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• Single cell RNAseq FASTQ files of three PDAC organoid lines (P28, P40, P47) using SORT-seq

  This dataset contains raw FASTQ files from single cell RNA sequencing of three patient-derived pancreatic ductal adenocarcinoma (PDAC) organoid lines (P28, P40, P47) cultured under standard conditions. For each line, single cells were FACS sorted into 384-well capture plates, with each well containing a 50nl droplet of barcoded primers. Plates were processed following an adapted SORT-seq protocol, and cDNA libraries were generated using CEL-Seq2 with TruSeq small RNA primers (Illumina). Sequencing was performed on an Illumina NextSeq500 platform using paired-end reads (read 1: 26 cycles, index read: 6 cycles, read 2: 60 cycles). Data are controlled-access and intended for single cell transcriptomic analysis of PDAC organoid heterogeneity and WNT pathway activity.

  Dataset EGAD50000002220

• Multi-site tumor sampling highlights molecular intra-tumor heterogeneity in malignant pleural mesothelioma

  Background:Malignant pleural mesothelioma (MPM) is a heterogeneous cancer. Better knowledge of molecular and cellular intra-tumor heterogeneity throughout the thoracic cavity is required to develop efficient therapies. This study focuses on molecular intra-tumor heterogeneity using the largest series to date in MPM and is the first to report on the multi-omics profiling of a substantial series of multi-site tumor samples. Methods:Intra-tumor heterogeneity was investigated in 16 patients from whom biopsies were taken at distinct anatomical sites. The paired biopsies collected from apex, side wall, costo-diaphragmatic or highest metabolic sites as well as 5 derived cell lines were screened using targeted sequencing. Whole exome sequencing, RNA sequencing, and DNA methylation were performed on a subset of the cohort for deep characterization. Molecular classification, recently defined histo-molecular gradients, and cell populations of the tumor microenvironment were assessed. Results:Sequencing analysis identified heterogeneous variants notably in NF2, a key tumor suppressor gene of mesothelial carcinogenesis. Subclonal tumor populations were shared among paired biopsies, suggesting a polyclonal dissemination of the tumor. Transcriptome analysis highlighted dysregulation of cell adhesion and extracellular matrix pathways, linked to changes in histo-molecular gradient proportions between anatomic sites. Methylome analysis revealed contribution of epigenetic mechanisms in two patients. Finally, significant changes in the expression of immune mediators and genes related to immunological synapse, as well as differential infiltration of immune populations in the tumor environment were observed and led to a switch from a hot to a cold immune profile in three patients. Conclusions:This comprehensive analysis reveals patient-dependent spatial intra-tumor heterogeneity at the genetic, transcriptomic and epigenetic levels and in the immune landscape of the tumor microenvironment. Results support

  Study EGAS00001005328

• Transcriptional Profiling of Macrophages In Situ in Metastatic Melanoma Reveals Localization-Dependent Phenotypes and Function

  Therapeutic interventions modulating immune function at the tumor site could improve outcomes in cancer. Here we analyzed metastatic melanoma, a tumor type highly responsive to T-cell based therapies, and found that the tumor-infiltrating T cells are localized closely to CD14+ monocytes/macrophages rather than to melanoma cells. Using novel immunofluorescence-guided laser capture micro-dissection, we analyzed transcriptomes of CD3+ T cells, CD14+ monocytes/macrophages and melanoma cells in non-dissociated tissue. CD14+ cells localized in tumors beds displayed specific transcriptional signatures distinct from CD14+ cells that were localized in tumor stroma.The 8 distinct metastatic melanoma samples are from the Cooperative Human Tissue Network (CHTN). 64 RNA-seq files are available from 16 intraCD14+, 16 stromaCD14+, 16 cancer cells, 8 intraCD3+ and 8 stromaCD3+ cells. 2 LCM harvests were completed for the CD14+ and cancer cells on two sections of the samples. One harvest was done for the CD3+ samples.The processed data (TPM/raw counts tables) are available on GEO under GSE180124. 

  Study phs002564

• Genomic Profiling of Sequentially Acquired Metastatic Sites from an "Exceptional Responder" Lung Adenocarcinoma Patient Reveals Extensive Genomic Heterogeneity and Novel Somatic Variants

  We employed next-generation sequencing to identify somatic alterations in multiple metastatic sites from an "exceptional responder" lung adenocarcinoma patient during his seven year course of ERBB2-directed therapies. The degree of heterogeneity was unprecedented, with ~1% similarity between somatic alterations of the lung and lymph nodes. One novel translocation, PLAG1-ACTA2, present in both sites, up-regulated ACTA2 expression. ERBB2, the predominant driver oncogene, was amplified in both sites, more pronounced in the lung, and harbored an L869R mutation in the lymph node. Functional studies demonstrated increased proliferation, migration, metastasis, and resistance to ERBB2-directed therapy due to L869R mutation and increased migration due to ACTA2 overexpression. Within the lung, a nonfunctional CDK12, due to a novel G879V mutation, correlated with down-regulation of DNA damage response genes, causing genomic instability, and sensitivity to chemotherapy. We propose a model whereby a sub-clone metastasized early from the primary site and evolved independently in lymph nodes.

