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1.
EBioMedicine ; 74: 103649, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34814051

RESUMO

BACKGROUND: Shotgun metagenomics has been used clinically for diagnosing infectious diseases. However, most technical assessments have been limited to individual sets of reference standards, experimental workflows, and laboratories. METHODS: A reference panel and performance metrics were designed and used to examine the performance of shotgun metagenomics at 17 laboratories in a coordinated collaborative study. We comprehensively assessed the reliability, key performance determinants, reproducibility, and quantitative potential. FINDINGS: Assay performance varied significantly across sites and microbial classes, with a read depth of 20 millions as a generally cost-efficient assay setting. Results of mapped reads by shotgun metagenomics could indicate relative and intra-site (but not absolute or inter-site) microbial abundance. INTERPRETATION: Assay performance was significantly impacted by the microbial type, the host context, and read depth, which emphasizes the importance of these factors when designing reference reagents and benchmarking studies. Across sites, workflows and platforms, false positive reporting and considerable site/library effects were common challenges to the assay's accuracy and quantifiability. Our study also suggested that laboratory-developed shotgun metagenomics tests for pathogen detection should aim to detect microbes at 500 CFU/mL (or copies/mL) in a clinically relevant host context (10^5 human cells/mL) within a 24h turn-around time, and with an efficient read depth of 20M. FUNDING: This work was supported by National Science and Technology Major Project of China (2018ZX10102001).


Assuntos
Bactérias/isolamento & purificação , Doenças Transmissíveis/diagnóstico , Fungos/isolamento & purificação , Metagenômica/instrumentação , Metagenômica/métodos , Bactérias/classificação , Bactérias/genética , Benchmarking , China , Fungos/classificação , Fungos/genética , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Laboratórios , Metagenômica/normas , Reprodutibilidade dos Testes , Análise de Sequência de DNA , Fluxo de Trabalho
2.
BMC Microbiol ; 21(1): 263, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34592929

RESUMO

BACKGROUND: Microbiome big data from population-scale cohorts holds the key to unleash the power of microbiomes to overcome critical challenges in disease control, treatment and precision medicine. However, variations introduced during data generation and processing limit the comparisons among independent studies in respect of interpretability. Although multiple databases have been constructed as platforms for data reuse, they are of limited value since only raw sequencing files are considered. DESCRIPTION: Here, we present MetaGeneBank, a standardized database that provides details on sample collection and sequencing, and abundances of genes, microbiota and molecular functions for 4470 raw sequencing files (over 12 TB) collected from 16 studies covering over 10 types of diseases and 14 countries using a unified data-processing pipeline. The incorporation of tools that enable browsing and searching with descriptive attributes, gene sequences, microbiota and functions makes the database user-friendly. We found that the source of specimen contributes more than sequencing centers or platforms to the variations of microbiota. Special attention should be paid when re-analyzing sequencing files from different countries. CONCLUSIONS: Collectively, MetaGeneBank provides a gateway to utilize the untapped potential of gut metagenomic data in helping fighting against human diseases. With the continuous updating of the database in terms of data volume, data types and sample types, MetaGeneBank would undoubtedly be the benchmarking database in the future in respect of data reuse, and would be valuable in translational science.


Assuntos
Bases de Dados Genéticas , Fezes/microbiologia , Metagenômica/instrumentação , Microbioma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
3.
Viruses ; 13(3)2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803225

RESUMO

Experiments in which complex virome sequencing data is generated remain difficult to explore and unpack for scientists without a background in data science. The processing of raw sequencing data by high throughput sequencing workflows usually results in contigs in FASTA format coupled to an annotation file linking the contigs to a reference sequence or taxonomic identifier. The next step is to compare the virome of different samples based on the metadata of the experimental setup and extract sequences of interest that can be used in subsequent analyses. The viromeBrowser is an application written in the opensource R shiny framework that was developed in collaboration with end-users and is focused on three common data analysis steps. First, the application allows interactive filtering of annotations by default or custom quality thresholds. Next, multiple samples can be visualized to facilitate comparison of contig annotations based on sample specific metadata values. Last, the application makes it easy for users to extract sequences of interest in FASTA format. With the interactive features in the viromeBrowser we aim to enable scientists without a data science background to compare and extract annotation data and sequences from virome sequencing analysis results.


