Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system
Identifieur interne : 000A63 ( Main/Exploration ); précédent : 000A62; suivant : 000A64Analytical 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 [République tchèque] ; Milan Macek [République tchèque] ; Attila Horváth [Hongrie] ; István BaloghSource :
- BMC Genomics [ 1471-2164 ] ; 2018.
Descripteurs français
- KwdFr :
- MESH :
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.
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
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
The online version of this article (10.1186/s12864-018-4544-x) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1186/s12864-018-4544-x
PubMed: 29466940
PubMed Central: 5822529
Affiliations:
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Le document en format XML
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<author><name sortKey="Krulisova, Veronika" sort="Krulisova, Veronika" uniqKey="Krulisova V" first="Veronika" last="Krulisova">Veronika Krulisova</name>
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<author><name sortKey="Horvath, Attila" sort="Horvath, Attila" uniqKey="Horvath A" first="Attila" last="Horváth">Attila Horváth</name>
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<author><name sortKey="Balogh, Istvan" sort="Balogh, Istvan" uniqKey="Balogh I" first="István" last="Balogh">István Balogh</name>
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<series><title level="j">BMC Genomics</title>
<idno type="eISSN">1471-2164</idno>
<imprint><date when="2018">2018</date>
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<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="KwdFr" xml:lang="fr"><term>Analyse de séquence d'ADN ()</term>
<term>Humains</term>
<term>Mucoviscidose (génétique)</term>
<term>Plasmides</term>
<term>Protéine CFTR (génétique)</term>
<term>Séquences répétées en tandem</term>
<term>Séquençage nucléotidique à haut débit ()</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="génétique" xml:lang="fr"><term>Mucoviscidose</term>
<term>Protéine CFTR</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>
<keywords scheme="MESH" xml:lang="fr"><term>Analyse de séquence d'ADN</term>
<term>Humains</term>
<term>Plasmides</term>
<term>Séquences répétées en tandem</term>
<term>Séquençage nucléotidique à haut débit</term>
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<front><div type="abstract" xml:lang="en"><sec><title>Background</title>
<p id="Par1">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.</p>
</sec>
<sec><title>Results</title>
<p id="Par2">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 <italic>CFTR</italic>
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.</p>
</sec>
<sec><title>Conclusions</title>
<p id="Par3">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 <italic>CFTR</italic>
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.</p>
</sec>
<sec><title>Electronic supplementary material</title>
<p>The online version of this article (10.1186/s12864-018-4544-x) contains supplementary material, which is available to authorized users.</p>
</sec>
</div>
</front>
<back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Templeton, Ar" uniqKey="Templeton A">AR Templeton</name>
</author>
<author><name sortKey="Clark, Ag" uniqKey="Clark A">AG Clark</name>
</author>
<author><name sortKey="Weiss, Km" uniqKey="Weiss K">KM Weiss</name>
</author>
<author><name sortKey="Nickerson, Da" uniqKey="Nickerson D">DA Nickerson</name>
</author>
<author><name sortKey="Boerwinkle, E" uniqKey="Boerwinkle E">E Boerwinkle</name>
</author>
<author><name sortKey="Sing, Cf" uniqKey="Sing C">CF Sing</name>
</author>
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