ArticleBMC genomics2018
Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system.
Article in BMC genomics, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers, 1 of them a synthesis that pooled it.
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
12 citing papers in PubMed, 1 synthesis or guideline pooled it, 46 citations in OpenAlex.
- Improved high-throughput MHC typing for non-model species using long-read sequencing.Molecular ecology resources · 2022Pooled it
- Molecular and pharmacological evaluation of rare, cystic fibrosis-causing missense mutations of the CFTR channel.The Journal of physiology · 2025Article
- A detailed analysis of second and third-generation sequencing approaches for accurate length determination of short tandem repeats and homopolymers.Nucleic acids research · 2025Article
- The Evolution of Next-Generation Sequencing Technologies.Methods in molecular biology (Clifton, N.J.) · 2025Review
- The performance of homopolymer detection using dichromatic and tetrachromatic fluorogenic next-generation sequencing platforms.BMC genomics · 2024Article
- Tatajuba: exploring the distribution of homopolymer tracts.NAR genomics and bioinformatics · 2022Article
- Spatio-temporal dynamics of intra-host variability in SARS-CoV-2 genomes.Nucleic acids research · 2022Article
- Assessment of Microsatellite Instability from Next-Generation Sequencing Data.Advances in experimental medicine and biology · 2022Article
- A Novel Algorithm for Detecting Microsatellite Instability Based on Next-Generation Sequencing Data.Frontiers in oncology · 2022Article
- SARS-CoV-2 genetic variations associated with COVID-19 pathogenicity.Microbial genomics · 2021Article
- Durable Near-Complete Response to Olaparib Plus Temozolomide and Radiation in a Patient WithJCO precision oncology · 2020Article
- Polymorphisms in Fc Gamma Receptors and Susceptibility to Malaria in an Endemic Population.Frontiers in immunology · 2020Review
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
9 authors at 2 institutions in 2 countries.
Funding
Abstract
backgroundCurrent 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.
resultsIn 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.
conclusionsOur 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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.