@article{MTMT:34293998, title = {SnakeLines: integrated set of computational pipelines for sequencing reads}, url = {https://m2.mtmt.hu/api/publication/34293998}, author = {Budis, Jaroslav and Krampl, Werner and Kucharik, Marcel and Hekel, Rastislav and Goga, Adrian and Sitarcik, Jozef and Lichvar, Michal and Smol'ak, David and Boehmer, Miroslav and Balaz, Andrej and Duris, Frantisek and Gazdarica, Juraj and Soltys, Katarina and Turna, Jan and Radvanszky, Jan and Szemes, Tomas}, doi = {10.1515/jib-2022-0059}, journal-iso = {Journal of Integrative Bioinformatics}, journal = {Journal of Integrative Bioinformatics}, unique-id = {34293998}, issn = {1613-4516}, abstract = {With the rapid growth of massively parallel sequencing technologies, still more laboratories are utilising sequenced DNA fragments for genomic analyses. Interpretation of sequencing data is, however, strongly dependent on bioinformatics processing, which is often too demanding for clinicians and researchers without a computational background. Another problem represents the reproducibility of computational analyses across separated computational centres with inconsistent versions of installed libraries and bioinformatics tools. We propose an easily extensible set of computational pipelines, called SnakeLines, for processing sequencing reads; including mapping, assembly, variant calling, viral identification, transcriptomics, and metagenomics analysis. Individual steps of an analysis, along with methods and their parameters can be readily modified in a single configuration file. Provided pipelines are embedded in virtual environments that ensure isolation of required resources from the host operating system, rapid deployment, and reproducibility of analysis across different Unix-based platforms. SnakeLines is a powerful framework for the automation of bioinformatics analyses, with emphasis on a simple set-up, modifications, extensibility, and reproducibility. The framework is already routinely used in various research projects and their applications, especially in the Slovak national surveillance of SARS-CoV-2.}, keywords = {reproducibility; FRAMEWORK; Virtual environment; Massively parallel sequencing; Computational pipeline}, year = {2023} } @article{MTMT:33903397, title = {Single-cell whole-genome sequencing, haplotype analysis in prenatal diagnosis of monogenic diseases}, url = {https://m2.mtmt.hu/api/publication/33903397}, author = {Chang, Liang and Jiao, Haining and Chen, Jiucheng and Wu, Guanlin and Liu, Ping and Li, Rong and Guo, Jianying and Long, Wenqing and Tang, Xiaojian and Lu, Bingjie and Xu, Haibin and Wu, Han}, doi = {10.26508/lsa.202201761}, journal-iso = {LIFE SCI ALLIANCE}, journal = {LIFE SCIENCE ALLIANCE}, volume = {6}, unique-id = {33903397}, abstract = {Monogenic inherited diseases are common causes of congenital disabilities, leading to severe economic and mental burdens on affected families. In our previous study, we demonstrated the validity of cell-based noninvasive prenatal testing (cbNIPT) in prenatal diagnosis by single-cell targeted sequencing. The present research further explored the feasibility of single-cell whole-genome sequencing (WGS) and haplotype analysis of various monogenic diseases with cbNIPT. Four families were recruited: one with inherited deafness, one with hemophilia, one with large vestibular aqueduct syndrome (LVAS), and one with no disease. Circulating trophoblast cells (cTBs) were obtained from maternal blood and analyzed by single-cell 15X WGS. Haplotype analysis showed that CFC178 (deafness family), CFC616 (hemo-philia family), and CFC111 (LVAS family) inherited haplotypes from paternal and/or maternal pathogenic loci. Amniotic fluid or fetal villi samples from the deafness and hemophilia families confirmed these results. WGS performed better than targeted sequencing in genome coverage, allele dropout (ADO), and false-positive (FP) ratios. Our findings suggest that cbNIPT by WGS and haplotype analysis have great potential for use in prenatally diagnosing various monogenic diseases.}, year = {2023}, eissn = {2575-1077}, orcid-numbers = {Chang, Liang/0000-0001-8028-2287} } @article{MTMT:34293999, title = {Non-Invasive Prenatal Testing (NIPT): Reliability, Challenges, and Future Directions}, url = {https://m2.mtmt.hu/api/publication/34293999}, author = {Jayashankar, Siva Shantini and Nasaruddin, Muhammad Luqman and Hassan, Muhammad Faiz and Dasrilsyah, Rima Anggrena and Shafiee, Mohamad Nasir and Ismail, Noor Akmal Shareela and Alias, Ekram}, doi = {10.3390/diagnostics13152570}, journal-iso = {DIAGNOSTICS}, journal = {DIAGNOSTICS}, volume = {13}, unique-id = {34293999}, issn = {2075-4418}, abstract = {Non-invasive prenatal testing was first discovered in 1988; it was primarily thought to be able to detect common aneuploidies, such as Patau syndrome (T13), Edward Syndrome (T18), and Down syndrome (T21). It comprises a simple technique involving the analysis of cell-free foetal DNA (cffDNA) obtained through maternal serum, using advances in next-generation sequencing. NIPT has shown promise as a simple and low-risk screening test, leading various governments and private organizations worldwide to dedicate significant resources towards its integration into national healthcare initiatives as well as the formation of consortia and research studies aimed at standardizing its implementation. This article aims to review the reliability of NIPT while discussing the current challenges prevalent among different communities worldwide.