PURPOSE: We set out to develop a publicly available tool that could accurately diagnose spinal muscular atrophy (SMA) in exome, genome, or panel sequencing data sets aligned to a GRCh37, GRCh38, or T2T reference genome. METHODS: The SMA Finder algorithm detects the most common genetic causes of SMA by evaluating reads that overlap the c.840 position of the SMN1 and SMN2 paralogs. It uses these reads to determine whether an individual most likely has 0 functional copies of SMN1. RESULTS: We developed SMA Finder and evaluated it on 16,626 exomes and 3911 genomes from the Broad Institute Center for Mendelian Genomics, 1157 exomes and 8762 panel samples from Tartu University Hospital, and 198,868 exomes and 198,868 genomes from the UK Biobank. SMA Finder's false-positive rate was below 1 in 200,000 samples, its positive predictive value was greater than 96%, and its true-positive rate was 29 out of 29. Most of these SMA diagnoses had initially been clinically misdiagnosed as limb-girdle muscular dystrophy. CONCLUSION: Our extensive evaluation of SMA Finder on exome, genome, and panel sequencing samples found it to have nearly 100% accuracy and demonstrated its ability to reduce diagnostic delays, particularly in individuals with milder subtypes of SMA. Given this accuracy, the common misdiagnoses identified here, the widespread availability of clinical confirmatory testing for SMA, and the existence of treatment options, we propose that it is time to add SMN1 to the American College of Medical Genetics list of genes with reportable secondary findings after genome and exome sequencing.
- MeSH
- algoritmy MeSH
- exom genetika MeSH
- genom lidský genetika MeSH
- genomika metody MeSH
- lidé MeSH
- protein přežití motorických neuronů 1 genetika MeSH
- protein přežití motorických neuronů 2 genetika MeSH
- sekvenční analýza DNA metody MeSH
- sekvenování exomu MeSH
- spinální svalová atrofie * genetika diagnóza MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Mezinárodní panel odborníků vydal společné konsenzuální stanovisko k možnostem klinického využití analýzy střevního mikrobiomu s jednoznačným závěrem: Mikrobiom je důležitou složkou lidského těla a jeho stav nezpochybnitelně souvisí s lidským zdravím. Metodika analýzy složení mikrobiomu však dosud nebyla standardizována a interpretace výsledků vzhledem ke zdraví jedince zatím není dostatečně průkazná. Terapeutické poradenství na základě výsledku testování mikrobiomu se důrazně nedoporučuje. Pro široké použití v klinické praxi je nutný další výzkum.
The international panel of experts has issued a joint consensus opinion on the possibilities of clinical use of gut microbiome analysis with an unequivocal conclusion: The microbiome is an important component of the human body, and its condition is indisputably related to human health. However, the methodology of microbiome composition analysis has not yet been standardized and the interpretation of the results regarding the health of the individual is not yet sufficiently conclusive. Therapeutic counseling based on the results of microbiome testing is strongly discouraged. Further research is required for widespread use in clinical practice.
BACKGROUND: Glioblastoma is the commonest malignant brain tumor and has a very poor prognosis. Reduced expression of the MGMT gene (10q26.3), influenced primarily by the methylation of two differentially methylated regions (DMR1 and DMR2), is associated with a good response to temozolomide treatment. However, suitable methods for detecting the methylation of the MGMT gene promoter and setting appropriate cutoff values are debated. RESULTS: A cohort of 108 patients with histologically and genetically defined glioblastoma was retrospectively examined with methylation-specific Sanger sequencing (sSeq) and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) methods. The DMR2 region was methylated in 29% of samples, whereas DMR1 was methylated in 12% of samples. Methylation detected with the MS-MLPA method using probes MGMT_215, MGMT_190, and MGMT_124 from the ME012-A1 kit (located in DMR1 and DMR2) correlated with the methylation of the corresponding CpG dinucleotides detected with sSeq (p = 0.005 for probe MGMT_215; p < 0.001 for probe MGMT_190; p = 0.016 for probe MGMT_124). The threshold for methylation detection with the MS-MLPA method was calculated with a ROC curve analysis and principal components analysis of the data obtained with the MS-MLPA and sSeq methods, yielding a weighted value of 0.362. Thus, methylation of the MGMT gene promoter was confirmed in 36% of samples. These patients had statistically significantly better overall survival (p = 0.003). CONCLUSIONS: Our results show that the threshold for methylation detection with the MS-MLPA method determined here is useful from a diagnostic perspective because it allows the stratification of patients who will benefit from specific treatment protocols, including temozolomide. Detailed analysis of the MGMT gene promoter enables the more-precise and personalized treatment of patients with glioblastoma.