Assuntos
Biologia Computacional/métodos , Metagenômica/métodos , Anotação de Sequência Molecular/métodos , Viroma/genética , Sequência de Bases/genética , Análise de Dados , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/instrumentação , Software
4.
Microbiol Res ; 247: 126727, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33652267

RESUMO

The MinION single-molecule sequencing system has been attracting the attention of the community of microbiologists involved in the conservation of cultural heritage. The use of MinION for the conservation of cultural heritage is extremely recent, but surprisingly the only few applications available have been exploring many different substrates: stone, textiles, paintings and wax. The use of MinION sequencing is mainly used to address the metataxonomy (with special emphasis on non-cultivable microorganisms) with the effort to identify species involved in the degradation of the substrates. In this review, we show the current applications available on different artworks, showing how this technology can be a useful tool for microbiologists and conservators also in light of its low cost and the easy chemistry.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Microbiota/genética , Bactérias/classificação , Bactérias/genética , DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Metagenômica/instrumentação , Pinturas , Análise de Sequência de DNA , Têxteis
5.
Nat Rev Microbiol ; 18(12): 705-716, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33024311

RESUMO

Viruses are extremely diverse and modulate important biological and ecological processes globally. However, much of viral diversity remains uncultured and yet to be discovered. Several powerful culture-independent tools, in particular metagenomics, have substantially advanced virus discovery. Among those tools is single-virus genomics, which yields sequenced reference genomes from individual sorted virus particles without the need for cultivation. This new method complements virus culturing and metagenomic approaches and its advantages include targeted investigation of specific virus groups and investigation of genomic microdiversity within viral populations. In this Review, we provide a brief history of single-virus genomics, outline how this emergent method has facilitated advances in virus ecology and discuss its current limitations and future potential. Finally, we address how this method may synergistically intersect with other single-virus and single-cell approaches.


Assuntos
Biologia Computacional/métodos , Genoma Viral , Metagenoma , Metagenômica/métodos , Vírion/genética , Vírus/genética , Animais , Organismos Aquáticos , Ciclo do Carbono , Técnicas de Cultura de Células , Biologia Computacional/instrumentação , Variação Genética , Humanos , Metagenômica/instrumentação , Pinças Ópticas , Vírion/metabolismo , Vírion/ultraestrutura , Vírus/classificação , Vírus/metabolismo , Vírus/ultraestrutura
6.
Microbiome ; 8(1): 48, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245390

RESUMO

BACKGROUND: Metagenomics is revolutionizing the study of microorganisms and their involvement in biological, biomedical, and geochemical processes, allowing us to investigate by direct sequencing a tremendous diversity of organisms without the need for prior cultivation. Unicellular eukaryotes play essential roles in most microbial communities as chief predators, decomposers, phototrophs, bacterial hosts, symbionts, and parasites to plants and animals. Investigating their roles is therefore of great interest to ecology, biotechnology, human health, and evolution. However, the generally lower sequencing coverage, their more complex gene and genome architectures, and a lack of eukaryote-specific experimental and computational procedures have kept them on the sidelines of metagenomics. RESULTS: MetaEuk is a toolkit for high-throughput, reference-based discovery, and annotation of protein-coding genes in eukaryotic metagenomic contigs. It performs fast searches with 6-frame-translated fragments covering all possible exons and optimally combines matches into multi-exon proteins. We used a benchmark of seven diverse, annotated genomes to show that MetaEuk is highly sensitive even under conditions of low sequence similarity to the reference database. To demonstrate MetaEuk's power to discover novel eukaryotic proteins in large-scale metagenomic data, we assembled contigs from 912 samples of the Tara Oceans project. MetaEuk predicted >12,000,000 protein-coding genes in 8 days on ten 16-core servers. Most of the discovered proteins are highly diverged from known proteins and originate from very sparsely sampled eukaryotic supergroups. CONCLUSION: The open-source (GPLv3) MetaEuk software (https://github.com/soedinglab/metaeuk) enables large-scale eukaryotic metagenomics through reference-based, sensitive taxonomic and functional annotation. Video abstract.


