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.
- MeSH
- DNA genetika MeSH
- exom MeSH
- genom lidský MeSH
- genomika MeSH
- lidé MeSH
- metagenomika * MeSH
- sekvenování celého genomu MeSH
- sekvenování exomu MeSH
- sliny * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Non-invasive prenatal tests for the detection of fetal aneuploidies are predominantly based on the analysis of cell-free DNA (cfDNA) from the plasma of pregnant women by next-generation sequencing. The development of alternative tests for routine genetic laboratories is therefore desirable. Multiplex digital droplet PCR was used to detect 16 amplicons from chromosome 21 and 16 amplicons from chromosome 18 as the reference. Two fluorescently labeled lock nucleic acid probes were used for the detection of reaction products. The required accuracy was achieved by examining 12 chips from each patient using Stilla technology. The plasma cfDNA of 26 pregnant women with euploid pregnancies and 16 plasma samples from pregnancies with trisomy 21 were analyzed to determine the cutoff value for sample classification. The test was validated in a blind study on 30 plasma samples from pregnant patients with a risk for trisomy 21 ranging from 1:4 to 1:801. The results were in complete agreement with the results of the invasive diagnostic procedure (sensitivity, specificity, PPV, and NPV of 100%). Low cost, and speed of analysis make it a potential screening method for implementation into the clinical workflow to support the combined biochemical and ultrasound results indicating a high risk for trisomy 21.
- MeSH
- aneuploidie MeSH
- Downův syndrom * diagnóza genetika MeSH
- lidé MeSH
- polymerázová řetězová reakce MeSH
- prenatální diagnóza metody MeSH
- těhotenství MeSH
- trizomie MeSH
- volné cirkulující nukleové kyseliny * genetika MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
We investigated the possible associations between leukocyte telomere length, therapy outcomes, and clinicopathological features in patients with colorectal cancer. Additionally, telomerase reverse transcriptase (TERT) expression was evaluated. Telomere length was measured using singleplex qPCR in 478 consecutive leukocyte DNA samples from 198 patients. Blood was drawn at diagnosis prior to any therapy and then at 6-month intervals for 18 months. Following diagnosis, the telomeres gradually shortened during the course of the treatment regardless of the patient's age. The most pronounced decrease was observed 12 months after the diagnosis (p < 0.0001). Based on tumor localization, the decrease in telomere length one year after the diagnosis followed different trajectories (p = 0.03). In patients treated with adjuvant therapy, telomere length correlated with the time elapsed after completion of therapy (p = 0.03). TERT expression did not correlate with the telomere length; however, it was higher in women than men (1.35-fold, 95% CI 1.11-1.65, p = 0.003) and in smokers than non-smokers (1.27-fold, 95% CI 1.01-1.61, p = 0.04). Leukocyte telomere length declines naturally during aging, but the accelerated shortening observed in our patients was age-independent. Telomere length manifestly reflected chemotherapy impact and could be linked to therapy toxicity.
- Publikační typ
- časopisecké články MeSH
Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV) represents an autoimmunity disease characterized by high mortality. For successful treatment, the detailed knowledge of its complex pathogenesis and the set of biomarkers for differential diagnostics are desired. Analysis of molecular content of small urinary extracellular vesicles (uEV) offers the possibility to find markers in the form of microRNAs (miRNAs) and study the pathways involved in pathogenesis. We used next-generation sequencing in the first preliminary study to detect the miRNAs with altered expression in uEVs of patients with AAV in comparison with age-matched controls. We confirmed the results using single-target quantitative polymerase chain reaction tests on different sets of samples and found five miRNAs (miR-30a-5p, miR-31-3p, miR-99a-5p, miR-106b-5p, miR-182-5p) with highly elevated levels in uEVs of patients. We performed the comparison of their targets with the differentially expressed proteins in uEVs of patients included in the first phase. We realized that upregulated miRNAs and proteins in uEVs in AAV patients target different biological pathways. The only overlap was detected in pathways regulating the actin cytoskeleton assembly and thus potentially affecting the glomerular functions. The associations of upregulated miRNAs with pathways that were neglected as components of complex AAV pathogenesis, e.g., the epidermal growth factor receptor signaling pathway, were found.