  Study phs001159

• WES in HCCOs with varying Doxorubicin resistance

  We aimed to identify somatic mutations that could explain the resistance to Doxorubicin. Therefor, we purified Exomes from liver biopsies and HCC organoids using Agilent SureSelectXT (clinical research exome, HS2 V8, or Human all Exon V6 Cosmic) and performed WES. Matched non-tumoral liver biopsies served as the control for germline mutations. 12 of these samples were previously reported. Sequence reads were aligned to the reference human genome GRCh38 using Burrows-Wheeler Aligner (BWA, v0.7.17). Duplicate removal, base quality adjustment, mutation calling (Mutect2, intervals: SureSelectXT_HS_V8_S33266340, germline-recourse: gnomad.exomes.r2.1.1.sites.liftover_grch38, Panel-of-Normal: 1000g_pon.hg38), filtering of Mutect calls, and functional annotation (Funcotator, funcotator_dataSources.v1.7.20200521g) were performed using the Genome Analysis Toolkit (GATK, v4.4) according to the Broad Institute best practices in Somatic short variant discovery workflows. Mutations with a functional annotation of Deletion, Insertion, Non-stop, Splice-Site, Start-Codon and Missense mutations were kept. A list of 43 commonly mutated genes in HCC was analyzed in the HCC organoids [TCGA, Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma. Cell, 2017. 169(7): p. 1327-1341.e23].

  Study EGAS50000000043

• Systemic Inflammation and Lymphocyte Activation Precede Rheumatoid Arthritis

  The Allen Institute for Immunology-University of California, San Diego-University of Colorado Transition to Rheumatoid Arthritis (ALTRA) project is a prospective longitudinal cohort study to understand the pre-clinical development of rheumatoid arthritis (RA). We performed cross-sectional and longitudinal multi-omics analyses of peripheral immunity in the anti-citrullinated protein autoantibody-positive (ACPA+) at-risk for RA period. We provide FASTQ files from (1) single-cell RNA sequencing (scRNA-seq) of ACPA+ at-risk (n=45), ACPA+ early RA (n=11), and site-matched ACPA- controls (n=38); (2) scRNA-seq of longitudinal samples from ACPA+ at-risk who developed clinically active RA during our study (n=16 participants, n=75 samples) and ACPA- controls (n=29 subjects, n=89 samples); (3) trimodal TEA-seq of a subset of ACPA+ at-risk (n=7) and ACPA- controls (n=5). Using these data, we characterize pathogenesis prior to clinical RA diagnosis and suggest the at-risk state exhibits substantial immune alterations that could potentially be targeted for early intervention to delay or prevent autoimmunity.

  Study phs003944

• Exome-seq, RNA-Seq, SNP array profiling of gastric tumor samples and cell lines.

  Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying genomic alterations remain poorly understood. Here we performed exome and transcriptome sequencing and SNP array assays to characterize 51 primary gastric tumors and 32 cell lines. Meta-analysis of the exome data and previously published data sets revealed 24 significantly mutated genes in MSS tumors, and 16 in MSI tumors. More than half of the patients in our collection could potentially benefit from targeted therapies. We identified 55 splice-site mutations accompanied by aberrant splicing products, in addition to mutation-independent differential isoform usage in tumors. The ZAK kinase isoform TV1 is preferentially upregulated in gastric tumors and cell lines relative to normal samples. This pattern is also observed in colorectal, bladder, and breast cancers. Overexpression of this particular isoform activated multiple cancer-related transcription factor reporters, while depletion of ZAK in gastric cell lines inhibited proliferation. These results reveal the spectrum of genomic and transcriptomic alterations in gastric cancer, and identify isoform-specific oncogenic properties of ZAK.

  Study EGAS00001000736

• Recursive splicing in long vertebrate genes

  Recent studies have shown that expression of many genes with exceptionally long introns in the mammalian brain can be perturbed by regulatory factors linked to neurodevelopmental or neurodegenerative disorders1-3, suggesting unique regulatory mechanisms. Here we identify functional recursive splice sites (RSS) in long introns of genes expressed in the brain. These RSS are highly conserved in genes with extreme length across diverse vertebrate species and permit step-wise removal of long introns via recursive splicing. Recursive splicing requires initial definition of a “recursive exon” that is located downstream of RSS, and most often contains premature stop codons. Moreover, we show that RSS create a splicing switch driven by splice site competition in order to distinguish primary mRNA isoforms from alternative isoforms that are prevalent in long genes. The recursive exon is not detectable in the dominant mRNA isoform due to recursive splicing, but is completely included when cryptic promoters or other cryptic exons are used. Thus, by coupling inclusion of recursive exons with the use of cryptic elements, RSS act to distinguish new mRNA isoforms emerging from long genes.

