Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols.
- MeSH
- Metagenomics * MeSH
- Viruses * genetics MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Publication type
- Journal Article MeSH
Fungi are major ecological players in both terrestrial and aquatic environments by cycling organic matter and channelling nutrients across trophic levels. High-throughput sequencing (HTS) studies of fungal communities are redrawing the map of the fungal kingdom by hinting at its enormous - and largely uncharted - taxonomic and functional diversity. However, HTS approaches come with a range of pitfalls and potential biases, cautioning against unwary application and interpretation of HTS technologies and results. In this Review, we provide an overview and practical recommendations for aspects of HTS studies ranging from sampling and laboratory practices to data processing and analysis. We also discuss upcoming trends and techniques in the field and summarize recent and noteworthy results from HTS studies targeting fungal communities and guilds. Our Review highlights the need for reproducibility and public data availability in the study of fungal communities. If the associated challenges and conceptual barriers are overcome, HTS offers immense possibilities in mycology and elsewhere.
Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
Nestr.
Cirkulující mikroRNA (miRNA) jsou nové perspektivní semi-invazivní molekulární markery nádorů různých tkání. O jejich deregulaci u myelodysplastického syndromu (MDS) není známo mnoho informací. V současnosti je diagnostika MDS založena na morfologickém průkazu dysplázie v kostní dřeni. V navrhovaném projektu chceme využít sekvenování nové generace pro screening cirkulujících miRNA v krevní plazmě pacientů s MDS. Studiem profilů plasmatických miRNA i) u neléčených pacientů s odlišnými subtypy onemocnění, ii) v různých kategoriích rizika a iii) u pacientů v průběhu léčby chceme vytipovat cirkulující miRNA, jejichž hladina je asociovaná s průběhem choroby. Celogenomová analýza, po které bude následovat validační fáze na úrovni jednotlivých vybraných miRNA pomocí digitálního PCR, si klade za cíl nalézt nové semi-invazivní molekulární markery vhodné pro monitorování pacientů s MDS a tím v důsledku přispět k prevenci progrese onemocnění, zvýšení přežití a zároveň ke zlepšení komfortu pacientů.; Circulating microRNAs (miRNAs) are new promising semi-invasive molecular markers of various types of cancer. However, little information is known about their deregulation in myelodysplastic syndromes (MDS). Nowadays, the diagnosis of MDS is based on morphological evidence of bone marrow dysplasia. In the proposed project, we will employ next-generation sequencing for the screening of circulating miRNAs in plasma samples from MDS patients. Comparison of plasma miRNA profiles i) in untreated MDS patients with various disease subtypes, ii) in different risk category, and iii) in MDS patients during treatment will enable to select circulating miRNAs with altered levels associated with the course of the disease. The genome-wide analysis followed by a validation phase performed by digital PCR on the level of particular preselected miRNAs aims to identify novel semi-invasive molecular markers suitable for monitoring of MDS patients, finally contributing to prevention of the disease progression, increase of survival, and improving comfort of the patients.
- MeSH
- Biomarkers blood MeSH
- Circulating MicroRNA blood adverse effects MeSH
- Plasma MeSH
- Humans MeSH
- Myelodysplastic Syndromes diagnosis MeSH
- High-Throughput Nucleotide Sequencing methods MeSH
- Check Tag
- Humans MeSH
- Conspectus
- Patologie. Klinická medicína
- NML Fields
- genetika, lékařská genetika
- hematologie a transfuzní lékařství
- NML Publication type
- závěrečné zprávy o řešení grantu AZV MZ ČR
Motivation: Modern molecular methods have increased our ability to describe microbial communities. Along with the advances brought by new sequencing technologies, we now require intensive computational resources to make sense of the large numbers of sequences continuously produced. The software developed by the scientific community to address this demand, although very useful, require experience of the command-line environment, extensive training and have steep learning curves, limiting their use. We created SEED 2, a graphical user interface for handling high-throughput amplicon-sequencing data under Windows operating systems. Results: SEED 2 is the only sequence visualizer that empowers users with tools to handle amplicon-sequencing data of microbial community markers. It is suitable for any marker genes sequences obtained through Illumina, IonTorrent or Sanger sequencing. SEED 2 allows the user to process raw sequencing data, identify specific taxa, produce of OTU-tables, create sequence alignments and construct phylogenetic trees. Standard dual core laptops with 8 GB of RAM can handle ca. 8 million of Illumina PE 300 bp sequences, ca. 4 GB of data. Availability and implementation: SEED 2 was implemented in Object Pascal and uses internal functions and external software for amplicon data processing. SEED 2 is a freeware software, available at http://www.biomed.cas.cz/mbu/lbwrf/seed/ as a self-contained file, including all the dependencies, and does not require installation. Supplementary data contain a comprehensive list of supported functions. Supplementary information: Supplementary data are available at Bioinformatics online.