}, keywords = {ANEUPLOIDY; TRISOMY; pregnancy; obstetrics; Next-generation sequencing; NIPT; prenatal testing; cell-free foetal DNA (cffDNA)}, year = {2023}, eissn = {2075-4418}, orcid-numbers = {Dasrilsyah, Rima Anggrena/0000-0002-3419-8313; Ismail, Noor Akmal Shareela/0000-0003-2642-885X} } @article{MTMT:33305082, title = {Advanced techniques for gene heterogeneity research: Single-cell sequencing and on-chip gene analysis systems}, url = {https://m2.mtmt.hu/api/publication/33305082}, author = {Dong, Zaizai and Wang, Yu and Yin, Dedong and Hang, Xinxin and Pu, Lei and Zhang, Jianfu and Geng, Jia and Chang, Lingqian}, doi = {10.1002/VIW.20210011}, journal-iso = {View}, journal = {View}, volume = {3}, unique-id = {33305082}, issn = {2688-268X}, abstract = {Gene heterogeneity leads to the differences in cellular behaviors in a wide range, such as tumor drug-resistant mutation, epithelial-mesenchymal transition, and migration, posing significant challenges to the development of biomedicine. Traditional gene analysis methods, such as polymerase chain reaction, employ a mass of cells as the gene source, resulting in that the gene properties from a specific single cell are hidden in massive gene information. Recent decades have seen the emerging single-cell gene analysis techniques with their unprecedented opportunities to study gene heterogeneity with high precision and high throughput. In this review, we summarized the state-of-the-art techniques for single-cell sequencing and on-chip gene analysis systems. The principles of each technique are introduced in detail, with the focus on the application scenarios in gene heterogeneity research. Looking forward, we also introduced the challenges in current technologies and point out the future direction for facilitating the technical improvement and clinical applications of single-cell gene analysis techniques.}, keywords = {single cell; single-cell sequencing; gene heterogeneity; on-chip gene analysis}, year = {2022}, eissn = {2688-268X} } @article{MTMT:33903398, title = {Non-intuitive trends of fetal fraction development related to gestational age and fetal gender, and their practical implications for non-invasive prenatal testing}, url = {https://m2.mtmt.hu/api/publication/33903398}, author = {Forgacova, Natalia and Gazdarica, Juraj and Budis, Jaroslav and Kucharik, Marcel and Sekelska, Martina and Szemes, Tomas}, doi = {10.1016/j.mcp.2022.101870}, journal-iso = {MOL CELL PROBE}, journal = {MOLECULAR AND CELLULAR PROBES}, volume = {66}, unique-id = {33903398}, issn = {0890-8508}, abstract = {Discovery of fetal cell-free DNA fragments in maternal blood revolutionized prenatal diagnostics. Although noninvasive prenatal testing (NIPT) is already a matured screening test with high specificity and sensitivity, the accurate estimation of the proportion of fetal fragments, called fetal fraction, is crucial to avoid false-negative results. In this study, we collected 6999 samples from women undergoing NIPT testing with a single male fetus to demonstrate the influence of fetal fraction by the maternal and fetal characteristics. We show several fetal fraction discrepancies that contradict the generally presented conventional view. At first, the fetal fraction is not consistently rising with the maturity of the fetus due to a drop in 15 weeks of maturation. Secondly, the male samples have a lower fetal fraction than female fetuses, arguably due to the smaller gonosomal chromosomes. Finally, we discuss not only the possible reasons why this inconsistency exists but we also outline why these differences have not yet been identified and published. We demonstrate two non-intuitive trends to better comprehend the fetal fraction development and more precise selection of patients with sufficient fetal fraction for accurate testing.}, keywords = {Prenatal Diagnosis; Genetic Testing; fetal fraction; non-invasive prenatal testing}, year = {2022}, eissn = {1096-1194} } @article{MTMT:32295169, title = {Repurposing non-invasive prenatal testing data: Population study of single nucleotide variants associated with colorectal cancer and Lynch syndrome}, url = {https://m2.mtmt.hu/api/publication/32295169}, author = {Forgacova, Natalia and Gazdarica, Juraj and Budis, Jaroslav and Radvanszky, Jan and Szemes, Tomas}, doi = {10.3892/ol.2021.13040}, journal-iso = {ONCOL LETT}, journal = {ONCOLOGY LETTERS}, volume = {22}, unique-id = {32295169}, issn = {1792-1074}, abstract = {In our previous work, genomic data generated through non-invasive prenatal testing (NIPT) based on low-coverage massively parallel whole-genome sequencing of total plasma DNA of pregnant women in Slovakia was described as a valuable source of population specific data. In the present study, these data were used to determine the population allele frequency of common risk variants located in genes associated with colorectal cancer (CRC) and Lynch syndrome (LS). Allele frequencies of identified variants were compared with six world populations to detect significant differences between populations. Finally, variants were interpreted, functional consequences were searched for and clinical significance of variants was investigated using publicly available databases. Although the present study did not identify any pathogenic variants associated with CRC or LS in the Slovak population using NIPT data, significant differences were observed in the allelic frequency of risk CRC variants previously reported in genome-wide association studies and common variants located in genes associated with LS. As Slovakia is one of the leading countries with the highest incidence of CRC among male patients in the world, there is a need for studies dedicated to investigating the cause of such a high incidence of CRC in Slovakia. The present study also assumed that extensive cross-country data aggregation of NIPT results would represent an unprecedented source of information concerning human genome variation in cancer research.