  Study EGAS00001001170

• Epigenetic and metabolomic data from type 2 diabetes adolescents

  Objective: Rates of type 2 diabetes (T2D) among adolescents are on the rise. Epigenetic changes could be associated with the metabolic alterations in adolescents with T2D. Methods: We performed a cross-sectional integrated analysis of DNA methylation data from peripheral blood mononuclear cells with serum metabolomic data from First Nation adolescents with T2D and controls participating in the Improving Renal Complications in Adolescents with type 2 diabetes through REsearch (iCARE) cohort study, to explore the molecular changes in adolescents with T2D. Results: Our analysis showed that 43 serum metabolites and 36 differentially methylated regions (DMR) were associated with T2D. Several DMRs were located near the transcriptional start site of genes with established roles in metabolic disease and associated with altered serum metabolites (e.g. glucose, leucine, and gamma-glutamylisoleucine). These included the free fatty acid receptor-1 (FFAR1), upstream transcription factor-2 (USF2), and tumor necrosis factor-related protein-9 (C1QTNF9), among others. Conclusions: We identified DMRs and metabolites that merit further investigation to determine their significance in controlling gene expression and metabolism which could define T2D risk in adolescents.

  Study EGAS00001003816

• An essential role for MYB in driving oncogenic EVI1 expression in enhancer-rearranged leukemias

  In acute myeloid leukemia (AML) with inv(3)(q21;q26) or t(3;3)(q21;q26), a translocated GATA2 enhancer drives oncogenic expression of EVI1. We generated an EVI1-GFP AML model and applied an unbiased CRISPR/Cas9 enhancer scan to uncover sequence motifs essential for EVI1 transcription. Using this approach, we pinpoint a single regulatory element in the translocated GATA2 enhancer that is critically required for aberrant EVI1 expression while being dispensable for GATA2 expression from its endogenous locus. This element contains a DNA binding motif for the transcription factor MYB that heavily occupies this site specifically at the translocated allele. MYB knockout as well as peptidomimetic blockade of p300-dependent MYB function resulted in downregulation of EVI1 but not of GATA2. Targeting MYB or mutating its DNA-binding motif within the GATA2 enhancer resulted in myeloid differentiation and cell death, suggesting that interference with MYB-driven EVI1 transcription provides a potential entry point for therapy of inv(3)/t(3;3) AMLs.

  Study EGAS00001004839

• An Isogenic Cell Line Panel for Sequence-based Screening of Targeted Anti-cancer Drugs

  We describe the creation and characterization of an isogenic cell line panel representing common cancer pathways, with multiple features optimized for high-throughput screening. More than 1,800 cell lines from three normal human cells were generated using CRISPR-technologies. Surprisingly, we discovered most of these lines did not result in complete gene inactivation, despite integration of sgRNA at the desired genomic site. However, a subset of the lines harbored true, biallelic disruptions of the targeted tumor suppressor gene, yielding a final panel of 100 well-characterize lines covering 19 pathways frequently subject to loss of function in cancers. This panel included genetic markers optimized for sequence-based ratiometric assays for drug-based screening assays. To illustrate the potential utility of this panel, we developed a multiplexed high-throughput screen that identified Wee1 inhibitor MK-1775 as a selective growth inhibitor of cells with inactivation of TP53. These cell lines and screening approach should prove useful for researchers studying a variety of cellular and biochemical phenomena

  Study EGAS00001005974

• Genomic analysis and evolutionary modeling of breast and larynx cancer, based on specimens from Polish population

  We explore the genomic characteristics of the original dataset of breast and larynx cancer specimens of several cell types from the collection of the Department of Applied Radiology of the Maria Sklodowska-Curie National Research Institute of Oncology, Krakow (Poland) Branch and Institute of Human Genetics Polish Academy of Sciences. DNA from paired tissue samples from primary tumor locations and concurrent metastasis to regional lymph nodes was sequenced and matched to controls. A range of genomic analyses was carried out, such as estimation of variant allele frequencies (VAF) and copy number variation (CNV). They show that subsets of variants from primary tumors are present in metastatic lymph nodes and that the CNV pattern is similar, but not identical to that known in the literature. In addition to that, we fit the VAF data to theoretical site frequency spectra predicted by multiple models found in the literature. Analysis suggests existence of secondary advantageous subclones arising on the clonal background as well as of a number of neutral or slightly deleterious passenger mutations.

  Study EGAS00001006456

• The Mutational Landscape of CTCL and Sezary Syndrome

  Sezary syndrome is a leukemic and aggressive form of cutaneous T-cell lymphoma (CTCL) resulting from the malignant transformation of skin-homing central memory CD4+ T cells. To identify new genetic alterations involved in Sezary syndrome and CTCL transformation we performed whole-exome sequencing of tumor-normal sample pairs from 26 Sezary syndrome and 16 CTCL patients. These analyses revealed a distinctive pattern of somatic copy number alterations in Sezary syndrome including highly prevalent recurrent chromosomal deletions involving the TP53, RB1, PTEN, DNMT3A, and CDKN1B tumor suppressor genes. Mutation analysis identified a broad spectrum of somatic mutations involving key genes involved in epigenetic regulation (TET2, CREBBP, MLL3, BRD9, SMARCA4 and CHD3) and signaling, including mutations in MAPK1, BRAF, CARD11 and PRKG1 driving increased MAPK, NFKB and NFAT activity upon T-cell receptor stimulation. Collectively, our findings provide new insights into the genetics of Sezary syndrome and CTCL and support the development of personalized therapies targeting key oncogenically activated signaling pathways for the treatment of these diseases.