Assuntos
Algoritmos , Eucariotos/genética , Metagenômica/métodos , Microbiota , Anotação de Sequência Molecular/métodos , Biologia Computacional/métodos , Bases de Dados Genéticas , Ensaios de Triagem em Larga Escala , Metagenoma , Metagenômica/instrumentação , Análise de Sequência de DNA/métodos
7.
BMC Biol ; 18(1): 37, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32264902

RESUMO

Metagenomics studies leverage genomic reference databases to generate discoveries in basic science and translational research. However, current microbial studies use disparate reference databases that lack consistent standards of specimen inclusion, data preparation, taxon labelling and accessibility, hindering their quality and comprehensiveness, and calling for the establishment of recommendations for reference genome database assembly. Here, we analyze existing fungal and bacterial databases and discuss guidelines for the development of a master reference database that promises to improve the quality and quantity of omics research.


Assuntos
Bactérias/genética , Bases de Dados Genéticas/normas , Fungos/genética , Metagenômica/normas , Metagenômica/instrumentação
8.
Sci Rep ; 10(1): 5125, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198413

RESUMO

Nanopore based DNA-sequencing delivers long reads, thereby simplifying the decipherment of bacterial communities. Since its commercial appearance, this technology has been assigned several attributes, such as its error proneness, comparatively low cost, ease-of-use, and, most notably, aforementioned long reads. The technology as a whole is under continued development. As such, benchmarks are required to conceive, test and improve analysis protocols, including those related to the understanding of the composition of microbial communities. Here we present a dataset composed of twelve different prokaryotic species split into four samples differing by nucleic acid quantification technique to assess the specificity and sensitivity of the MinION nanopore sequencer in a blind study design. Taxonomic classification was performed by standard taxonomic sequence classification tools, namely Kraken, Kraken2 and Centrifuge directly on reads. This allowed taxonomic assignments of up to 99.27% on genus level and 92.78% on species level, enabling true-positive classification of strains down to 25,000 genomes per sample. Full genomic coverage is achieved for strains abundant as low as 250,000 genomes per sample under our experimental settings. In summary, we present an evaluation of nanopore sequence processing analysis with respect to microbial community composition. It provides an open protocol and the data may serve as basis for the development and benchmarking of future data processing pipelines.


Assuntos
Bactérias/genética , Benchmarking/métodos , Genoma Bacteriano/genética , Tipagem Molecular/métodos , Sequenciamento por Nanoporos/métodos , Análise de Sequência de DNA/métodos , Bactérias/classificação , Sequência de Bases , DNA Bacteriano/genética , Metagenômica/instrumentação , Metagenômica/métodos , Sequenciamento por Nanoporos/instrumentação
9.
Sci Rep ; 10(1): 2985, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076089

RESUMO

Identification of bacteria causing tissue infections can be comprehensive and, in the cases of non- or slow-growing bacteria, near impossible with conventional methods. Performing shotgun metagenomic sequencing on bacterial DNA extracted directly from the infected tissue may improve time to diagnosis and targeted treatment considerably. However, infected tissue consists mainly of human DNA (hDNA) which hampers bacterial identification. In this proof of concept study, we present a modified version of the Ultra-Deep Microbiome Prep kit for DNA extraction procedure, removing additional human DNA. Tissue biopsies from 3 patients with orthopedic implant-related infections containing varying degrees of Staphylococcus aureus were included. Subsequent DNA shotgun metagenomic sequencing using Oxford Nanopore Technologies' (ONT) MinION platform and ONTs EPI2ME WIMP and ARMA bioinformatic workflows for microbe and antibiotic resistance genes identification, respectively. The modified DNA extraction protocol led to an additional ~10-fold reduction of human DNA while preserving S. aureus DNA. Including the DNA sequencing and bioinformatics analyses, the presented protocol has the potential of identifying the infection-causing pathogen in infected tissue within 7 hours after biopsy. However, due to low number of S. aureus reads, positive identification of antibiotic resistance genes was not possible.


Assuntos
DNA Bacteriano/isolamento & purificação , Metagenômica/instrumentação , Kit de Reagentes para Diagnóstico , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/isolamento & purificação , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biópsia , Farmacorresistência Bacteriana/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Metagenoma/genética , Sequenciamento por Nanoporos , Estudo de Prova de Conceito , Infecções Relacionadas à Prótese/diagnóstico , Infecções Relacionadas à Prótese/tratamento farmacológico , Infecções Relacionadas à Prótese/microbiologia , Infecções Relacionadas à Prótese/patologia , Análise de Sequência de DNA , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética
10.
Mol Ecol Resour ; 20(1): 256-267, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31293086