}, keywords = {colorectal cancer; Lynch syndrome; non-invasive prenatal testing; low-coverage massively parallel whole genome sequencing}, year = {2021}, eissn = {1792-1082} } @article{MTMT:32411369, title = {Privacy-preserving storage of sequenced genomic data}, url = {https://m2.mtmt.hu/api/publication/32411369}, author = {Hekel, Rastislav and Budis, Jaroslav and Kucharik, Marcel and Radvanszky, Jan and Pos, Zuzana and Szemes, Tomas}, doi = {10.1186/s12864-021-07996-2}, journal-iso = {BMC GENOMICS}, journal = {BMC GENOMICS}, volume = {22}, unique-id = {32411369}, issn = {1471-2164}, abstract = {Background The current and future applications of genomic data may raise ethical and privacy concerns. Processing and storing of this data introduce a risk of abuse by potential offenders since the human genome contains sensitive personal information. For this reason, we have developed a privacy-preserving method, named Varlock providing secure storage of sequenced genomic data. We used a public set of population allele frequencies to mask the personal alleles detected in genomic reads. Each personal allele described by the public set is masked by a randomly selected population allele with respect to its frequency. Masked alleles are preserved in an encrypted confidential file that can be shared in whole or in part using public-key cryptography. Results Our method masked the personal variants and introduced new variants detected in a personal masked genome. Alternative alleles with lower population frequency were masked and introduced more often. We performed a joint PCA analysis of personal and masked VCFs, showing that the VCFs between the two groups cannot be trivially mapped. Moreover, the method is reversible and personal alleles in specific genomic regions can be unmasked on demand. Conclusion Our method masks personal alleles within genomic reads while preserving valuable non-sensitive properties of sequenced DNA fragments for further research. Personal alleles in the desired genomic regions may be restored and shared with patients, clinics, and researchers. We suggest that the method can provide an additional security layer for storing and sharing of the raw aligned reads.}, keywords = {Personal data; Genomic privacy; Genomic reads}, year = {2021}, eissn = {1471-2164} } @article{MTMT:32159983, title = {Copy number variant detection with low-coverage whole-genome sequencing represents a viable alternative to the conventional array-cgh}, url = {https://m2.mtmt.hu/api/publication/32159983}, author = {Kucharík, M. and Budiš, J. and Hýblová, M. and Minárik, G. and Szemes, T.}, doi = {10.3390/diagnostics11040708}, journal-iso = {DIAGNOSTICS}, journal = {DIAGNOSTICS}, volume = {11}, unique-id = {32159983}, issn = {2075-4418}, abstract = {Copy number variations (CNVs) represent a type of structural variant involving alterations in the number of copies of specific regions of DNA that can either be deleted or duplicated. CNVs contribute substantially to normal population variability, however, abnormal CNVs cause numerous genetic disorders. At present, several methods for CNV detection are applied, ranging from the conventional cytogenetic analysis, through microarray-based methods (aCGH), to next-generation sequencing (NGS). In this paper, we present GenomeScreen, an NGS-based CNV detection method for low-coverage, whole-genome sequencing. We determined the theoretical limits of its accuracy and obtained confirmation in an extensive in silico study and in real patient samples with known genotypes. In theory, at least 6 M uniquely mapped reads are required to detect a CNV with the length of 100 kilobases (kb) or more with high confidence (Z-score > 7). In practice, the in silico analysis required at least 8 M to obtain >99% accuracy (for 100 kb deviations). We compared GenomeScreen with one of the currently used aCGH methods in diagnostic laboratories, which has mean resolution of 200 kb. GenomeScreen and aCGH both detected 59 deviations, while GenomeScreen furthermore detected 134 other (usually) smaller variations. When compared to aCGH, overall performance of the proposed GenemoScreen tool is comparable or superior in terms of accuracy, turn-around time, and cost-effectiveness, thus providing reasonable benefits, particularly in a prenatal diagnosis setting. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.}, keywords = {Adult; ARTICLE; human; Genotype; controlled study; theoretical study; Prenatal Diagnosis; Copy number variation; cost effectiveness analysis; computer model; high throughput sequencing; Whole genome sequencing; CNV detection; ACGH replacement; CNV detection comparison; Low-coverage WGS}, year = {2021}, eissn = {2075-4418} } @article{MTMT:31710298, title = {Validation of Copy Number Variants Detection from Pregnant Plasma Using Low-Pass Whole-Genome Sequencing in Noninvasive Prenatal Testing-Like Settings}, url = {https://m2.mtmt.hu/api/publication/31710298}, author = {Hyblova, Michaela and Harsanyova, Maria and Nikulenkov-Grochova, Diana and Kadlecova, Jitka and Kucharik, Marcel and Budis, Jaroslav and Minarik, Gabriel}, doi = {10.3390/diagnostics10080569}, journal-iso = {DIAGNOSTICS}, journal = {DIAGNOSTICS}, volume = {10}, unique-id = {31710298}, issn = {2075-4418}, 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.}, keywords = {CNV detection; Noninvasive prenatal testing (NIPT); low-pass whole-genome sequencing}, year = {2020}, eissn = {2075-4418}, orcid-numbers = {Budis, Jaroslav/0000-0002-8667-6655} } @article{MTMT:31710299, title = {Non-invasive prenatal testing (NIPT) by low coverage genomic sequencing: Detection limits of screened chromosomal microdeletions}, url = {https://m2.mtmt.hu/api/publication/31710299}, author = {Kucharik, Marcel and Gnip, Andrej and Hyblova, Michaela and Budis, Jaroslav and Strieskova, Lucia and Harsanyova, Maria and Pos, Ondrej and Kubiritova, Zuzana and Radvanszky, Jan and Minarik, Gabriel and Szemes, Tomas}, doi = {10.1371/journal.pone.0238245}, journal-iso = {PLOS ONE}, journal = {PLOS ONE}, volume = {15}, unique-id = {31710299}, issn = {1932-6203}, 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 anin-silicostudy 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. Thein-silicostudy 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 within-silicoresults, 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.