  Study phs000994

• Genetics of Glucose Regulation in Gestation and Growth (Gen3G) Cohort - Placenta Transcriptomics RNA Sequencing

  The overarching goals of Genetics of Glucose regulation in Gestation and Growth (Gen3G) are to increase our understanding of biological, environmental, and genetic determinants of glucose regulation during pregnancy and their impact on fetal/child development. Gen3G is a prospective cohort study: we initially recruited 1,024 pregnant women between 2010-2013 at Blood Sampling in Pregnancy Clinic during the first trimester of pregnancy (median 9 weeks of gestation) in Sherbrooke, Québec, Canada. We assessed 898 pregnant women at second trimester (median 26 weeks of gestation), when participants completed a 75g oral glucose tolerance test (OGTT) as clinically indicated for screening of gestational diabetes mellitus (GDM). We collected data for 854 mother-child dyads at delivery (from medical records), in addition to placenta and/or cord blood samples in majority of newborns.The overall goal of the grant that supported data that is included in this dbGaP submission was to discover novel placental factors that regulate glucose metabolism in pregnancy and predict GDM by conducting genome-wide transcriptomics (RNA and miRNA) in carefully collected placenta samples from Gen3G.

  Study phs003151

• Blood-based monitoring of relapsed/refractory cHL patients predict responses to PD-1 blockade treatment

  Hodgkin lymphoma (cHL) patients are uniquely susceptible to treatment with programmed cell death-1 (PD-1) treatment. However, monitoring with FDG-PET/CT, the current response detection standard, is challenging. Here, we address the potential applicability of tissue biomarkers and blood-based biomarkers in PD-1 treated cHL patients. We evaluated 9p24.1/PD-L1/PD-L2 alterations, and expression of PD-L1 and antigen presentation molecules (HLA class I/II) in 9 tissue biopsies of 4 patients. In addition, we assessed circulating tumor DNA (ctDNA), extracellular vesicle associated miRNAs (EV-miRNAs), soluble PD-L1 and serum TARC (sTARC) in 26 longitudinal blood samples of these patients during PD-1 treatment. We found that the predictive power of tissue biomarkers is dependent on the timing of the biopsy . The dynamics of blood-based biomarkers: sTARC, EV-miRNAs and ctDNA corresponded well immunotherapy treatment response. Therefore, blood-based biomarkers are promising monitoring strategies in PD-1 treated cHL patients and should be further explored in clinical trials or observational studies.

  Study EGAS00001005894

• Molecular stratification of endometrioid ovarian carcinoma predicts clinical outcome

  Endometrioid ovarian carcinoma (EnOC) is an under-investigated type of ovarian carcinoma. Here, we report the largest genomic study of EnOCs to date, performing whole exome sequencing of 112 cases following rigorous pathological assessment. High frequencies of mutation were detected in CTNNB1(43%), PIK3CA(43%), ARID1A(36%), PTEN(29%), TP53(26%) and SOX8(19%), a novel target of recurrent mutation in EnOC. POLE and mismatch repair protein-encoding genes were mutated at lower frequency (6%, 18%) with significant co-occurrence. A molecular taxonomy was constructed using a novel algorithm (PRISTINE), identifying clinically distinct EnOC subtypes: TP53m cases demonstrated greater genomic complexity, were frequently FIGO stage III/IV at diagnosis (48%) and incompletely debulked (44%), and demonstrated inferior survival; conversely, CTNNB1m cases demonstrated low complexity and excellent clinical outcome, were predominantly stage I/II at diagnosis (89%) and completely resected (87%). Tumour complexity provides further resolution within the TP53wt/CTNNB1wt group. Moreover, we identify the WNT, MAPK/RAS and PI3K pathways as good candidate targets for molecular therapeutics in EnOC.

  Study EGAS00001004366

• Single-cell RNA-seq of celiac disease-specific plasma cells

  Disease-specific plasma cells (PCs) reactive with transglutaminase 2 (TG2) or deamidated gluten peptides (DGP) are abundant in celiac disease (CeD) gut lesions. Their contribution toward CeD pathogenesis is unclear. We assessed expression of markers associated with PC longevity in 15 untreated and 26 treated CeD patients in addition to 13 non-CeD controls, and performed RNA-sequencing with clonal inference and transcriptomic analysis of 3251 single PCs. We observed antigen-dependent V-gene selection and stereotypic antibodies. Generation of recombinant DGP-specific antibodies revealed a key role of a heavy-chain residue that displays polymorphism, suggesting that immunoglobulin gene polymorphisms may influence CeD-specific antibody responses. We identified transcriptional differences between CeD-specific vs non-disease-specific PCs and between short-lived vs long-lived PCs. The short-lived CD19+CD45+ phenotype dominated in untreated and short-term-treated CeD, in particular among disease-specific PCs but also in the general PC population. Thus, the disease lesion of untreated CeD is characterized by massive accumulation of short-lived PCs that are not only directed against disease-specific antigens.

