CovidSeniorV1, Main, Exploration, bibRecord, 000585

Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing.

Identifieur interne : 000585 ( Main/Exploration ); précédent : 000584; suivant : 000586

Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing.

Auteurs : Federica Alessandrini [Italie] ; Sara Caucci [Italie] ; Valerio Onofri [Italie] ; Filomena Melchionda [Italie] ; Adriano Tagliabracci [Italie] ; Patrizia Bagnarelli [Italie] ; Laura Di Sante [Italie] ; Chiara Turchi [Italie] ; Stefano Menzo [Italie]

Source :

Genes [ 2073-4425 ] ; 2020.

RBID : pubmed:32806776

Descripteurs français

KwdFr :
- Adulte (MeSH), Adulte d'âge moyen (MeSH), Amorces ADN (normes), Animaux (MeSH), Betacoronavirus (génétique), Betacoronavirus (pathogénicité), Cellules Vero (MeSH), Femelle (MeSH), Génome viral (MeSH), Humains (MeSH), Infections à coronavirus (virologie), Mâle (MeSH), Pandémies (MeSH), Pneumopathie virale (virologie), Réaction de polymérisation en chaîne (méthodes), Réaction de polymérisation en chaîne (normes), Sujet âgé (MeSH), Sujet âgé de 80 ans ou plus (MeSH), Séquençage du génome entier (méthodes), Séquençage du génome entier (normes).
MESH :
- génétique : Betacoronavirus.
- méthodes : Réaction de polymérisation en chaîne, Séquençage du génome entier.
- normes : Amorces ADN, Réaction de polymérisation en chaîne, Séquençage du génome entier.
- pathogénicité : Betacoronavirus.
- virologie : Infections à coronavirus, Pneumopathie virale.
- Adulte, Adulte d'âge moyen, Animaux, Cellules Vero, Femelle, Génome viral, Humains, Mâle, Pandémies, Sujet âgé, Sujet âgé de 80 ans ou plus.

English descriptors

KwdEn :
- Adult (MeSH), Aged (MeSH), Aged, 80 and over (MeSH), Animals (MeSH), Betacoronavirus (genetics), Betacoronavirus (pathogenicity), Chlorocebus aethiops (MeSH), Coronavirus Infections (virology), DNA Primers (standards), Female (MeSH), Genome, Viral (MeSH), Humans (MeSH), Male (MeSH), Middle Aged (MeSH), Pandemics (MeSH), Pneumonia, Viral (virology), Polymerase Chain Reaction (methods), Polymerase Chain Reaction (standards), Vero Cells (MeSH), Whole Genome Sequencing (methods), Whole Genome Sequencing (standards).
MESH :
- chemical , standards : DNA Primers.
- genetics : Betacoronavirus.
- methods : Polymerase Chain Reaction, Whole Genome Sequencing.
- pathogenicity : Betacoronavirus.
- standards : Polymerase Chain Reaction, Whole Genome Sequencing.
- virology : Coronavirus Infections, Pneumonia, Viral.
- Adult, Aged, Aged, 80 and over, Animals, Chlorocebus aethiops, Female, Genome, Viral, Humans, Male, Middle Aged, Pandemics, Vero Cells.

Abstract

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.

DOI: 10.3390/genes11080929
PubMed: 32806776
PubMed Central: PMC7463572

Affiliations:

Italie

Links toward previous steps (curation, corpus...)

to stream Main, to step Corpus: 000611
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Le document en format XML