}, year = {2020}, eissn = {1932-6203} } @article{MTMT:31461958, title = {Sequencing Shorter cfDNA Fragments Decreases the False Negative Rate of Non-invasive Prenatal Testing}, url = {https://m2.mtmt.hu/api/publication/31461958}, author = {Xue, Ying and Zhao, Guodong and Qiao, Longwei and Lu, Jiafeng and Yu, Bin and Wang, Ting}, doi = {10.3389/fgene.2020.00280}, journal-iso = {FRONT GENET}, journal = {FRONTIERS IN GENETICS}, volume = {11}, unique-id = {31461958}, abstract = {Circulating fetal cell-free DNA (cfDNA) is generally shorter than maternal cfDNA. Size selection of shorter cfDNA in total cfDNA could significantly increase the fetal fraction, but there are few reports of using this method to decrease the false negative rate for NIPT. In this study, nine false negative cases were retrospectively analyzed by NIPT retesting and E-gel based size-selection NIPT and the fetal cfDNA fraction in maternal total cfDNA was evaluated by calculating the proportion of reads from chromosome Y. Fetal placenta karyotypes were confirmed by CNVplex assays to analysis the reasons for false negative cases. Of the 81,601 pregnancies who underwent NIPT, nine false negative cases (0.01%) were found. Of eight retested cases, two (25%) had positive NIPT retest results, and five (62.5%) had positive size-selection NIPT results. For fetal cfDNA fraction, 100% cases had improvement after size-selection NIPT compared with the initial NIPT and retest results, and the fetal cfDNA fraction growth ratio ranged from 99 to 359%. For one twin pregnancy with one T18 fetus, size selection improved the fetal cfDNA fraction to 23.10%, and successfully detected the T18 fetus in NIPT. Placental tissue analysis results for two cases indicated both had confined placental mosaicism (CPM), which was confirmed with size-selection NIPT. In conclusion, size selection can significantly enrich the fetal cfDNA fraction and decrease the false negative rate of NIPT, especially for CPM and twin pregnancies.}, keywords = {NIPT; False negative; confined placental mosaicism; TWIN PREGNANCIES; shorter cfDNA fragments; size selection}, year = {2020}, eissn = {1664-8021} } @article{MTMT:30728593, title = {Non-invasive prenatal testing as a valuable source of population specific allelic frequencies}, url = {https://m2.mtmt.hu/api/publication/30728593}, author = {Budis, Jaroslav and Gazdarica, Juraj and Radvanszky, Jan and Harsanyova, Maria and Gazdaricova, Iveta and Strieskova, Lucia and Frno, Richard and Duris, Frantisek and Minarik, Gabriel and Sekelska, Martina and Nagy, Bálint and Szemes, Tomas}, doi = {10.1016/j.jbiotec.2019.04.026}, journal-iso = {J BIOTECHNOL}, journal = {JOURNAL OF BIOTECHNOLOGY}, volume = {299}, unique-id = {30728593}, issn = {0168-1656}, abstract = {Low-coverage massively parallel genome sequencing for non-invasive prenatal testing (NIPT) of common aneuploidies is one of the most rapidly adopted and relatively low-cost DNA tests. Since aggregation of reads from a large number of samples allows overcoming the problems of extremely low coverage of individual samples, we describe the possible re-use of the data generated during NIPT testing for genome scale population specific frequency determination of small DNA variants, requiring no additional costs except of those for the NIPT test itself. We applied our method to a data set comprising of 1501 original NIPT test results and evaluated the findings on different levels, from in silico population frequency comparisons up to wet lab validation analyses using a gold-standard method based on Sanger sequencing. The revealed high reliability of variant calling and allelic frequency determinations suggest that these NIPT data could serve as valuable alternatives to large scale population studies even for smaller countries around the world.}, keywords = {non-invasive prenatal testing; Low-coverage massively parallel whole-genome sequencing; Population specific allelic frequencies}, year = {2019}, eissn = {1873-4863}, pages = {72-78}, orcid-numbers = {Duris, Frantisek/0000-0001-8985-4122; Nagy, Bálint/0000-0002-0295-185X} } @article{MTMT:31064987, title = {Detection of Solid Tumor Molecular Residual Disease(MRD) Using Circulating Tumor DNA (ctDNA)}, url = {https://m2.mtmt.hu/api/publication/31064987}, author = {Chin, Re-I and Chen, Kevin and Usmani, Abul and Chua, Chanelle and Harris, Peter K. and Binkley, Michael S. and Azad, Tej D. and Dudley, Jonathan C. and Chaudhuri, Aadel A.}, doi = {10.1007/s40291-019-00390-5}, journal-iso = {MOL DIAGN THER}, journal = {MOLECULAR DIAGNOSIS & THERAPY}, volume = {23}, unique-id = {31064987}, issn = {1177-1062}, abstract = {Circulating tumor DNA (ctDNA) is a component of cell-free DNA that is shed by malignant tumors into the bloodstream and other bodily fluids. Levels of ctDNA are typically low, particularly in patients with localized disease, requiring highly sophisticated methods for detection and quantification. Multiple liquid biopsy methods have been developed for ctDNA analysis in solid tumor malignancies and are now enabling detection and assessment of earlier stages of disease, post-treatment molecular residual disease (MRD), resistance to targeted systemic therapy, and tumor mutational burden. Understanding ctDNA biology, mechanisms of release, and clearance and size characteristics, in conjunction with the application of molecular barcoding and targeted error correction, have increased the sensitivity and specificity of ctDNA detection techniques. Combinatorial approaches including integration of ctDNA data withcirculating protein biomarkers may further improve assay sensitivity and broaden the scope of ctDNA applications. Circulating viral DNA may be utilized to monitor disease in some virally induced malignancies. In spite of increasingly accurate methods of ctDNA detection, results need to be interpreted with caution given that somatic mosaicisms such as clonal hematopoiesis of indeterminate potential (CHIP) may give rise to genetic variants in the bloodstream unrelated to solid tumors, and the limited concordance observed between different commercial platforms. Overall, highly precise ctDNA detection and quantification methods have the potential to transform clinical practice via non-invasive monitoring of solid tumor malignancies, residual disease detection at earlier timepoints than standard clinical and/or imaging surveillance, and treatment personalization based on real-time assessment of the tumor genomic landscape.