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.
Highlights
Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors
While the pyrosequencing-based next generation sequencing (NGS) system was able to detect poly-A 6-mers reliably, detection rates fell under 75% for poly-C, poly-G and poly-T 6-mers
Despite the great variability in sequencing accuracy, we found usable excellent sensitivity and specificity, because all benign and pathogenic variants were confirmed by pyrosequencing using the in-house designed primer set both in HP and non-HP containing regions
Summary
Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. 1.43 million HPs ( known as mononucleotide microsatellites) exist in the human exome, with the size of 4-mer and up. This means that an average of eight such HP sequences can be found in every exon. HP sequences composed of A:T base pairs are over-represented in the human genome compared to G:C HPs [3,4,5]. Both pairs show structural stability [6, 7], these loci in the genome are highly mutagenic and have been characterized as hotspots for length change mutations [8,9,10], which has, presumably, contributed to their reduced occurrence in the exome over time
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