Sekvenování DNA patří již řadu let ke standardním postupům při molekulárně-genetických analýzách biologického materiálu. V medicíně nachází široké uplatnění, zejména v oblasti diagnostiky dědičných chorob a nádorových onemocnění, přičemž rozvoj DNA diagnostiky byl významně podpořen zveřejněním sekvence lidského genomu v roce 2001. V posledních několika letech dochází k rychlému technologickému rozvoji nových sekvenačních technologií, který umožnil vznik sekvenátorů nové generace („tzv. New Generation Sequencing“). Nové technologie založené na principu masivního paralelního sekvenování (např. Roche/454, Illumina Genome Analyzer IIx, Life Technologies SOLiD 3 a další) umožňují zásadní navýšení kapacity sekvenátorů a výrazné snížení ceny. Tento významný technologický pokrok umožnil rozvoj celogenomového sekvenování včetně analýz individuálních lidských genomů a nastartoval rozvoj personální genomiky. První osekvenované individuální lidské genomy patřily významným genetikům J. C. Venterovi (2007) a J. D. Watsonovi (2008), avšak rychle následovaly sekvenační analýzy dalších jedinců z různých etnik, které přinesly podstatné informace o interpersonálních rozdílech ve struktuře genomů (byly např. charakterizovány nukleotidové polymorfismy, delece a amplifikace úseků DNA). První významné aplikovatelné výsledky již přineslo sekvenování genomů nádorových buněk, např. akutní myeloidní leukémie. Ačkoli v současné době ještě nejsme schopni interpretovat význam všech detekovaných variant genomu, znamená možnost sekvenování individuálních lidských genomů zásadní zlom v DNA diagnostice i celé medicíně.
DNA sequencing has become a standard method widely used in molecular genetic analysis of biological materials. Its use in medicine is widespread, especially in diagnostics of inherited disorders and cancer related diseases. Development of DNA diagnostics has been strongly accelerated by publication of the human genome sequence in 2001. During the last few years one can observe rapid development of novel sequencing technologies, which have led to the introduction of so called „New Generation Sequencing“. These new technologies based on principles of massive parallel sequencing (e.g. Roche/454, Illumina Genome Analyzer IIx, Life Technologies SOLiD 3 and others) enable a massive increase of sequencing capacity and in parallel also a fundamental decrease of costs. This major technological breakthrough allowed development of the whole-genome sequencing including analyses of individual human genomes. It also started the era of personal genomics. The first sequenced individual human genomes belonged to famous geneticists J. C. Venter (2007) and J. D. Watson (2008), but they were rapidly followed by sequencing analyses of other individuals from various ethnic groups. These studies brought substantial information about interpersonal differences in genome structure (through characterization of nucleotide polymorphisms, DNA deletions and amplifications etc.). Sequencing of cancer cell genomes, e.g. acute myeloid leukemia has already brought first important clinically relevant results. Although currently we are still unable to interpret the relevance of all detected genome variants, it is obvious, that the possibility to sequence individual human genomes represents a fundamental breakthrough not only in DNA diagnostics but also in clinical medicine.
... Background 2 -- 2.1 Growth in vims genomic sequencing 2 -- 2.2 Growth in vims genomic applications 2 ... ... for genomic applications 7 -- 3. ... ... Practical considerations when implementing a virus genomic sequencing programme 8 -- 3.1 Planning a sequencing ... ... , partial consensus sequences and raw sequence data 12 -- 4.4 Platforms for sharing 13 -- 5. ... ... Practical guidance on technical aspects of genomic sequencing and analysis of SARS-CoV-2 30 -- 6.1 Genome ...
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- MeSH
- Betacoronavirus MeSH
- COVID-19 MeSH
- Disease Outbreaks MeSH
- Genome, Viral MeSH
- Public Health Surveillance MeSH
- Whole Genome Sequencing MeSH
- Conspectus
- Veřejné zdraví a hygiena
- NML Fields
- veřejné zdravotnictví
- virologie
- NML Publication type
- publikace WHO
2nd ed. x, 193 s., il.
Identification of genomic variability in population plays an important role in the clinical diagnostics of human genetic diseases. Thanks to rapid technological development in the field of massive parallel sequencing technologies, also known as next-generation sequencing (NGS), complex genomic analyses are now easier and cheaper than ever before, which consequently leads to more effective utilization of these techniques in clinical practice. However, interpretation of data from NGS is still challenging due to several issues caused by natural variability of DNA sequences in human populations. Therefore, development and realization of projects focused on description of genetic variability of local population (often called "national or digital genome") with a NGS technique is one of the best approaches to address this problem. The next step of the process is to share such data via publicly available databases. Such databases are important for the interpretation of variants with unknown significance or (likely) pathogenic variants in rare diseases or cancer or generally for identification of pathological variants in a patient's genome. In this paper, we have compiled an overview of published results of local genome sequencing projects from United Kingdom and Europe together with future plans and perspectives for newly announced ones.