- MeSH
- CpG ostrůvky genetika MeSH
- DNA modifikační methylasy * genetika MeSH
- dospělí MeSH
- enzymy opravy DNA * genetika MeSH
- glioblastom * genetika farmakoterapie MeSH
- lidé středního věku MeSH
- lidé MeSH
- metylace DNA * genetika MeSH
- nádorové supresorové proteiny * genetika MeSH
- nádory mozku * genetika MeSH
- promotorové oblasti (genetika) * genetika MeSH
- retrospektivní studie MeSH
- sekvenční analýza DNA metody MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- temozolomid terapeutické užití MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
- validační studie MeSH
Allelic variability in the adaptive immune receptor loci, which harbor the gene segments that encode B cell and T cell receptors (BCR/TCR), is of critical importance for immune responses to pathogens and vaccines. Adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread in immunology research making it the most readily available source of information about allelic diversity in immunoglobulin (IG) and T cell receptor (TR) loci. Here, we present a novel algorithm for extrasensitive and specific variable (V) and joining (J) gene allele inference, allowing the reconstruction of individual high-quality gene segment libraries. The approach can be applied for inferring allelic variants from peripheral blood lymphocyte BCR and TCR repertoire sequencing data, including hypermutated isotype-switched BCR sequences, thus allowing high-throughput novel allele discovery from a wide variety of existing data sets. The developed algorithm is a part of the MiXCR software. We demonstrate the accuracy of this approach using AIRR-seq paired with long-read genomic sequencing data, comparing it to a widely used algorithm, TIgGER. We applied the algorithm to a large set of IG heavy chain (IGH) AIRR-seq data from 450 donors of ancestrally diverse population groups, and to the largest reported full-length TCR alpha and beta chain (TRA and TRB) AIRR-seq data set, representing 134 individuals. This allowed us to assess the genetic diversity within the IGH, TRA, and TRB loci in different populations and to establish a database of alleles of V and J genes inferred from AIRR-seq data and their population frequencies with free public access through VDJ.online database.
- MeSH
- alely * MeSH
- algoritmy * MeSH
- genetická variace MeSH
- lidé MeSH
- receptory antigenů B-buněk genetika imunologie MeSH
- receptory antigenů T-buněk genetika imunologie MeSH
- sekvenční analýza DNA metody MeSH
- software * MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Germline DNA testing using the next-gene-ration sequencing (NGS) technology has become the analytical standard for the diagnostics of hereditary diseases, including cancer. Its increasing use places high demands on correct sample identification, independent confirmation of prioritized variants, and their functional and clinical interpretation. To streamline these processes, we introduced parallel DNA and RNA capture-based NGS using identical capture panel CZECANCA, which is routinely used for DNA analysis of hereditary cancer predisposition. Here, we present the analytical workflow for RNA sample processing and its analytical and diagnostic performance. Parallel DNA/RNA analysis allowed credible sample identification by calculating the kinship coefficient. The RNA capture-based approach enriched transcriptional targets for the majority of clinically relevant cancer predisposition genes to a degree that allowed analysis of the effect of identified DNA variants on mRNA processing. By comparing the panel and whole-exome RNA enrichment, we demonstrated that the tissue-specific gene expression pattern is independent of the capture panel. Moreover, technical replicates confirmed high reproducibility of the tested RNA analysis. We concluded that parallel DNA/RNA NGS using the identical gene panel is a robust and cost-effective diagnostic strategy. In our setting, it allows routine analysis of 48 DNA/RNA pairs using NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) in a single run with sufficient coverage to analyse 226 cancer predisposition and candidate ge-nes. This approach can replace laborious Sanger confirmatory sequencing, increase testing turnaround, reduce analysis costs, and improve interpretation of the impact of variants by analysing their effect on mRNA processing.
