Unveiling microbial diversity: harnessing long-read sequencing technology

Nature Methods, Год журнала: 2024, Номер 21(6), С. 954 - 966

Опубликована: Апрель 30, 2024

Язык: Английский

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Recommendations for the introduction of metagenomic high-throughput sequencing in clinical virology, part I: Wet lab procedure

Journal of Clinical Virology, Год журнала: 2020, Номер 134, С. 104691 - 104691

Опубликована: Ноя. 18, 2020

Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in variety sample types and infectious syndromes. mHTS still its early stages translating into clinical application. To support development, implementation standardization procedures virus diagnostics, European Society Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim ENNGS to bring together professionals involved viral diagnostics share methodologies experiences, develop application recommendations. This manuscript aims provide practical recommendations wet lab necessary give development validation laboratory methods, including quality assurance, control assessment protocols.

Язык: Английский

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Recommendations for the introduction of metagenomic next-generation sequencing in clinical virology, part II: bioinformatic analysis and reporting

Journal of Clinical Virology, Год журнала: 2021, Номер 138, С. 104812 - 104812

Опубликована: Март 26, 2021

Metagenomic next-generation sequencing (mNGS) is an untargeted technique for determination of microbial DNA/RNA sequences in a variety sample types from patients with infectious syndromes. mNGS still its early stages broader translation into clinical applications. To further support the development, implementation, optimization and standardization procedures virus diagnostics, European Society Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim ENNGS to bring together professionals involved viral diagnostics share methodologies experiences, develop application guidelines. Following publication Recommendations introduction virology, part I: wet lab procedure this journal, current manuscript aims provide practical recommendations bioinformatic analysis data reporting results clinicians.

Язык: Английский

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BugSeq: a highly accurate cloud platform for long-read metagenomic analyses

BMC Bioinformatics, Год журнала: 2021, Номер 22(1)

Опубликована: Март 25, 2021

Abstract Background As the use of nanopore sequencing for metagenomic analysis increases, tools capable performing long-read taxonomic classification (ie. determining composition a sample) in fast and accurate manner are needed. Existing were either designed short-read data (eg. Centrifuge), take days to analyse modern sequencer outputs MetaMaps) or suffer from suboptimal accuracy CDKAM). Additionally, all require command line expertise do not scale cloud. Results We present BugSeq, novel, highly classifier reads. evaluate BugSeq on simulated data, mock microbial communities real clinical samples. On ZymoBIOMICS Even Log communities, (F1 = 0.95 at species level) offers better read than MetaMaps 0.89–0.94) fraction time. significantly improves Centrifuge 0.79–0.93) CDKAM 0.91–0.94) while offering competitive run times. When applied 41 samples patients with lower respiratory tract infections, produces greater concordance microbiological culture qPCR compared “What’s In My Pot” analysis. Conclusion is deployed cloud easy scalable analyses. freely available non-commercial https://bugseq.com/free .

Язык: Английский

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The human “contaminome”: bacterial, viral, and computational contamination in whole genome sequences from 1000 families

Scientific Reports, Год журнала: 2022, Номер 12(1)

Опубликована: Июнь 14, 2022

Abstract The unmapped readspace of whole genome sequencing data tends to be large but is often ignored. We posit that it contains valuable signals both human infection and contamination. Using poorly aligned reads from sequences (WGS) over 1000 families nearly 5000 individuals, we present insights into common viral, bacterial, computational contamination plague studies. several notable results: (1) In addition known contaminants such as Epstein-Barr virus phiX, blood lymphocyte cell lines contain many other contaminants, likely originating storage, prep, pipelines. (2) Sequencing plate biological sample source a strongly influence profile. And, (3) Y-chromosome fragments not on the reference commonly mismap bacterial genomes. Both experiment-derived prominent in next-generation data. Such can compromise results WGS well metagenomics studies, standard protocols for identifying removing should developed ensure fidelity sequencing-based

Язык: Английский

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The Skin Microbiome: Current Techniques, Challenges, and Future Directions

Microorganisms, Год журнала: 2023, Номер 11(5), С. 1222 - 1222

Опубликована: Май 6, 2023

Skin acts as a barrier that promotes the colonization of bacteria, fungi, archaea, and viruses whose membership function may differ depending on various specialized niches or micro-environments skin. The group microorganisms inhabiting skin, also known skin microbiome, offers protection against pathogens while actively interacting with host's immune system. Some members microbiome can act opportunistic pathogens. is influenced by factors such site, birth mode, genetics, environment, products, conditions. association(s) health disease has (have) been identified characterized via culture-dependent culture-independent methods. Culture-independent methods (such high-throughput sequencing), in particular, have expanded our understanding microbiome's role maintaining promoting disease. However, intrinsic challenges associated low microbial biomass high host content samples hindered advancements field. In addition, limitations current collection extraction biases derived from sample preparation analysis significantly results conclusions many studies. Therefore, present review discusses technical processing samples, advantages disadvantages sequencing approaches, potential future areas focus for

Язык: Английский

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Evaluation of the Impact of Concentration and Extraction Methods on the Targeted Sequencing of Human Viruses from Wastewater

Environmental Science & Technology, Год журнала: 2024, Номер 58(19), С. 8239 - 8250

