3743 results for "RNA AND sequencing OR transcriptome"

in 15.20 milliseconds.

• Molecular_risk_stratification_in_patients_with_T1_colorectal_cancer_WES

  The introduction of bowel cancer screening has led to a significant increase in the proportion of patients being diagnosed with asymptomatic, early-stage colorectal cancer (CRC). Although the majority of these patients are successfully treated with surgery alone, a small proportion of patients have ‘born-to-be-bad’ aggressive lesions with early dissemination leading to distant metastases. Current standard of care histological assessment is unable to distinguish between these aggressive versus non-aggressive early lesions which is essential to provide appropriate clinical management decisions. This study aims to carry out molecular and histological profiling of approximately 300 T1 CRCs in order to develop a molecular stratifier based on the risk of relapse in early-invasive CRC. This novel T1 cohort will represent the world’s largest molecularly characterised T1 cohort of samples, with digital pathology assessment alongside whole exome sequencing, copy number variation analysis and 3’ RNA-seq. This data will be used to generate a robust panel of molecular and/or histological markers applicable to formalin-fixed paraffin embedded (FFPE) archival tissue which discriminates between T1 lesions based on risk of relapse, which will ultimately be used to inform clinical management of CRC at the earliest stages of the disease.

  Study EGAS00001005734

• Identification of Germline Monoallelic Mutations in IKZF2 in Patients with Immune Dysregulation.

  Helios, encoded by IKZF2, is a member of the Ikaros family of transcription factors with pivotal roles in T-follicular helper, NK- and T-regulatory cell physiology. Somatic IKZF2 mutations are frequently found in lymphoid malignancies. Although germline mutations in IKZF1 and IKZF3, encoding Ikaros and Aiolos, have recently been identified in patients with phenotypically similar immunodeficiency syndromes, the effect of germline mutations in IKZF2 on human hematopoiesis and immunity remains enigmatic. We identified germline IKZF2 mutations (one nonsense (p.R291X)- and 4 distinct missense variants) in six patients with systemic lupus erythematosus, immune thrombocytopenia or EBV-associated hemophagocytic lymphohistiocytosis. Patients exhibited hypogammaglobulinemia, decreased number of T-follicular helper and NKcells. Single-cell RNA sequencing of PBMCs from the patient carrying the R291X variant revealed upregulation of pro-inflammatory genes associated with T-cell receptor activation and T-cell exhaustion. Functional assays revealed the inability of HeliosR291X to homodimerize and bind target DNA as dimers. Moreover, proteomic analysis by proximity-dependent Biotin Identification revealed aberrant interaction of 3/5 Helios mutants with core components of the NuRD complex conveying HELIOS mediated epigenetic and transcriptional dysregulation.

  Study EGAS00001005874

• Patient-tailored design for selective co-inhibition of leukemic cell subpopulations

  The extensive primary and secondary drug resistance in many cancer types requires rational approaches to design personalized and selective combinatorial therapies that do not only show synergistic effect in overall cancer cell killing but also result in minimal toxic side effects on non-malignant cells. To address the combinatorial explosion in the number of relevant combinations, we implemented a machine learning approach that prioritizes patient-customized drug combinations with a desired synergy-efficacy-toxicity balance by combining single-cell RNA-sequencing with ex vivo single-agent testing in scarce patient-derived primary cells. When applied to two diagnostic and two refractory AML patient cases, each with a different genetic background, our integrated approach accurately predicted patient-specific combinations that were shown to result not only in synergistic cancer cell co-inhibition but were also capable of targeting specific AML cell subpopulations that emerge in differing stages of disease pathogenesis or treatment regimens. Our data-driven approach provides an unbiased means for systematic identification of personalized combinatorial regimens that selectively co-inhibit leukemic cells while avoiding inhibition of non-malignant cells, and highlight the relevance of considering cell heterogeneity for personalized cancer therapy.