- MeSH
- ANCA-asociované vaskulitidy * genetika MeSH
- biologické markery MeSH
- extracelulární vezikuly * genetika MeSH
- ledviny MeSH
- lidé MeSH
- mikro RNA * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Structural variants (SVs) represent an important source of genetic variation. One of the most critical problems in their detection is breakpoint uncertainty associated with the inability to determine their exact genomic position. Breakpoint uncertainty is a characteristic issue of structural variants detected via short-read sequencing methods and complicates subsequent population analyses. The commonly used heuristic strategy reduces this issue by clustering/merging nearby structural variants of the same type before the data from individual samples are merged. RESULTS: We compared the two most used dissimilarity measures for SV clustering in terms of Mendelian inheritance errors (MIE), kinship prediction, and deviation from Hardy-Weinberg equilibrium. We analyzed the occurrence of Mendelian-inconsistent SV clusters that can be collapsed into one Mendelian-consistent SV as a new measure of dataset consistency. We also developed a new method based on constrained clustering that explicitly identifies these types of clusters. CONCLUSIONS: We found that the dissimilarity measure based on the distance between SVs breakpoints produces slightly better results than the measure based on SVs overlap. This difference is evident in trivial and corrected clustering strategy, but not in constrained clustering strategy. However, constrained clustering strategy provided the best results in all aspects, regardless of the dissimilarity measure used.
- MeSH
- genom lidský * MeSH
- genomika MeSH
- lidé MeSH
- nejistota MeSH
- shluková analýza MeSH
- strukturální variace genomu * MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND AND OBJECTIVES: The aim of the study was to optimize routine non-invasive prenatal detection of fetal RHD gene from plasma of RhD-negative pregnant women (the median of gestational age was 25 weeks, range 10-38) to detect RhD materno-fetal incompatibility and to avoid the redundant immunoprophylaxis. MATERIALS AND METHODS: Initially only one exon of RHD gene (exon 10) was investigated in 281 plasma samples (144 verified after delivery), in the second phase three RHD exons (5, 7, 10) were analyzed in 246 samples of plasma and maternal genomic DNA (204 verified) by real-time PCR method. Detection of Y-chromosomal sequence DYS-14 and five X-chromosomal insertion/deletion polymorphisms was used to confirm the fetal cfDNA detectability in plasma. Specific polymorphisms in RHD gene were detected by sequence-specific primer PCR in nine samples. RESULTS: When only the RHD exon 10 was tested, 2·8% of verified samples were false positive and 3·5% false negative. With three RHD exons (5, 7, 10) and maternal genomic DNA testing, only one case was false negative (0·5%). Nine samples were inconclusive due to RHD-positive results in maternal genomic DNA. These samples were analyzed for specific mutations in RHD gene. Combination of both methods for fetal cfDNA verification succeeded in 75% of tested group. CONCLUSION: Implementation of analysis of three RHD exons and maternal genomic DNA to routine practice lowers dramatically the ratio of false positive and negative results. This method enables more accurate determination of fetal RHD status with the reduction of unnecessary medical care and RhD immunoprophylaxis.
- MeSH
- DNA MeSH
- genotyp MeSH
- kojenec MeSH
- krevní skupiny - systém Rh-Hr * genetika MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- lidé MeSH
- plod MeSH
- prenatální diagnóza * MeSH
- těhotenství MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The digital polymerase chain reaction (dPCR) multiplexing method can simultaneously detect and quantify closely related deoxyribonucleic acid sequences in complex mixtures. The dPCR concept is continuously improved by the development of microfluidics and micro- and nanofabrication, and different complex techniques are introduced. In this review, we introduce dPCR techniques based on sample compartmentalization, droplet- and chip-based systems, and their combinations. We then discuss dPCR multiplexing methods in both laboratory research settings and advanced or routine clinical applications. We focus on their strengths and weaknesses with regard to the character of biological samples and to the required precision of such analysis, as well as showing recently published work based on those methods. Finally, we envisage possible future achievements in this field.