RESUMO

The accurate quantification of eukaryotic species abundances from bulk samples remains a key challenge for community ecology and environmental biomonitoring. We resolve this challenge by combining shotgun sequencing, mapping to reference DNA barcodes or to mitogenomes, and three correction factors: (a) a percent-coverage threshold to filter out false positives, (b) an internal-standard DNA spike-in to correct for stochasticity during sequencing, and (c) technical replicates to correct for stochasticity across sequencing runs. The SPIKEPIPE pipeline achieves a strikingly high accuracy of intraspecific abundance estimates (in terms of DNA mass) from samples of known composition (mapping to barcodes R2  = .93, mitogenomes R2  = .95) and a high repeatability across environmental-sample replicates (barcodes R2  = .94, mitogenomes R2  = .93). As proof of concept, we sequence arthropod samples from the High Arctic, systematically collected over 17 years, detecting changes in species richness, species-specific abundances, and phenology. SPIKEPIPE provides cost-efficient and reliable quantification of eukaryotic communities.


Assuntos
Código de Barras de DNA Taxonômico/métodos , Eucariotos/classificação , Eucariotos/genética , Metagenômica/métodos , Animais , Biodiversidade , DNA/genética , Metagenômica/instrumentação
11.
Genes (Basel) ; 10(11)2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31703372

RESUMO

Microbial communities in remote locations remain under-studied. This is particularly true on glaciers and icecaps, which cover approximately 11% of the Earth's surface. The principal reason for this is the inaccessibility of most of these areas due to their extreme isolation and challenging environmental conditions. While remote research stations have significantly lowered the barrier to studying the microbial communities on icecaps, their use has led to a bias for data collection in the near vicinity of these institutions. Here, miniaturisation of a DNA sequencing lab suitable for off-grid metagenomic studies is demonstrated. Using human power alone, this lab was transported across Europe's largest ice cap (Vatnajökull, Iceland) by ski and sledge. After 11 days of unsupported polar-style travel, a metagenomic study of a geothermal hot spring gorge was conducted on the remote northern edge of the ice cap. This tent-based metagenomic study resulted in over 24 h of Nanopore sequencing, powered by solar power alone. This study demonstrates the ability to conduct DNA sequencing in remote locations, far from civilised resources (mechanised transport, external power supply, internet connection, etc.), whilst greatly reducing the time from sample collection to data acquisition.


Assuntos
Expedições , Camada de Gelo/microbiologia , Metagenoma , Metagenômica/métodos , Microbiota , Sequenciamento por Nanoporos/métodos , Energia Solar , Fontes de Energia Elétrica , Islândia , Metagenômica/instrumentação , Sequenciamento por Nanoporos/instrumentação
12.
Curr Biol ; 29(11): R401-R402, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31163139

RESUMO

At a time of unprecedented impacts on marine biodiversity, scientists are rapidly becoming persuaded by the potential of screening large swathes of the oceans through the retrieval, amplification and sequencing of trace DNA fragments left behind by marine organisms; an approach known as 'environmental DNA' (eDNA) [1]. In trying to circumvent the many challenges associated with water filtration and DNA isolation from environmental samples, significant investment is being made in high-tech solutions, such as automated underwater vehicles and robots [2]. Here, instead, we explored a simpler, alternative option, based on the recovery of eDNA from sponges (phylum Porifera), the planet's most effective water-filterers. We obtained sponge samples from Mediterranean and Antarctic surveys, extracted total DNA from their tissues, and obtained tens of thousands of fish DNA reads via metabarcoding, which were able to clearly distinguish samples from the two regions. One Antarctic sample yielded hundreds of reads from chinstrap penguin (Pygoscelis antarcticus) and Weddell seal (Leptonychotes weddellii). We argue that this 'natural sampler DNA' (nsDNA) approach is poised to become a powerful, affordable, universal tool for aquatic biodiversity monitoring globally.


