Publications

@article{Kvapilova2024,

title = {Validated WGS and WES protocols proved saliva-derived gDNA as an equivalent to blood-derived gDNA for clinical and population genomic analyses},

author = {K. Kvapilova and P. Misenko and J. Radvanszky and O. Brzon and J. Budis and J. Gazdarica and O. Pos and M. Korabecna and M. Kasny and T. Szemes and P. Kvapil and J. Paces and Z. Kozmik},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85185401063&doi=10.1186%2fs12864-024-10080-0&partnerID=40&md5=005d098ebc4c002ab39a2a6b70f9512d},

doi = {10.1186/s12864-024-10080-0},

issn = {14712164},

year  = {2024},

date = {2024-01-01},

urldate = {2024-01-01},

journal = {BMC Genomics},

volume = {25},

number = {1},

publisher = {BioMed Central Ltd},

abstract = {Background: Whole exome sequencing (WES) and whole genome sequencing (WGS) have become standard methods in human clinical diagnostics as well as in population genomics (POPGEN). Blood-derived genomic DNA (gDNA) is routinely used in the clinical environment. Conversely, many POPGEN studies and commercial tests benefit from easy saliva sampling. Here, we evaluated the quality of variant call sets and the level of genotype concordance of single nucleotide variants (SNVs) and small insertions and deletions (indels) for WES and WGS using paired blood- and saliva-derived gDNA isolates employing genomic reference-based validated protocols. Methods: The genomic reference standard Coriell NA12878 was repeatedly analyzed using optimized WES and WGS protocols, and data calls were compared with the truth dataset published by the Genome in a Bottle Consortium. gDNA was extracted from the paired blood and saliva samples of 10 participants and processed using the same protocols. A comparison of paired blood–saliva call sets was performed in the context of WGS and WES genomic reference-based technical validation results. Results: The quality pattern of called variants obtained from genomic-reference-based technical replicates correlates with data calls of paired blood–saliva-derived samples in all levels of tested examinations despite a higher rate of non-human contamination found in the saliva samples. The F1 score of 10 blood-to-saliva-derived comparisons ranged between 0.8030–0.9998 for SNVs and between 0.8883–0.9991 for small-indels in the case of the WGS protocol, and between 0.8643–0.999 for SNVs and between 0.7781–1.000 for small-indels in the case of the WES protocol. Conclusion: Saliva may be considered an equivalent material to blood for genetic analysis for both WGS and WES under strict protocol conditions. The accuracy of sequencing metrics and variant-detection accuracy is not affected by choosing saliva as the gDNA source instead of blood but much more significantly by the genomic context, variant types, and the sequencing technology used. © The Author(s) 2024.},

keywords = {Single nucleotide variants, Validation},

pubstate = {published},

tppubtype = {article}

}

@article{Hyblova2020,

title = {Validation of copy number variants detection from pregnant plasma using low-pass whole-genome sequencing in noninvasive prenatal testing-like settings},

author = {M Hyblova and M Harsanyova and D Nikulenkov-Grochova and J Kadlecova and M Kucharik and J Budis and G Minarik},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85090251668&doi=10.3390%2fdiagnostics10080569&partnerID=40&md5=b34f5ed83d4c332d97149be1644573c0},

doi = {10.3390/diagnostics10080569},

issn = {20754418},

year  = {2020},

date = {2020-01-01},

journal = {Diagnostics},

volume = {10},

number = {8},

publisher = {MDPI AG},

abstract = {Detection of copy number variants as an integral part of noninvasive prenatal testing is increasingly used in clinical practice worldwide. We performed validation on plasma samples from 34 pregnant women with known aberrations using cell-free DNA sequencing to evaluate the sensitivity for copy number variants (CNV) detection using an in-house CNV fraction-based detection algorithm. The sensitivity for CNVs smaller than 3 megabases (Mb), larger than 3Mb, and overall was 78.57%, 100%, and 90.6%, respectively. Regarding the fetal fraction, detection sensitivity in the group with a fetal fraction of less than 10% was 57.14%, whereas there was 100% sensitivity in the group with fetal fraction exceeding 10%. The assay is also capable of indicating whether the origin of an aberration is exclusively fetal or fetomaternal/maternal. This validation demonstrated that a CNV fraction-based algorithm was applicable and feasible in clinical settings as a supplement to testing for common trisomies 21, 18, and 13. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).},

keywords = {Copy number variation, Fetal fraction, Non-invasive prenatal testing, Validation},

pubstate = {published},

tppubtype = {article}

}

@article{Kucharik2020,

title = {Non-invasive prenatal testing (NIPT) by low coverage genomic sequencing: Detection limits of screened chromosomal microdeletions},