}, year = {2019}, eissn = {1179-2000}, pages = {311-331} } @article{MTMT:31064983, title = {Adaptable Model Parameters in Non-Invasive Prenatal Testing Lead to More Stable Predictions}, url = {https://m2.mtmt.hu/api/publication/31064983}, author = {Gazdarica, Juraj and Budis, Jaroslav and Duris, Frantisek and Turna, Jan and Szemes, Tomas}, doi = {10.3390/ijms20143414}, journal-iso = {INT J MOL SCI}, journal = {INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, volume = {20}, unique-id = {31064983}, issn = {1661-6596}, abstract = {Recent advances in massively parallel shotgun sequencing opened up new options for affordable non-invasive prenatal testing (NIPT) for fetus aneuploidy from DNA material extracted from maternal plasma. Tests typically compare chromosomal distributions of a tested sample with a control set of healthy samples with unaffected fetuses. Deviations above certain threshold levels are concluded as positive findings. The main problem with this approach is that the variance of the control set is dependent on the number of sequenced fragments. The higher the amount, the more precise the estimation of actual chromosomal proportions is. Testing a sample with a highly different number of sequenced reads as used in training may thus lead to over- or under-estimation of their variance, and so lead to false predictions. We propose the calculation of a variance for each tested sample adaptively, based on the actual number of its sequenced fragments. We demonstrate how it leads to more stable predictions, mainly in real-world diagnostics with the highly divergent inter-sample coverage.}, keywords = {Statistical models; z-score; non-invasive prenatal testing}, year = {2019}, eissn = {1422-0067} } @article{MTMT:31064982, title = {Combination of Fetal Fraction Estimators Based on Fragment Lengths and Fragment Counts in Non-Invasive Prenatal Testing}, url = {https://m2.mtmt.hu/api/publication/31064982}, author = {Gazdarica, Juraj and Hekel, Rastislav and Budis, Jaroslav and Kucharik, Marcel and Duris, Frantisek and Radvanszky, Jan and Turna, Jan and Szemes, Tomas}, doi = {10.3390/ijms20163959}, journal-iso = {INT J MOL SCI}, journal = {INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, volume = {20}, unique-id = {31064982}, issn = {1661-6596}, abstract = {The reliability of non-invasive prenatal testing is highly dependent on accurate estimation of fetal fraction. Several methods have been proposed up to date, utilizing different attributes of analyzed genomic material, for example length and genomic location of sequenced DNA fragments. These two sources of information are relatively unrelated, but so far, there have been no published attempts to combine them to get an improved predictor. We collected 2454 single euploid male fetus samples from women undergoing NIPT testing. Fetal fractions were calculated using several proposed predictors and the state-of-the-art SeqFF method. Predictions were compared with the reference Y-based method. We demonstrate that prediction based on length of sequenced DNA fragments may achieve nearly the same precision as the state-of-the-art methods based on their genomic locations. We also show that combination of several sample attributes leads to a predictor that has superior prediction accuracy over any single approach. Finally, appropriate weighting of samples in the training process may achieve higher accuracy for samples with low fetal fraction and so allow more reliability for subsequent testing for genomic aberrations. We propose several improvements in fetal fraction estimation with a special focus on the samples most prone to wrong conclusion.}, keywords = {DNA; Statistical methods; NIPT; fetal fraction; maternal serum screening; fetal cells}, year = {2019}, eissn = {1422-0067}, orcid-numbers = {Radvanszky, Jan/0000-0001-9657-1198} } @article{MTMT:3420877, title = {Combining count- and length-based z-scores leads to improved predictions in non-invasive prenatal testing}, url = {https://m2.mtmt.hu/api/publication/3420877}, author = {Jaroslav, Budis and Juraj, Gazdarica and Jan, Radvanszky and Gabor, Szucs and Marcel, Kucharik and Lucia, Strieskova Iveta and Gazdaricova, Iveta and Maria, Harsanyova and Frantisek, Duris and Gabriel, Minarik and Martina, Sekelska and Nagy, Bálint and Jan, Turna and Tomas, Szemes}, doi = {10.1093/bioinformatics/bty806}, journal-iso = {BIOINFORMATICS}, journal = {BIOINFORMATICS}, volume = {35}, unique-id = {3420877}, issn = {1367-4803}, abstract = {Motivation: Non-invasive prenatal testing or NIPT is currently among the top researched topic in obstetric care. While the performance of the current state-of-the-art NIPT solutions achieve high sensitivity and specificity, they still struggle with a considerable number of samples that cannot be concluded with certainty. Such uninformative results are often subject to repeated blood sampling and re-analysis, usually after two weeks, and this period may cause a stress to the future mothers as well as increase the overall cost of the test. Results: We propose a supplementary method to traditional z-scores to reduce the number of such uninformative calls. The method is based on a novel analysis of the length profile of circulating cell free DNA which compares the change in such profiles when random-based and length-based elimination of some fragments is performed. The proposed method is not as accurate as the standard z-score; however, our results suggest that combination of these two independent methods correctly resolves a substantial portion of healthy samples with an uninformative result. Additionally, we discuss how the proposed method can be used to identify maternal aberrations, thus reducing the risk of false positive and false negative calls. Availability and implementation: The open-source code of the proposed methods, together with test data, is freely available for non-commercial users at github web page https://github.com/jbudis/lambda.