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Article ; Online: Agnostic Sequencing for Detection of Viral Pathogens.

Gauthier, Nick P G / Chorlton, Samuel D / Krajden, Mel / Manges, Amee R

2023 Volume 36, Issue 1, Page(s) e0011922

Abstract: The advent of next-generation sequencing (NGS) technologies has expanded our ability to detect and analyze microbial genomes and has yielded novel molecular approaches for infectious disease diagnostics. While several targeted multiplex PCR and NGS-based ...

Abstract	The advent of next-generation sequencing (NGS) technologies has expanded our ability to detect and analyze microbial genomes and has yielded novel molecular approaches for infectious disease diagnostics. While several targeted multiplex PCR and NGS-based assays have been widely used in public health settings in recent years, these targeted approaches are limited in that they still rely on
MeSH term(s)	Viruses/genetics ; High-Throughput Nucleotide Sequencing/methods ; Bacteria/genetics ; Metagenomics/methods ; Genome, Viral/genetics
Language	English
Publishing date	2023-02-27
Publishing country	United States
Document type	Journal Article ; Review ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	645015-5
ISSN	1098-6618 ; 0893-8512
ISSN (online)	1098-6618
ISSN	0893-8512
DOI	10.1128/cmr.00119-22
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Article ; Online: Validation of an automated, end-to-end metagenomic sequencing assay for agnostic detection of respiratory viruses.

Gauthier, Nick P G / Chan, Wilson / Locher, Kerstin / Smailus, Duane / Coope, Robin / Charles, Marthe / Jassem, Agatha / Kopetzky, Jennifer / Chorlton, Samuel D / Manges, Amee R

The Journal of infectious diseases

2024

Abstract: Background: Current molecular diagnostics are limited in the number and type of detectable pathogens. Metagenomic next generation sequencing (mNGS) is an emerging, and increasingly feasible, pathogen-agnostic diagnostic approach. Translational barriers ... ...

Abstract	Background: Current molecular diagnostics are limited in the number and type of detectable pathogens. Metagenomic next generation sequencing (mNGS) is an emerging, and increasingly feasible, pathogen-agnostic diagnostic approach. Translational barriers prohibit the widespread adoption of this technology in clinical laboratories. We validate an end-to-end mNGS assay for detection of respiratory viruses. Our assay is optimized to reduce turnaround time, lower cost-per-sample, increase throughput, and deploy secure and actionable bioinformatic results. Methods: We validated our assay using residual nasopharyngeal swab specimens from Vancouver General Hospital (n = 359), RT-PCR-positive, or negative for Influenza, SARS-CoV-2, and RSV. We quantified sample stability, assay precision, the effect of background nucleic acid levels, and analytical limits of detection. Diagnostic performance metrics were estimated. Results: We report that our mNGS assay is highly precise, semi-quantitative, with analytical limits of detection ranging from 103-104 copies/mL. Our assay is highly specific (100%) and sensitive (61.9% Overall: 86.8%; RT-PCR Ct < 30). Multiplexing capabilities enable processing of up to 55-specimens simultaneously on an Oxford Nanopore GridION device, with results reported within 12-hours. Conclusions: This study outlines the diagnostic performance and feasibility of mNGS for respiratory viral diagnostics, infection control, and public health surveillance. We addressed translational barriers to widespread mNGS adoption.
Language	English
Publishing date	2024-05-02
Publishing country	United States
Document type	Journal Article
ZDB-ID	3019-3
ISSN	1537-6613 ; 0022-1899
ISSN (online)	1537-6613
ISSN	0022-1899
DOI	10.1093/infdis/jiae226
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Article ; Online: A Molecular Analysis of Microalgae from Around the Globe to Revise Raphidonema (Trebouxiophyceae, Chlorophyta).