Assuntos
Organismos Aquáticos , Biodiversidade , Código de Barras de DNA Taxonômico/métodos , DNA Ambiental/análise , Poríferos , Animais , Regiões Antárticas , Organismos Aquáticos/genética , Código de Barras de DNA Taxonômico/instrumentação , Mar Mediterrâneo , Metagenômica/instrumentação , Oceanos e Mares
13.
ACS Nano ; 13(6): 6540-6549, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31179687

RESUMO

In-field molecular diagnosis of plant diseases via nucleic acid amplification is currently limited by cumbersome protocols for extracting and isolating pathogenic DNA from plant tissues. To address this challenge, a rapid plant DNA extraction method was developed using a disposable polymeric microneedle (MN) patch. By applying MN patches on plant leaves, amplification-assay-ready DNA can be extracted within a minute from different plant species. MN-extracted DNA was used for direct polymerase chain reaction amplification of plant plastid DNA without purification. Furthermore, using this patch device, extraction of plant pathogen DNA ( Phytophthora infestans) from both laboratory-inoculated and field-infected leaf samples was performed for detection of late blight disease in tomato. MN extraction achieved 100% detection rate of late blight infections for samples after 3 days of inoculation when compared to the conventional gold standard cetyltrimethylammonium bromide (CTAB)-based DNA extraction method and 100% detection rate for all blind field samples tested. This simple, cell-lysis-free, and purification-free DNA extraction method could be a transformative approach to facilitate rapid sample preparation for molecular diagnosis of various plant diseases directly in the field.


Assuntos
Código de Barras de DNA Taxonômico/métodos , DNA Fúngico/química , Phytophthora/genética , Doenças das Plantas/microbiologia , Código de Barras de DNA Taxonômico/instrumentação , DNA Fúngico/genética , Solanum lycopersicum/microbiologia , Metagenômica/instrumentação , Metagenômica/métodos , Agulhas , Phytophthora/patogenicidade , Folhas de Planta/química , Folhas de Planta/microbiologia
14.
Microbiome ; 7(1): 44, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30898140

RESUMO

BACKGROUND: Wastewater treatment plants (WWTPs) are recognized as hotspots for horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). Despite our understanding of the composition and distribution of ARGs in WWTPs, the genetic location, host, and fate of ARGs remain largely unknown. RESULTS: In this study, we combined Oxford Nanopore and Illumina metagenomics sequencing to comprehensively uncover the resistome context of influent, activated sludge, and effluent of three WWTPs and simultaneously track the hosts of the ARGs. The results showed that most of the ARGs detected in all compartments of the WWTPs were carried by plasmids. Transposons and integrons also showed higher prevalence on plasmids than on the ARG-carrying chromosome. Notably, integrative and conjugative elements (ICEs) carrying five types of ARGs were detected, and they may play an important role in facilitating the transfer of ARGs, particularly for tetracycline and macrolide-lincosamide-streptogramin (MLS). A broad spectrum of ARGs carried by plasmids (29 subtypes) and ICEs (4 subtypes) was persistent across the WWTPs. Host tracking showed a variety of antibiotic-resistant bacteria in the effluent, suggesting the high potential for their dissemination into receiving environments. Importantly, phenotype-genotype analysis confirmed the significant role of conjugative plasmids in facilitating the survival and persistence of multidrug-resistant bacteria in the WWTPs. At last, the consistency in the quantitative results for major ARGs types revealed by Nanopore and Illumina sequencing platforms demonstrated the feasibility of Nanopore sequencing for resistome quantification. CONCLUSION: Overall, these findings substantially expand our current knowledge of resistome in WWTPs, and help establish a baseline analysis framework to study ARGs in the environment.


Assuntos
Bactérias/classificação , Resistência Microbiana a Medicamentos , Metagenômica/instrumentação , Análise de Sequência de DNA/instrumentação , Águas Residuárias/microbiologia , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Transferência Genética Horizontal , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Lincosamidas/farmacologia , Macrolídeos/farmacologia , Metagenômica/métodos , Nanoporos , Filogenia , Análise de Sequência de DNA/métodos , Esgotos/microbiologia , Estreptograminas/farmacologia , Tetraciclina/farmacologia
15.
Nat Rev Genet ; 20(6): 341-355, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30918369

RESUMO

Clinical metagenomic next-generation sequencing (mNGS), the comprehensive analysis of microbial and host genetic material (DNA and RNA) in samples from patients, is rapidly moving from research to clinical laboratories. This emerging approach is changing how physicians diagnose and treat infectious disease, with applications spanning a wide range of areas, including antimicrobial resistance, the microbiome, human host gene expression (transcriptomics) and oncology. Here, we focus on the challenges of implementing mNGS in the clinical laboratory and address potential solutions for maximizing its impact on patient care and public health.


