Nanopore metagenomic sequencing for detection and characterization of SARS-CoV-2 in clinical samples
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 Sequen...
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| creator | Gauthier, Nick P. G Nelson, Cassidy Bonsall, Michael B Locher, Kerstin Charles, Marthe MacDonald, Clayton Krajden, Mel Chorlton, Samuel D Manges, Amee R |
| description | 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. |
| doi_str_mv | 10.1371/journal.pone.0259712 |
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G ; Nelson, Cassidy ; Bonsall, Michael B ; Locher, Kerstin ; Charles, Marthe ; MacDonald, Clayton ; Krajden, Mel ; Chorlton, Samuel D ; Manges, Amee R</creator><contributor>Kalendar, Ruslan</contributor><creatorcontrib>Gauthier, Nick P. G ; Nelson, Cassidy ; Bonsall, Michael B ; Locher, Kerstin ; Charles, Marthe ; MacDonald, Clayton ; Krajden, Mel ; Chorlton, Samuel D ; Manges, Amee R ; Kalendar, Ruslan</creatorcontrib><description>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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0259712</identifier><identifier>PMID: 34793508</identifier><language>eng</language><publisher>San Francisco: Public Library of Science</publisher><subject>Analysis ; Assaying ; Bioinformatics ; Biology and life sciences ; Coronaviruses ; COVID-19 ; Crosstalk ; Diagnostic systems ; Disease control ; DNA sequencing ; Genomes ; Hospitals ; Infections ; Laboratories ; Medicine ; Medicine and health sciences ; Metagenomics ; Methods ; Next-generation sequencing ; Nucleotide sequencing ; Pandemics ; Pathogens ; Pathology ; Performance assessment ; Phylogeny ; Polymerase chain reaction ; Public health ; Research and Analysis Methods ; RNA polymerase ; Severe acute respiratory syndrome ; Severe acute respiratory syndrome coronavirus 2 ; Supervision ; Viral diseases ; Viral infections ; Viruses ; Zoology</subject><ispartof>PloS one, 2021-11, Vol.16 (11), p.e0259712-e0259712</ispartof><rights>COPYRIGHT 2021 Public Library of Science</rights><rights>2021 Gauthier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2021 Gauthier et al 2021 Gauthier et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c669t-f841f3a0a4009a20c32efd209b5004801b08e55cbf21189c5ffb9fbffd3e88a43</citedby><cites>FETCH-LOGICAL-c669t-f841f3a0a4009a20c32efd209b5004801b08e55cbf21189c5ffb9fbffd3e88a43</cites><orcidid>0000-0001-9891-8546 ; 0000-0003-2462-7249 ; 0000-0003-2567-8996</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601544/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8601544/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2102,2928,23866,27924,27925,53791,53793</link.rule.ids></links><search><contributor>Kalendar, Ruslan</contributor><creatorcontrib>Gauthier, Nick P. G</creatorcontrib><creatorcontrib>Nelson, Cassidy</creatorcontrib><creatorcontrib>Bonsall, Michael B</creatorcontrib><creatorcontrib>Locher, Kerstin</creatorcontrib><creatorcontrib>Charles, Marthe</creatorcontrib><creatorcontrib>MacDonald, Clayton</creatorcontrib><creatorcontrib>Krajden, Mel</creatorcontrib><creatorcontrib>Chorlton, Samuel D</creatorcontrib><creatorcontrib>Manges, Amee R</creatorcontrib><title>Nanopore metagenomic sequencing for detection and characterization of SARS-CoV-2 in clinical samples</title><title>PloS one</title><description>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.