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1.
Commun Biol ; 5(1): 151, 2022 02 22.
Article in English | MEDLINE | ID: covidwho-1708032

ABSTRACT

A large gap remains between sequencing a microbial community and characterizing all of the organisms inside of it. Here we develop a novel method to taxonomically bin metagenomic assemblies through alignment of contigs against a reference database. We show that this workflow, BugSplit, bins metagenome-assembled contigs to species with a 33% absolute improvement in F1-score when compared to alternative tools. We perform nanopore mNGS on patients with COVID-19, and using a reference database predating COVID-19, demonstrate that BugSplit's taxonomic binning enables sensitive and specific detection of a novel coronavirus not possible with other approaches. When applied to nanopore mNGS data from cases of Klebsiella pneumoniae and Neisseria gonorrhoeae infection, BugSplit's taxonomic binning accurately separates pathogen sequences from those of the host and microbiota, and unlocks the possibility of sequence typing, in silico serotyping, and antimicrobial resistance prediction of each organism within a sample. BugSplit is available at https://bugseq.com/academic .


Subject(s)
Algorithms , Bacteria/genetics , Computational Biology/methods , Metagenome/genetics , Metagenomics/methods , Nanopore Sequencing/methods , Bacteria/classification , COVID-19/epidemiology , COVID-19/prevention & control , COVID-19/virology , Humans , Internet , Pandemics/prevention & control , Reproducibility of Results , SARS-CoV-2/classification , SARS-CoV-2/genetics , SARS-CoV-2/physiology
2.
Adv Sci (Weinh) ; 8(23): e2102593, 2021 12.
Article in English | MEDLINE | ID: covidwho-1559092

ABSTRACT

Fast and accurate identification of microbial pathogens is critical for the proper treatment of infections. Traditional culture-based diagnosis in clinics is increasingly supplemented by metagenomic next-generation-sequencing (mNGS). Here, RNA/cDNA-targeted sequencing (meta-transcriptomics using NGS (mtNGS)) is established to reduce the host nucleotide percentage in clinic samples and by combining with Oxford Nanopore Technology (ONT) platforms (meta-transcriptomics using third-generation sequencing, mtTGS) to improve the sequencing time. It shows that mtNGS improves the ratio of microbial reads, facilitates bacterial identification using multiple-strategies, and discovers fungi, viruses, and antibiotic resistance genes, and displaying agreement with clinical findings. Furthermore, longer reads in mtTGS lead to additional improvement in pathogen identification and also accelerate the clinical diagnosis. Additionally, primary tests utilizing direct-RNA sequencing and targeted sequencing of ONT show that ONT displays important potential but must be further developed. This study presents the potential of RNA-targeted pathogen identification in clinical samples, especially when combined with the newest developments in ONT.


Subject(s)
Bronchoalveolar Lavage Fluid/microbiology , High-Throughput Nucleotide Sequencing/methods , Infections/genetics , Metagenomics/methods , RNA/genetics , Sequence Analysis, RNA/methods , Aged , Bronchoalveolar Lavage/methods , Female , Humans , Male , Metagenome/genetics , Middle Aged
3.
NPJ Biofilms Microbiomes ; 7(1): 81, 2021 11 18.
Article in English | MEDLINE | ID: covidwho-1526078

ABSTRACT

The oral microbiome has been connected with lung health and may be of significance in the progression of SARS-CoV-2 infection. Saliva-based SARS-CoV-2 tests provide the opportunity to leverage stored samples for assessing the oral microbiome. However, these collection kits have not been tested for their accuracy in measuring the oral microbiome. Saliva is highly enriched with human DNA and reducing it prior to shotgun sequencing may increase the depth of bacterial reads. We examined both the effect of saliva collection method and sequence processing on measurement of microbiome depth and diversity by 16S rRNA gene amplicon and shotgun metagenomics. We collected 56 samples from 22 subjects. Each subject provided saliva samples with and without preservative, and a subset provided a second set of samples the following day. 16S rRNA gene (V4) sequencing was performed on all samples, and shotgun metagenomics was performed on a subset of samples collected with preservative with and without human DNA depletion before sequencing. We observed that the beta diversity distances within subjects over time was smaller than between unrelated subjects, and distances within subjects were smaller in samples collected with preservative. Samples collected with preservative had higher alpha diversity measuring both richness and evenness. Human DNA depletion before extraction and shotgun sequencing yielded higher total and relative reads mapping to bacterial sequences. We conclude that collecting saliva with preservative may provide more consistent measures of the oral microbiome and depleting human DNA increases yield of bacterial sequences.