Yakimovich, Kurt M / Gauthier, Nick P G / Engstrom, Casey B / Leya, Thomas / Quarmby, Lynne M

Journal of phycology

2021 Volume 57, Issue 5, Page(s) 1419–1432

Abstract: We isolated five microalgal strains from alpine snow near Vancouver, Canada, which display morphological features suggestive of the genera Koliella and Raphidonema. Due to variations in cell size and shape, we could not make a clear delimitation based on ...

Abstract	We isolated five microalgal strains from alpine snow near Vancouver, Canada, which display morphological features suggestive of the genera Koliella and Raphidonema. Due to variations in cell size and shape, we could not make a clear delimitation based on morphology. We proceeded to a molecular analysis and included 22 strains from the CCCryo culture collection, previously identified as members of four closely related genera: Raphidonema, Koliella, Stichococcus, and Pseudochlorella. For greater taxonomic context in our phylogenetic analysis, we also obtained authentic strains for the type species of Koliella and Pseudochlorella, but were unable to find one for Raphidonema. To examine generic boundaries, we did a phylogenetic analysis on the rbcL gene for all strains, establishing distinct lineages. Our novel isolates fell within Raphidonema, and so we analyzed the ITS2 gene of all Raphidonema strains to delimit species. To support species delimitations, we did a Compensatory Base Change analysis using the secondary structure of the ITS2 gene to assist in aligning the sequence. We also computed a maximum likelihood phylogenetic tree to examine species clades of Raphidonema. We assigned epitypes for two Raphidonema species based on the best morphological match to strains in the ITS2 clades. We then amended their diagnoses so they can be more reliably identified using DNA sequence data. We also propose two new species, R. catena and R. monicae, that formed their own species clades according to our ITS2 analysis.
MeSH term(s)	Canada ; Chlorophyta/genetics ; Microalgae/genetics ; Phylogeny ; Sequence Analysis, DNA
Language	English
Publishing date	2021-06-25
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	281226-5
ISSN	1529-8817 ; 0022-3646
ISSN (online)	1529-8817
ISSN	0022-3646
DOI	10.1111/jpy.13183
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Article ; Online: Alterations in the nasopharyngeal microbiome associated with SARS-CoV-2 infection status and disease severity.

Gauthier, Nick P G / Locher, Kerstin / MacDonald, Clayton / Chorlton, Samuel D / Charles, Marthe / Manges, Amee R

PloS one

2022 Volume 17, Issue 10, Page(s) e0275815

Abstract: ... using the BugSeq 16S analysis pipeline.: Results: We found significant beta (PERMANOVA p < 0.05 ... but not alpha (Kruskal-Wallis p > 0.05) diversity differences in the nasopharyngeal microbiota ...

Abstract	Objectives: The COVID-19 pandemic and ensuing public health emergency has emphasized the need to study SARS-CoV-2 pathogenesis. The human microbiome has been shown to regulate the host immune system and may influence host susceptibility to viral infection, as well as disease severity. Several studies have assessed whether compositional alterations in the nasopharyngeal microbiota are associated with SARS-CoV-2 infection. However, the results of these studies were varied, and many did not account for disease severity. This study aims to examine whether compositional differences in the nasopharyngeal microbiota are associated with SARS-CoV-2 infection status and disease severity. Methods: We performed Nanopore full-length 16S rRNA sequencing on 194 nasopharyngeal swab specimens from hospitalized and community-dwelling SARS-CoV-2-infected and uninfected individuals. Sequence data analysis was performed using the BugSeq 16S analysis pipeline. Results: We found significant beta (PERMANOVA p < 0.05), but not alpha (Kruskal-Wallis p > 0.05) diversity differences in the nasopharyngeal microbiota among our study groups. We identified several differentially abundant taxa associated with SARS-CoV-2 infection status and disease severity using ALDEx2. Finally, we observed a trend towards higher abundance of Enterobacteriaceae in specimens from hospitalized SARS-CoV-2-infected patients. Conclusions: This study identified several alterations in the nasopharyngeal microbiome associated with SARS-CoV-2 infection status and disease severity. Understanding the role of the microbiome in infection susceptibility and severity may open new avenues of research for disease prevention and treatment.
MeSH term(s)	COVID-19 ; Humans ; Microbiota ; Nasopharynx ; Pandemics/prevention & control ; RNA, Ribosomal, 16S/genetics ; SARS-CoV-2 ; Severity of Illness Index
Chemical Substances	RNA, Ribosomal, 16S
Language	English
Publishing date	2022-10-14
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0275815
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Article ; Online: BugSplit enables genome-resolved metagenomics through highly accurate taxonomic binning of metagenomic assemblies