}, year = {2019}, eissn = {1460-2059}, pages = {1284-1291}, orcid-numbers = {Nagy, Bálint/0000-0002-0295-185X} } @article{MTMT:31064980, title = {Identification of Structural Variation from NGS-Based Non-Invasive Prenatal Testing}, url = {https://m2.mtmt.hu/api/publication/31064980}, author = {Pos, Ondrej and Budis, Jaroslav and Kubiritova, Zuzana and Kucharik, Marcel and Duris, Frantisek and Radvanszky, Jan and Szemes, Tomas}, doi = {10.3390/ijms20184403}, journal-iso = {INT J MOL SCI}, journal = {INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, volume = {20}, unique-id = {31064980}, issn = {1661-6596}, abstract = {Copy number variants (CNVs) are an important type of human genome variation, which play a significant role in evolution contribute to population diversity and human genetic diseases. In recent years, next generation sequencing has become a valuable tool for clinical diagnostics and to provide sensitive and accurate approaches for detecting CNVs. In our previous work, we described a non-invasive prenatal test (NIPT) based on low-coverage massively parallel whole-genome sequencing of total plasma DNA for detection of CNV aberrations >= 600 kbp. We reanalyzed NIPT genomic data from 5018 patients to evaluate CNV aberrations in the Slovak population. Our analysis of autosomal chromosomes identified 225 maternal CNVs (47 deletions; 178 duplications) ranging from 600 to 7820 kbp. According to the ClinVar database, 137 CNVs (60.89%) were fully overlapping with previously annotated variants, 66 CNVs (29.33%) were in partial overlap, and 22 CNVs (9.78%) did not overlap with any previously described variant. Identified variants were further classified with the AnnotSV method. In summary, we identified 129 likely benign variants, 13 variants of uncertain significance, and 83 likely pathogenic variants. In this study, we use NIPT as a valuable source of population specific data. Our results suggest the utility of genomic data from commercial CNV analysis test as background for a population study.}, keywords = {Next generation sequencing; COPY NUMBER VARIANTS; population study; non-invasive prenatal testing}, year = {2019}, eissn = {1422-0067}, orcid-numbers = {Pos, Ondrej/0000-0003-2491-2285; Budis, Jaroslav/0000-0002-8667-6655; Radvanszky, Jan/0000-0001-9657-1198} } @article{MTMT:31064979, title = {Experimental factors are associated with fetal fraction in size selection noninvasive prenatal testing}, url = {https://m2.mtmt.hu/api/publication/31064979}, author = {Qiao, Longwei and Mao, Jun and Liu, Minjuan and Liu, Yinghua and Song, Xiaoyan and Tang, Hui and Zhang, Qing and Li, Hong and Lu, Yaojuan and Liang, Yuting and Wang, Ting}, journal-iso = {AM J TRANSL RES}, journal = {AMERICAN JOURNAL OF TRANSLATIONAL RESEARCH}, volume = {11}, unique-id = {31064979}, issn = {1943-8141}, abstract = {Every year, 4-6 million pregnant women undergo noninvasive prenatal testing (NIPT), which is used world-wide for fetal aneuploidy screening. Adequate fetal cell-free DNA (cfDNA) is the critically important factor to ensure high sensitivity and specificity. In this study, we sought to increase the fetal fraction by adjusting experimental factors in the size selection for NIPT. CfDNA was extracted from 1495 pregnant women at 12-26 weeks of gestation for sequencing of shorter cfDNA NIPT (< 140 bp). Multivariable linear regression models were used to evaluate the association between experimental factors and fetal fraction. Nomograms for the likelihood of high fetal fraction (> 20%) were constructed according to significant factors in multivariable regression models. Our results suggested that cfDNA and library concentrations were negatively correlated with fetal fraction, and uniquely mapped reads were positively correlated with fetal fraction. Lower cfDNA and library concentrations, shorter cfDNA fragments, and higher uniquely mapped reads may be more conducive to obtaining higher fetal fractions. Furthermore, we constructed easy-to-use nomograms incorporating the maternal, fetal characteristics and experimental factors to precisely predict the probability of high fetal fraction with an area under the curve (AUC) of 0.773 (95% confidence interval: 0.749-0.797). Collectively, our maternal plasma cfDNA-based nomograms consider experimental factors that can be adjusted and may improve a laboratory's ability to obtain higher fetal cfDNA concentrations.}, keywords = {Noninvasive prenatal testing; fetal fraction; experimental factors}, year = {2019}, eissn = {1943-8141}, pages = {6370-6381} } @article{MTMT:31579246, title = {Result of Prospective Validation of the Trisomy Test((R)) for the Detection of Chromosomal Trisomies}, url = {https://m2.mtmt.hu/api/publication/31579246}, author = {Sekelska, Martina and Izsakova, Anita and Kubosova, Katarina and Tilandyova, Petra and Csekes, Erika and Kuchova, Zaneta and Hyblova, Michaela and Harsanyova, Maria and Kucharik, Marcel and Budis, Jaroslav and Szemes, Tomas and Minarik, Gabriel}, doi = {10.3390/diagnostics9040138}, journal-iso = {DIAGNOSTICS}, journal = {DIAGNOSTICS}, volume = {9}, unique-id = {31579246}, issn = {2075-4418}, abstract = {Noninvasive prenatal testing (NIPT) is one of the most common prenatal screening tests used worldwide. Trisomy Test (R) 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 (R) is fully comparable with other commercial NIPT tests available worldwide.