author = {M Kucharik and A Gnip and M Hyblova and J Budis and L Strieskova and M Harsanyova and O Pös and Z Kubiritova and J Radvanszky and G Minarik and T Szemes},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85089990305&doi=10.1371%2fjournal.pone.0238245&partnerID=40&md5=56850bc0c9e9e5e266a3538da3dd5bed},

doi = {10.1371/journal.pone.0238245},

issn = {19326203},

year  = {2020},

date = {2020-01-01},

journal = {PLoS ONE},

volume = {15},

number = {8 August},

publisher = {Public Library of Science},

abstract = {To study the detection limits of chromosomal microaberrations in non-invasive prenatal testing with aim for five target microdeletion syndromes, including DiGeorge, Prader-Willi/Angelman, 1p36, Cri-Du-Chat, and Wolf-Hirschhorn syndromes. We used known cases of pathogenic deletions from ISCA database to specifically define regions critical for the target syndromes. Our approach to detect microdeletions, from whole genome sequencing data, is based on sample normalization and read counting for individual bins. We performed both an in-silico study using artificially created data sets and a laboratory test on mixed DNA samples, with known microdeletions, to assess the sensitivity of prediction for varying fetal fractions, deletion lengths, and sequencing read counts. The in-silico study showed sensitivity of 79.3% for 10% fetal fraction with 20M read count, which further increased to 98.4% if we searched only for deletions longer than 3Mb. The test on laboratory-prepared mixed samples was in agreement with in-silico results, while we were able to correctly detect 24 out of 29 control samples. Our results suggest that it is possible to incorporate microaberration detection into basic NIPT as part of the offered screening/diagnostics procedure, however, accuracy and reliability depends on several specific factors. Copyright: © 2020 Kucharik et al.},

keywords = {Copy number variation, Fetal fraction, Non-invasive prenatal testing, Validation},

pubstate = {published},

tppubtype = {article}

}

@article{Sekelska2019,

title = {Result of prospective validation of the trisomy Test® for the detection of chromosomal trisomies},

author = {M Sekelska and A Izsakova and K Kubosova and P Tilandyova and E Csekes and Z Kuchova and M Hyblova and M Harsanyova and M Kucharik and J Budis and T Szemes and G Minarik},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85076805864&doi=10.3390%2fdiagnostics9040138&partnerID=40&md5=ffdda3394730383358ea467fee4a90f8},

doi = {10.3390/diagnostics9040138},

issn = {20754418},

year  = {2019},

date = {2019-01-01},

journal = {Diagnostics},

volume = {9},

number = {4},

publisher = {MDPI AG},

abstract = {Noninvasive prenatal testing (NIPT) is one of the most common prenatal screening tests used worldwide. Trisomy Test® belongs to NIPT tests based on low-coverage whole-genome sequencing. In our prospective study, 7279 samples of pregnant women collected during approximately two years were analyzed. In this cohort, 117 positive cases for trisomies 21, 18, and 13 were reported. An in-house designed bioinformatic pipeline and proprietary biostatistical approach was used for the detection of trisomies. The pooled sensitivity and specificity of our test reached 99.12% and 99.94%, respectively. The proportion of repeatedly uninformative results after repeated blood draws was 1.11%. Based on the presented results, we can confirm that the Trisomy Test® is fully comparable with other commercial NIPT tests available worldwide. © 2019 by the authors.},

keywords = {Aneuploidy, Fetal fraction, Non-invasive prenatal testing, Validation},

pubstate = {published},

tppubtype = {article}

}

@article{Kubiritova201964,

title = {On the critical evaluation and confirmation of germline sequence variants identified using massively parallel sequencing},

author = {Z Kubiritova and M Gyuraszova and E Nagyova and M Hyblova and M Harsanyova and J Budis and R Hekel and J Gazdarica and F Duris and L Kadasi and T Szemes and J Radvanszky},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-85064435175&doi=10.1016%2fj.jbiotec.2019.04.013&partnerID=40&md5=175358cc48df08933da3da830780ad66},

