Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system.
Identifieur interne : 000995 ( PubMed/Corpus ); précédent : 000994; suivant : 000996Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system.
Auteurs : Gergely Ivády ; Lászl Madar ; Erika Dzsudzsák ; Katalin Koczok ; János Kappelmayer ; Veronika Krulisova ; Milan Macek ; Attila Horváth ; István BaloghSource :
- BMC genomics [ 1471-2164 ] ; 2018.
English descriptors
- KwdEn :
- MESH :
- chemical , genetics : Cystic Fibrosis Transmembrane Conductance Regulator.
- genetics : Cystic Fibrosis.
- methods : High-Throughput Nucleotide Sequencing, Sequence Analysis, DNA.
- Humans, Plasmids, Tandem Repeat Sequences.
Abstract
Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. Although pyro- and ion semiconductor sequencing both have the advantage of delivering long and high quality reads, problems might occur when sequencing homopolymer-containing regions, since the repeating identical bases are going to incorporate during the same synthesis cycle, which leads to uncertainty in base calling. The aim of this study was to evaluate the analytical performance of a pyrosequencing-based next-generation sequencing system in detecting homopolymer sequences using homopolymer-preintegrated plasmid constructs and human DNA samples originating from patients with cystic fibrosis.
DOI: 10.1186/s12864-018-4544-x
PubMed: 29466940
Links to Exploration step
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University Hospital Motol, Charles University, Prague, Czech Republic.</nlm:affiliation></affiliation></author><author><name sortKey="Macek, Milan" sort="Macek, Milan" uniqKey="Macek M" first="Milan" last="Macek">Milan Macek</name><affiliation><nlm:affiliation>Department of Biology and Medical Genetics, Second Faculty of Medicine and University Hospital Motol, Charles University, Prague, Czech Republic.</nlm:affiliation></affiliation></author><author><name sortKey="Horvath, Attila" sort="Horvath, Attila" uniqKey="Horvath A" first="Attila" last="Horváth">Attila Horváth</name><affiliation><nlm:affiliation>Genomic Medicine and Bioinformatic Core Facility, University of Debrecen, Debrecen, Hungary.</nlm:affiliation></affiliation></author><author><name sortKey="Balogh, Istvan" sort="Balogh, Istvan" uniqKey="Balogh I" first="István" last="Balogh">István Balogh</name><affiliation><nlm:affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, 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sort="Madar, Laszl" uniqKey="Madar L" first="Lászl" last="Madar">Lászl Madar</name><affiliation><nlm:affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary.</nlm:affiliation></affiliation></author><author><name sortKey="Dzsudzsak, Erika" sort="Dzsudzsak, Erika" uniqKey="Dzsudzsak E" first="Erika" last="Dzsudzsák">Erika Dzsudzsák</name><affiliation><nlm:affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary.</nlm:affiliation></affiliation></author><author><name sortKey="Koczok, Katalin" sort="Koczok, Katalin" uniqKey="Koczok K" first="Katalin" last="Koczok">Katalin Koczok</name><affiliation><nlm:affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary.</nlm:affiliation></affiliation></author><author><name sortKey="Kappelmayer, Janos" sort="Kappelmayer, Janos" uniqKey="Kappelmayer J" first="János" last="Kappelmayer">János Kappelmayer</name><affiliation><nlm:affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary.</nlm:affiliation></affiliation></author><author><name sortKey="Krulisova, Veronika" sort="Krulisova, Veronika" uniqKey="Krulisova V" first="Veronika" last="Krulisova">Veronika Krulisova</name><affiliation><nlm:affiliation>Department of Biology and Medical Genetics, Second Faculty of Medicine and University Hospital Motol, Charles University, Prague, Czech Republic.</nlm:affiliation></affiliation></author><author><name sortKey="Macek, Milan" sort="Macek, Milan" uniqKey="Macek M" first="Milan" last="Macek">Milan Macek</name><affiliation><nlm:affiliation>Department of Biology and Medical Genetics, Second Faculty of Medicine and University Hospital Motol, Charles University, Prague, Czech Republic.</nlm:affiliation></affiliation></author><author><name sortKey="Horvath, Attila" sort="Horvath, Attila" uniqKey="Horvath A" first="Attila" last="Horváth">Attila Horváth</name><affiliation><nlm:affiliation>Genomic Medicine and Bioinformatic Core Facility, University of Debrecen, Debrecen, Hungary.