}, keywords = {NIPT; low-coverage whole-genome sequencing; Trisomy Test (R)}, year = {2019}, eissn = {2075-4418}, orcid-numbers = {Kucharik, Marcel/0000-0002-5373-8282; Budis, Jaroslav/0000-0002-8667-6655; Szemes, Tomas/0000-0002-0900-2534} } @article{MTMT:31064985, title = {Ultracentrifugation enrichment protocol followed by total RNA sequencing allows assembly of the complete mitochondrial genome}, url = {https://m2.mtmt.hu/api/publication/31064985}, author = {Strieskova, Lucia and Gazdaricova, Iveta and Kajsik, Michal and Soltys, Katarina and Budis, Jaroslav and Pos, Ondrej and Lickova, Martina and Klempa, Boris and Szemes, Tomas}, doi = {10.1016/j.jbiotec.2019.04.019}, journal-iso = {J BIOTECHNOL}, journal = {JOURNAL OF BIOTECHNOLOGY}, volume = {299}, unique-id = {31064985}, issn = {0168-1656}, abstract = {The mitochondrial genome is an independent genetic system in each eukaryotic cell outside the nuclear genome. Mitochondrial DNA ( mtDNA) appears in high copy number within one cell, unlike nuclear DNA, which exists in two copies. But nevertheless, mtDNA represent only small part of total cellular DNA what causes problematic analysis and identification of relevant mutations. While most researchers tend to overlook it because of its small size, the mitochondrial genome contains genes that are essential for cellular energetics and survival. Because of the increased awareness on the importance of metabolism and bioenergetics in a wide variety of human diseases, more and more mtDNA studies were performed. Mitochondrial genome research has established the connection between mtDNA and a wide variety of diseases such as cancer or neurodegenerative disorders. At the present time, several methods are known, that allow sequencing of mtDNA. However, genomic analysis is often complicated due to the low content of mtDNA compared to nuclear DNA. For this reason, we have designed a new approach to obtaining the genomic mitochondrial sequence. We chose RNA based sequencing. Since human mtDNA does not contain introns, the reconstruction of whole mitochondrial genome through RNA sequencing seems to be effective. Our method is based on total RNA sequencing coupled with simple ultracentrifugation protocol and de novo assembly. Following our protocol, we were able to assemble a complete mammalian mitochondrial genome with a length of 16,505 bp and an average coverage of 156. The method is a relatively simple and inexpensive which could help in the further research or diagnostics of mtDNA-based diseases.}, keywords = {Ultracentrifugation; mitochondrial genome; RNA sequencing}, year = {2019}, eissn = {1873-4863}, pages = {8-12}, orcid-numbers = {Pos, Ondrej/0000-0003-2491-2285; Klempa, Boris/0000-0002-6931-1224} } @article{MTMT:31064984, title = {Non-invasive prenatal testing to detect chromosome aneuploidies in 57,204 pregnancies}, url = {https://m2.mtmt.hu/api/publication/31064984}, author = {Xue, Ying and Zhao, Guodong and Li, Hong and Zhang, Qin and Lu, Jiafeng and Yu, Bin and Wang, Ting}, doi = {10.1186/s13039-019-0441-5}, journal-iso = {MOL CYTOGENET}, journal = {MOLECULAR CYTOGENETICS}, volume = {12}, unique-id = {31064984}, issn = {1755-8166}, abstract = {BackgroundNon-invasive prenatal testing (NIPT) has been widely used to detect common fetal chromosome aneuploidies, such as trisomy 13, 18, and 21 (T13, T18, and T21), and has expanded to sex chromosome aneuploidies (SCAs) during recent years, but few studies have reported NIPT detection of rare fetal chromosome aneuploidies (RCAs). In this study, we evaluated the clinical practical performance of NIPT to analyze all 24 chromosome aneuploidies among 57,204 pregnancies in the Suzhou area of China.MethodsThis was a retrospective analysis of prospectively collected NIPT data from two next-generation sequencing (NGS) platforms (Illumina and Proton) obtained from The Affiliated Suzhou Hospital of Nanjing Medical University. NIPT results were validated by karyotyping or clinical follow-up.ResultsNIPT using the Illumina platform identified 586 positive cases; fetal karyotyping and follow-up results validated 178T21 cases, 49T18 cases, 4T13 cases, and 52 SCAs. On the Proton platform, 270 cases were positive during NIPT. Follow-up confirmed 85T21 cases, 17T18 cases, 4T13 cases, 28 SCAs, and 1 fetal chromosome 22 aneuploidy case as true positives. There were 5 false-negative results, including 4T21 and 1T18 cases. The NGS platforms showed similar sensitivities and positive predictive values (PPVs) in detecting T21, T18, T13 and SCAs (p>0.01). However, the Proton platform showed better specificity in detecting 45, X and the Illumina platform had better specificity in detecting T13 (p<0.01). The major factor contributing to NIPT false-positives on the Illumina platform was false SCAs cases (65.11%). Maternal chromosome aneuploidies, maternal cancers, and confined placental mosaicism caused discordant results between fetal karyotyping and NIPT.ConclusionNIPT with NGS showed good performance for detecting T13, T18, and T21. The Proton platform had better performance for detecting SCAs, but the NIPT accuracy rate for detecting RCAs was insufficient.