  Study EGAS00001004614

• Targeted therapy of advanced parathyroid carcinoma guided by genomic and transcriptomic profiling (hipo_021)

  Parathyroid carcinoma (PC) is an orphan malignancy with a high risk of recurrence after surgery. Tumor-directed systemic treatments for PC are not established. We used whole-genome and RNA sequencing in four patients with advanced PC to identify molecular alterations that could guide clinical management. In two cases, the genomic and transcriptomic profiles provided targets for experimental therapies that resulted in biochemical response and prolonged disease stabilization: (i) immune checkpoint inhibition with pembrolizumab based on high tumor mutational burden and a single-base substitution signature associated with APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) overactivation; (ii) multi-receptor tyrosine kinase inhibition with lenvatinib due to overexpression of FGFR1 and RET and, later in the course of disease, PARP inhibition with olaparib prompted by signs of defective homologous recombination DNA repair. In addition, our data provided new insights into the molecular landscape of PC with respect to the genome-wide footprints of specific mutational processes and pathogenic germline alterations. These data underscore the potential of comprehensive molecular analyses to improve care for patients with ultra-rare cancers based on insight into disease biology.

  Study EGAS00001006747

• The impact of BNT162b2 mRNA vaccine against SARS-CoV-2 on adaptive and innate immune responses

  The mRNA-based BNT162b2 protects against severe disease and mortality caused by SARS-CoV-2 through induction of specific antibody and T-cell responses. Much less is known about its broad effects on immune responses against other pathogens. In the present study, we investigated the specific adaptive immune responses induced by BNT162b2 vaccination against various SARS-CoV-2 variants, as well as its effects on the responsiveness of human immune cells upon stimulation with heterologous viral, bacterial, and fungal pathogens. BNT162b2 vaccination induced effective humoral and cellular immunity against SARS-CoV-2 that started to wane after six months. We also observed long-term transcriptional changes in immune cells after vaccination, as assessed by RNA sequencing. Additionally, vaccination with BNT162b2 modulated innate immune responses as measured by the production of inflammatory cytokines when stimulated with various microbial stimuli other than SARS-CoV-2, including higher IL-1/IL-6 release and decreased production of IFN-α. Altogether, these data expand our knowledge regarding the overall immunological effects of this new class of vaccines and underline the need of additional studies to elucidate their effects on both innate and adaptive immune responses.

  Study EGAS00001006818

• Single-cell profiling of co-cultures of GSCCs and macrophages

  Glioblastoma (GBM) remains the most malignant primary brain tumor, with a median survival rarely exceeding 2 years. Tumor heterogeneity and an immunosuppressive microenvironment are key factors contributing to the poor response rates of current therapeutic approaches. GBM-associated macrophages (GAMs) often exhibit immunosuppressive features that promote tumor progression. However, their dynamic interactions with GBM tumor cells remain poorly understood. Here, we used patient-derived GBM stem cell cultures and combined single-cell RNA sequencing of GAM-GBM co-cultures and real-time in vivo monitoring of GAM-GBM interactions in orthotopic zebrafish xenograft models to provide insight into the cellular, molecular, and spatial heterogeneity. Our analyses revealed substantial heterogeneity across GBM patients in GBM-induced GAM polarization and the ability to attract and activate GAMs – features that correlated with patient survival. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock-out experiments in zebrafish subsequently identified LGALS1 as a primary regulator of immunosuppression. Overall, our work highlights that GAM-GBM interactions can be studied in a clinically relevant way using co-cultures and avatar models, while offering new opportunities to identify promising immune-modulating targets.

  Study EGAS00001007482

• SINGLE-CELL RNA SEQUENCING Single-cell RNA sequencing was performed on 13 ‘mild-moderate’ and 10 ‘critical’ COVID19 PBMC samples

  Summary Epidemiological and clinical reports have indicated that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we performed cytokine and multiplex immune profiling in mild-moderate and critically ill COVID-19 patients. Hypercytokinemia in COVID-19 differed from the IFN-γ-driven cytokine storm in macrophage activation syndrome, and was more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling showed that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Expression of antigen presenting machinery was also reduced in critical disease. Furthermore, we found that neutrophils contributed to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together these findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

