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• RNA alignment

  RNAseq aligned to hg38.p14 using nfcore/RNA-seq

  Dataset EGAD50000000428

• EXOME_ARRAY_ANALYSIS

  EXOME ARRAY ANALYSIS OF ADVERSE REACTIONS TO FLUOROPYRIMIDINE-BASED THERAPY FOR GASTROINTESTINAL CANCER

  Dataset EGAD00010001499

• Smoking-dependent expression alterations in nasal epithelium reveal immune impairment linked to germline variation and lung cancer risk

  Study EGAS00001006137

• Genetic and Phenotypic Analysis of Multiple Sclerosis in Hispanics

  We sought to identify MS susceptibility variants specific to the Major Histocompatibility Complex and to assess the effect of ancestral risk modification within 2652 Latinx and Hispanic individuals, as well as 2435 Black and African American individuals ascertained in collaboration with the Alliance for Research in Hispanic MS (ARHMS). All DNA samples were obtained through whole blood extraction and were genotyped on the MS Chip, an Illumina Infinium custom genotyping array that contains targeted and dense coverage of the extended MHC, specifically designed for imputation. Classical HLA alleles, SNPs, and amino acid residues were imputed simultaneously from genotyped SNPs using HLA-TAPAS and a multi-ethnic reference panel of 2504 samples from the 1000 Genomes Project. We have identified several novel susceptibility alleles which are rare in European populations including HLA-B*53:01, and we have identified an independent role for HLA-DRB1*15:01 and HLA-DQB1*06:02 on MS risk. We found a decrease in Native American ancestry in MS cases vs controls across the MHC, peaking near the previously identified MICB locus; and we have identified several susceptibility variants for which MS risk is modified by global or local ancestry. We are making data on all genotyped and imputed SNPs, classical HLA alleles, and amino acid residues available through dbGaP for the Hispanic and African American samples utilized in these analyses.

  Study phs003105

• High Density SNP Association Analysis of Lung Cancer

  This research builds upon an extensive resource of a case-control study that has been ongoing at the UT MD Anderson Cancer Center since 1991. To identify risk variants for lung cancer, we conducted a genome-wide association study. Cases are newly diagnosed, histologically-confirmed patients presenting at MD Anderson Cancer and who had not previously received treatment other than surgery. Controls are healthy individuals seen for routine care at Kelsey-Seybold Clinics, the largest physician group-practice plan in the Houston Metropolitan area. This lung GWAS led to the identification of a susceptibility locus for lung cancer at 15q25.1. We used data from 315,450 tagging SNPs in 1,154 current and former (ever) smoking cases of European ancestry and 1,137 frequency-matched, ever-smoking controls from Houston, Texas in the discovery followed by the replication of the ten SNPs most significantly associated with lung cancer in an additional 711 cases and 632 controls from Texas and 2,013 cases and 3,062 controls from the UK. Two SNPs, rs1051730 and rs8034191, were significantly associated with risk of lung cancer with combined analysis yielded odds ratios of 1.32 (P < 1X10-17) for both SNPs. These two SNPs mapped to a region of strong linkage disequilibrium within 15q25.1 containing PSMA4 and the nicotinic acetylcholine receptor subunit genes CHRNA3 and CHRNA5. (Nat Genet. 2008 May;40(5):616-22. PMID:18385676)

  Study phs000753

• Evaluation of Hybridization Capture versus Amplicon-based Methods for Whole Exome Sequencing

  Next generation sequencing has aided characterization of genomic variation. While whole genome sequencing may capture all possible mutations, whole exome sequencing is more cost-effective and captures most phenotype-altering mutations. Initial strategies for exome enrichment utilized a hybridization-based capture approach. Recently, amplicon-based methods were designed to simplify preparation and utilize smaller DNA inputs. We appraised two hybridization capture-based and two amplicon-based whole exome sequencing methods, utilizing both Illumina and Ion Torrent sequencers, comparing on-target alignment, uniformity, and variant calling. While the amplicon methods had higher on-target rates, the hybridization capture-based approaches showed better uniformity. All methods identified many of the same single nucleotide variants, but each amplicon-based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform-specific variant calling algorithms. All methods demonstrated effective copy number variant calling when compared against a single nucleotide polymorphism array. This study illustrates some differences between various whole exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach.