  Study EGAS00001004623

• MicroRNAs, Hypertension and End Organ Damage in Humans

  Study 1) Profiling of microRNAs in plasma of patients with hypertension complicated or not by metabolic syndrome or chronic kidney disease Hypertension (HTN) or high blood pressure is associated with subclinical target organ damage such as cardiac, vascular and kidney injury, which may lead to complications and death. In this study, we investigated circulating microRNAs as biomarkers of target organ damage in HTN patients. We profiled circulating microRNAs by RNA sequencing in platelet-poor plasma of normotensive subjects and patients with HTN complicated or not by metabolic syndrome (MetS) or chronic kidney disease (CKD) (n=15 each group). Differentially expressed microRNAs were identified with a threshold of false discovery rate <0.1. Differentially expressed microRNAs were identified uniquely to associate with HTN (8), MetS (1) or CKD (13), and 8 were similarly differentially expressed in different groups. This study identified the association of differentially expressed circulating microRNAs with target organ damage in HTN patients, which could have some pathophysiological and therapeutic implications. Study 2) Profiling of microRNAs in gluteal subcutaneous small arteries of patients with hypertension complicated or not by chronic kidney disease Hypertension (HTN) is associated with vascular damage characterized by endothelial dysfunction and vascular remodeling and stiffening, which contributes to kidney injury leading to chronic kidney disease (CKD). MicroRNAs are short non-coding RNAs which repress/degrade target mRNAs. The microRNA role in vascular injury in HTN remains unclear. In this study, we aimed to identify differentially expressed microRNAs in small arteries of patients with HTN associated or not with CKD, in order to shed light on the pathophysiological molecular mechanisms. Normotensive subjects and HTN patients associated or not with CKD grades 3-4 were studied (n=15-16). Small arteries were isolated from subcutaneous gluteal biopsies, RNAs were extracted, and small and total RNA sequencing was performed by Illumina HiSeq-2500. Differentially expressed genes were identified with a P<0.05 and fold change (FC) >1.3. Differentially expressed microRNAs and mRNAs were identified uniquely to associate with HTN (microRNAs: 10, mRNAs: 68), CKD (microRNAs: 68, mRNAs: 395), and both groups (microRNAs: 2, mRNAs: 32). This study identified differentially expressed microRNAs and mRNAs in small arteries with target organ damage in HTN, which could have some pathophysiological and therapeutic implications.

  Study phs002389

• Cergentis FFPE-TLC sequencing of colorectal carcinoma

  In this study, formalin-fixed paraffin-embedded targeted locus capture (FFPE-TLC) sequencing is used as a novel technology for targeted detection of tumor-specific genomic structural variants (SVs) in the primary tumor of 29 colorectal cancer patients with metastatic disease. The tumor region was macrodissected and sequenced for 29 patients, and the "normal" region of the same slide was macrodissected and sequenced for 8 of these patients. SVs were found in the common fragile site (CFS)-associated genes MACROD2, PRKN, FHIT and WWOX as well as SVs caused by three LINE transposable elements. Tumor-specificity of selected SVs was independently verified by droplet digital PCR of tumor tissue DNA and their applicability as plasma circulating tumor DNA biomarkers was demonstrated. This dataset contains the hg19 reference alignment of the FFPE-TLC sequences for all 29 colorectal cancers and 8 adjacent normals.

  Dataset EGAD50000000618

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• IMCISION DNAseq

  Whole-exome sequencing of 32 primary head and neck squamous cell carcinoma samples prior to treatment with neoadjuvant anti-PD-1 (n=6) or anti-PD-1 + anti-CTLA-4 (n=26) immunotherapy. DNA quantity used: 50ng. Library Preparation Kit: Twist Human Core Exome Plus (Twist Bioscience). Sequencing parameters: NovaSeq 6000, 2x 100 bp. File type: fastQ.