  Study EGAS00001005039

• Genetic characterization of B-cell prolymphocytic leukemia: a hierarchical prognostic model involving MYC and TP53 abnormalities - RNA-seq

  B-cell prolymphocytic leukemia (B-PLL) is a rare disease, whose molecular pathology is largely unknown. We report here the cytogenetic and molecular findings in a large series of 34 B-PLL. Karyotype (K) was complex (≥3 abnormalities) in 73%, and highly complex (HCK≥5) in 45%. The most frequent chromosomal aberrations were: translocation targeting the MYC gene [t(MYC)] (62%), 17p deletion including TP53 gene (38%), trisomy 18/18q (30%), 13q14 deletion (29%), trisomy 3 (24%), trisomy 12 (24%) and 8p deletion (23%). Whole-Exome Sequencing performed in 16 patients revealed recurrent mutations in TP53 (6/16, 38%), MYD88 (n=4), BCOR (n=4), MYC (n=3), SF3B1 (n=3), FAT1 (n=3), SETD2 (n=2), CHD2 (n=2), CXCR4 (n=2) and BCLAF1 (n=2). The main group of patients (21/34, 62%) had a t(MYC) associated with a higher percentage of prolymphocytes (p=0.03), CD38 expression (p<0.001), lower K complexity (p=0.0004), mutations in MYC and in genes involved in RNA metabolism and chromatin remodeling. Principal component analysis of gene expression data showed that patients with t(MYC) clustered together. A second group with MYC gain (5/34, 15%), was associated with HCK≥5 (p=0,01) and trisomy 3 (p=0,008). Altogether, 26/34 patients (76%) had a MYC activation, translocation or gain, that were mutually exclusive. We identified 3 distinct cytogenetic prognostic groups (p=0.0006): lower risk: absence of MYC activation (median not reached); intermediate risk: MYC activation without del17p (125 months); high risk: MYC activation + del17p (11 months). Our results show that cytogenetic analysis is a useful diagnostic tool in B-PLL that improves prognostic stratification.

  Study EGAS00001003274

• Bulk-RNA Sequencing of high-grade pancreatic and non-pancreatic Neuroendocrine Neoplasms

  Therapeutic decisions in oncology depend on a precise pathological classification of individual neoplasms. Recent years have seen an intensification of research activities aimed at the extraction of clinically relevant information from patient-derived 'omics' data based on Machine-Learning models. However, a comprehensive training of Machine-Learning models requires sufficiently large numbers of training samples, which are usually not available for rare cancer types. The problem is worsened when individual tissues segregate into different cancer subtypes, as their discrimination would require even more training samples. Methods: Here, we report on a new data-augmentation technique to support the training of Machine-Learning models on ‘omics’ data from pancreatic neuroendocrine neoplasms (panNEN). PanNENs display all properties described above: Only about 2-3% of all pancreatic neoplasms are neuroendocrine and they fall into different subtypes with distinctly different prognosis, which makes the precise classification of such samples both difficult and important for therapy decisions. The approach reconstructs a given transcriptome based on healthy pancreatic cell type signatures and creates a Machine-Learning model that integrates the observed reconstruction error and predicted cell type proportions as features. Results: A benchmark of the deconvolution model predictions with the ground-truth found that the model could efficiently predict the sample grading, disease-related patient survival time, and differentiate between different subtypes in four panNEN and one mixed panNEN and non-pancreatic NENs dataset. We compared the predictive performance of the deconvolution-trained model to that of a model trained directly on the transcriptomic data, under inclusion of the Ki-67 classification gold standard biomarker, and found the performances to be comparable. Conclusion: Our approach serves as a data-augmentation technique to facilitate the training of Machine Learning models for rare cancer types via utilization of data from healthy origin. Additionally, the deconvolution results were clinically interpretable and further research can render the deconvolution approach an effective complementary asset for the clinical classification of neoplasms.