Subject(s)
Microbiota/genetics , Saliva/microbiology , Adult , Bacteria/genetics , COVID-19/genetics , DNA/genetics , DNA, Bacterial/genetics , Female , Humans , Male , Metagenome/genetics , Metagenomics/methods , Middle Aged , RNA, Ribosomal, 16S/genetics , SARS-CoV-2/pathogenicity , Sequence Analysis, DNA/methods
4.
Viruses ; 13(10)2021 10 06.
Article in English | MEDLINE | ID: covidwho-1460085

ABSTRACT

According to various estimates, only a small percentage of existing viruses have been discovered, naturally much less being represented in the genomic databases. High-throughput sequencing technologies develop rapidly, empowering large-scale screening of various biological samples for the presence of pathogen-associated nucleotide sequences, but many organisms are yet to be attributed specific loci for identification. This problem particularly impedes viral screening, due to vast heterogeneity in viral genomes. In this paper, we present a new bioinformatic pipeline, VirIdAl, for detecting and identifying viral pathogens in sequencing data. We also demonstrate the utility of the new software by applying it to viral screening of the feces of bats collected in the Moscow region, which revealed a significant variety of viruses associated with bats, insects, plants, and protozoa. The presence of alpha and beta coronavirus reads, including the MERS-like bat virus, deserves a special mention, as it once again indicates that bats are indeed reservoirs for many viral pathogens. In addition, it was shown that alignment-based methods were unable to identify the taxon for a large proportion of reads, and we additionally applied other approaches, showing that they can further reveal the presence of viral agents in sequencing data. However, the incompleteness of viral databases remains a significant problem in the studies of viral diversity, and therefore necessitates the use of combined approaches, including those based on machine learning methods.


Subject(s)
Alphacoronavirus/isolation & purification , Betacoronavirus/isolation & purification , Chiroptera/virology , Genome, Viral/genetics , Metagenome/genetics , Alphacoronavirus/classification , Alphacoronavirus/genetics , Animals , Betacoronavirus/classification , Betacoronavirus/genetics , Chiroptera/genetics , Computational Biology/methods , Feces/virology , High-Throughput Nucleotide Sequencing , Metagenomics/methods , Moscow , Phycodnaviridae/classification , Phycodnaviridae/genetics , Phycodnaviridae/isolation & purification , Sequence Analysis, DNA
5.
Expert Rev Mol Diagn ; 21(11): 1139-1146, 2021 11.
Article in English | MEDLINE | ID: covidwho-1450340

ABSTRACT

INTRODUCTION: Meningoencephalitis patients are often severely impaired and benefit from early etiological diagnosis, though many cases remain without identified cause. Metagenomics as pathogen agnostic approach can result in additional etiological findings; however, the exact diagnostic yield when used as a secondary test remains unknown. AREAS COVERED: This review aims to highlight recent advances with regard to wet and dry lab methodologies of metagenomic testing and technical milestones that have been achieved. A selection of procedures currently applied in accredited diagnostic laboratories is described in more detail to illustrate best practices. Furthermore, a meta-analysis was performed to assess the additional diagnostic yield utilizing metagenomic sequencing in meningoencephalitis patients. Finally, the remaining challenges for successful widespread implementation of metagenomic sequencing for the diagnosis of meningoencephalitis are addressed in a future perspective. EXPERT OPINION: The last decade has shown major advances in technical possibilities for using mNGS in diagnostic settings including cloud-based analysis. An additional advance may be the current established infrastructure of platforms for bioinformatic analysis of SARS-CoV-2, which may assist to pave the way for global use of clinical metagenomics.


