Amplicon based NGS of human CD4 and CD8 T cells
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ABSTRACT:
PROVIDER: EGAS00001004139 | EGA |
REPOSITORIES: EGA
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Project description:Genes of the major histocompatibility complex (MHC) code for cell surface proteins essential for adaptive immunity. They show the most outstanding genetic diversity in vertebrates, which has been connected with various fitness traits and thus with the long-term persistence of populations. In this study, polymorphism of the MHC class II DRB locus was investigated in chamois with Single-Strand Conformation Polymorphism (SSCP)/Sanger genotyping and Ion Torrent S5 next-generation sequencing (NGS). From eight identified DRB variants in 28 individuals, five had already been described, and three were new, undescribed alleles. With conventional SSCP/Sanger sequencing, we were able to detect seven alleles, all of which were also detected with NGS. We found inconsistencies in the individual genotypes between the two methods, which were mainly caused by allelic dropout in the SSCP/Sanger method. Six out of 28 individuals were falsely classified as homozygous with SSCP/Sanger analysis. Overall, 25% of the individuals were identified as genotyping discrepancies between the two methods. Our results show that NGS technologies are better performing in sequencing highly variable regions such as the MHC, and they also have a higher detection capacity, thus allowing a more accurate description of the genetic composition, which is crucial for evolutionary and population genetic studies.
| S-EPMC7552744 | biostudies-literature
Project description:Motivation:PCR-based DNA enrichment followed by massively parallel sequencing is a straightforward and cost effective method to sequence genes up to high depth. The full potential of amplicon-based sequencing assays is currently not achieved as analysis methods do not take into account the source amplicons of the detected variants. Tracking the source amplicons has the potential to identify systematic biases, enhance variant calling and improve the designs of future assays. Results:We present Nimbus, a software suite for the analysis of amplicon-based sequencing data. Nimbus includes tools for data pre-processing, alignment, single nucleotide polymorphism (SNP), insertion and deletion calling, quality control and visualization. Nimbus can detect SNPs in its alignment seeds and reduces alignment issues by the usage of decoy amplicons. Tracking the amplicons throughout analysis allows easy and fast design optimization by amplicon performance comparison. It enables detection of probable false positive variants present in a single amplicon from real variants present in multiple amplicons and provides multiple sample visualization. Nimbus was tested using HaloPlex Exome datasets and outperforms other callers for low-frequency variants. The variants called by Nimbus were highly concordant between twin samples and SNP-arrays. The Nimbus suite provides an end-to-end solution for variant calling, design optimization and visualization of amplicon-derived next-generation sequencing datasets. Availability and implementation:https://github.com/erasmus-center-for-biomics/Nimbus. Supplementary information:Supplementary data are available at Bioinformatics online.
| S-EPMC6084620 | biostudies-literature
Project description:OBJECTIVE:Model trajectories of CD4+ and CD8+ cell counts after starting combination antiretroviral therapy (ART) and use the model to predict trends in these counts and the CD4+?:?CD8+ ratio. DESIGN:Cohort study of antiretroviral-naïve HIV-positive adults who started ART after 1997 (ART Cohort Collaboration) with more than 6 months of follow-up data. METHODS:We jointly estimated CD4+ and CD8+ cell count trends and their correlation using a bivariate random effects model, with linear splines describing their population trends, and predicted the CD4+?:?CD8+ ratio trend from this model. We assessed whether CD4+ and CD8+ cell count trends and the CD4+?:?CD8+ ratio trend varied according to CD4+ cell count at start of ART (baseline), and, whether these trends differed in patients with and without virological failure more than 6 months after starting ART. RESULTS:A total of 39?979 patients were included (median follow-up was 53 months). Among patients with baseline CD4+ cell count at least 50 cells/?l, predicted mean CD8+ cell counts continued to decrease between 3 and 15 years post-ART, partly driving increases in the predicted mean CD4+?:?CD8+ ratio. During 15 years of follow-up, normalization of the predicted mean CD4+?:?CD8+ ratio (to >1) was only observed among patients with baseline CD4+ cell count at least 200 cells/?l. A higher baseline CD4+ cell count predicted a shorter time to normalization. CONCLUSION:Declines in CD8+ cell count and increases in CD4+?:?CD8+ ratio occurred up to 15 years after starting ART. The likelihood of normalization of the CD4+?:?CD8+ ratio is strongly related to baseline CD4+ cell count.
| S-EPMC5991182 | biostudies-literature
Project description:Genetic heterogeneity can provide tumors with opportunities for therapy evasion, however the degree of genetic heterogeneity within metastatic melanomas has not been thoroughly investigated. We therefore isolated DNA from different regions of formalin fixed paraffin embedded metastatic melanoma tissue samples and subjected them to amplicon sequencing-based profiling of mutations in a panel of well known cancer genes using the Ion Ampliseq Cancer Panel.