  Study EGAS00001004861

• Single-cell RNA-sequencing of CD19 Chimeric Antigen Receptor (CAR) T-cells (CD19 CAR T-cells) from a Phase I clinical study in paediatric ALL: CARPALL

  Chimeric antigen receptor (CAR)-modified T-cells have become established as an effective treatment of haematological cancers. In the context of relapsed and refractory childhood pre-B cell acute lymphoblastic leukaemia (B ALL), CD19 targeting CAR T-cells often induce durable remissions. Previously, we generated a novel low-affinity CAR incorporating a CD19-specific single-chain variable fragment (scFV) called CAT, displaying a faster off-rate of interaction than the FMC63 CD19 binder used in prior clinical studies. Here, we systematically analysed CD19 CAR T-cells of ten children with relapsed or refractory B ALL enrolled in the CARPALL trial (NCT02443831). To characterize persisting CD19 CAR T-cells, we performed high throughput single-cell gene expression and T-cell receptor (TCR) sequencing of infusion products and serial blood and bone marrow samples up to five years post-infusion. We isolated CAR T-cells from peripheral blood or bone marrow by flow cytometry for CD3 and CAR expression, prior to single cell sequencing (Chromium 10X) platform.

  Dataset EGAD00001010018

• Whole genome sequencing data from tumor and normal samples

  Whole genome sequencing FASTQ files from tumor and normal samples from 202 patients. All experiments reported in this article were performed in the Clinical Laboratory Improvement Amendments (CLIA)-certified and College of American Pathologists (CAP)-accredited laboratories at Personalis Inc., as guided by the Association for Molecular Pathology (AMP) and CAP’s joint recommendations52. Preferred input material consisting of FFPE slides (enabling H&E and macrodissection which is part of the standard NeXT Personal process) was not available for the majority (94%) of tumor samples. Most tumor samples (72%) were processed from FFPE curls, with the remaining processed from pre-extracted DNA and fresh frozen material. Where FFPE material on slide was available, tumor sections were macrodissected to increase tumor content, with a minimum allowable cellularity threshold of 20% determined by pathological review. The tumor cellularity threshold of 20% was chosen based on prior work demonstrating that no significant correlation is observed between tumor purity and assay limit of detection (LOD) at this threshold31. Genomic DNA was isolated from tumor and normal samples and WGS libraries prepared as previously described31. Genomic DNA was isolated from matched tumor and normal samples using the Qiagen AllPrep DNA/RNA FFPE Tissue Kit or the QIAamp DNA Mini Kit (QIAGEN, Germantown, MD, USA) using internally optimized workflows. WGS sequencing libraries were prepared with 120-500 ng of acoustically sheared genomic DNA (Covaris LLC, Woburn, MA, USA) using the KAPA HyperPrep Kit (Roche Sequencing Solutions, Pleasanton, CA, USA) and customized methods. Libraries were cleaned-up using AMPure XP beads and then quantified using the KAPA Library Quantification Kit (Roche Sequencing Solutions, Pleasanton, CA, USA), before being sequenced to 30X depth of coverage using a NovaSeq 6000 instrument (Illumina, San Diego, CA, USA).

  Dataset EGAD50000001909

• Access to "A Spatially Resolved Single-Cell Atlas of the Human Fetal Olfactory System"

  The human nasal region arises from neural crest and placodal lineages, yet its early development remains poorly understood owing to limited fetal tissue access and structural complexity. Here we present an integrated single-nucleus and spatial transcriptomic atlas of the human fetal nasal region, generated from 10 fetuses (males and females) between 7 and 12 post-conceptional weeks. Single-nucleus RNA sequencing resolved 32 distinct cell types, while integration with multiplexed error-robust fluorescence in situ hybridization (MERFISH) enabled spatial and temporal mapping of gene expression dynamics across the olfactory epithelium (OE) and adjacent tissues. We identify novel markers of olfactory sensory neuron differentiation and pathways governing epithelial patterning and OE morphogenesis. Notably, spatially organized expression of 169 olfactory receptor genes reveals molecular evidence for the “one neuron-one receptor” principle during the first trimester. Together, this work establishes the first molecular and spatial framework of early human olfactory development and provides a foundational resource for studies of sensory neurogenesis and congenital disorders.