}, keywords = {PERFORMANCE; Next generation sequencing; NIPT; Chromosome aneuploidies}, year = {2019}, eissn = {1755-8166} } @article{MTMT:27053562, title = {Residual disease detection using targeted parallel sequencing predicts relapse in cytogenetically normal acute myeloid leukemia}, url = {https://m2.mtmt.hu/api/publication/27053562}, author = {Gaksch, Lukas and Kashofer, Karl and Heitzer, Ellen and Quehenberger, Franz and Daga, Shruti and Hofer, Sybille and Halbwedl, Iris and Graf, Ricarda and Krisper, Nina and Hoefler, Gerald and Zebisch, Armin and Sill, Heinz and Woelfler, Albert}, doi = {10.1002/ajh.24922}, journal-iso = {AM J HEMATOL}, journal = {AMERICAN JOURNAL OF HEMATOLOGY}, volume = {93}, unique-id = {27053562}, issn = {0361-8609}, year = {2018}, eissn = {1096-8652}, pages = {23-30}, orcid-numbers = {Sill, Heinz/0000-0003-0993-4371} } @article{MTMT:27529099, title = {A method for improving the accuracy of non-invasive prenatal screening by cell-free foetal DNA size selection}, url = {https://m2.mtmt.hu/api/publication/27529099}, author = {He, Q Z and Wu, X J and He, Q Y and Xiang, J J and Zhang, C H and Lu, L and Wang, T and Li, H}, doi = {10.1080/09674845.2018.1468152}, journal-iso = {BRIT J BIOMED SCI}, journal = {BRITISH JOURNAL OF BIOMEDICAL SCIENCE}, volume = {75}, unique-id = {27529099}, issn = {0967-4845}, year = {2018}, eissn = {2474-0896}, pages = {133-138} } @article{MTMT:30559117, title = {Enhanced detection of circulating tumor DNA by fragment size analysis}, url = {https://m2.mtmt.hu/api/publication/30559117}, author = {Mouliere, Florent and Chandrananda, Dineika and Piskorz, Anna M. and Moore, Elizabeth K. and Morris, James and Ahlborn, Lise Barlebo and Mair, Richard and Goranova, Teodora and Marass, Francesco and Heider, Katrin and Wan, Jonathan C. M. and Supernat, Anna and Hudecova, Irena and Gounaris, Ioannis and Ros, Susana and Jimenez-Linan, Mercedes and Garcia-Corbacho, Javier and Patel, Keval and Ostrup, Olga and Murphy, Suzanne and Eldridge, Matthew D. and Gale, Davina and Stewart, Grant D. and Burge, Johanna and Cooper, Wendy N. and van der Heijden, Michiel S. and Massie, Charles E. and Watts, Colin and Corrie, Pippa and Pacey, Simon and Brindle, Kevin M. and Baird, Richard D. and Mau-Sorensen, Morten and Parkinson, Christine A. and Smith, Christopher G. and Brenton, James D. and Rosenfeld, Nitzan}, doi = {10.1126/scitranslmed.aat4921}, journal-iso = {SCI TRANSL MED}, journal = {SCIENCE TRANSLATIONAL MEDICINE}, volume = {10}, unique-id = {30559117}, issn = {1946-6234}, abstract = {Existing methods to improve detection of circulating tumor DNA (ctDNA) have focused on genomic alterations but have rarely considered the biological properties of plasma cell-free DNA (cfDNA). We hypothesized that differences in fragment lengths of circulating DNA could be exploited to enhance sensitivity for detecting the presence of ctDNA and for noninvasive genomic analysis of cancer. We surveyed ctDNA fragment sizes in 344 plasma samples from 200 patients with cancer using low-pass whole-genome sequencing (0.4x). To establish the size distribution of mutant ctDNA, tumor-guided personalized deep sequencing was performed in 19 patients. We detected enrichment of ctDNA in fragment sizes between 90 and 150 bp and developed methods for in vitro and in silico size selection of these fragments. Selecting fragments between 90 and 150 bp improved detection of tumor DNA, with more than twofold median enrichment in >95% of cases and more than fourfold enrichment in >10% of cases. Analysis of size-selected cfDNA identified clinically actionable mutations and copy number alterations that were otherwise not detected. Identification of plasma samples from patients with advanced cancer was improved by predictive models integrating fragment length and copy number analysis of cfDNA, with area under the curve (AUC) >0.99 compared to AUC <0.80 without fragmentation features. Increased identification of cfDNA from patients with glioma, renal, and pancreatic cancer was achieved with AUC > 0.91 compared to AUC < 0.5 without fragmentation features. Fragment size analysis and selective sequencing of specific fragment sizes can boost ctDNA detection and could complement or provide an alternative to deeper sequencing of cfDNA.}, year = {2018}, eissn = {1946-6242}, orcid-numbers = {Mouliere, Florent/0000-0001-7043-0514; Chandrananda, Dineika/0000-0002-8834-9500; Marass, Francesco/0000-0002-8993-7320; Heider, Katrin/0000-0003-4035-1668; Ros, Susana/0000-0002-5451-1880} } @article{MTMT:27055633, title = {Genomics-based non-invasive prenatal testing for detection of fetal chromosomal aneuploidy in pregnant women}, url = {https://m2.mtmt.hu/api/publication/27055633}, author = {Badeau, Mylene and Lindsay, Carmen and Blais, Jonatan and Nshimyumukiza, Leon and Takwoingi, Yemisi and Langlois, Sylvie and Legare, France and Giguere, Yves and Turgeon, Alexis F and Witteman, William and Rousseau, Francois}, doi = {10.1002/14651858.CD011767.pub2}, journal-iso = {COCHRANE DATABASE SYST REV}, journal = {COCHRANE DATABASE OF SYSTEMATIC REVIEWS}, unique-id = {27055633}, issn = {1361-6137}, year = {2017}, eissn = {1469-493X} } @article{MTMT:26929154, title = {A comparison of Illumina and Ion Torrent sequencing platforms in the context of differential gene expression}, url = {https://m2.mtmt.hu/api/publication/26929154}, author = {Lahens, Nicholas F and Ricciotti, Emanuela and Smirnova, Olga and Toorens, Erik and Kim, Eun Ji and Baruzzo, Giacomo and Hayer, Katharina E and Ganguly, Tapan and Schug, Jonathan and Grant, Gregory R}, doi = {10.1186/s12864-017-4011-0}, journal-iso = {BMC GENOMICS}, journal = {BMC GENOMICS}, volume = {18}, unique-id = {26929154}, issn = {1471-2164}, year = {2017}, eissn = {1471-2164}, orcid-numbers = {Baruzzo, Giacomo/0000-0001-6129-5007} } @article{MTMT:27231043, title = {In silico size selection is effective in reducing false positive NIPS cases of monosomy X that are due to maternal mosaic monosomy X}, url = {https://m2.mtmt.hu/api/publication/27231043}, author = {Shubina, J and Trofimov, DY and Barkov, IY and Stupko, OK and Goltsov, AY and Mukosey, IS and Tetruashvili, NK and Kim, LV and Bakharev, VA and Karetnikova, NA and Kochetkova, TO and Krasheninnikova, RV and Bystritskiy, AA and Sukhikh, GT}, doi = {10.1002/pd.5178}, journal-iso = {PRENATAL DIAG}, journal = {PRENATAL DIAGNOSIS}, volume = {37}, unique-id = {27231043}, issn = {0197-3851}, year = {2017}, eissn = {1097-0223}, pages = {1305-1310} }