Induja Chandrakumar / Nick P. G. Gauthier / Cassidy Nelson / Michael B. Bonsall / Kerstin Locher / Marthe Charles / Clayton MacDonald / Mel Krajden / Amee R. Manges / Samuel D. Chorlton

Communications Biology, Vol 5, Iss 1, Pp 1-

2022 Volume 10

Abstract: A new computational method, BugSplit, teases out individual species’ genomes from metagenomic samples. The authors show that BugSplit is able to identify the presence of a novel coronavirus in COVID-19 patients using a database from 2019 predating the ... ...

Abstract	A new computational method, BugSplit, teases out individual species’ genomes from metagenomic samples. The authors show that BugSplit is able to identify the presence of a novel coronavirus in COVID-19 patients using a database from 2019 predating the pandemic and can separate host and pathogen sequences in other clinical samples with much higher specificity and accuracy than competing tools.
Keywords	Biology (General) ; QH301-705.5
Language	English
Publishing date	2022-02-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
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Article ; Online: BugSplit enables genome-resolved metagenomics through highly accurate taxonomic binning of metagenomic assemblies.

Chandrakumar, Induja / Gauthier, Nick P G / Nelson, Cassidy / Bonsall, Michael B / Locher, Kerstin / Charles, Marthe / MacDonald, Clayton / Krajden, Mel / Manges, Amee R / Chorlton, Samuel D

Communications biology

2022 Volume 5, Issue 1, Page(s) 151

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 ... ...

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 .
MeSH term(s)	Algorithms ; Bacteria/classification ; Bacteria/genetics ; COVID-19/epidemiology ; COVID-19/prevention & control ; COVID-19/virology ; Computational Biology/methods ; Humans ; Internet ; Metagenome/genetics ; Metagenomics/methods ; Nanopore Sequencing/methods ; Pandemics/prevention & control ; Reproducibility of Results ; SARS-CoV-2/classification ; SARS-CoV-2/genetics ; SARS-CoV-2/physiology
Language	English
Publishing date	2022-02-22
Publishing country	England
Document type	Journal Article
ISSN	2399-3642
ISSN (online)	2399-3642
DOI	10.1038/s42003-022-03114-4
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Article: A Molecular Analysis of Microalgae from Around the Globe to Revise Raphidonema (Trebouxiophyceae, Chlorophyta)

Yakimovich, Kurt M. / Gauthier, Nick P.G. / Engstrom, Casey B. / Leya, Thomas / Quarmby, Lynne M.