2013-09-08 | E-MTAB-1794 | biostudies-arrayexpress
Project description:The study of gene expression as an intermediate phenotype in associating genotype to disease is growing rapidly. The success of this approach depends heavily on the availability of accurate, high throughput platforms for assaying expression levels of genome-wide gene sets. We evaluated a new platform for gene expression, Phalanx Human OneArray (HOA), in 90 HapMap cell lines (CEU panel) for which Illumina BeadChip data were available (Stranger et al. 2007, PMID: 17873874). We found high reproducibility between technical HOA replicates (R = 0.99) and strong inter-platform correlation based on 2,458 sequence-matched probes (R = 0.87). We next performed an association analysis between gene expression levels and HapMap SNPs in the 60 unrelated CEU parents [SNP data downloaded directly from HapMap project (http://www.hapmap.org/)(HapMap phase II/Rel # 22)]. Among the 4356 genes (HOA array) showing variable expression, 122 (2.8%) had a significant cis association (p-value < 0.001) with at least one SNP. Of the 1751 genes showing variable expression on both platforms, 84 had a significant cis association with 36.9% of the genes (31/84) present in both data sets, 39.3 % (33/84) only in HOA, and 23.8% (20/84) only in Illumina. A subset of these 84 eQTLs overlap or are in strong linkage disequilibrium (LD) with reported complex disease/trait susceptibility loci. Combining the results of the two platforms provides a more comprehensive eQTL database (30% increase), which may help to accelerate our understanding of the molecular basis of quantitative traits and diseases. We quantified gene expression levels in the 90 HapMap cell lines (CEU panel) using Phalanx HOA for which Illumina BeadChip data were available (Stranger et al. 2007). We used the same set of RNA samples employed in Stranger et al 2007 (see GEO Series GSE6536, CEU panel; raw data were re-analyzed, ie, re-normalized, thereby generating new values) which allowed us to undertake a detailed comparison to the Illumina expression platform and profiled the 60 parent samples at Industrial Technology Research Institute (ITRI), Taiwan and the 30 children samples at Wellcome Trust Sanger Institute (WTSI), United Kingdom. We performed two in vitro transcription (IVT) reactions for each RNA sample and three technical replicates per IVT with exception of seven samples (15CP, 33CP, 43CP, 44CP, 48CP, 56CP, 60CP) that had two technical replicates for one of the two IVT reactions. *** This Series represents the Phalanx HOA data. *** File "info.pass.parent.txt" (a description of the relationship between an array and a sample and its replicates) is linked below as a supplementary file.
2012-04-29 | E-GEOD-15905 | biostudies-arrayexpress
Project description:The gut microbiota in chicken has long been studied, mostly from the perspective of growth performance. However, there are some immunological studies regarding gut homeostasis in chicken. Although CD4+CD25+ T cells are reported to act as regulatory T cells (Tregs) in chicken, there have been no studies showing the relationship between gut microbiota and Tregs. Therefore, we established a model for 'antibiotics (ABX)-treated chickens' through administration of an antibiotic cocktail consisting of ampicillin, gentamycin, neomycin, metronidazole, and vancomycin in water for 7 days. CD4+CD8-CD25+ and CD4+CD8+CD25+ T cells in cecal tonsils were significantly decreased in this model. Gram-positive bacteria, especially Clostridia, was responsible for the changes in CD4+CD8-CD25+ or CD4+CD8+CD25+ T cells in cecal tonsils. Feeding ABX-treated chickens with acetate recovered CD4+CD8-CD25+ and CD4+CD8+CD25+ T cells in cecal tonsils. GPR43, a receptor for acetate, was highly expressed in CD4+CD8-CD25+ T cells. In conclusion, our study demonstrated that the gut microbiota can regulate the population of CD4+CD8-CD25+ and CD4+CD8+CD25+ T cells, and that acetate is responsible for the induction of CD4+CD8-CD25+ T cells in cecal tonsils via GPR43.