Assuntos
Doenças Transmissíveis/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ciência de Laboratório Médico/métodos , Metagenoma , Metagenômica/métodos , Animais , Antibacterianos/uso terapêutico , Bactérias/genética , Bactérias/isolamento & purificação , Doenças Transmissíveis/diagnóstico , Doenças Transmissíveis/microbiologia , Doenças Transmissíveis/virologia , DNA/genética , DNA/isolamento & purificação , Farmacorresistência Bacteriana Múltipla/genética , Fungos/genética , Fungos/isolamento & purificação , Helmintos/genética , Helmintos/isolamento & purificação , Interações Hospedeiro-Patógeno , Humanos , Ciência de Laboratório Médico/instrumentação , Metagenômica/instrumentação , Saúde Pública/tendências , Vírus/genética , Vírus/isolamento & purificação
16.
PLoS One ; 14(1): e0206194, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30629604

RESUMO

Preparation of high-quality sequencing libraries is a costly and time-consuming component of metagenomic next generation sequencing (mNGS). While the overall cost of sequencing has dropped significantly over recent years, the reagents needed to prepare sequencing samples are likely to become the dominant expense in the process. Furthermore, libraries prepared by hand are subject to human variability and needless waste due to limitations of manual pipetting volumes. Reduction of reaction volumes, combined with sub-microliter automated dispensing of reagents without consumable pipette tips, has the potential to provide significant advantages. Here, we describe the integration of several instruments, including the Labcyte Echo 525 acoustic liquid handler and the iSeq and NovaSeq Illumina sequencing platforms, to miniaturize and automate mNGS library preparation, significantly reducing the cost and the time required to prepare samples. Through the use of External RNA Controls Consortium (ERCC) spike-in RNAs, we demonstrated the fidelity of the miniaturized preparation to be equivalent to full volume reactions. Furthermore, detection of viral and microbial species from cell culture and patient samples was also maintained in the miniaturized libraries. For 384-well mNGS library preparations, we achieved cost savings of over 80% in materials and reagents alone, and reduced preparation time by 90% compared to manual approaches, without compromising quality or representation within the library.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Microquímica/métodos , Análise de Sequência de RNA/métodos , Automação Laboratorial , Redução de Custos , Estudos de Viabilidade , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Metagenômica/economia , Metagenômica/instrumentação , Microquímica/economia , Microquímica/instrumentação , Análise de Sequência de RNA/economia , Análise de Sequência de RNA/instrumentação
17.
Viruses ; 10(8)2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30110939

RESUMO

There has been an increase in the quest for metagenomics as an approach for the identification and study of the diversity of human viruses found in aquatic systems, both for their role as waterborne pathogens and as water quality indicators. In the last few years, environmental viral metagenomics has grown significantly and has enabled the identification, diversity and entire genome sequencing of viruses in environmental and clinical samples extensively. Prior to the arrival of metagenomics, traditional molecular procedures such as the polymerase chain reaction (PCR) and sequencing, were mostly used to identify and classify enteric viral species in different environmental milieu. After the advent of metagenomics, more detailed reports have emerged about the important waterborne viruses identified in wastewater treatment plant effluents and surface water. This paper provides a review of methods that have been used for the concentration, detection and identification of viral species from different environmental matrices. The review also takes into consideration where metagenomics has been explored in different African countries, as well as the limitations and challenges facing the approach. Procedures including sample processing, experimental design, sequencing technology, and bioinformatics analysis are discussed. The review concludes by summarising the current thinking and practices in the field and lays bare key issues that those venturing into this field need to consider and address.


Assuntos
Biologia Computacional/métodos , Genoma Viral , Metagenômica/métodos , Vírus/genética , África , Bibliometria , Biologia Computacional/estatística & dados numéricos , Água Doce/virologia , Água Subterrânea/virologia , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Metagenômica/instrumentação , Água do Mar/virologia , Viroses/diagnóstico , Viroses/virologia , Vírus/classificação , Vírus/isolamento & purificação , Águas Residuárias/virologia
18.
Methods ; 142: 89-99, 2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29684640