Journal of phycology. 2021 Oct., v. 57, no. 5

2021

Abstract	We isolated five microalgal strains from alpine snow near Vancouver, Canada, which display morphological features suggestive of the genera Koliella and Raphidonema. Due to variations in cell size and shape, we could not make a clear delimitation based on morphology. We proceeded to a molecular analysis and included 22 strains from the CCCryo culture collection, previously identified as members of four closely related genera: Raphidonema, Koliella, Stichococcus, and Pseudochlorella. For greater taxonomic context in our phylogenetic analysis, we also obtained authentic strains for the type species of Koliella and Pseudochlorella, but were unable to find one for Raphidonema. To examine generic boundaries, we did a phylogenetic analysis on the rbcL gene for all strains, establishing distinct lineages. Our novel isolates fell within Raphidonema, and so we analyzed the ITS2 gene of all Raphidonema strains to delimit species. To support species delimitations, we did a Compensatory Base Change analysis using the secondary structure of the ITS2 gene to assist in aligning the sequence. We also computed a maximum likelihood phylogenetic tree to examine species clades of Raphidonema. We assigned epitypes for two Raphidonema species based on the best morphological match to strains in the ITS2 clades. We then amended their diagnoses so they can be more reliably identified using DNA sequence data. We also propose two new species, R. catena and R. monicae, that formed their own species clades according to our ITS2 analysis.
Keywords	Raphidonema ; Stichococcus ; algology ; genes ; microalgae ; new species ; phylogeny ; snow ; statistical analysis ; Canada
Language	English
Dates of publication	2021-10
Size	p. 1419-1432.
Publishing place	John Wiley & Sons, Ltd
Document type	Article
Note	JOURNAL ARTICLE
ZDB-ID	281226-5
ISSN	1529-8817 ; 0022-3646
ISSN (online)	1529-8817
ISSN	0022-3646
DOI	10.1111/jpy.13183
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Alterations in the Nasopharyngeal Microbiome Associated with SARS-CoV-2 Infection Status and Disease Severity

Gauthier, Nick P.G / Locher, Kerstin / MacDonald, Clayton / Chorlton, Samuel D / Charles, Marthe / Manges, Amee R.

medRxiv

Abstract: ... using the BugSeq 16S analysis pipeline. Results: We found significant beta (PERMANOVA p < 0.05), but not ... alpha (Kruskal-Wallis p > 0.05) diversity differences in the nasopharyngeal microbiota among our study ...

Abstract	Objectives: The COVID-19 pandemic and ensuing public health emergency has emphasized the need to study SARS-CoV-2 pathogenesis. The human microbiome has been shown to regulate the host immune system and may influence host susceptibility to viral infection, as well as disease severity. Several studies have assessed whether compositional alterations in the nasopharyngeal microbiota are associated with SARS-CoV-2 infection. However, the results of these studies were varied, and many did not account for disease severity. This study aims to examine whether compositional differences in the nasopharyngeal microbiota are associated with SARS-CoV-2 infection status and disease severity. Methods: We performed Nanopore full-length 16S rRNA sequencing on 194 nasopharyngeal swab specimens from hospitalized and community-dwelling SARS-CoV-2-infected and uninfected individuals. Sequence data analysis was performed using the BugSeq 16S analysis pipeline. Results: We found significant beta (PERMANOVA p < 0.05), but not alpha (Kruskal-Wallis p > 0.05) diversity differences in the nasopharyngeal microbiota among our study groups. We identified several differentially abundant taxa associated with SARS-CoV-2 infection status and disease severity using ALDEx2. Finally, we observed a trend towards higher abundance of Enterobacteriaceae in specimens from hospitalized SARS-CoV-2-infected patients. Conclusions: This study identified several alterations in the nasopharyngeal microbiome associated with SARS-CoV-2 infection status and disease severity. Understanding the role of the microbiome in infection susceptibility and severity may open new avenues of research for disease prevention and treatment.
Keywords	covid19
Language	English
Publishing date	2022-06-13
Publisher	Cold Spring Harbor Laboratory Press
Document type	Article ; Online
DOI	10.1101/2022.06.13.22276358
Database	COVID19

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Article ; Online: Nanopore metagenomic sequencing for detection and characterization of SARS-CoV-2 in clinical samples.

Gauthier, Nick P G / Nelson, Cassidy / Bonsall, Michael B / Locher, Kerstin / Charles, Marthe / MacDonald, Clayton / Krajden, Mel / Chorlton, Samuel D / Manges, Amee R

PloS one

2021 Volume 16, Issue 11, Page(s) e0259712

Abstract: Objectives: The COVID-19 pandemic has underscored the need for rapid novel diagnostic strategies. Metagenomic Next-Generation Sequencing (mNGS) may allow for the detection of pathogens that can be missed in targeted assays. The goal of this study was to ...