</nlm:affiliation></affiliation></author><author><name sortKey="Balogh, Istvan" sort="Balogh, Istvan" uniqKey="Balogh I" first="István" last="Balogh">István Balogh</name><affiliation><nlm:affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary. balogh@med.unideb.hu.</nlm:affiliation></affiliation></author></analytic><series><title level="j">BMC genomics</title><idno type="eISSN">1471-2164</idno><imprint><date when="2018" type="published">2018</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Cystic Fibrosis (genetics)</term><term>Cystic Fibrosis Transmembrane Conductance Regulator (genetics)</term><term>High-Throughput Nucleotide Sequencing (methods)</term><term>Humans</term><term>Plasmids</term><term>Sequence Analysis, DNA (methods)</term><term>Tandem Repeat Sequences</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Cystic Fibrosis Transmembrane Conductance Regulator</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Cystic Fibrosis</term></keywords><keywords scheme="MESH" qualifier="methods" xml:lang="en"><term>High-Throughput Nucleotide Sequencing</term><term>Sequence Analysis, DNA</term></keywords><keywords scheme="MESH" xml:lang="en"><term>Humans</term><term>Plasmids</term><term>Tandem Repeat Sequences</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. Although pyro- and ion semiconductor sequencing both have the advantage of delivering long and high quality reads, problems might occur when sequencing homopolymer-containing regions, since the repeating identical bases are going to incorporate during the same synthesis cycle, which leads to uncertainty in base calling. The aim of this study was to evaluate the analytical performance of a pyrosequencing-based next-generation sequencing system in detecting homopolymer sequences using homopolymer-preintegrated plasmid constructs and human DNA samples originating from patients with cystic fibrosis.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">29466940</PMID><DateCompleted><Year>2018</Year><Month>08</Month><Day>30</Day></DateCompleted><DateRevised><Year>2019</Year><Month>12</Month><Day>10</Day></DateRevised><Article PubModel="Electronic"><Journal><ISSN IssnType="Electronic">1471-2164</ISSN><JournalIssue CitedMedium="Internet"><Volume>19</Volume><Issue>1</Issue><PubDate><Year>2018</Year><Month>02</Month><Day>21</Day></PubDate></JournalIssue><Title>BMC genomics</Title><ISOAbbreviation>BMC Genomics</ISOAbbreviation></Journal><ArticleTitle>Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system.</ArticleTitle><Pagination><MedlinePgn>158</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1186/s12864-018-4544-x</ELocationID><Abstract><AbstractText Label="BACKGROUND">Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. Although pyro- and ion semiconductor sequencing both have the advantage of delivering long and high quality reads, problems might occur when sequencing homopolymer-containing regions, since the repeating identical bases are going to incorporate during the same synthesis cycle, which leads to uncertainty in base calling. The aim of this study was to evaluate the analytical performance of a pyrosequencing-based next-generation sequencing system in detecting homopolymer sequences using homopolymer-preintegrated plasmid constructs and human DNA samples originating from patients with cystic fibrosis.</AbstractText><AbstractText Label="RESULTS">In the plasmid system average correct genotyping was 95.8% in 4-mers, 87.4% in 5-mers and 72.1% in 6-mers. Despite the experienced low genotyping accuracy in 5- and 6-mers, it was possible to generate amplicons with more than a 90% adequate detection rate in every homopolymer tract. When homopolymers in the CFTR gene were sequenced average accuracy was 89.3%, but varied in a wide range (52.2 - 99.1%). In all but one case, an optimal amplicon-sequencing primer combination could be identified. In that single case (7A tract in exon 14 (c.2046_2052)), none of the tested primer sets produced the required analytical performance.</AbstractText><AbstractText Label="CONCLUSIONS">Our results show that pyrosequencing is the most reliable in case of 4-mers and as homopolymer length gradually increases, accuracy deteriorates. With careful primer selection, the NGS system was able to correctly genotype all but one of the homopolymers in the CFTR gene. In conclusion, we configured a plasmid test system that can be used to assess genotyping accuracy of NGS devices and developed an accurate NGS assay for the molecular diagnosis of CF using self-designed primers for amplification and sequencing.