  Dataset EGAD00001008139

• Distribution of data using Live Distribution

  Live Distribution Welcome to the documentation for using the Live Distribution feature for distributing data files securely through the EGA platform. This guide will walk you through the process of downloading encrypted files and decrypting them using Crypt4GH. Please follow the steps below to ensure a smooth experience. Before Downloading Create an EGA user. Make sure that you have the permissions to download the dataset of interest. In case you don’t have access, request access to the dataset. Add your Crypt4GH-compatible public key to your EGA account. Please allow a few hours for your public key to be synced with your profile. Afterwards, you will be able to connect to your EGA outbox using the SFTP protocol. Download Graphical User Interface (GUI) You can use any GUI that supports SFTP connections, such as FileZilla, an open-source FTP client. For Filezilla as your GUI, follow these steps to download files: Open FileZilla and access Site Manager (File > Site Manager). Create a new connection with the following settings (Figure 1): Protocol: SFTP - SSH File Transfer Protocol Host: __EGA_OUTBOX_DOMAIN__ Logon Type: Key file User: your EGA username Key file: path/to/your/private_key Figure 1: Process of establishing a new connection to __EGA_OUTBOX_DOMAIN__ using a key file as the logon method in FileZilla. The figure showcases the FileZilla version 3.52.2 operating on IOS v11.2.3. By following the depicted steps, users can create a secure and efficient connection to the EGA outbox, ensuring seamless data transfers. Click Connect to access your Outbox. This folder serves as your storage space within the EGA cloud, containing files accessible for download in a secure way. Browse the remote directory on the right side of the FileZilla screen. Select the files you wish to download, right-click, and choose Download (Figure 2). Figure 2: Step-by-step process of manually downñoad files from __EGA_OUTBOX_DOMAIN__ using FileZilla, with FileZilla version 3.52.2 operating on IOS v11.2.3. The figure demonstrates how users can downñoad data from the EGA outbox to their local storage by following the depicted steps SFTP command line You can also use the SFTP command line to securely download files from the EGA Outbox. Using SFTP command line client in Linux/Unix Open a terminal window Enter the following command to connect: sftp username@hostname Enter your EGA password To see a list of available sftp commands type help sftp> put – Upload file sftp> get – Download file sftp> cd path – Change remote directory to ‘path’ sftp> pwd – Display remote working directory sftp> lcd path – Change the local directory to ‘path’ sftp> lpwd – Display local working directory sftp> ls – Display the contents of the remote working directory sftp> lls – Display the contents of the local working directory Type get command to download files. For example: get encrypted_file.c4gh Use the bye command to close the connection (SFTP session). Convenient SSH settings Include the following settings in your SSH config file, located in ~/.ssh/config Host __EGA_OUTBOX_DOMAIN__ EGA-outbox hostname __EGA_OUTBOX_DOMAIN__ User username IdentityFile path/to/the/private/key Replace username and path/to/the/private/key with the appropriate settings, and you will be able to connect to the __EGA_OUTBOX_DOMAIN__ simply using sftp EGA-outbox. How to decrypt Files archived at the EGA are encrypted based on Crypt4GH. Hence, to decrypt the files you need to install Crypt4GH. You can install a python implementation of it, with pip install crypt4gh or directly from the Github repository pip install git+https://github.com/EGA-archive/crypt4gh.git After installing Crypt4GH, decrypt files using the following command: crypt4gh decrypt --sk /path/private/key < encrypted_file.c4gh > decrypted_filename The command reads the encrypted file from stdin (with <) and output the decrypted version to stdout (with >). Replace encrypted_file.c4gh and decrypted_filename with the appropriate filenames but make sure to not use the same filename for both reading and writing because your SHELL would then truncate both files before you even read or write. Frequently Asked Questions What username should I use to log in to my outbox? The authentication process for logging in to the EGA website, as well as accessing your inbox and outbox, requires the use of your username, not your email address. Therefore, if you registered a username different from your email address when creating your EGA account, you must use that username to log in. If you have forgotten your registered username, please, contact our Helpdesk team for assistance. I see that some files in my dataset have 'unavailable' as extension. What should I do? Within your Outbox, you'll find a list of all the datasets available for download. Occasionally, certain files may be marked as "unavailable". These unavailable files can be identified by the "unavailable" extension added to their filenames (e.g. filename.fastq.gz.unavailable.c4gh). If you encounter an unavailable file that you need, please reach out to our Helpdesk. We'll promptly work on making the file accessible for download as soon as possible. Specific to using keys Can I access one EGA account from different devices? Yes, you can access your account from different devices by linking several public keys to your EGA account. Each device can generate a unique public-private key pair, and the corresponding public keys can be linked to the same account. This way, you can use different public keys on different devices and still have access to the same account and data. I have several keys and I don't remember which one is which When generating SSH keys, it's a good practice to add a comment using the -C flag. This will allow you to add a descriptive tag to your key, making it easier to identify later on. Here's an example command that generates an SSH key with a comment: ssh-keygen -t ed25519 -C work-pass In this example, we're generating an ed25519 SSH key with the comment work-pass. Once you have multiple keys with different comments, you can use the comments to easily identify each key. To view the comments for your existing SSH keys, you can use the following command: ssh-keygen -l -f /path/to/key This will display the key fingerprint and the associated comment. By checking the comments, you should be able to identify which key is which. What if I can't find my SSH keys for uploading files with a key file, and how can I use new keys? If you can't find your SSH keys, don't worry - you can make new ones. To do this, open your terminal or command prompt and type a command to make a new SSH key. You can pick a name for the key, and choose a password to keep it safe. After making the key, you can add the new key to your account or server where you want to upload files using the key file. This usually involves copying and pasting the key's "public" (e.g. file.pub) part to the right place. If you lose track of the key again, just make a new one and add it again. Keep in mind that SSH keys belong to you and your computer, so if you switch computers or accounts, you'll need to make new keys. I don't want to type the passphrase every time I use the key. What can I do? You can use an ssh-agent to avoid typing the passphrase every time you use the key. An ssh-agent is a program that stores your private keys in memory and provides them to ssh when needed. You can add your key to the ssh-agent using the command ssh-add followed by the path to your key file.Here's an example of the steps to follow: Open a terminal window.Start the ssh-agent by typing the command eval $(ssh-agent).Add your key to the ssh-agent by typing the command ssh-add [key filepath]. For instance, if your key file is located in the home directory with the name mykey, the command will look like this: ssh-add ~/mykey After adding your, key to the ssh-agent, you should be able to use ssh without having to enter your passphrase every time. Can I use my password for authentication (without my private key)? If you prefer to use your username and password for authentication instead of your private key, you can still do so. When using a Graphical User Interface (GUI) such as FileZilla, you can select Ask for password as your Logon Type (Figure 3). This option will prompt you to enter your password when you click Connect, instead of using your private key. Figure 3: This option will prompt you to enter your password when you click "Connect", instead of using your private key. Figure 3: Process of establishing a new connection to __EGA_OUTBOX_DOMAIN__ using your password as the logon method in FileZilla. The figure showcases the FileZilla version 3.52.2 operating on IOS v11.2.3. By following the depicted steps, users can create a secure and efficient connection to the inbox, ensuring seamless data transfers. It's worth noting that using a password for authentication can be less secure than using an SSH key, as passwords can be more easily compromised through various means. However, if you choose to use your password for authentication, selecting "Ask for password" as your Logon Type is a good way to do so securely via a GUI. Why is it better to use my key and not my password? SSH keys for authentication is generally considered to be more secure and convenient than using passwords. SSH keys are more difficult to crack than passwords, and they can be restricted to specific users and machines, giving you more control over access. Once you set up your SSH keys, you can use them to authenticate quickly and easily, without having to enter a password every time. This makes automation of tasks, such as uploading encrypted files, much simpler. Additionally, SSH keys provide better logging, allowing you to keep track of who is accessing your systems and when. All in all, using SSH keys is a good practice for improving security and convenience in your authentication process.