</description><subject>Analysis</subject><subject>Assaying</subject><subject>Bioinformatics</subject><subject>Biology and life sciences</subject><subject>Coronaviruses</subject><subject>COVID-19</subject><subject>Crosstalk</subject><subject>Diagnostic systems</subject><subject>Disease control</subject><subject>DNA sequencing</subject><subject>Genomes</subject><subject>Hospitals</subject><subject>Infections</subject><subject>Laboratories</subject><subject>Medicine</subject><subject>Medicine and health sciences</subject><subject>Metagenomics</subject><subject>Methods</subject><subject>Next-generation sequencing</subject><subject>Nucleotide sequencing</subject><subject>Pandemics</subject><subject>Pathogens</subject><subject>Pathology</subject><subject>Performance assessment</subject><subject>Phylogeny</subject><subject>Polymerase chain reaction</subject><subject>Public health</subject><subject>Research and Analysis Methods</subject><subject>RNA polymerase</subject><subject>Severe acute respiratory syndrome</subject><subject>Severe acute respiratory syndrome coronavirus 2</subject><subject>Supervision</subject><subject>Viral diseases</subject><subject>Viral infections</subject><subject>Viruses</subject><subject>Zoology</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2021</creationdate><recordtype>article</recordtype><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNk12L1DAUhoso7rr6DwQLgujFjEmTtOmNMAx-DCwu7OjehtP0pJOhk4xJK-qvN7NTZUf2QnKRcPLkTc6bc7LsOSVzyir6duvH4KCf773DOSlEXdHiQXZOa1bMyoKwh3fWZ9mTGLeECCbL8nF2xnhVM0HkedZ-Buf3PmC-wwE6dH5ndR7x24hOW9flxoe8xQH1YL3LwbW53kAAPWCwv-A26E2-XlyvZ0t_Myty63LdW2c19HmE3b7H-DR7ZKCP-GyaL7KvH95_WX6aXV59XC0XlzNdlvUwM5JTw4AAJ6SGgmhWoGkLUjeCEC4JbYhEIXRjCkplrYUxTW0aY1qGUgJnF9mLo-6-91FNBkWVvKmJ4IyQRKyOROthq_bB7iD8VB6sug340CkIg9U9KsEoENa0WCHnpCpBCyZAk6Zi2NbCJK13021js8NWoxsC9CeipzvOblTnvytZEir44bmvJ4Hgk99xUDsbNfY9OPTj8d1Ucl6JhL78B70_u4nqICVgnfHpXn0QVYtSMsKFpDJR83uoNFpMf5-qydgUPznw5uRAYgb8MXQwxqhW6-v_Z69uTtlXd9gNQj9sou_HQ1HFU5AfQR18jAHNX5MpUYdm-OOGOjSDmpqB_Qasqvqu</recordid><startdate>20211118</startdate><enddate>20211118</enddate><creator>Gauthier, Nick P. 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G</au><au>Nelson, Cassidy</au><au>Bonsall, Michael B</au><au>Locher, Kerstin</au><au>Charles, Marthe</au><au>MacDonald, Clayton</au><au>Krajden, Mel</au><au>Chorlton, Samuel D</au><au>Manges, Amee R</au><au>Kalendar, Ruslan</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Nanopore metagenomic sequencing for detection and characterization of SARS-CoV-2 in clinical samples</atitle><jtitle>PloS one</jtitle><date>2021-11-18</date><risdate>2021</risdate><volume>16</volume><issue>11</issue><spage>e0259712</spage><epage>e0259712</epage><pages>e0259712-e0259712</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><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.</abstract><cop>San Francisco</cop><pub>Public Library of Science</pub><pmid>34793508</pmid><doi>10.1371/journal.pone.0259712</doi><tpages>e0259712</tpages><orcidid>https://orcid.org/0000-0001-9891-8546</orcidid><orcidid>https://orcid.org/0000-0003-2462-7249</orcidid><orcidid>https://orcid.org/0000-0003-2567-8996</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Analysis Assaying Bioinformatics Biology and life sciences Coronaviruses COVID-19 Crosstalk Diagnostic systems Disease control DNA sequencing Genomes Hospitals Infections Laboratories Medicine Medicine and health sciences Metagenomics Methods Next-generation sequencing Nucleotide sequencing Pandemics Pathogens Pathology Performance assessment Phylogeny Polymerase chain reaction Public health Research and Analysis Methods RNA polymerase Severe acute respiratory syndrome Severe acute respiratory syndrome coronavirus 2 Supervision Viral diseases Viral infections Viruses Zoology |
| title | Nanopore metagenomic sequencing for detection and characterization of SARS-CoV-2 in clinical samples |
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