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001-es BibID:BIBFORM030851
Első szerző:Bubán Tamás (belgyógyász, gasztroenterológus)
Cím:Detection of internal tandem duplications in the FLT3 gene by different electrophoretic methods / Tamás Bubán, Katalin Koczok, Róza Földesi, Gabriella Szabó, Andrea Sümegi, Miklós Tanyi, László Szerafin, Miklós Udvardy, János Kappelmayer, Péter Antal-Szalmás
Dátum:2011
Megjegyzések:Abstract Background: In acute myeloid leukemia (AML), the internal tandem duplication (ITD) in the juxtamembrane domain of the FLT3 (Fms-like tyrosine kinase 3) gene is one of the most frequent genetic alterations associated with poor prognosis. Methods: A complex evaluation of the analytical properties of the three most frequently used detection methods - PCR followed by agarose (AGE), polyacrylamide (PAGE) or capillary electrophoresis (CE) - was performed on 95 DNA samples obtained from 73 AML patients. Results: All the three methods verified the presence of a mutant allele in 20 samples from 18 patients. AGE and PAGE could detect the presence of 1%-2% mutant allele, while the detection limit of CE was 0.28%. However, acceptable reproducibility (inter-assay CV <25%) of the mutant allele rate determination was only achievable above 1.5% mutant/total allele rate. The reproducibility of the ITD size determination by CE was much better, but the ITD size calculated by PeakScanner or GeneScan analysis was 7% lower as compared to values obtained by DNA sequencing. The presence of multiple ITD was over-estimated by PAGE and AGE due to the formation of heteroduplexes. Conclusions: This study suggests the use of PCR+CE in the diagnostics and the follow-up of AML patients. The data further supports the importance of proper analytical evaluation of home-made molecular biological diagnostic tests.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
electrophoresis
FLT3
ITD
egyetemen (Magyarországon) készült közlemény
Megjelenés:Clinical Chemistry and Laboratory Medicine. - 50 : 2 (2011), p. 301-310. -
További szerzők:Koczok Katalin (1979-) (labororvos) Földesi Róza (1967-) (klinikai laboratóriumi kutató, PhD hallgató) Szabó Gabriella Sümegi Andrea (1969-) (biológus) Tanyi Miklós (1968-) (sebész) Szerafin László (1958-) (belgyógyászat, haematológia, klinikai onkológia szakorvos) Udvardy Miklós (1947-) (belgyógyász, haematológus) Kappelmayer János (1960-) (laboratóriumi szakorvos) Antal-Szalmás Péter (1968-) (laboratóriumi szakorvos)
Pályázati támogatás:TÁMOP-4.2.1/B-09/1/KONV-2010-0007
TÁMOP
Celluláris hematológia - immunológia
Internet cím:DOI
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001-es BibID:BIBFORM072517
035-os BibID:(cikkazonosító)158 (WoS)000426305200003 (Scopus)85042425048
Első szerző:Ivády Gergely (laboratóriumi szakorvos)
Cím:Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system / Ivády Gergely, Madar László, Dzsudzsák Erika, Koczok Katalin, Kappelmayer János, Krulisova Veronika, Macek Milan, Horváth Attila, Balogh István
Dátum:2018
ISSN:1471-2164
Megjegyzések: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.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Pyrosequencing
Homopolymer detection
Cystic fibrosis
Megjelenés:BMC Genomics. - 19 (2018), p. 1-8. -
További szerzők:Madar László (1972-) (klinikai laboratóriumi kutató) Dzsudzsák Erika Koczok Katalin (1979-) (labororvos) Kappelmayer János (1960-) (laboratóriumi szakorvos) Krulisova, Veronika Macek Jr., Milan Horváth Attila (1988-) (programtervező informatikus) Balogh István (1972-) (molekuláris biológus, genetikus)
Pályázati támogatás:K109076
OTKA
GINOP-2.3.2-15-2016-00039
GINOP
Internet cím:Szerző által megadott URL
DOI
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