  Documentation access/download/files/live-outbox

• How to upload Crypt4GH files

  Uploading files Users who hold an ega-box-XXX account can upload files using either INBOX or FTP. Users who have a Submitter role associated with their email will only be able to upload files using INBOX. Before uploading your files, please make sure that any files that will be uploaded to EGA do not use special characters in their naming convention, such as # ? ( ) [ ] / \ = + < > : ; " ' , * ^ | &. This can cause issues with the archiving process, leading to problems for end users. The EGA is a shared, public service with limited storage. To manage the available resources, we enforce a limit of 10TB per submission account at any one time. If you exceed this limit, a “permission denied” message will be displayed. This will prevent you from uploading more files, but connecting to your inbox.For submissions larger than 10TB, please perform uploads in 10TB batches: register all the metadata and then finalise the submission. Upload the next batch of files and repeat the same metadata registration and finalisation process until you have completed the file upload. Further information can be found in the SP documentation. INBOX FTP The INBOX is only compatible with files encrypted using the Crypt4gh tool Before uploading If you are not a registered EGA user, you will first need an EGA user account. Please note that it may take a few days for your account to be activated, as it needs to be vouched for by the EGA Helpdesk. Once your account is validated, you will be able to request a submitter role. [Optional] Meanwhile, you can create and add your public key to your EGA account profile. This option is not available for old submission accounts (e.g., ega-box-NNN). As soon as you have been granted a submitter role, you will be able to connect with your username and password to the EGA inbox using the SFTP protocol. If you have also registered a public key in your profile, you can also connect using this key. To upload files to your account, you can use the graphical user interface (GUI) or the command line. Graphical User Interface (GUI)We recommend using FileZilla, a free, open-source FTP client. However, you can use any other GUI that allows connecting over the SFTP protocol. For FileZilla as your GUI, follow these steps to upload files: Create a new connection in Site Manager (File > Site Manager) and select the following options (Figure 1): Protocol: SFTP - SSH File Transfer ProtocolHost: __EGA_INBOX_DOMAIN__Logon Type: Key fileUser: your EGA usernameKey file: Path/to/your/private_keyFigure 1: Process of establishing a new connection to __EGA_INBOX_DOMAIN__ using a key file as the logon method in FileZilla. The figure showcases the FileZilla version 3.52.2 operating on IOS v11.2.3. By following the depicted steps, users can create a secure and efficient connection to the inbox, ensuring seamless data transfers.Click Connect, and you will log in remotely to your home directory. You can think of this folder as a storage "in the EGA cloud" in which you will add your files for the EGA. The uploading area has three folders:To-encrypt: Files uploaded in this folder will be encrypted automatically on the fly.Encrypted: Files uploaded in this folder must already be encrypted with Crypt4gh. Upload your files here if your connection is unstable or you have problems completing the upload into-encrypt.Etc: This folder contains two files that allow the server to show you your username and group instead of some internal numbers. Please do not upload files here; otherwise, you will obtain a permission denied error. Find the files you want to upload by browsing your local storage (left side of your screen in FileZilla). Select all the files you want to upload, then right-click on them and select Upload (Figure 2). Figure 2: Step-by-step process of manually uploading files to __EGA_INBOX_DOMAIN__ using FileZilla, with FileZilla version 3.52.2 operating on IOS v11.2.3. The figure demonstrates how users can transfer data from their local storage to the "EGA cloud" by following the depicted steps Please note that regardless of which folder you upload your files in, both folders (to-encrypt, encrypted) will point to the same path (/) (Figure 3). Therefore, you will see your files in both folders. Figure 3: Both folders, to-encrypt and encrypted, point to the same path (/)" If your connection is unstable, please encrypt your files first using Crypt4gh. Then upload them to the ‘encrypted’ folder. The example above shows how to connect to __EGA_INBOX_DOMAIN__ using the private key. However, if you prefer to log in using your credentials, you can do so. Please go to the Frequently Asked Questions (FAQs)  for more information. SFTP command line To upload files securely to your private area of the EGA, you can use SFTP(Secure File Transfer Protocol) with your favorite FTP client. Here's what you need to know to get started: Connect to the target host __EGA_INBOX_DOMAIN__. This is the new hostname for the EGA SFTP service. Log in with your EGA username and key files (or password). Upload files to your private EGA inbox to ensure that only you can access the files. By following these steps, you can securely upload your files to the EGA for safe storage and sharing. Using the SFTP command line client in Linux/Unix Open a terminal and type sftp username@hostnameEnter your EGA passwordTo see a list of available SFTP commands, type helpsftp> put – Upload filesftp> get – Download filesftp> cd path – Change remote directory to ‘path’sftp> pwd – Display remote working directorysftp> lcd path – Change the local directory to ‘path’sftp> lpwd – Display local working directorysftp> ls – Display the contents of the remote working directorysftp> lls – Display the contents of the local working directoryType the "put" command to upload files. For example: put *.bamUse the bye command to close the connection (SFTP session). After uploading- Once you have uploaded files to the inbox, please bear in mind that the checksum needs to be calculated, which can take up to two days. You will only be able to link your files to a run/analysis once the encrypted checksum has been calculated.