  Dac EGAC50000000688

• Genetics of gene expression in human macrophage response to Salmonella

  Understanding how cells sense and respond to their environment, and how these responses are modulated by genetic variation, are fundamental biological problems, particularly for understanding how pathogenic organisms invade and manipulate the cells of the human immune system. Macrophages recognize and respond to many important human pathogens including HIV-1, Mycobacteria tuberculosis and Salmonella. This study will focus on the cellular response of human macrophages to Salmonella infection and how this response is modulated by the genetic bacground of the individual as well as additional pro-inflammatory stimulus (interferon-gamma priming). We will acquire 100 human induced pluripotent stem cell lines from the HipSci project, differentiate the cells in vitro into macrophages and expose them to four environmental conditions: (i) no stimulation, (ii) interferon-gamma (18h), (iii) Salmonella typhimurium SL1344 (5h), (iv) interferon-gamma (18h) + Salmonella (5h).Subsequently, we will isolate RNA from the samples for sequencing.

  Dataset EGAD00001003204

• Single-cell and bulk RNA-sequencing of nivolumab-treated glioblastoma

  Glioblastoma is a highly aggressive type of brain tumour for which there is no curative treatment available. Immunotherapies have shown limited responses in unselected glioblastoma patients, and it is currently not well understood what separates the few responders from the non-responders at the cellular and molecular level. Here we investigate the phenotypic and transcriptional evolutionary dynamics at single-cell resolution during nivolumab immune checkpoint treatment of eight glioblastoma patients enrolled in a translational phase I/II clinical trial.

  Study EGAS00001007110

• Exceptional Responders Initiative

  The Exceptional Responders Initiative is a pilot study to investigate the underlying molecular factors driving exceptional treatment responses of cancer patients to drug therapies. Study researchers will examine molecular profiles of tumors from patients either enrolled in a clinical trial for an investigational drug(s) and who achieved an exceptional response relative to other trial participants, or who achieved an exceptional response to a non-investigational chemotherapy. An exceptional response is defined as achievement of either a complete response or a partial response for at least 6 months duration in a trial or treatment where the overall response rate is < 10%. The hope is to discover underlying molecular features that can be further investigated and may eventually predict benefit from a given drug or class of drugs for a particular patient. This pilot project will successfully characterize approximately 100 cases of tumor tissue and, when available, case-matched germline DNA. All samples will undergo whole exome sequencing, and cases with sufficient nucleic acids will undergo additional analyses (e.g. whole genome sequencing, mRNA-sequencing, mi RNA sequencing, promoter methylation analysis, SNP etc). Each case will be annotated with demographic and clinical information, along with follow-up information minimally sufficient to correlate molecular profiles with response. Both retrospective and prospective collections will be considered. The project will also accept sequencing data and clinical data from patients who have had sequencing performed outside of this project. All data will be de-identified and placed in a controlled access database so other investigators may use them for additional insights. Clinically annotated tissue specimens meeting the criteria will be provided by groups participating in the Exceptional Cases Initiative to a Biospecimen Core Resource (BCR), which will perform quality control on the tissues, and will use a standard operating procedure to isolate nucleic acids. The nucleic acids will be shipped to a sequencing center to perform whole exome sequencing and analysis. These findings will be made available to the broader cancer research community in a controlled access database.

  Study phs001145

• Single cell RNA sequencing of synovial B cells in early Rheumatoid Arthritis

  This dataset includes 3432 paired single cell sequencing fastq files derived from synovial B cells of 5 early Rheumatoid Arthritis patients. Libraries were prepared using the SmartSeq2 protocol. All wells contained ERCC spike-ins. Libraries were sequenced on an Illumina NovaSeq instrument with 2x100bp paired-end reads yielding a median of 2.5M reads/well. Sample aliases ending on 368 and 384 represent empty control wells. Sample aliases starting with P11417_4 and P13157_1 belong to ACPA- patient A7, sample aliases starting with P11417_5 and P11417_6 belong to ACPA+ patient A1, sample aliases starting with P13157_2 belong to ACPA- patient A3, sample aliases starting with P13157_3 and P13157_6 belong to ACPA+ patient A2, sample aliases starting with P13157_4 and P13157_5 belong to ACPA- patient A4.