We provide the complete sequence of a virus tentatively named "Tetranychus urticae-associated picorna-like virus 1PK13" (TuaPV1-PK13) obtained from the high-throughput sequencing of a symptomless apple leaf sample. Although the virus sequence was originally derived from apple leaves, the data suggest that the virus is associated with the two-spotted mite Tetranychus urticae.
BACKGROUND: Genomic sequence assemblies are key tools for a broad range of gene function and evolutionary studies. The diploid amphibian Xenopus tropicalis plays a pivotal role in these fields due to its combination of experimental flexibility, diploid genome, and early-branching tetrapod taxonomic position, having diverged from the amniote lineage ~360 million years ago. A genome assembly and a genetic linkage map have recently been made available. Unfortunately, large gaps in the linkage map attenuate long-range integrity of the genome assembly. RESULTS: We laser dissected the short arm of X. tropicalis chromosome 7 for next generation sequencing and computational mapping to the reference genome. This arm is of particular interest as it encodes the sex determination locus, but its genetic map contains large gaps which undermine available genome assemblies. Whole genome amplification of 15 laser-microdissected 7p arms followed by next generation sequencing yielded ~35 million reads, over four million of which uniquely mapped to the X. tropicalis genome. Our analysis placed more than 200 previously unmapped scaffolds on the analyzed chromosome arm, providing valuable low-resolution physical map information for de novo genome assembly. CONCLUSION: We present a new approach for improving and validating genetic maps and sequence assemblies. Whole genome amplification of 15 microdissected chromosome arms provided sufficient high-quality material for localizing previously unmapped scaffolds and genes as well as recognizing mislocalized scaffolds.
- MeSH
- Chromosomes genetics MeSH
- Genomics MeSH
- Nucleic Acid Hybridization MeSH
- Lasers * MeSH
- Chromosome Mapping MeSH
- Microdissection * MeSH
- Sequence Analysis, DNA methods MeSH
- Nucleic Acid Amplification Techniques MeSH
- High-Throughput Nucleotide Sequencing methods MeSH
- Xenopus genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Understanding the complex Entamoeba communities in the mammalian intestine has been, to date, complicated by the lack of a suitable approach for molecular detection of multiple variants co-occurring in mixed infections. Here, we report on the application of a high throughput sequencing approach based on partial 18S rDNA using the Illumina MiSeq platform. We describe, to our knowledge, for the first time, the Entamoeba communities in humans, free-ranging western lowland gorillas and central chimpanzees living in the Dja Faunal Reserve in Cameroon. We detected 36 Entamoeba haplotypes belonging to six haplotype clusters, containing haplotypes possessing high and low host specificity. Most of the detected haplotypes belonged to commensal Entamoeba, however, the pathogenic species (Entamoeba histolytica and Entamoeba nuttalli) were also detected. We observed that some Entamoeba haplotypes are shared between humans and other hosts, indicating their zoonotic potential. The findings are important not only for understanding the epidemiology of amoebiasis in humans in rural African localities, but also in the context of wild great ape conservation.