- MeSH
- biosenzitivní techniky * MeSH
- polymerázová řetězová reakce MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Rozvoj molekulárně genetických technik, provázený vývojem bioinformatických přístupů a rozšířením genealogie jako hobby, přináší nové možnosti také forenzní genetice. V přehledném článku jsou porovnány dnes již klasické metody genotypizace a určování příbuznosti, založené na analýze polymorfismů typu krátkých tandemových repetic, s možnostmi investigativní genetické genealogie, která využívá genotypizaci založenou na analýze stovek tisíců jednonukleotidových polymorfimů, bioinformatiky a sdílení profilů jednotlivců v rámci veřejných databází.
The development of molecular genetic techniques, coupled with the development of bioinformatic approaches and the expansion of genealogy as a hobby, also brings new possibilities for forensic genetics. The review article compares today's classical methods of genotyping and determination of kinship based on the analysis of polymorphisms such as short tandem repeats, with the possibilities of investigative genetic genealogy, which uses genotyping based on analysis of hundreds of thousands of single nucleotide polymorphisms, bioinformatics and sharing individual profiles within public databases. We describe the principles of these laboratory techniques and the interpretation of their results. We compare these new approaches with the current practice in forensic laboratories. We stress the importance of the introduction of whole genome sequencing into forensic genetics. Whole genome sequencing allows due its ability to work with degraded DNA samples the preparation of proper material for investigative genetic genealogy which has already proven its usefulness in successfully solving serial murder cases as well as in identifying unknown victims of violent crimes. In addition to methodological approaches and achievements, the limits and ethical aspects of this new field of forensic genetics are also mentioned.
In families with X-linked recessive diseases, foetal sex is determined prenatally by detection of Y-chromosomal sequences in cell-free foetal DNA (cffDNA) in maternal plasma. The same procedure is used to confirm the cffDNA presence during non-invasive prenatal RhD incompatibility testing but there are no generally accepted markers for the detection of cffDNA fraction in female-foetus bearing pregnancies. We present a methodology allowing the detection of paternal X-chromosomal alleles on maternal background and the confirmation of female sex of the foetus by positive amplification signals. Using digital droplet PCR (ddPCR) we examined X-chromosomal INDEL (insertion/deletion) polymorphisms: rs2307932, rs16397, rs16637, rs3048996, rs16680 in buccal swabs of 50 females to obtain the population data. For all INDELs, we determined the limits of detection for each ddPCR assay. We examined the cffDNA from 63 pregnant women bearing Y-chromosome negative foetuses. The analysis with this set of INDELs led to informative results in 66.67% of examined female-foetus bearing pregnancies. Although the population data predicted higher informativity (74%) we provided the proof of principle of this methodology. We successfully applied this methodology in prenatal diagnostics in a family with Wiscott-Aldrich syndrome and in pregnancies tested for the risk of RhD incompatibility.
- MeSH
- analýza určování pohlaví metody MeSH
- dospělí MeSH
- genetické testování MeSH
- lidé MeSH
- lidské chromozomy X genetika MeSH
- mutace INDEL * MeSH
- plod chemie metabolismus MeSH
- polymerázová řetězová reakce metody MeSH
- polymorfismus genetický * MeSH
- prenatální diagnóza metody MeSH
- těhotenství MeSH
- volné cirkulující nukleové kyseliny analýza genetika MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
PCR has become one of the most valuable techniques currently used in bioscience, diagnostics and forensic science. Here we review the history of PCR development and the technologies that have evolved from the original PCR method. Currently, there are two main areas of PCR utilization in bioscience: high-throughput PCR systems and microfluidics-based PCR devices for point-of-care (POC) applications. We also discuss the commercialization of these techniques and conclude with a look into their modifications and use in innovative areas of biomedicine. For example, real-time reverse transcription PCR is the gold standard for SARS-CoV-2 diagnoses. It could also be used for POC applications, being a key component of the sample-to-answer system.
- MeSH
- Betacoronavirus genetika izolace a purifikace MeSH
- COVID-19 MeSH
- design vybavení MeSH
- klinické laboratorní techniky přístrojové vybavení metody MeSH
- koronavirové infekce diagnóza virologie MeSH
- lidé MeSH
- mikrofluidní analytické techniky přístrojové vybavení metody MeSH
- pandemie MeSH
- polymerázová řetězová reakce přístrojové vybavení metody MeSH
- SARS-CoV-2 MeSH
- testování na COVID-19 MeSH
- virová pneumonie diagnóza virologie MeSH
- vyšetření u lůžka MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