  Dataset EGAD00001009076

• Renal_habitat_WXS

  This project aims at correlating genetic diversity (as well as transcriptomic, metabolic and immune diversity) observed in multiple targeted tumour biopsies and circulating tumour DNA with heterogeneity on multiparametric MRI. To this end, 20 patients with RCC (15 patients with localised/locally advanced disease and 5 with mRCC) will undergo multiparametric MRI including metabolic assessment by 13C-MRSI as well as measurements of tumour perfusion, oxygenation and water diffusion. Based on unique combinations of these imaging parameters, tumour habitats will be defined through cluster mapping. Intra-operative biopsy and immediate freezing of tumour cores from different imaging habitats will be performed. Multiregional ex-vivo tumour biopsies will be spatially registered to the MR data by using 3D printed tumour moulds generated from high-resolution volumetric sequences. These moulds allow slicing tumour specimens in identical planes to the MR acquisition and increase the accuracy of tissue sampling. The biopsies will then undergo molecular profiling such as metabolomic and RNA sequencing (including mRNA immune profiling), as well as DNA sequencing to assess driver and clonal mutations in different tumour habitats and guide design of appropriate cfDNA probes. The targeted cfDNA capture panel will be employed to determine the ability of cfDNA to capture the full genetic diversity observed in the primary tumour and to relate cfDNA to various imaging habitats in both renal and pancreatic cancer. Data integration will be undertaken between imaging habitats and molecular profiles for each region.

  Study EGAS00001003703

• Identification of driver oncogenes in scirrhous-type gastric cancer cell lines

  Scirrhous type gastric cancer (SGC) is one of the most intractable cancer subtypes in human, and its therapeutic targets have been rarely identified to date. Exploration of somatic mutations in the SGC genome with the next generation sequencers has been hampered by low tumor contents by the markedly increased fibrous tissues. Thus, SGC cell lines may be useful resources to search for novel oncogenes in. Here we have conducted whole exome sequencing and RNA sequencing on two SGC cell lines, OCUM-8 and OCUM-9. Interestingly, most of the mutations thus identified have not been reported. In OCUM-8 cells, a novel CD44-IGF1R fusion gene is discovered, the protein product of which ligates the amino-terminus of CD44 to the transmembrane and the tyrosine-kinase domains of IGF1R. Further, both of CD44 and IGF1R are markedly amplified in the OCUM-8 genome and abundantly expressed. CD44-IGF1R has a transforming ability, and the suppression of its kinase activity leads to rapid cell death of OCUM-8. On the other hand, OCUM-9 seems to possess multiple oncogenic events in its genome. The MET gene is highly amplified, and the treatment of OCUM-9 with crizotinib, a MET inhibitor, results in cell death. Further, a novel BORCS5-ETV6 fusion gene is identified in the OCUM-9 genome. BORCS5-ETV6 possesses oncogenic activity, and suppression of its message partially inhibits cell growth. Prevalence of these novel fusion genes among SGC awaits further investigation, but we validate the significance of cell lines as appropriate reagents for detailed genomic analyses of SGC.

  Study JGAS000179

• Genomic Analysis of Extra-Nodal Natural Killer/T-Cell Lymphoma (ENKTCL)