- MeSH
- Genomics methods MeSH
- Humans MeSH
- Neoplasms * genetics MeSH
- Whole Genome Sequencing MeSH
- High-Throughput Nucleotide Sequencing * methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
- Geographicals
- United Kingdom MeSH
Endosymbiotic relationships between eukaryotic and prokaryotic cells are common in nature. Endosymbioses between two eukaryotes are also known; cyanobacterium-derived plastids have spread horizontally when one eukaryote assimilated another. A unique instance of a non-photosynthetic, eukaryotic endosymbiont involves members of the genus Paramoeba, amoebozoans that infect marine animals such as farmed fish and sea urchins. Paramoeba species harbor endosymbionts belonging to the Kinetoplastea, a diverse group of flagellate protists including some that cause devastating diseases. To elucidate the nature of this eukaryote-eukaryote association, we sequenced the genomes and transcriptomes of Paramoeba pemaquidensis and its endosymbiont Perkinsela sp. The endosymbiont nuclear genome is ~9.5 Mbp in size, the smallest of a kinetoplastid thus far discovered. Genomic analyses show that Perkinsela sp. has lost the ability to make a flagellum but retains hallmark features of kinetoplastid biology, including polycistronic transcription, trans-splicing, and a glycosome-like organelle. Mosaic biochemical pathways suggest extensive 'cross-talk' between the two organisms, and electron microscopy shows that the endosymbiont ingests amoeba cytoplasm, a novel form of endosymbiont-host communication. Our data reveal the cell biological and biochemical basis of the obligate relationship between Perkinsela sp. and its amoeba host, and provide a foundation for understanding pathogenicity determinants in economically important Paramoeba.
In 2016, guidelines for diagnostic Next Generation Sequencing (NGS) have been published by EuroGentest in order to assist laboratories in the implementation and accreditation of NGS in a diagnostic setting. These guidelines mainly focused on Whole Exome Sequencing (WES) and targeted (gene panels) sequencing detecting small germline variants (Single Nucleotide Variants (SNVs) and insertions/deletions (indels)). Since then, Whole Genome Sequencing (WGS) has been increasingly introduced in the diagnosis of rare diseases as WGS allows the simultaneous detection of SNVs, Structural Variants (SVs) and other types of variants such as repeat expansions. The use of WGS in diagnostics warrants the re-evaluation and update of previously published guidelines. This work was jointly initiated by EuroGentest and the Horizon2020 project Solve-RD. Statements from the 2016 guidelines have been reviewed in the context of WGS and updated where necessary. The aim of these recommendations is primarily to list the points to consider for clinical (laboratory) geneticists, bioinformaticians, and (non-)geneticists, to provide technical advice, aid clinical decision-making and the reporting of the results.
- MeSH
- Exome * MeSH
- Genome, Human * MeSH
- Polymorphism, Single Nucleotide MeSH
- Humans MeSH
- Whole Genome Sequencing MeSH
- High-Throughput Nucleotide Sequencing methods MeSH
- Rare Diseases diagnosis genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.
- MeSH
- Benchmarking * MeSH
- Datasets as Topic MeSH
- Humans MeSH
- Mutation MeSH
- DNA Mutational Analysis standards MeSH
- Cell Line, Tumor MeSH
- Breast Neoplasms genetics MeSH
- Reference Standards MeSH
- Reproducibility of Results MeSH
- Whole Genome Sequencing standards MeSH
- High-Throughput Nucleotide Sequencing standards MeSH
- Germ Cells MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Research Support, N.I.H., Extramural MeSH
Tuberculosis (TB) is considered one of the most serious infectious diseases worldwide. Effective control of tuberculosis infection involves multiple steps, such as reliable detection, treatment, an epidemiological control as a part of case management, and further surveillance and monitoring of TB spread in the human population. Due to the accelerating advances in molecular biology, especially in DNA sequencing, in the past decade, the application of these methods has become crucial for TB evolution studies, differentiation of Mycobacterium tuberculosis genotypes, and their distribution. Currently, several molecular genetic methods are available. The oldest typing methods (e.g., IS6110-RFLP, spoligotyping, and MIRU-VNTR) can discover the chain of transmission to the patient. Currently, whole genome sequencing facilitates is furthermore able to identify the source of infection, the transmission trays among individuals sharing the same isolate, as well as determination of the TB evolution and its resistance to antituberculotic agents. It is obvious that this technique will become a new gold standard in genotyping methods in tuberculosis molecular epidemiological studies. In this article, molecular genetic typing methods with a special focus on whole genome sequencing and data management are reviewed.