doi = {10.1016/j.jbiotec.2019.04.013},

issn = {01681656},

year  = {2019},

date = {2019-01-01},

journal = {Journal of Biotechnology},

volume = {298},

pages = {64-75},

publisher = {Elsevier B.V.},

abstract = {Although massively parallel sequencing (MPS) is becoming common practice in both research and routine clinical care, confirmation requirements of identified DNA variants using alternative methods are still topics of debate. When evaluating variants directly from MPS data, different read depth statistics, together with specialized genotype quality scores are, therefore, of high relevance. Here we report results of our validation study performed in two different ways: 1) confirmation of MPS identified variants using Sanger sequencing; and 2) simultaneous Sanger and MPS analysis of exons of selected genes. Detailed examination of false-positive and false-negative findings revealed typical error sources connected to low read depth/coverage, incomplete reference genome, indel realignment problems, as well as microsatellite associated amplification errors leading to base miss-calling. However, all these error types were identifiable with thorough manual revision of aligned reads according to specific patterns of distributions of variants and their corresponding reads. Moreover, our results point to dependence of both basic quantitative metrics (such as total read counts, alternative allele read counts and allelic balance) together with specific genotype quality scores on the used bioinformatics pipeline, stressing thus the need for establishing of specific thresholds for these metrics in each laboratory and for each involved pipeline independently. © 2019 Elsevier B.V.},

keywords = {Genetic testing, Single nucleotide variants, Validation, Variant calling},

pubstate = {published},

tppubtype = {article}

}

@article{Minarik2015,

title = {Utilization of benchtop next generation sequencing platforms ion torrent PGM and miseq in noninvasive prenatal testing for chromosome 21 trisomy and testing of impact of in silico and physical size selection on its analytical performance},

author = {G Minarik and G Repiska and M Hyblova and E Nagyova and K Soltys and J Budis and F Duris and R Sysak and M G Bujalkova and B Vlkova-Izrael and O Biro and B Nagy and T Szemes},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-84956618019&doi=10.1371%2fjournal.pone.0144811&partnerID=40&md5=ba19c1dabbdb0f2c2d3718fd27e8fc9c},

doi = {10.1371/journal.pone.0144811},

issn = {19326203},

year  = {2015},

date = {2015-01-01},

journal = {PLoS ONE},

volume = {10},

number = {12},

publisher = {Public Library of Science},

abstract = {Objectives The aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods. Methods Samples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation. The impact of in silico and physical size selection on analytical performance of the test was studied. Results Using a z score value of 3 as the cut-off, 98.11% - 100% (104-106/106) specificity and 100% (24/24) sensitivity and 99.06% - 100% (105-106/106) specificity and 100% (24/24) sensitivity were observed for Ion Torrent PGM and MiSeq, respectively. After in silico based size selection both platforms reached 100% specificity and sensitivity. Following the physical size selection z scores of tested trisomic samples increased significantlyâ€"p = 0.0141 and p = 0.025 for Ion Torrent PGM and MiSeq, respectively. Conclusions Noninvasive prenatal testing for chromosome 21 trisomy with the utilization of benchtop NGS systems led to results equivalent to previously published studies performed on highto- ultrahigh throughput NGS systems. The in silico size selection led to higher specificity of the test. Physical size selection performed on isolated DNA led to significant increase in z scores. The observed results could represent a basis for increasing of cost effectiveness of the test and thus help with its penetration worldwide. © 2015 Minarik et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.},

keywords = {Aneuploidy, Non-invasive prenatal testing, Size selection, Validation},

pubstate = {published},

tppubtype = {article}

}

@article{Repiská20131185,

title = {Selection of the optimal manual method of cell free fetal DNA isolation from maternal plasma},

author = {G Repiská and T Sedláčková and T Szemes and P Celec and G Minárik},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-84882355382&doi=10.1515%2fcclm-2012-0418&partnerID=40&md5=f476653212c66d4f5b9ec534582f5921},

doi = {10.1515/cclm-2012-0418},

issn = {14346621},

year  = {2013},

date = {2013-01-01},

journal = {Clinical Chemistry and Laboratory Medicine},

volume = {51},

number = {6},

pages = {1185-1189},

abstract = {Background: The cell free fetal DNA (cffDNA) present in plasma of pregnant women represents an important alternative source of DNA for non-invasive prenatal diagnosis. Due to the low quantity and increased fragmentation of cffDNA, the choice of DNA extraction method is a crucial step for downstream analyses. Methods: In our study, the three spin column-based kits for isolation of cffDNA [DNA Blood Mini Kit (DBM), DSP Virus Kit (DSP) and Circulating Nucleic Acid (CNA) Kit] were compared. Original and optimized protocol were used in comparison and applied in the two phases of the study. Results: A statistically significant difference in performance of the kits was determined based on the comparison of genomic equivalents per mL (GEq/mL) values (p < 0.0001). The GEq/mL of isolated DNA was significantly higher using CNA and DSP Kits than DBM Kit. The CNA Kit and DSP Kit did not significantly differ in the GEq/mL values, although all tested samples isolated with CNA Kit showed higher values. Conclusions: According to our results the commonly used DBM Kit could be successfully replaced with CNA or DSP Kits. The replacement could be beneficial in qualitative as well quantitative tests (e.g., gender determination, aneu ploidy detection) when the isolation yield limits subsequent analyses. However, there is an important decision to be made when switching DBM Kit for DSP or CNA Kits. The price of DBM Kit is two and six times lower than DSP and CNA Kits, respectively.},