  In this study, we performed extensive genetic profiling on 209 extra-nodal natural killer/T-cell lymphoma (ENKTCL) nasal-type cases, including 174 Chinese from four hospitals in China, and 31 Hispanic and 4 Caucasian patients from City of Hope and Eberhard Karls University of Tübingen. All of the cases were reviewed by experienced hematopathologists to confirm diagnosis. Written informed consents from patients were reviewed and approved by the institutional review board of each institution. We deposited targeted exon sequencing data of a selected 333-gene panel on the ENKTCL cases in dbGaP. DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissues with an AllPrep DNA/RNA FFPE Kit. A custom platform (Agilent SureSelect) was used to capture target exons from genomic DNA of ENKTCL tumors. DNA was fragmented to 200bp using a Covaris S220 Focused-ultrasonicator and was end-repaired and ligated to Illumina adaptors with a KAPA Hyper Prep Kit. Ligated products were purified and amplified with a 7-cycle PCR. Exome capture from the PCR products was performed using a SureSelect Target Enrichment Kit. The captured exomes were further amplified with an 8-cycle PCR and purified for cluster generation using the cBot cluster generation system with a HiSeq PE Cluster Kit V4. 100bp paired-end reads were generated from an Illumina HiSeq 2500 system with a HiSeq SBS Kit V4.

  Study phs002925

• The Haplotype-Resolved Genome and Epigenome of the Aneuploid HeLa Cancer Cell Line

  The genomic architecture of HeLa remains largely unexplored beyond its karyotype, partly because like many cancers, its extensive aneuploidy renders such analyses challenging. We carried out haplotype-resolved whole-genome sequencing of the HeLa CCL-2 strain, examined point- and indel-mutation variations, mapped copy-number variations and loss of heterozygosity regions, and phased variants across full chromosome arms. We also investigated variation and copy-number profiles for HeLa S3 and eight additional strains. We find that HeLa is relatively stable in terms of point variation, with few new mutations accumulating after early passaging. Haplotype resolution facilitated reconstruction of an amplified, highly rearranged region of chromosome 8q24.21 at which integration of the human papilloma virus type 18 (HPV-18) genome occurred and that is likely to be the event that initiated tumorigenesis. We combined these maps with RNA-seq and ENCODE Project data sets to phase the HeLa epigenome. This revealed strong, haplotype specific activation of the proto-oncogene MYC by the integrated HPV-18 genome approximately 500 kilobases upstream, and enabled global analyses of the relationship between gene dosage and expression. These data provide an extensively phased, high-quality reference genome for past and future experiments relying on HeLa, and demonstrate the value of haplotype resolution for characterizing cancer genomes and epigenomes. Reprinted from Adey et. al. 2013, Nature, with permission from Nature Publishing Group.

  Study phs000642

• Coding and non-coding drivers of mantle cell lymphoma identified through exome and genome sequencing

  Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we re-sequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent non-coding mutations surrounding a single exon of the HNRNPH1 gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent non-coding mutations in disrupting an auto-regulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly implicate a role for aberrant regulation of mRNA processing in MCL pathobiology.

  Study EGAS00001004289

• DNA and RNA sequencing of single human haploid germ cells

  Our goal was to look at allele-specific gene expression in single spermatids from two human donors. We first genotyped and phased each donor using a combination of linked-read sequencing (one sample per donor, 10X Chromium), and single haploid whole genome sequencing (53 spermatids for the first donor, and 19 for the second). We then sequenced mRNA from single spermatids (302 from the first donor, and 164 from the second), using the Smart-Seq2 protocol. We used these gene expression data, in combination with the haplotype data, to look for gene products that exhibit allele-specific bias reflecting the haploid genotype of the cell. We find a large class of human genes that show such allele-specific bias in spermatids, indicating that these gene products are not fully shared across the cytoplasmic bridges that connect spermatids. We term these genes “genoinformative markers” (GIMs).

