequencing data, comprehensive comparisons of these methods are limited. Results: In this study, we use the largest-to-date set of laboratory-generated and simulated controls across 846 species to evaluate the performance of 11 metagenomic classifiers. Tools were characterized on the basis of their ability to identify taxa at the genus, species, and strain levels, quantify relative abundances of taxa, and classify individual reads to the species level. Strikingly, the number of species identified by the 11 tools can differ by over three orders of magnitude on the same datasets. Various strategies can ameliorate taxonomic misclassification, including abundance filtering, ensemble approaches, and tool intersection. Nevertheless, these strategies were often insufficient to completely eliminate false positives from environmental samples, which are especially important where they concern medically relevant species. Overall, pairing tools with different classification strategies (k-mer, alignment, marker) can combine their respective advantages. Conclusions: This study provides positive and negative controls, titrated standards, and a guide for selecting tools for metagenomic analyses by comparing ranges of precision, accuracy, and recall. We show that proper experimental design and analysis parameters can reduce false positives, provide greater resolution of species in complex metagenomic samples, and improve the interpretation of results.
We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacter... more We present two standards developed by the Genomic Standards Consortium (GSC) for reporting bacterial and archaeal genome sequences. Both are extensions of the Minimum Information about Any (x) Sequence (MIxS). The standards are the Minimum Information about a Single Amplified Genome (MISAG) and the Minimum Information about a Metagenome-Assembled Genome (MIMAG), including, but not limited to, assembly quality, and estimates of genome completeness and contamination. These standards can be used in combination with other GSC checklists, including the Minimum Information about a Genome Sequence (MIGS), Minimum Information about a Metagenomic Sequence (MIMS), and Minimum Information about a Marker Gene Sequence (MIMARKS). Community-wide adoption of MISAG and MIMAG will facilitate more robust comparative genomic analyses of bacterial and archaeal diversity.
Journal of biomolecular techniques : JBT, Apr 10, 2017
The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Res... more The Extreme Microbiome Project (XMP) is a project launched by the Association of Biomolecular Resource Facilities Metagenomics Research Group (ABRF MGRG) that focuses on whole genome shotgun sequencing of extreme and unique environments using a wide variety of biomolecular techniques. The goals are multifaceted, including development and refinement of new techniques for the following: 1) the detection and characterization of novel microbes, 2) the evaluation of nucleic acid techniques for extremophilic samples, and 3) the identification and implementation of the appropriate bioinformatics pipelines. Here, we highlight the different ongoing projects that we have been working on, as well as details on the various methods we use to characterize the microbiome and metagenome of these complex samples. In particular, we present data of a novel multienzyme extraction protocol that we developed, called Polyzyme or MetaPolyZyme. Presently, the XMP is characterizing sample sites around the wo...
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