- MeSH
- Entamoebiasis epidemiology parasitology veterinary MeSH
- Entamoeba * MeSH
- Gorilla gorilla parasitology MeSH
- Humans MeSH
- Ape Diseases epidemiology parasitology MeSH
- Pan troglodytes parasitology MeSH
- Intestinal Diseases, Parasitic epidemiology parasitology veterinary MeSH
- High-Throughput Nucleotide Sequencing * MeSH
- Conservation of Natural Resources MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Africa MeSH
In the light of recent advances in sequencing, which is becoming increasingly available, the microarray technology has partly assumed a complementary and application role. The last two decades, however, brought into application a genuine microarray approaches and technologies, especially population (VIPTM) chips, enabling one to costeffectively screen populations of tens of thousands of individuals (from humans to microorganisms) and thus effectively generate solutions for personalized medicine as well as multi-patient studies and therapies of neurodegenerative diseases. The paper summarizes basic and advanced contemporary microarray platforms and biomarkers useful for the research and diagnostics of neurodegenerative diseases including the microarray state of the art population (VIPTM) chips. Since the development and function of the nervous system is highly dependent on the products of gene expression, individual genotype and phenotype, the described diagnostic tools will likely contribute to a highly demanded elucidation of the molecular mechanisms and therapeutic solutions for neurodegenerative diseases.
- Keywords
- Ames test,
- MeSH
- Cellulose, Oxidized MeSH
- Pharmaceutic Aids MeSH
- Pharmaceutical Preparations MeSH
- Humans MeSH
- Nutrigenomics MeSH
- DNA Damage MeSH
- High-Throughput Screening Assays MeSH
- Mutagenicity Tests MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Equipment and Supplies MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
UNLABELLED: In an attempt to explore infectious agents associated with nasopharyngeal carcinomas (NPCs), we employed our high-throughput RNA sequencing (RNA-seq) analysis pipeline, RNA CoMPASS, to investigate the presence of ectopic organisms within a number of NPC cell lines commonly used by NPC and Epstein-Barr virus (EBV) researchers. Sequencing data sets from both CNE1 and HONE1 were found to contain reads for human papillomavirus 18 (HPV-18). Subsequent real-time reverse transcription-PCR (RT-PCR) analysis on a panel of NPC cell lines identified HPV-18 in CNE1 and HONE1 as well as three additional NPC cell lines (CNE2, AdAH, and NPC-KT). Further analysis of the chromosomal integration arrangement of HPV-18 in NPCs revealed patterns identical to those observed in HeLa cells. Clustering based on human single nucleotide variation (SNV) analysis of two separate HeLa cell lines and several NPC cell lines demonstrated two distinct clusters with CNE1, as well as HONE1 clustering with the two HeLa cell lines. In addition, duplex-PCR-based genotyping showed that CNE1, CNE2, and HONE1 do not have a HeLa cell-specific L1 retrotransposon insertion, suggesting that these three HPV-18(+) NPC lines are likely products of a somatic hybridization with HeLa cells, which is also consistent with our RNA-seq-based gene level SNV analysis. Taking all of these findings together, we conclude that a widespread HeLa contamination may exist in many NPC cell lines, and authentication of these cell lines is recommended. Finally, we provide a proof of concept for the utility of an RNA-seq-based approach for cell authentication. IMPORTANCE: Nasopharyngeal carcinoma (NPC) cell lines are important model systems for analyzing the complex life cycle and pathogenesis of Epstein-Barr virus (EBV). Using an RNA-seq-based approach, we found HeLa cell contamination in several NPC cell lines that are commonly used in the EBV and related fields. Our data support the notion that contamination resulted from somatic hybridization with HeLa cells, likely occurring at the point of cell line establishment. Given the rarity of NPCs, the long history of NPC cell lines, and the lack of rigorous cell line authentication, it is likely that the actual prevalence and impact of HeLa cell contamination on the EBV field might be greater. We therefore recommend cell line authentication prior to performing experiments using NPC cell lines to avoid inaccurate conclusions. The novel RNA-seq-based cell authentication approach reported here can serve as a comprehensive method for validating cell lines.
- MeSH
- Genome * MeSH
- HeLa Cells chemistry MeSH
- DNA Contamination MeSH
- Humans MeSH
- Cell Line, Tumor chemistry MeSH
- Nasopharyngeal Neoplasms genetics MeSH
- Polymerase Chain Reaction MeSH
- Sequence Analysis, RNA MeSH
- High-Throughput Nucleotide Sequencing MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, N.I.H., Extramural MeSH