RESUMO

Assembly of reference-quality genomes from next-generation sequencing data is a key challenge in genomics. Recently, we and others have shown that Hi-C data can be used to address several outstanding challenges in the field of genome assembly. This principle has since been developed in academia and industry, and has been used in the assembly of several major genomes. In this paper, we explore the central principles underlying Hi-C-based assembly approaches, by quantitatively defining and characterizing three invariant Hi-C interaction patterns on which these approaches can build: Intrachromosomal interaction enrichment, distance-dependent interaction decay and local interaction smoothness. Specifically, we evaluate to what degree each invariant pattern holds on a single locus level in different species, cell types and Hi-C map resolutions. We find that these patterns are generally consistent across species and cell types but are affected by sequencing depth, and that matrix balancing improves consistency of loci with all three invariant patterns. Finally, we overview current Hi-C-based assembly approaches in light of these invariant patterns and demonstrate how local interaction smoothness can be used to easily detect scaffolding errors in extremely sparse Hi-C maps. We suggest that simultaneously considering all three invariant patterns may lead to better Hi-C-based genome assembly methods.


Assuntos
Mapeamento Cromossômico/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Modelos Genéticos , Anotação de Sequência Molecular/métodos , Animais , Mapeamento Cromossômico/instrumentação , DNA/química , DNA/genética , Genoma/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Imageamento Tridimensional/instrumentação , Imageamento Tridimensional/métodos , Metagenômica/instrumentação , Modelos Estatísticos , Imagem Molecular/instrumentação , Imagem Molecular/métodos , Conformação de Ácido Nucleico , Análise de Sequência de DNA/instrumentação , Análise de Sequência de DNA/métodos
19.
Elife ; 62017 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-28678007

RESUMO

Metagenomics and single-cell genomics have enabled genome discovery from unknown branches of life. However, extracting novel genomes from complex mixtures of metagenomic data can still be challenging and represents an ill-posed problem which is generally approached with ad hoc methods. Here we present a microfluidic-based mini-metagenomic method which offers a statistically rigorous approach to extract novel microbial genomes while preserving single-cell resolution. We used this approach to analyze two hot spring samples from Yellowstone National Park and extracted 29 new genomes, including three deeply branching lineages. The single-cell resolution enabled accurate quantification of genome function and abundance, down to 1% in relative abundance. Our analyses of genome level SNP distributions also revealed low to moderate environmental selection. The scale, resolution, and statistical power of microfluidic-based mini-metagenomics make it a powerful tool to dissect the genomic structure of microbial communities while effectively preserving the fundamental unit of biology, the single cell.


Assuntos
Fontes Termais/microbiologia , Dispositivos Lab-On-A-Chip , Metagenômica/instrumentação , Metagenômica/métodos , Microfluídica/instrumentação , Microfluídica/métodos , Biologia Computacional/métodos , Estados Unidos
20.
PLoS One ; 12(7): e0181452, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28715458

RESUMO

Metagenetics is a rapid and taxonomically comprehensive method for revealing community structures within environmental samples, based on large amounts of sequence data produced by high-throughput sequencers. Because community structures of planktonic copepods are important in the ocean owing to their high diversity and abundance, a metagenetic analysis of the 28S D2 region using Roche 454 was previously developed. However, the Illumina MiSeq platform with a high sequence output is being used more frequently in metagenetics, and non-calanoid copepods have not previously been fully evaluated. Here, we evaluated an Illumina MiSeq-based metagenetic analysis using a mock community and field-collected samples that were examined in a previous study using Roche 454, and the community structure, including non-calanoid copepods, was compared among morphological and metagenetic analyses. We removed a singleton read and applied an appropriate abundance threshold to remove erroneous Molecular Operational Taxonomic Units (MOTUs) with low-abundance sequences in the MiSeq-based analysis. Results showed that the copepod community was successfully characterized using Illumina MiSeq. Higher-quality sequences were obtained using MiSeq than by Roche 454, which reduced the overestimation of diversity, especially at a strict 99% similarity threshold for MOTU clustering. Taxonomic compositions in terms of both biomass and presence/absence of species, including non-calanoids, were more appropriately represented in the MiSeq- than in Roche 454-based analysis. Our data showed that metagenetic analysis using Illumina MiSeq is more useful for revealing copepod communities than Roche 454, owing to the lower cost and higher quality.


Assuntos
Copépodes/genética , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Metagenoma/genética , Metagenômica/instrumentação , Plâncton/genética , Animais , Biodiversidade , Classificação , Biologia Computacional , Oceanos e Mares
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