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Ivády</LastName><ForeName>Gergely</ForeName><Initials>G</Initials><AffiliationInfo><Affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Madar</LastName><ForeName>László</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Dzsudzsák</LastName><ForeName>Erika</ForeName><Initials>E</Initials><AffiliationInfo><Affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Koczok</LastName><ForeName>Katalin</ForeName><Initials>K</Initials><AffiliationInfo><Affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Division of Clinical Genetics, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Kappelmayer</LastName><ForeName>János</ForeName><Initials>J</Initials><AffiliationInfo><Affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Krulisova</LastName><ForeName>Veronika</ForeName><Initials>V</Initials><AffiliationInfo><Affiliation>Department of Biology and Medical Genetics, Second Faculty of Medicine and University Hospital Motol, Charles University, Prague, Czech Republic.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Macek</LastName><ForeName>Milan</ForeName><Initials>M</Initials><Suffix>Jr</Suffix><AffiliationInfo><Affiliation>Department of Biology and Medical Genetics, Second Faculty of Medicine and University Hospital Motol, Charles University, Prague, Czech Republic.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Horváth</LastName><ForeName>Attila</ForeName><Initials>A</Initials><AffiliationInfo><Affiliation>Genomic Medicine and Bioinformatic Core Facility, University of Debrecen, Debrecen, Hungary.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Balogh</LastName><ForeName>István</ForeName><Initials>I</Initials><AffiliationInfo><Affiliation>Department of Laboratory Medicine, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary. balogh@med.unideb.hu.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Division of Clinical Genetics, University of Debrecen, Nagyerdei krt. 98, Debrecen, H-4032, Hungary. balogh@med.unideb.hu.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>K109076</GrantID><Agency>Országos Tudományos Kutatási Alapprogramok</Agency><Country>International</Country></Grant><Grant><GrantID>00064203</GrantID><Agency>Ministerstvo Zdravotnictví Ceské Republiky</Agency><Country>International</Country></Grant><Grant><GrantID>CZ.2.16/3.1.00/24022OPPK</GrantID><Agency>European Regional Development Fund Prague</Agency><Country>International</Country></Grant><Grant><GrantID>NF-CZ11-PDP-3-003-2014, LM2015091 and COST-LD14073</GrantID><Agency>Norway Grants</Agency><Country>International</Country></Grant><Grant><GrantID>UNKP-16-3 IV/4</GrantID><Agency>New National Excellence Program of the Ministry of Human Capacities</Agency><Country>International</Country></Grant><Grant><GrantID>GINOP-2.3.2-15-2016-00039</GrantID><Agency>Ministry of National Economy, Hungary</Agency><Country>International</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType><PublicationType UI="D023361">Validation Study</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2018</Year><Month>02</Month><Day>21</Day></ArticleDate></Article><MedlineJournalInfo><Country>England</Country><MedlineTA>BMC Genomics</MedlineTA><NlmUniqueID>100965258</NlmUniqueID><ISSNLinking>1471-2164</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="C505032">CFTR protein, human</NameOfSubstance></Chemical><Chemical><RegistryNumber>126880-72-6</RegistryNumber><NameOfSubstance UI="D019005">Cystic Fibrosis Transmembrane Conductance Regulator</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D003550" MajorTopicYN="N">Cystic Fibrosis</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D019005" MajorTopicYN="N">Cystic Fibrosis Transmembrane Conductance Regulator</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D059014" MajorTopicYN="N">High-Throughput Nucleotide Sequencing</DescriptorName><QualifierName UI="Q000379" MajorTopicYN="Y">methods</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D006801" MajorTopicYN="N">Humans</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D010957" MajorTopicYN="N">Plasmids</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017422" MajorTopicYN="N">Sequence Analysis, 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| HfdSelect -Kh $EXPLOR_AREA/Data/PubMed/Corpus/biblio.hfd \
| NlmPubMed2Wicri -a MersV1
| This area was generated with Dilib version V0.6.33. |  |