  Study phs000938

• Skin Microbiome in Disease States: Atopic Dermatitis and Immunodeficiency

  The NIH Intramural Skin Microbiome Consortium (NISMC) is a collaboration of investigators with primary expertise in genomics, bioinformatics, large-scale DNA sequencing, dermatology, immunology, allergy, infectious disease, and clinical microbiology. Atopic dermatitis (AD, "eczema") is a chronic relapsing skin disorder that affects ~15% of U.S. children and is associated with $1 billion of medical costs annually. AD is characterized by dry, itchy skin, infiltrated with immune cells. Colonization by Staphylococcus aureus (S. aureus) is ten-fold more common in AD patients and is associated with disease flares. We hypothesize that, in addition to S. aureus, AD may also be associated with additional novel microbes and/or selective shifts of commensal microbes that are relevant to disease progression. The NISMC seeks to define the microbiota that resides in and on the skin and nares of three patient groups, all of whom have eczematous lesions and are currently seen at the NIH Clinical Center: (1) AD patients; (2) Wiskott-Aldrich syndrome (WAS) patients; and (3) Hyper IgE syndrome (HIES) syndrome patients. Examination of the microbiome of patients with WAS or HIES syndromes, both rare immunodeficiencies, will advance our understanding of how an individual's immune system shapes their cutaneous microbial community. We are performing a prospective longitudinal study that follows these groups of patient thorough the cycles of eczema flares, ascertaining clinical data and samples at each stage.

  Study phs000266

• Memorial Sloan Kettering (MSKCC) Single Cell Mutational Profiling in Myeloid Malignancies

  Myeloid malignancies have previously been characterized by large scale bulk molecular profiling studies on patient samples. These studies, while clinically informative, are unable to delineate mutation order as well as clonal architecture and complexity. To investigate the clonal framework of myeloid malignancies, the Single Cell Mutational Profiling in Myeloid Malignancies study was designed. This study performed sequencing of 148 samples from 117 adult patients diagnosed with clonal hematopoiesis (n = 21), myeloproliferative neoplasms (MPN; n = 14), and acute myeloid leukemia (AML; n = 82) between 20 and 87 years of age. Sequencing of all samples was performed using the Tapestri single cell DNA sequencing platform from Mission Bio, Inc. The platform utilizes a custom amplicon panel covering 109 amplicons across 31 genes frequently mutated in myeloid malignancies (CH, MPN, AML). Using the single cell DNA sequencing data, we were capable of elucidating clonal architecture and clonal complexity in each sample. In a subset of samples, we also performed simultaneous single cell DNA sequencing and cell surface protein expression analysis using the Tapestri DNA + Protein platform. This study allowed us to determine the expression of 7 cell surface proteins (CD3, CD11b, CD19, CD34, CD38, CD45RA, and CD90) at single cell resolution. We performed DNA+ Protein sequencing on samples from CH patients (n = 7) as well as AML patients (n = 17).

  Study phs002049

• Genomic Analyses in Neoadjuvant Immunotherapy-Treated Head and Neck Cancers

  This is a phase 2 multicenter interventional trial involving the preoperative (neoadjuvant) administration of pembrolizumab, an anti-programmed cell death protein-1 (anti-PD-1) agent, to patients with pathologically confirmed, surgically resectable advanced head and neck squamous cell carcinoma (HNSCC). Participating centers were Dana-Farber/Harvard Cancer Center, Alvin J. Siteman Cancer Center, Washington University and Memorial Sloan Kettering Cancer Center. The trial (NCT02296684) consisted of two cohorts: Cohort 1 enrolled and treated patients received one dose of pembrolizumab prior to surgical resection with curative intent, and adjuvant pembrolizumab was planned for patients with high risk pathological features (positive margins and/or extranodal extension) in the surgical specimen. Cohort 2 enrolled and treated patients received 2 doses of pembrolizumab prior to surgical resection with curative intent. Adjuvant treatment was not dictated by the trial protocol for Cohort 2. The primary endpoint was rate of at least 50% pathological tumor response (pTR), defined as the presence of tumor necrosis/keratinous debris in the tumor bed following neoadjuvant pembrolizumab. pTR was graded as follows: pTR0 (<10%), pTR1 (10-49%) and pTR2 (≥50%). Baseline, pre-treatment tumor biopsy and post-neoadjuvant pembrolizumab surgically resected tumor specimens were dissociated, and CD3+CD45+ cells were analyzed for T cell receptor (TCR) expression and 5' gene expression analysis using single cell sequencing technologies for a subset of patients.