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<term>Aged (MeSH)</term>
<term>Aged, 80 and over (MeSH)</term>
<term>Animals (MeSH)</term>
<term>Betacoronavirus (genetics)</term>
<term>Betacoronavirus (pathogenicity)</term>
<term>Chlorocebus aethiops (MeSH)</term>
<term>Coronavirus Infections (virology)</term>
<term>DNA Primers (standards)</term>
<term>Female (MeSH)</term>
<term>Genome, Viral (MeSH)</term>
<term>Humans (MeSH)</term>
<term>Male (MeSH)</term>
<term>Middle Aged (MeSH)</term>
<term>Pandemics (MeSH)</term>
<term>Pneumonia, Viral (virology)</term>
<term>Polymerase Chain Reaction (methods)</term>
<term>Polymerase Chain Reaction (standards)</term>
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<term>Animaux (MeSH)</term>
<term>Betacoronavirus (génétique)</term>
<term>Betacoronavirus (pathogénicité)</term>
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<term>Femelle (MeSH)</term>
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<term>Sujet âgé (MeSH)</term>
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<term>Pneumopathie virale</term>
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<term>Adulte d'âge moyen</term>
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<term>Cellules Vero</term>
<term>Femelle</term>
<term>Génome viral</term>
<term>Humains</term>
<term>Mâle</term>
<term>Pandémies</term>
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<front><div type="abstract" xml:lang="en">Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.</div>
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<ELocationID EIdType="pii" ValidYN="Y">E929</ELocationID>
<ELocationID EIdType="doi" ValidYN="Y">10.3390/genes11080929</ELocationID>
<Abstract><AbstractText>Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.</AbstractText>
</Abstract>
<AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Alessandrini</LastName>
<ForeName>Federica</ForeName>
<Initials>F</Initials>
<AffiliationInfo><Affiliation>Legal Medicine Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Torrette, 60126 Ancona, Italy.</Affiliation>
</AffiliationInfo>
</Author>
<Author ValidYN="Y"><LastName>Caucci</LastName>
<ForeName>Sara</ForeName>
<Initials>S</Initials>
<AffiliationInfo><Affiliation>Virology Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Torrette, 60126 Ancona, Italy.</Affiliation>
</AffiliationInfo>
</Author>
<Author ValidYN="Y"><LastName>Onofri</LastName>
<ForeName>Valerio</ForeName>
<Initials>V</Initials>
<AffiliationInfo><Affiliation>Legal Medicine Unit, AOU Ospedali Riuniti, Torrette, 60126 Ancona, Italy.</Affiliation>
</AffiliationInfo>
</Author>
<Author ValidYN="Y"><LastName>Melchionda</LastName>
<ForeName>Filomena</ForeName>
<Initials>F</Initials>
<AffiliationInfo><Affiliation>Legal Medicine Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Torrette, 60126 Ancona, Italy.</Affiliation>
</AffiliationInfo>
</Author>
<Author ValidYN="Y"><LastName>Tagliabracci</LastName>
<ForeName>Adriano</ForeName>
<Initials>A</Initials>
<AffiliationInfo><Affiliation>Legal Medicine Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Torrette, 60126 Ancona, Italy.</Affiliation>
</AffiliationInfo>
</Author>
<Author ValidYN="Y"><LastName>Bagnarelli</LastName>
<ForeName>Patrizia</ForeName>
<Initials>P</Initials>
<AffiliationInfo><Affiliation>Virology Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Torrette, 60126 Ancona, Italy.</Affiliation>
</AffiliationInfo>
</Author>
<Author ValidYN="Y"><LastName>Di Sante</LastName>
<ForeName>Laura</ForeName>
<Initials>L</Initials>
<AffiliationInfo><Affiliation>Virology Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Torrette, 60126 Ancona, Italy.</Affiliation>
</AffiliationInfo>
</Author>
<Author ValidYN="Y"><LastName>Turchi</LastName>
<ForeName>Chiara</ForeName>
<Initials>C</Initials>
<Identifier Source="ORCID">0000-0001-9211-810X</Identifier>
<AffiliationInfo><Affiliation>Legal Medicine Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Torrette, 60126 Ancona, Italy.</Affiliation>
</AffiliationInfo>
</Author>
<Author ValidYN="Y"><LastName>Menzo</LastName>
<ForeName>Stefano</ForeName>
<Initials>S</Initials>
<Identifier Source="ORCID">0000-0002-4425-3750</Identifier>
<AffiliationInfo><Affiliation>Virology Unit, Department of Biomedical Sciences and Public Health, Polytechnic University of Marche, Ancona, Torrette, 60126 Ancona, Italy.</Affiliation>
</AffiliationInfo>
</Author>
</AuthorList>
<Language>eng</Language>
<PublicationTypeList><PublicationType UI="D023362">Evaluation Study</PublicationType>
<PublicationType UI="D016428">Journal Article</PublicationType>
<PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType>
</PublicationTypeList>
<ArticleDate DateType="Electronic"><Year>2020</Year>
<Month>08</Month>
<Day>12</Day>
</ArticleDate>
</Article>
<MedlineJournalInfo><Country>Switzerland</Country>
<MedlineTA>Genes (Basel)</MedlineTA>
<NlmUniqueID>101551097</NlmUniqueID>
<ISSNLinking>2073-4425</ISSNLinking>
</MedlineJournalInfo>
<ChemicalList><Chemical><RegistryNumber>0</RegistryNumber>