keywords = {Liquid biopsy, Non-invasive prenatal testing, Validation},

pubstate = {published},

tppubtype = {article}

}

@article{Sedlackova2013,

title = {Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods},

author = {T Sedlackova and G Repiska and P Celec and T Szemes and G Minarik},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-84874073176&doi=10.1186%2f1480-9222-15-5&partnerID=40&md5=723b0e9568bb2d83456bbc497a945237},

doi = {10.1186/1480-9222-15-5},

issn = {14809222},

year  = {2013},

date = {2013-01-01},

journal = {Biological Procedures Online},

volume = {15},

number = {1},

abstract = {Background: Specific applications and modern technologies, like non-invasive prenatal testing, non-invasive cancer diagnostic and next generation sequencing, are currently in the focus of researchers worldwide. These have common characteristics in use of highly fragmented DNA molecules for analysis. Hence, for the performance of molecular methods, DNA concentration is a crucial parameter; we compared the influence of different levels of DNA fragmentation on the accuracy of DNA concentration measurements. Results: In our comparison, the performance of the currently most commonly used methods for DNA concentration measurement (spectrophotometric, fluorometric and qPCR based) were tested on artificially fragmented DNA samples. In our comparison, unfragmented and three specifically fragmented DNA samples were used. According to our results, the level of fragmentation did not influence the accuracy of spectrophotometric measurements of DNA concentration, while other methods, fluorometric as well as qPCR-based, were significantly influenced and a decrease in measured concentration was observed with more intensive DNA fragmentation. Conclusions: Our study has confirmed that the level of fragmentation of DNA has significant impact on accuracy of DNA concentration measurement with two of three mostly used methods (PicoGreen and qPCR). Only spectrophotometric measurement was not influenced by the level of fragmentation, but sensitivity of this method was lowest among the three tested. Therefore if it is possible the DNA quantification should be performed with use of equally fragmented control DNA. © 2013 Sedlackova et al.; licensee BioMed Central Ltd.},

keywords = {Liquid biopsy, Non-invasive prenatal testing, Validation},

pubstate = {published},

tppubtype = {article}

}

@article{Vlková2010b,

title = {Advances in the research of fetal DNA in maternal plasma for noninvasive prenatal diagnostics},

author = {B Vlková and T Szemes and G Minárik and J Turňa and P Celec},

url = {https://www.scopus.com/inward/record.uri?eid=2-s2.0-77954303943&partnerID=40&md5=f8502e85159556021100dec5321b7865},

issn = {12341010},

year  = {2010},

date = {2010-01-01},

journal = {Medical Science Monitor},

volume = {16},

number = {4},

pages = {RA85-RA91},

abstract = {Molecular analysis of fetal DNA present in the maternal circulation allows noninvasive, early, and precise determination of fetal genetic status in prenatal diagnostics. The most common clinical applications, i.e. prenatal gender determination and fetal RhD genotyping, are possible already in the first trimester using specialized protocols for DNA isolation from plasma and subsequent realtime PCR detection. Recent advances in molecular techniques enable other applications of fetal DNA purified from maternal plasma samples. Chromosomal abnormalities (e.g. trisomy 21) can be diagnosed by digital PCR, which offers higher accuracy in quantifying DNA sequences than standard real-time PCR. Digital PCR, but also MALDI-TOF, are suitable for detecting point mutations, widening the spectrum of applications to monogenic diseases. The ongoing lowering of costs for massively parallel sequencing might lead to replacement of most of the other currently used approaches. Adopting specialized protocols for the purification of fragmented circulating fetal DNA and improving the bioinformatic analysis of raw data can bring us closer to sequencing the fetal genome as the ultimate goal of prenatal DNA diagnostics, with wide-ranging medical applications. The discussion and solution of ethical issues beyond early fetal gender or paternity determination is hanging just behind the rapid technical progress of noninvasive prenatal DNA diagnostics. © Med Sci Monit, 2010.},

keywords = {Liquid biopsy, Non-invasive prenatal testing, Review, Validation},

pubstate = {published},

tppubtype = {article}

}