  Study phs002864

• Center for Common Disease Genomics [CCDG] - Cardiovascular: Genetics of Coronary Heart Disease - Characterizaton of Coronary Prone Pedigrees

  The primary cohort includes samples from 499 men and 154 women who survived a myocardial infarction, percutaneous transluminal angioplasty, or coronary artery bypass grafting (CABG) by age 55 years for men or age 65 years for women. Patients were seen at least six months after their acute event. Samples from another 488 men and 541 women that showed a nearly identical age, gender, and measured blood pressure distribution were collected as population-based controls. These controls tended to live in urban Salt Lake City, where the vast majority of the population is Caucasian. Blood samples were collected in the morning after 12h to 16h of fasting and prepared according to guidelines of the Lipid Research Clinic's program Manual of Laboratory Operations. All samples have serum, DNA and/or white blood cells, and a host of measurements (e.g. standard clinical chemistries, LDL and HDL cholesterol, triglycerides, glucose, HbA1C, insulin, blood pressures, anthropometric data, information on medical conditions and drug use, etc.). Additional details about the cohort or sample preparation were published previously. Here we present in total 1,475 patients that were successfully sequenced. This number includes 228 women, 419 men, and 7 of unreported gender survivors as well as 339 women and 482 men that served as population-based controls.

  Study phs001901

• BrainCloud: Data from human postmortem brain procurement for the neuropathology section

  This study explores the temporal dynamics and genetic control of transcription and DNA methylation in the human dorsolateral prefrontal cortex in postmortem tissue. This study examines 269 subjects for gene expression (version 1) and 108 subjects for DNA methylation (version 2). The subjects are normal controls without neuropathological and neuropsychiatric diagnosis and range in age from fetal weeks 14-20 through old age (>80). We discover fast changes in gene expression occurring during early brain development. Later in life, the changes are considerably slower. Many genes reverse pattern of expression between fetal and early postnatal development. We identify thousands of strong associations of SNPs with gene expression. We examine DNA methylation in ~14,500 genes at ~27,000 CpG loci focused on 5' promoter regions. The fastest changes in DNA methylation also occur during the prenatal period, slow down markedly after birth and continue to slow further with aging. DNA methylation is strongly associated with genotypic variants and correlates with expression of a subset of genes. DNA for genotyping was obtained from the cerebella and applied to either Illumina 650K or 1 million BeadArrays - only genotypes common to both platforms are analyzed here. Genotypes were called using BeadExpress software. Doi: 10.1038/nature10524 Nature, 478:519-524, 2011; doi:10.1016/j.ajhg.2011.12.020, AJHG 90, 1-13, Feb 10, 2012. The methylation data can be downloaded at: BrainCloud.

  Study phs000417

• Alzheimer's Disease Genetics Consortium (ADGC) Genome Wide Association Study -NIA Alzheimer's Disease Centers Cohort

  The National Institute on Aging (NIA) Alzheimer's Disease Centers (ADCs) cohort includes subjects ascertained and evaluated by the clinical and neuropathology cores of the 29 NIA-funded ADCs. Data collection is coordinated by the National Alzheimer's Coordinating Center (NACC). NACC coordinates collection of phenotype data from the 29 ADCs, cleans all data, coordinates implementation of definitions of AD cases and controls, and coordinates collection of samples. The ADC cohort consists of autopsy-confirmed and clinically-confirmed AD cases, and cognitively normal elders (CNEs) with complete neuropathology data who were older than 60 years at age of death, and living CNEs evaluated using the Uniform dataset (UDS) protocol who were documented to not have mild cognitive impairment (MCI) and were between 60 and 100 years of age at assessment. ADCs sent frozen tissue from autopsied subjects and DNA samples from some autopsied subjects and from living subjects to the National Cell Repository for Alzheimer's Disease (NCRAD). DNA was prepared by NCRAD for genotyping and sent to the genotyping site at Children's Hospital of Philadelphia. ADC samples were genotyped and analyzed in separate batches. [Reprinted from AC Naj et al. Common variants at MS4A4/MS4A6E, CD2AP, CD33 and EPHA1 are associated with late-onset Alzheimer's disease. Nature Genetics 43, 436-441 (2011). doi:10.1038/ng.801. PMID: 21460841.]