<NameOfSubstance UI="D017931">DNA Primers</NameOfSubstance>
</Chemical>
</ChemicalList>
<SupplMeshList><SupplMeshName Type="Disease" UI="C000657245">COVID-19</SupplMeshName>
<SupplMeshName Type="Organism" UI="C000656484">severe acute respiratory syndrome coronavirus 2</SupplMeshName>
</SupplMeshList>
<CitationSubset>IM</CitationSubset>
<MeshHeadingList><MeshHeading><DescriptorName UI="D000328" MajorTopicYN="N">Adult</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D000368" MajorTopicYN="N">Aged</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D000369" MajorTopicYN="N">Aged, 80 and over</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D000818" MajorTopicYN="N">Animals</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D000073640" MajorTopicYN="N">Betacoronavirus</DescriptorName>
<QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName>
<QualifierName UI="Q000472" MajorTopicYN="N">pathogenicity</QualifierName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D002522" MajorTopicYN="N">Chlorocebus aethiops</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D018352" MajorTopicYN="N">Coronavirus Infections</DescriptorName>
<QualifierName UI="Q000821" MajorTopicYN="Y">virology</QualifierName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D017931" MajorTopicYN="N">DNA Primers</DescriptorName>
<QualifierName UI="Q000592" MajorTopicYN="N">standards</QualifierName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D005260" MajorTopicYN="N">Female</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D016679" MajorTopicYN="N">Genome, Viral</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D006801" MajorTopicYN="N">Humans</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D008297" MajorTopicYN="N">Male</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D008875" MajorTopicYN="N">Middle Aged</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D058873" MajorTopicYN="N">Pandemics</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D011024" MajorTopicYN="N">Pneumonia, Viral</DescriptorName>
<QualifierName UI="Q000821" MajorTopicYN="Y">virology</QualifierName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D016133" MajorTopicYN="N">Polymerase Chain Reaction</DescriptorName>
<QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName>
<QualifierName UI="Q000592" MajorTopicYN="N">standards</QualifierName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D014709" MajorTopicYN="N">Vero Cells</DescriptorName>
</MeshHeading>
<MeshHeading><DescriptorName UI="D000073336" MajorTopicYN="N">Whole Genome Sequencing</DescriptorName>
<QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName>
<QualifierName UI="Q000592" MajorTopicYN="N">standards</QualifierName>
</MeshHeading>
</MeshHeadingList>
<KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="Y">COVID-19</Keyword>
<Keyword MajorTopicYN="Y">SARS-CoV-2</Keyword>
<Keyword MajorTopicYN="Y">massively parallel sequencing</Keyword>
<Keyword MajorTopicYN="Y">nasopharyngeal swab</Keyword>
<Keyword MajorTopicYN="Y">viral genome</Keyword>
</KeywordList>
</MedlineCitation>
<PubmedData><History><PubMedPubDate PubStatus="received"><Year>2020</Year>
<Month>07</Month>
<Day>01</Day>
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<PubMedPubDate PubStatus="revised"><Year>2020</Year>
<Month>07</Month>
<Day>30</Day>
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<PubMedPubDate PubStatus="accepted"><Year>2020</Year>
<Month>08</Month>
<Day>10</Day>
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<Day>19</Day>
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<affiliations><list><country><li>Italie</li>
</country>
</list>
<tree><country name="Italie"><noRegion><name sortKey="Alessandrini, Federica" sort="Alessandrini, Federica" uniqKey="Alessandrini F" first="Federica" last="Alessandrini">Federica Alessandrini</name>
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<name sortKey="Bagnarelli, Patrizia" sort="Bagnarelli, Patrizia" uniqKey="Bagnarelli P" first="Patrizia" last="Bagnarelli">Patrizia Bagnarelli</name>
<name sortKey="Caucci, Sara" sort="Caucci, Sara" uniqKey="Caucci S" first="Sara" last="Caucci">Sara Caucci</name>
<name sortKey="Di Sante, Laura" sort="Di Sante, Laura" uniqKey="Di Sante L" first="Laura" last="Di Sante">Laura Di Sante</name>
<name sortKey="Melchionda, Filomena" sort="Melchionda, Filomena" uniqKey="Melchionda F" first="Filomena" last="Melchionda">Filomena Melchionda</name>
<name sortKey="Menzo, Stefano" sort="Menzo, Stefano" uniqKey="Menzo S" first="Stefano" last="Menzo">Stefano Menzo</name>
<name sortKey="Onofri, Valerio" sort="Onofri, Valerio" uniqKey="Onofri V" first="Valerio" last="Onofri">Valerio Onofri</name>
<name sortKey="Tagliabracci, Adriano" sort="Tagliabracci, Adriano" uniqKey="Tagliabracci A" first="Adriano" last="Tagliabracci">Adriano Tagliabracci</name>
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Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing.

Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing.

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