  Study phs002279

• Investigating Delayed-Onset Drug Hypersensitivity Reactions Prospectively

  Delayed-type drug hypersensitivity reactions (dtDHR) are significant causes of morbidity and mortality, but the origin, phenotype and function of pathogenic T cells across the spectrum of severity are unknown. Research has been hampered by several barriers. We leveraged recent technical advancements to study skin-resident memory T cells (TRM) versus recruited T cell subsets in the pathogenesis of severe systemic forms of disease, Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS), and skin-limited disease, morbilliform drug eruption (MDE). As part of the overarching study, we used single cell RNA sequencing (scRNA-seq) with CITE sequencing and TCR sequencing on prospectively collected skin and blood samples from patients with SJS/TEN and MDE patients. We compared this to healthy controls obtained from discarded skin from surgeries and discarded blood collars from a blood donation center. These data are included here in dbGaP database. The overarching study also performed bulk transcriptional profiling and microscopic analysis of clinical skin samples, as well as a mouse model of MDE (PMID: 39042477). Together, the data supported clonal expansion and recruitment of cytotoxic CD8+ T cells (identified using the mean normalized expression of NKG7, GNLY, GZMA, GZMB, and PRF1) from circulation into skin, along with both expanded and non-expanded cytotoxic CD8+ skin TRM in SJS/TEN whereas MDE displayed a cytotoxic T cell profile without expansion or recruitment of cytotoxic CD8+ T cells from circulation implicating TRM as the potential cause of disease. Concurrently, MDE displayed a pro-Treg signature, highlighted by CCR8 and TNFRSF4 (OX40), which promotes Treg survival compared to SJS/TEN.

  Study phs003344

• Mutational patterns and regulatory networks in epigenetic subgroups of meningioma (H033)

  DNA methylation patterns delineate clinically relevant subgroups of meningioma. We previously established the six meningioma methylation classes (MC) benign-1, 2, 3, intermediate-A, B and malignant. Here, we set out to identify subgroup-specific mutational patterns and pathway regulation. Whole-genome-sequencing was performed on 62 samples across all MCs and WHO grades from 62 patients with matched blood control, including 40 sporadic and 22 radiation-induced (Mrad) meningiomas. RNA sequencing was added for 18 cases and chromatin-immunoprecipitation for the enhancer mark H3K27ac followed by sequencing (ChIP-seq) for 16 samples. Besides the known mutations in meningioma, structural alterations were found to contribute to the spectrum of mechanisms inactivating NF2 in sporadic meningioma similar to previous reports for Mrad. Aberrations of DMD were found to be enriched in MCs with NF2 mutations and DMD was among the most differentially upregulated genes in NF2 mutant compared to NF2 wild-type cases. The mutational signature AC3 was detected in both sporadic meningioma and Mrad, but distributed across the genome in sporadic cases and enriched near genomic breakpoints in Mrad. In general, the malignant MC presented a significantly higher exposure to AC3 and higher genomic instability beyond the mutational load than the other MCs. Pathway analysis of the ChIP-seq clusters revealed that the FOXM1 is most differentially activated in high-grade cases, along with super enhancer recruitment near HOX genes and respective upregulation of expression. This data further elucidate the biological mechanisms differentiating meningiomas of different WHO grade and epigenetic subgroups and suggest leveraging the genomic instability as novel therapeutic targets.

  Study EGAS00001003481

• Characterisation of the genetic landscape of cutaneous leiomyoma and leiomyosarcoma - Leiomyoma WES

  Cutaneous leiomyoma (cLM) and leiomyosarcoma (cLMS) are rare benign and malignant soft tissue neoplasms showing smooth muscle differentiation, respectively, that arise from mesenchymal cells in the dermis and subcutis. Through whole exome sequencing of cLM and cLMS cases, we observed distinct differences between the somatic mutational profile of these tumour types. FH was identified as a driver gene in cLM with genetic alterations of FH occurring via somatic point mutation, somatic copy number loss, biallelic inactivation and germline point mutations. TP53 and RB1 were identified as driver genes in the cLMS cohort, with genetic alterations of TP53 occurring via somatic and germline point mutations, copy number loss and biallelic inactivation. Using RNA-sequencing, we identified recurrent gene fusions, including CRTC1/3-MAML2 in cLMS and a novel MYLK-MAP3K2 fusion. Analysis of the cell types present in the tumour microenvironment revealed a significantly increased presence of macrophages and decreased presence of myeloid dendritic cells in the cLMS cohort relative to the cLM cohort. Additionally, we identified common driver genes between cLMS and LMS from other sites. Thus, we provide the first in-depth profile of the genetic landscape of cLM and cLMS.

  Dataset EGAD00001014787