  Study phs000372

• NHLBI TOPMed: Best ADd-on Therapy Giving Effective Response (BADGER)

  BADGER is a 56-week randomized, double-blind, three-treatment, three-period cross-over trial that will evaluate the differential improvement in control that is achieved following three separate treatment interventions in children whose asthma is not acceptably controlled on a low dose of ICS (per NAEPP guidelines). All participants will enter an 8-week run-in period during which time they will receive a dose of 1x ICS (fluticasone 200 μg/day). During this 8-week time period, running 2-week averages to establish the lack of acceptable asthma control will be calculated. Thus, a child could qualify for randomization at any time during this 8-week run-in period. This approach should maximize both patient safety and successful enrollment. Children will continue to receive 1x ICS during the entire treatment phase. During each period of the treatment phase, they also will receive one add-on therapy in the form of LABA, LTRA or additional 1x ICS. The order of the add-on therapy assignment will be determined by randomization into one of six treatment sequences (order determined randomly). Each treatment period will be 16 weeks in length; the initial 4 weeks of each period will be considered to be the washout period for the previous treatment. The primary outcome measures will be frequency of asthma exacerbations, asthma control days, and FEV1.

  Study phs001728

• Immune Responses in Checkpoint Myocarditis Across Heart, Blood, and Tumor

  Immune checkpoint inhibitors (ICIs) are widely used in anti-cancer therapies, but they can cause morbid and potentially fatal immune-related adverse events (irAEs), such as ICI-related myocarditis (irMyocarditis). The pathogenesis of irMyocarditis and its relationship to anti-tumor immunity remain poorly understood. We sought to define immune responses in heart, tumor, and blood in irMyocarditis patients and controls by leveraging single‐cell RNA sequencing (scRNA‐seq) coupled with T-cell Receptor Sequencing (TCR-Seq). Our analysis demonstrated increased frequencies of cytotoxic T cells, inflammatory mononuclear phagocytes (MNPs), conventional dendritic cells (cDCs), and inflammatory fibroblasts in irMyocarditis heart tissue. Additionally, we revealed decreased frequencies of plasmacytoid dendritic cells, cDCs, and B lineage cells but an increased frequency of MNPs in the blood of irMyocarditis patients. Raw data for heart and blood can be found in dbGaP. All processed data and raw TCRseq data from heart tissue and tumor samples for this study can be found in GEO (Accession number GSE228597).Subject IDs with the prefix "SIC" were collected by the Severe Immunotherapy Complications (SIC) service at MGH due to suspicion of immune-related adverse events. Subject IDs with the prefix "donor" were collected by the MGH Melanoma Biobank at scheduled timepoints; this biobank systematically collects samples from patients at MGH at clinically relevant timepoints.

  Study phs003413

• Investigating Host Genetic Risk Factors for Tuberculosis in Highly Endemic South African Populations

  It is apparent from our recent population genetic and admixture mapping work 1–3 that a substantial part of the ancestral input into the South African Coloured (SAC) population is from the San or Khoe groups, which are not well represented in publicly available genetic databases. It is a reasonable assumption that the SAC population in the Western Cape may have derived more genetic input from the present KhoeSan population in the Northern Cape than the San residing in Namibia. It was concluded by our group that it is indeed the southern African KhoeSan group, the ≠Khomani, that best represent the KhoeSan contribution seen in the SAC 4. More recently, it has been shown that there are ancestry related increases in TB susceptibility, especially with increased Bantu-speaking African and KhoeSan ancestry 5. We aim to elucidate the epidemiological and human-host genetic risk factors for TB and the immunological pathways modulating TB infection. The proposed sub-study will enroll people evaluated for TB at Northern Cape community health clinics and their contacts from their households and/or community. We will conduct a demographic interview, collect saliva, which will be used for genetic analysis, and blood to determine latent TB infection status and capture immunological responses to mycobacterial infections at the cellular level and RNA sequencing.