Subject(s)
Genome, Viral/genetics , Meningoencephalitis/diagnosis , Meningoencephalitis/virology , Metagenome/genetics , Diagnostic Tests, Routine , Humans , Metagenomics/methods
7.
J Med Virol ; 93(3): 1786-1791, 2021 03.
Article in English | MEDLINE | ID: covidwho-1196491

ABSTRACT

Pangolin metagenomic data obtained from public databases were used to assemble partial or complete viral genomes showing genetic relationship to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Sendai virus, flavivirus, picornavirus, parvovirus, and genomovirus, respectively. Most of these virus genomes showed genomic recombination signals. Phylogeny based on the SARS-CoV-2-related virus sequences assembled in this study and those recently published indicated that pangolin SARS-CoV-2-related viruses were clustered into two sub-lineages according to geographic sampling sites. These findings suggest the need for further pangolin samples, from different countries, to be collected and analyzed for coronavirus to elucidate whether pangolins are intermittent hosts for SARS-CoV-2.


Subject(s)
COVID-19/virology , Genome, Viral/genetics , Metagenome/genetics , Pangolins/virology , SARS-CoV-2/genetics , Animals , Host Specificity/genetics , Metagenomics/methods , Phylogeny , Recombination, Genetic/genetics
8.
Sci Rep ; 10(1): 3963, 2020 03 03.
Article in English | MEDLINE | ID: covidwho-659769

ABSTRACT

The diversity of pathogens associated with acute respiratory infection (ARI) makes diagnosis challenging. Traditional pathogen screening tests have a limited detection range and provide little additional information. We used total RNA sequencing ("meta-transcriptomics") to reveal the full spectrum of microbes associated with paediatric ARI. Throat swabs were collected from 48 paediatric ARI patients and 7 healthy controls. Samples were subjected to meta-transcriptomics to determine the presence and abundance of viral, bacterial, and eukaryotic pathogens, and to reveal mixed infections, pathogen genotypes/subtypes, evolutionary origins, epidemiological history, and antimicrobial resistance. We identified 11 RNA viruses, 4 DNA viruses, 4 species of bacteria, and 1 fungus. While most are known to cause ARIs, others, such as echovirus 6, are rarely associated with respiratory disease. Co-infection of viruses and bacteria and of multiple viruses were commonplace (9/48), with one patient harboring 5 different pathogens, and genome sequence data revealed large intra-species diversity. Expressed resistance against eight classes of antibiotic was detected, with those for MLS, Bla, Tet, Phe at relatively high abundance. In summary, we used a simple total RNA sequencing approach to reveal the complex polymicrobial infectome in ARI. This provided comprehensive and clinically informative information relevant to understanding respiratory disease.


Subject(s)
Metagenome/genetics , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virology , Bacteria/classification , Bacteria/genetics , Bacteria/pathogenicity , DNA Viruses/classification , DNA Viruses/genetics , DNA Viruses/pathogenicity , Drug Resistance, Microbial/genetics , Female , Fungi/classification , Fungi/genetics , Fungi/pathogenicity , Humans , Male , Phylogeny , RNA Viruses/classification , RNA Viruses/genetics , RNA Viruses/pathogenicity , Viruses/classification , Viruses/genetics , Viruses/pathogenicity
9.
Arch Virol ; 166(1): 207-212, 2021 Jan.
Article in English | MEDLINE | ID: covidwho-848346

ABSTRACT

In this study, we analyzed the viral population in oropharyngeal samples from T. brasiliensis using a viral metagenomic approach. Genomes corresponding to members of the families Circoviridae, Genomoviridae, Herpesviridae, Paramyxoviridae, Coronaviridae, and Astroviridae were detected. This study provides the first preliminary understanding of the oropharyngeal virome of T. brasiliensis, which may guide the discovery and isolation of novel viruses in the future and highlights the need for continuing investigations in this regard.


Subject(s)
Chiroptera/virology , Metagenome/genetics , Oropharynx/virology , Viruses/genetics , Animals , Brazil , Metagenomics/methods , Phylogeny
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