Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F00216208%3A11130%2F18%3A10375363" target="_blank" >RIV/00216208:11130/18:10375363 - isvavai.cz</a>

Alternative codes found

RIV/00064203:_____/18:10375363

Result on the web

<a href="https://doi.org/10.1186/s12864-018-4544-x" target="_blank" >https://doi.org/10.1186/s12864-018-4544-x</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1186/s12864-018-4544-x" target="_blank" >10.1186/s12864-018-4544-x</a>

Result language

angličtina

Original language name

Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system

Original language description

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. 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. 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.

Czech name

—

Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10600 - Biological sciences

Project

<a href="/en/project/LM2015091" target="_blank" >LM2015091: National Center for Medical Genomic</a><br>

Continuities

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2018

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

BMC Genomics

ISSN

1471-2164

e-ISSN

—

Volume of the periodical

19

Issue of the periodical within the volume

1

Country of publishing house

GB - UNITED KINGDOM

Number of pages

8

Pages from-to

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UT code for WoS article

000426305200003

EID of the result in the Scopus database

2-s2.0-85042425048