  Study EGAS00001007850

• Mid-pass Whole-genome Sequencing in a Malagasy Cohort Uncovers Body Composition Associations

  The majority of human genomic research studies have been conducted in European-ancestry cohorts, reducing the likelihood of detecting potentially novel and globally impactful findings. Here, we present mid-pass whole-genome sequencing data and a genome-wide association study in a cohort of 264 self-reported Malagasy individuals from three locations on the island of Madagascar. We describe genetic variation in this Malagasy cohort, providing insight into the shared and unique patterns of genetic variation across the island. We observe phenotypic variation by location, and find high rates of hypertension particularly in the Southern Highlands sampling site as well as elevated self-reported malaria prevalence in the West Coast site relative to other sites. After filtering to a subset of 214 minimally-related individuals, we find a number of genetic associations with body composition traits, including many variants that are only observed in African populations or populations with admixed African ancestry from the 1000 Genomes Project. This study highlights the importance of including diverse populations in genomic research for the potential to gain novel insights, even with small cohort sizes. This project was conducted in partnership and consultation with local stakeholders in Madagascar and serves as an example of genomic research that prioritizes community engagement and that has potential impacts on our understanding of human health and disease.

  Study EGAS50000000496

• Transposable Elements Shape Stemness in Normal and Leukemic Hematopoiesis

  Despite most acute myeloid leukemia (AML) patients achieving complete remission after induction chemotherapy, two-thirds will relapse with fatal disease within five years. AML is organized as a cellular hierarchy sustained by leukemia stem cells (LSC) at the apex, with LSC properties directly linked to tumor progression, therapy failure, and disease relapse 1–5. Despite the central role of LSC in poor patient outcomes, little is known about the genetic determinants driving their stemness properties. As LSCs share many functional and molecular properties with normal hematopoietic stem cells (HSC) 6, we investigated accessible chromatin unique across normal hematopoietic and cancer cell states and identified transposable elements (TEs) as genetic determinants of both primitive populations in comparison with their downstream mature progeny. A clinically-relevant TE chromatin accessibility-based LSCTE121 signature was developed that enabled patient classification based on survival outcomes. Through functional assays, primitive cell specific-TE subfamilies were found to serve as docking sites for stem cell-associated regulators of genome topology or lineage-specific transcription factors, including LYL1 in LSCs. Finally, using chromatin editing tools, we establish that chromatin accessibility at LTR12C elements in LSCs are necessary to maintain stemness properties. Our work identifies TEs as genetic drivers of primitive versus mature cell states, where distinct TE subfamilies account for stemness properties in normal versus leukemic hematopoietic stem cells.

  Study EGAS50000001141

• Transposable Elements Shape Stemness in Normal and Leukemic Hematopoiesis

  Despite most acute myeloid leukemia (AML) patients achieving complete remission after induction chemotherapy, two-thirds will relapse with fatal disease within five years. AML is organized as a cellular hierarchy sustained by leukemia stem cells (LSC) at the apex, with LSC properties directly linked to tumor progression, therapy failure, and disease relapse 1–5. Despite the central role of LSC in poor patient outcomes, little is known about the genetic determinants driving their stemness properties. As LSCs share many functional and molecular properties with normal hematopoietic stem cells (HSC) 6, we investigated accessible chromatin unique across normal hematopoietic and cancer cell states and identified transposable elements (TEs) as genetic determinants of both primitive populations in comparison with their downstream mature progeny. A clinically-relevant TE chromatin accessibility-based LSCTE121 signature was developed that enabled patient classification based on survival outcomes. Through functional assays, primitive cell specific-TE subfamilies were found to serve as docking sites for stem cell-associated regulators of genome topology or lineage-specific transcription factors, including LYL1 in LSCs. Finally, using chromatin editing tools, we establish that chromatin accessibility at LTR12C elements in LSCs are necessary to maintain stemness properties. Our work identifies TEs as genetic drivers of primitive versus mature cell states, where distinct TE subfamilies account for stemness properties in normal versus leukemic hematopoietic stem cells.

  Study EGAS50000001147

• WES of 2 human osteosarcoma and corresponding cell lines

  Progress in the systemic control of osteosarcoma has been limited over the past decades thus indicating the urgent clinical need for the development of novel treatment strategies. Therefore, we have recently developed new preclinical models to study promising novel agents for the treatment of pediatric osteosarcoma. The checkpoint kinase (chk) inhibitor prexasertib (LY2606368) and its salt form (LSN2940930) have recently been shown to be active in adult and pediatric malignancies, including sarcoma. We have now tested the potency of prexasertib in clonogenic survival assays in two new lines of primary patient-derived osteosarcoma cells and in two established osteosarcoma cell lines as a single agent and in combination with cisplatin and the poly ADP-ribose polymerase (PARP) inhibitor talazoparib. Prexasertib alone results in strongly reduced clonogenic survival at low nanomolar concentrations and acts by affecting cell cycle progression, induction of apoptosis and induction of double-stranded DNA breakage at concentrations that are well below clinically tolerable and safe plasma concentrations. In combination with cisplatin and talazoparib, prexasertib acts in a synergistic fashion. Chk1 inhibition by prexasertib and its combination with the DNA damaging agent cisplatin and the PARP-inhibitor talazoparib thus emerges as a potential new treatment option for pediatric osteosarcoma which will now have to be tested in preclinical primary patient derived in vivo models and clinical studies.