Abstract	Objectives: The COVID-19 pandemic has underscored the need for rapid novel diagnostic strategies. Metagenomic Next-Generation Sequencing (mNGS) may allow for the detection of pathogens that can be missed in targeted assays. The goal of this study was to assess the performance of nanopore-based Sequence-Independent Single Primer Amplification (SISPA) for the detection and characterization of SARS-CoV-2. Methods: We performed mNGS on clinical samples and designed a diagnostic classifier that corrects for barcode crosstalk between specimens. Phylogenetic analysis was performed on genome assemblies. Results: Our assay yielded 100% specificity overall and 95.2% sensitivity for specimens with a RT-PCR cycle threshold value less than 30. We assembled 10 complete, and one near-complete genomes from 20 specimens that were classified as positive by mNGS. Phylogenetic analysis revealed that 10/11 specimens from British Columbia had a closest relative to another British Columbian specimen. We found 100% concordance between phylogenetic lineage assignment and Variant of Concern (VOC) PCR results. Our assay was able to distinguish between the Alpha and Gamma variants, which was not possible with the current standard VOC PCR being used in British Columbia. Conclusions: This study supports future work examining the broader feasibility of nanopore mNGS as a diagnostic strategy for the detection and characterization of viral pathogens.
MeSH term(s)	COVID-19/diagnosis ; Humans ; Metagenome ; Nanopore Sequencing/methods ; Pandemics ; SARS-CoV-2/isolation & purification ; Sensitivity and Specificity
Language	English
Publishing date	2021-11-18
Publishing country	United States
Document type	Evaluation Study ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0259712
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Article ; Online: Nanopore metagenomic sequencing for detection and characterization of SARS-CoV-2 in clinical samples.

Nick P G Gauthier / Cassidy Nelson / Michael B Bonsall / Kerstin Locher / Marthe Charles / Clayton MacDonald / Mel Krajden / Samuel D Chorlton / Amee R Manges

PLoS ONE, Vol 16, Iss 11, p e

2021 Volume 0259712

Abstract: Objectives The COVID-19 pandemic has underscored the need for rapid novel diagnostic strategies. Metagenomic Next-Generation Sequencing (mNGS) may allow for the detection of pathogens that can be missed in targeted assays. The goal of this study was to ... ...

Abstract	Objectives The COVID-19 pandemic has underscored the need for rapid novel diagnostic strategies. Metagenomic Next-Generation Sequencing (mNGS) may allow for the detection of pathogens that can be missed in targeted assays. The goal of this study was to assess the performance of nanopore-based Sequence-Independent Single Primer Amplification (SISPA) for the detection and characterization of SARS-CoV-2. Methods We performed mNGS on clinical samples and designed a diagnostic classifier that corrects for barcode crosstalk between specimens. Phylogenetic analysis was performed on genome assemblies. Results Our assay yielded 100% specificity overall and 95.2% sensitivity for specimens with a RT-PCR cycle threshold value less than 30. We assembled 10 complete, and one near-complete genomes from 20 specimens that were classified as positive by mNGS. Phylogenetic analysis revealed that 10/11 specimens from British Columbia had a closest relative to another British Columbian specimen. We found 100% concordance between phylogenetic lineage assignment and Variant of Concern (VOC) PCR results. Our assay was able to distinguish between the Alpha and Gamma variants, which was not possible with the current standard VOC PCR being used in British Columbia. Conclusions This study supports future work examining the broader feasibility of nanopore mNGS as a diagnostic strategy for the detection and characterization of viral pathogens.
Keywords	Medicine ; R ; Science ; Q
Subject code	572
Language	English
Publishing date	2021-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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