- MeSH
- Drug Resistance, Bacterial genetics MeSH
- Phylogeography MeSH
- Genome, Bacterial * MeSH
- Genotype MeSH
- Humans MeSH
- Molecular Epidemiology standards MeSH
- Molecular Typing methods standards MeSH
- Mycobacterium tuberculosis classification genetics MeSH
- Whole Genome Sequencing * MeSH
- Tuberculosis diagnosis epidemiology microbiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
Streptococcus pneumoniae (pneumokok) je grampozitivní kok vyvolávající jak neinvazivní, tak invazivní infekční onemocnění. Onemocnění vyvolaná pneumokokem mohou být preventabilní očkováním. Invazivní pneumokoková onemocnění (IPO) musí splňovat mezinárodní definici případu, jsou hlášena na národní i mezinárodní úrovni a v řadě zemí, včetně České republiky, jsou sledována v programu surveillance. Důležitou součástí surveillance IPO je sledování vyskytujících se sérotypů, hodnocení četnosti jednotlivých sérotypů v čase a v relaci k probíhajícím vakcinačním programům. Ve světe i v České republice je u pneumokoků stále častěji prováděna metoda celogenomové sekvenace (whole genome sequencing, WGS), která umožňuje určování sérotypu pneumokoků ze sekvenačních dat, dále přesnou analýzu jejich genetických vztahů a studium genů obsažených v jejich genomu. Celogenomová sekvenace umožňuje získávat spolehlivá a mezinárodně srovnatelná sekvenační data, která lze snadno sdílet. Sekvenační data jsou analyzována pomocí bioinformatických nástrojů, které vyžadují znalosti z oblasti přírodních věd s důrazem na genetiku a znalosti z oblasti bioinformatiky. V publikaci jsou představeny některé možnosti analýzy pneumokoka, kterými jsou sérotypizace, multilokusová sekvenační typizace (MLST), ribozomální MLST (rMLST), core genome MLST (cgMLST), whole genome MLST (wgMLST), single nucleotide polymorphism (SNP) analýza, určení Global Pneumococcal Sequence Cluster (GPSC), stanovení genů virulence a genů antibiotické rezistence. U metody WGS je prezentována strategie její aplikace v Evropě a realizace v praxi. Analýza pneumokoků metodou WGS představuje zlepšení v provádění surveillance IPO, kdy je sérotyp určován molekulárně geneticky, jsou prováděny další podrobnější typizace, získaná data lze snadno mezinárodně porovnávat a lze lépe hodnotit účinnost vakcinačních programů.
Streptococcus pneumoniae (pneumococcus) is a Gram-positive coccus causing both non-invasive and invasive infectious diseases. Pneumococcal diseases are vaccine preventable. Invasive pneumococcal diseases (IPD) meeting the international case definition are reported nationally and internationally and are subject to surveillance programmes in many countries, including the Czech Republic. An important part of IPD surveillance is the monitoring of causative serotypes and their frequency over time and in relation to ongoing vaccination programmes. In the world and in the Czech Republic, whole genome sequencing (WGS) is increasingly used for pneumococci, which allows for serotyping from sequencing data, precise analysis of their genetic relationships, and the study of genes present in their genome. Whole-genome sequencing enables the generation of reliable and internationally comparable data that can be easily shared. Sequencing data are analysed using bioinformatics tools that require knowledge in the field of natural sciences with an emphasis on genetics and expertise in bioinformatics. This publication presents some options for pneumococcal analysis, i.e., serotyping, multilocus sequence typing (MLST), ribosomal MLST (rMLST), core genome MLST (cgMLST), whole genome MLST (wgMLST), single nucleotide polymorphism (SNP) analysis, assignment to Global Pneumococcal Sequence Cluster (GPSC), and identification of virulence genes and antibiotic resistance genes. The WGS strategies and applications for Europe and WGS implementation in practice are presented. WGS analysis of pneumococci allows for improved IPD surveillance, thanks to molecular serotyping, more detailed typing, generation of internationally comparable data, and improved evaluation of the effectiveness of vaccination programmes.
- MeSH
- Molecular Biology methods MeSH
- Multilocus Sequence Typing classification methods MeSH
- Pneumococcal Infections microbiology MeSH
- Whole Genome Sequencing * methods standards MeSH
- Serogroup MeSH
- Serotyping classification methods MeSH
- Streptococcus pneumoniae * genetics isolation & purification MeSH
- Bacterial Typing Techniques classification MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Review MeSH