  Study EGAS00001003923

• Ultrasensitive detection of minor allele fractions in maternal samples based on microhaplotype analysis

  Background: Tools for quantifying and studying genetic variation in heterogeneous cell populations have a limited sensitivity. However, many biological problems require ultrasensitive genetic deconvolution of cell mixtures. The detection of fetal cells in cervical samples from pregnant women presents such a challenge. Methods: Using deep sequencing of microhaplotype loci and analysis of the error profiles in genetically pure and mixed samples, we increase the sensitivity for minor allele detection and demonstrate the potential for paternal allele detection in cervical samples from pregnant women. Results: Using artificial DNA mixtures, we show that unique alleles can be readily identified in mixtures with a ratio of 1 in 10,000 (0.01%). By applying this method on ten non-invasive cervical samples from pregnant women we could identify the presence of paternal alleles in half of the samples. Unexpectedly, we demonstrate those alleles not to be of fetal origin but of sperm. Conclusions: We developed a novel method that can deconvolute genetic mixtures to at least 0.01%, which can be applied to study minor allele fractions in genetic mixtures and, for example, could aid in trophoblast cell detection in maternal blood or cervix. Our results demonstrate the importance of discriminating fetal and paternal alleles when analyzing cervical fetal cells and may pinpoint a potential cause of erroneous results in previous trophoblast isolation studies.

  Study EGAS00001007057

• Exome and RNA sequencing of relapsed TCF3-PBX1 t(1;19) acute lymphoblastic leukemia

  TCF3-PBX1 (E2A-PBX1) is a recurrent gene fusion in B-cell precursor lymphoblastic leukemia (BCP-ALL), which is caused by the translocation t(1;19)(q23;p13). TCF3-PBX1 BCP-ALL patients typically benefit from chemotherapy; however, many relapse and subsequently develop resistant disease with few effective treatment options. Mechanisms driving disease progression and therapy resistance have not been studied in TCF3-PBX1 BCP-ALL. Here, we aimed to identify novel treatment options for TCF3-PBX1 BCP-ALL by profiling leukemia cells from a relapsed patient, and determine molecular mechanisms underlying disease pathogenesis and progression. By drug sensitivity testing of leukemic blasts from the index patient, control samples and TCF3-PBX1 positive and negative BCP-ALL cell lines, we identified the phosphatidylinositide 3-kinase delta (p110δ) inhibitor idelalisib as an effective treatment for TCF3-PBX1 BCP-ALL. This was further supported by evidence showing TCF3-PBX1 directly regulates expression of PIK3CD, the gene encoding p110δ. Other somatic mutations to TP53 and MTOR, as well as aberrant expression of CXCR4, may influence additional drug sensitivities specific to the index patient and accompanied progression of the disease. Our results suggest that idelalisib is a promising treatment option for patients with TCF3-PBX1 BCP-ALL while other drugs could be useful depending on the genetic context of individual patients.

  Study EGAS00001001876

• GoT2D: Genetics of Type 2 Diabetes, a study of the the genetic architecture of type 2 diabetes using low pass whole genome sequencing and high density SNP genotyping in 2,657 individuals.

  The Genetics of Type 2 Diabetes Consortium (GoT2D) is a collaboration between the University of Michigan, the Broad Institute and the Wellcome Trust Centre for Human Genetics. The overall aim is to extend upon recent efforts, such as genome-wide association studies (GWAS) and large scale meta-analyses. While they have proved successful at mapping genomic loci that influence human diseases, like type 2 diabetes, much of the heritability remains unexplained. In this study, we use next generation sequencing and genotyping technologies to query for lower frequency variants in the human genome. Thereby, allowing a deeper characterization of the spectrum of alleles associated with type 2 diabetes risk, and a better assessment of the genes that play a role in the etiology of type 2 diabetes development. We studied 1,326 T2D cases and 1,331 normoglycemic controls from Northern and Central Europe (Sweden, Finland, UK, and Germany). To efficiently characterize the entire genome sequence of each individual, we performed low-coverage (~5x) whole-genome sequencing, augmented by deep coverage (~100x) sequencing of the exome, and dense (2.5M) single nucleotide polymorphism (SNP) genotyping using the HumanOmni2.5 array. The data deposited in EGA will include all the Swedish, Finnish, UK, and German samples.