- MeSH
- DNA genetika MeSH
- genetická predispozice k nemoci * MeSH
- lidé MeSH
- nádory genetika diagnóza MeSH
- reprodukovatelnost výsledků MeSH
- RNA genetika MeSH
- sekvenční analýza DNA metody MeSH
- sekvenční analýza RNA metody MeSH
- vysoce účinné nukleotidové sekvenování * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
16S rRNA amplicon sequencing or, more recently, metatranscriptomic analysis are currently the only preferred methods for microbial profiling of samples containing a predominant ratio of human to bacterial DNA. However, due to the off-target amplification of human DNA, current protocols are inadequate for bioptic samples. Here we present an efficient, reliable, and affordable method for the bacteriome analysis of clinical samples human DNA content predominates. We determined the microbiota profile in a total of 40 human biopsies of the esophagus, stomach, and duodenum using 16S rRNA amplicon sequencing with the widely used 515F-806R (V4) primers targeting the V4 region, 68F-338R primers and a modified set of 68F-338R (V1-V2M) primers targeting the V1-V2 region. With the V4 primers, on average 70% of amplicon sequence variants (ASV) mapped to the human genome. On the other hand, this off-target amplification was absent when using the V1-V2M primers. Moreover, the V1-V2M primers provided significantly higher taxonomic richness and reproducibility of analysis compared to the V4 primers. We conclude that the V1-V2M 16S rRNA sequencing method is reliable, cost-effective, and applicable for low-bacterial abundant human samples in medical research.
- MeSH
- biopsie MeSH
- gastrointestinální trakt MeSH
- geny rRNA MeSH
- lidé MeSH
- mikrobiota * genetika MeSH
- reprodukovatelnost výsledků MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA metody MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Next-generation sequencing methods provide comprehensive data for the analysis of structural and functional analysis of the genome. The draft genomes with low contig number and high N50 value can give insight into the structure of the genome as well as provide information on the annotation of the genome. In this study, we designed a pipeline that can be used to assemble prokaryotic draft genomes with low number of contigs and high N50 value. We aimed to use combination of two de novo assembly tools (SPAdes and IDBA-Hybrid) and evaluate the impact of this approach on the quality metrics of the assemblies. The followed pipeline was tested with the raw sequence data with short reads (< 300) for a total of 10 species from four different genera. To obtain the final draft genomes, we firstly assembled the sequences using SPAdes to find closely related organism using the extracted 16 s rRNA from it. IDBA-Hybrid assembler was used to obtain the second assembly data using the closely related organism genome. SPAdes assembler tool was implemented using the second assembly, produced by IDBA-hybrid as a hint. The results were evaluated using QUAST and BUSCO. The pipeline was successful for the reduction of the contig numbers and increasing the N50 statistical values in the draft genome assemblies while preserving the coverage of the draft genomes.
- MeSH
- sekvenční analýza DNA metody MeSH
- vysoce účinné nukleotidové sekvenování * metody MeSH
- Publikační typ
- časopisecké články MeSH
Provázanost dermatologie a lékařské genetiky je nezanedbatelná. Lékařská genetika a molekulární biologie dnes umožňuje diagnostiku a genetickou prevenci pro široké spektrum onemocnění z oblasti genodermatóz. Součástí této problematiky je i oblast vzácných onemocnění, etické a právní souvislosti genetického poradenství a genetické diagnostiky, včetně postupů prenatálního a preimplantačního testování. V článku informujeme o současných možnostech genetických vyšetření genodermatóz a doporučených postupech.
The interconnection between dermatology and medical genetics is significant. Medical genetics and molecular biology nowadays enable diagnosis and genetic prevention for a wide range of genodermatoses. This includes the area of rare diseases, the ethical and legal implications of genetic counselling and genetic diagnosis, including prenatal and pre-implantation testing procedures. In this article, we report on current options for genetic testing of genodermatoses and recommended procedures.
- MeSH
- genetická onemocnění kůže * diagnóza genetika MeSH
- genetické poradenství MeSH
- genetické testování metody MeSH
- lidé MeSH
- prenatální diagnóza MeSH
- prognóza MeSH
- sekvenční analýza DNA metody MeSH
- vzácné nemoci MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
x
x
- MeSH
- diagnostické techniky molekulární klasifikace MeSH
- mikrobiální genetika metody MeSH
- nanopórové sekvenování metody MeSH
- sekvenční analýza DNA metody MeSH
- sekvenování celého genomu klasifikace metody MeSH
- techniky typizace bakterií metody MeSH
- vysoce účinné nukleotidové sekvenování * klasifikace metody přístrojové vybavení MeSH
- Publikační typ
- práce podpořená grantem MeSH
- přehledy MeSH