| S-EPMC5988814 | biostudies-literature
Project description:Follicular cell-derived thyroid cancers are heterogenous and morphological classification is a complex and highly specialized task. Hence, identification of somatic alterations could provide insights to tumor biology and serve as an add-on diagnostic tool. Furthermore, results from these add-on tools could point in the direction of a more personalized treatment strategy. In the present study we set out to identify and validate the somatic mutation profile in a sample-set of follicular cell-derived thyroid neoplasia. One-hundred-and-one archived formalin fixed paraffin embedded (FFPE) tissue samples from patients diagnosed with follicular cell-derived thyroid neoplasia were included, and upon DNA-extraction and qualitative measurements 99 samples were eligible for amplicon-based next-generation-sequencing. Libraries were generated using the TruSeq Amplicon Cancer Panel, followed by sequencing using a MiSeq. Upon data processing and variant filtering all variants were manually assessed to exclude false positive mutations in the final curated list. Moreover, hot-spot mutations were validated using an independent platform from Agilent. Each diagnostic group were correlated to mutation burden and individual mutations were classified according to recent guidelines for somatic mutation classification. Close to 100% of the archived FFPE samples were eligible for DNA-library preparation and amplicon sequencing based on DNA quality criterion. The distribution of mutations in the specific diagnostic groups resulted in a higher mutation frequency among the most dedifferentiated than in the groups with a more differentiated cell profile. Based on the distribution mutations across the samples and using hierarchical clustering, we generated four tentative mutational signatures; highly mutated tumors; tumors with mainly NRAS and TP53 mutations; BRAF mutated tumors and tumors with none or single sporadic mutations. Future studies including more samples and follow-up data may amend these signatures, however our results imply that morphological classification of follicular cell derived thyroid neoplasia could be supplemented with a somatic mutational signature. Taken together, broad screening of the somatic alterations in FFPE tissue of thyroid neoplasia is comprehensible and essential for future identification of possible treatment targets and personalized medicine.
| S-EPMC7105679 | biostudies-literature
Project description: Not available
| S-BSST1142 | biostudies-other
Project description:Next-generation sequencing (NGS) enables rapid identification of common and rare drug-resistant genetic variations from tuberculosis (TB) patients' sputum samples and MTB isolates. However, whether this technology is effective for formalin-fixed and paraffin-embedded (FFPE) tissues remains unclear. An amplicon-based targeted NGS sequencing panel was developed to predict susceptibility to 9 antituberculosis drugs, including 3 first-line drugs, by directly detecting FFPE tissues. A total of 178 tissue samples from TB patients who underwent phenotypic drug susceptibility test were retrospectively tested from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. Phenotypic drug susceptibility test results were used as the reference standard. We identified 22 high-quality mutations from 178 FFPE tissue samples, including 15 high+moderate+minimal confidence-level mutations associated with drug resistance (rpoB D435V, S450F/L; KatG S315T; inhA-fabG promoter c-15t; embB G406S, M306V; rpsL K43R, K88R, rrs a1401g, a514c; gyrA D94G/Y/A, A90V), 6 mutations not associated with resistance (rpoB D435Y, H445S, L430P, L452P; embB G406A/D), and one mutation site embB M306I defined as indeterminate. Compared to the phenotypic method, sensitivities (95% CI) for rifampicin, isoniazid, and ethambutol were 96% (79.65-99.90%), 93.55% (78.58-99.21%), and 71.43% (35.24-92.44%), respectively; while for second-line drugs, it varied from 23.53% (9.05-47.77%) for capreomycin to 86.84% (72.20-94.72%) for streptomycin. Specificities for all drugs were satisfactory (>94.51%). Therefore, important pathological FFPE tissue samples, despite partially degraded DNA, can be used as essential specimens for molecular diagnosis of drug resistant TB by amplicon-based targeted NGS technology. IMPORTANCE Amplicon-based targeted NGS technology focuses on a set of gene mutations of known or suspected associations with drug susceptibility in Mycobacterium tuberculosis (MTB). This method offers many benefits, such as low sequencing cost, easy customization, high throughput, shorter testing time and not culture dependent. Formalin-fixed and paraffin-embedded (FFPE) tissues are important pathological specimen in diagnosing tuberculous disease because they are noninfectious and provide excellent preservation of tissue morphology with low storage cost. However, the performance of amplicon-based targeted NGS method on FFPE samples has not been reported yet. Therefore, we evaluated the performance of this method using FFPE samples collected from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. We demonstrate that the amplicon-based targeted NGS method performs excellent on FFPE samples, and it can be applied to pathological diagnosis of drug resistant tuberculosis.
| S-EPMC8826733 | biostudies-literature
Project description:CD4 and CD8 T cells are vital components of the immune system. We found that histone deacetylase 3 (HDAC3) is critical for the development of CD4 T cells, as HDAC3-deficient DP thymocytes generate only CD8SP thymocytes in mice. In the absence of HDAC3, MHC Class II-restricted OT-II thymocytes are redirected to the CD8 cytotoxic lineage, which occurs with accelerated kinetics. Analysis of histone acetylation and RNA-seq reveals that HDAC3-deficient DP thymocytes are biased towards the CD8 lineage prior to positive selection. Commitment to the CD4 or CD8 lineage is determined by whether persistent TCR signaling or cytokine signaling predominates, respectively. Despite elevated IL-21R/γc/STAT5 signaling in HDAC3-deficient DP thymocytes, blocking IL-21R does not restore CD4 lineage commitment. Instead, HDAC3 binds directly to CD8-lineage promoting genes. Thus, HDAC3 is required to restrain CD8-lineage genes in DP thymocytes for the generation of CD4 T cells.
| S-EPMC6338460 | biostudies-literature