  Study EGAS00001001459

• Cell fate mapping of human glioblastoma reveals an invariant stem cell hierarchy pre- and post-treatment

  Human glioblastomas (GBMs) are thought to harbour a subpopulation of glioblastoma stem cells (GSCs) that initiate tumour formation and regrowth following treatment. However, the origin of their proliferative heterogeneity during tumour growth pre- and post-treatment remains poorly understood. Here we study the clonal evolution of DNA barcoded GBMs following their serial propagation in xenograft mouse models to define the fate behaviour of individual GBM cells. Through quantitative analysis of clone sizes scored across multiple passages, we show that the growth of GBM clones in vivo are consistent with a remarkably neutral process resulting from a conserved proliferative hierarchy. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, that in turn generates short-lived cells that lack proliferative ability. We also identify rare "outlier" clones that deviate from these dynamics, and further show that chemotherapy reproducibly facilitates the selective expansion of pre-existing drug-resistant GBM lineages. Finally, we show that coexisting, functionally distinct GSCs may be targeted separately with epigenetic compounds. These findings show that, independent of an evolving mutational signature, the clonal dynamics of human GBM can be surprisingly robust, with intratumoural heterogeneity derived primarily from the defined stochastic fate behaviour of tumour cells within a conserved proliferative hierarchy.

  Study EGAS00001002424

• Single cell sequencing reveals the origin and the order of mutation acquisition in T-cell acute lymphoblastic leukemia.

  Next-generation sequencing has provided a detailed overview of the various genomic lesions implicated in the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). Typically, 10 to 20 genomic lesions are found in T-ALL cells at diagnosis. However, it is currently unclear in which order these mutations are acquired and in which progenitor cells this is initiated. To address these questions, we used targeted single-cell sequencing of total bone marrow cells and CD34+CD38- multipotent progenitor cells for 4 T-ALL cases. Hierarchical clustering detected a dominant leukemia clone at diagnosis, accompanied by a few subclones harboring only a fraction of the mutations. We developed a graph-based algorithm to determine the order of mutation acquisition. Two of the four patients had an early event in a known oncogene (MED12, STAT5B) amongst various pre-leukemic events. Intermediate events included loss of 9p21 (CDKN2A/B) and acquisition of fusion genes, while NOTCH1 mutations were typically late events. Analysis of CD34+CD38- cells and myeloid progenitors revealed that in half of the cases somatic mutations were detectable in multipotent progenitor cells. We demonstrate that targeted single cell sequencing can elucidate the order of mutation acquisition in T-ALL and that T-ALL development can start in a multipotent progenitor cell.

  Study EGAS00001002830

• Using_genetics_to_identify_cell_types_and_mechanisms_underlying_susceptibility_to_primary_sclerosing_cholangitis

  Primary sclerosing cholangitis (PSC) is a T-cell mediated, chronic inflammatory condition of the biliary tree that is strongly associated with inflammatory bowel disease. Genome-wide association studies have identified 22 non-HLA genetic risk variants associated PSC. Identifying the genes impacted by these variants has proven difficult as the majority lie in non-coding regions of the genome. Knowledge of the genes and biological pathways these non-coding variants are perturbing is vital to understanding the disease biology. One means of assessing the impact of non-coding variants within disease associated loci upon genes is via colocalisation with eQTL. Many eQTL are cell-type specific, requiring the analysis of disease relevant cell types to detect colocalisation. We have collected PSC-relevant T-cell-subtypes from the peripheral blood of PSC patients via fluorescence activated cell sorting in preparation for RNA sequencing and mapping of eQTL. Samples were collected at the Norfolk and Norwich University Hopital, for which local ethical approval has been granted. Lysed cell samples will be transferred to WTSI and DNA/RNA will be extracted from lysed cell samples by T143 before genotyping (DNA) and custom library preparation and sequencing (RNA). This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Study EGAS00001002643