Опубликована: Май 1, 2024

Sequencing human viruses in wastewater is challenging due to their low abundance compared the total microbial background. This study impact of four virus concentration/extraction methods (Innovaprep, Nanotrap, Promega, and Solids extraction) on probe-capture enrichment for followed by sequencing. Different yielded distinct profiles. Innovaprep ultrafiltration (following solids removal) had highest sequencing sensitivity richness, resulting successful assembly several near-complete genomes. However, it was less sensitive detecting SARS-CoV-2 digital polymerase chain reaction (dPCR) Promega Nanotrap. Across all preparation methods, astroviruses polyomaviruses were most highly abundant viruses, rare. These findings suggest that success can be increased using reduce nontarget nucleic acids extract, though absolute concentration extracted acid, as indicated Qubit, targeted dPCR, may not directly related performance. Further, broadly panels capture viral diversity but risks losing signals specific low-abundance viruses. Overall, this highlights importance aligning wet lab bioinformatic with goals when employing from wastewater.

Язык: Английский

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Addressing the dynamic nature of reference data: a new nucleotide database for robust metagenomic classification

mSystems, Год журнала: 2025, Номер unknown

Опубликована: Март 20, 2025

ABSTRACT Accurate metagenomic classification relies on comprehensive, up-to-date, and validated reference databases. While the NCBI BLAST Nucleotide (nt) database, encompassing a vast collection of sequences from all domains life, represents an invaluable resource, its massive size—currently exceeding 10 12 nucleotides—and exponential growth pose significant challenges for researchers seeking to maintain current nt-based indices classification. Recognizing that no exist widely used Centrifuge classifier, last public version currently available was released in 2018, we addressed this critical gap by leveraging advanced high-performance computing resources. We present new Centrifuge-compatible nt databases, meticulously constructed using novel pipeline incorporating different quality control measures, including decontamination filtering. These measures demonstrably reduce spurious classifications, as shown through our reanalysis published data where Plasmodium annotations were dramatically reduced decontaminated highlighting how database can significantly impact research conclusions. Through temporal comparisons, also reveal approach minimizes inconsistencies taxonomic assignments stemming asynchronous updates between sequence taxonomy discrepancies are particularly evident taxa such Listeria monocytogenes Naegleria fowleri , accuracy varied across versions. made pre-built indexes, respond need open, robust, metagenomics. Applications environmental metagenomics, forensics, clinical which require comprehensive coverage, will benefit resource. Our work highlights importance treating databases dynamic entities, subject ongoing validation akin software development best practices. This is crucial ensuring reliability analysis, especially continue expand size complexity. IMPORTANCE Accurately identifying diverse microbes sample, whether human gut, soil or crime scene, fields ranging medicine science. Researchers rely DNA match sequenced fragments known microbial species. However, while vast, poses challenges. Its makes it difficult many use effectively with classifiers, contamination within identification. addresses these providing cleaned, updated, specifically optimized tool. resource reduces errors improves identification groups. Moreover, readily usable overcome barrier, enabling leverage full potential analysis. findings underscore treat emphasizing continuous versioning essential practices robust reproducible metagenomics research.

Язык: Английский

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Deep viral blood metagenomics reveals extensive anellovirus diversity in healthy humans

Scientific Reports, Год журнала: 2021, Номер 11(1)

Опубликована: Март 25, 2021

Abstract Human blood metagenomics has revealed the presence of different types viruses in apparently healthy subjects. By far, anelloviruses constitute viral family that is more frequently found human blood, although amplification biases and contaminations pose a major challenge this field. To investigate further, we subjected pooled plasma samples from 120 donors Spain to high-speed centrifugation, RNA DNA extraction, random amplification, massive parallel sequencing. Our results confirm extensive such samples, which represented nearly 97% total sequence reads obtained. We assembled 114 genomes belonging family, revealing remarkable diversity. Phylogenetic analysis ORF1 suggested 28 potentially novel anellovirus species, 24 were validated by Sanger sequencing discard artifacts. These findings underscore importance implementing efficient purification procedures enrich fraction as an essential step virome studies question pathological role anelloviruses.

Язык: Английский

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Improved diagnosis of viral encephalitis in adult and pediatric hematological patients using viral metagenomics

Journal of Clinical Virology, Год журнала: 2020, Номер 130, С. 104566 - 104566

Опубликована: Июль 31, 2020

Metagenomic sequencing is a powerful technique that enables detection of the full spectrum pathogens present in any specimen single test. Hence, metagenomics increasingly being applied for viruses clinical cases with suspected infections unknown etiology and large number relevant potential causes. This typically case patients presenting encephalitis, particular when immunity impaired by underlying disorders. In this study, viral has been to cohort hematological encephalitis origin. Because loads cerebrospinal fluid are generally low, technical performance metagenomic protocol enrichment capture probes targeting all known vertebrate sequences was studied. Subsequently, optimized Viral increased sequence read count on samples 100 - 10.000 fold, compared unenriched sequencing. five out 41 (12%) virus detected which had not current routine diagnostics. BK polyomavirus, hepatitis E virus, human herpes virus-6 Epstein Barr were identified unbiased approach. study demonstrated origin may benefit from early testing as step

Язык: Английский

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