- When linking your files to the 'Run' or 'Analysis', ensure that the file name matches the file path '/name' in the INBOX folder.- Please delete the files from your SFTP INBOX after all the runs/analyses have been registered and files are ingested (SP > Files > Files ingested). This will clear your inbox space an allow you to upload more files. This will also prevent the files from reappearing in your Submitter Portal inbox. Frequently Asked Questions Specific to the inbox What username should I use to log in to my inbox? The authentication process for logging in to the EGA website, as well as accessing your inbox and outbox, requires the use of your username.  If you have forgotten your registered username, please contact our Helpdesk team for assistance. How are checksums calculated in your inbox? If you encrypt the file beforehand and upload it to the "encrypted" folder, the unencrypted checksum will not be calculated until the file is ingested (i.e., until it is used in a run/analysis). If the file is uploaded to the "to-encrypt" folder, then both checksums are calculated.Please bear in mind that after files have been uploaded to the inbox, the checksum must be calculated, which can take from a few hours to two days.  Specific to using keys to authenticate Can I access one EGA account from different devices? Yes, you can access your account from different devices by linking several public keys to your EGA account. Each device can generate a unique public-private key pair, and the corresponding public keys can be linked to the same account. This way, you can use different public keys on different devices and still have access to the same account and data. I have several keys and I don't remember which one is which When generating SSH keys, it's a good practice to add a comment using the -C flag. This will allow you to add a descriptive tag to your key, making it easier to identify later on. Here's an example command that generates an SSH key with a comment: ssh-keygen -t ed25519 -C work-pass In this example, we're generating an ed25519 SSH key with the comment work-pass. Once you have multiple keys with different comments, you can use the comments to easily identify each key. To view the comments for your existing SSH keys, you can use the following command: ssh-keygen -l -f /path/to/key This will display the key fingerprint and the associated comment. By checking the comments, you should be able to identify which key is which. What if I can't find my SSH keys for uploading files with a key file, and how can I use new keys? If you can't find your SSH keys, don't worry - you can make new ones. To do this, open your terminal or command prompt and type a command to make a new SSH key. You can pick a name for the key, and choose a password to keep it safe. After making the key, you can add the new key to your account or server where you want to upload files using the key file. This usually involves copying and pasting the key's "public" (e.g. file.pub) part to the right place. If you lose track of the key again, just make a new one and add it again. Keep in mind that SSH keys belong to you and your computer, so if you switch computers or accounts, you'll need to make new keys. I don't want to type the passphrase every time I use the key. What can I do? You can use an ssh-agent to avoid typing the passphrase every time you use the key. An ssh-agent is a program that stores your private keys in memory and provides them to ssh when needed. You can add your key to the ssh-agent using the command ssh-add followed by the path to your key file.Here's an example of the steps to follow: Open a terminal window.Start the ssh-agent by typing the command eval $(ssh-agent).Add your key to the ssh-agent by typing the command ssh-add [key filepath]. For instance, if your key file is located in the home directory with the name mykey, the command will look like this: ssh-add ~/mykey After adding your, key to the ssh-agent, you should be able to use ssh without having to enter your passphrase every time. Can I use my password for authentication (without my private key)? If you prefer to use your username and password for authentication instead of your private key, you can still do so. When using a Graphical User Interface (GUI) such as FileZilla, you can select Ask for password as your Logon Type (Figure 3). This option will prompt you to enter your password when you click Connect, instead of using your private key. Figure 3: This option will prompt you to enter your password when you click "Connect", instead of using your private key. Figure 3: Process of establishing a new connection to __EGA_INBOX_DOMAIN__ using your password as the logon method in FileZilla. The figure showcases the FileZilla version 3.52.2 operating on IOS v11.2.3. By following the depicted steps, users can create a secure and efficient connection to the inbox, ensuring seamless data transfers. It's worth noting that using a password for authentication can be less secure than using an SSH key, as passwords can be more easily compromised through various means. However, if you choose to use your password for authentication, selecting "Ask for password" as your Logon Type is a good way to do so securely via a GUI. Why is it better to use my key and not my password? SSH keys for authentication is generally considered to be more secure and convenient than using passwords. SSH keys are more difficult to crack than passwords, and they can be restricted to specific users and machines, giving you more control over access. Once you set up your SSH keys, you can use them to authenticate quickly and easily, without having to enter a password every time. This makes automation of tasks, such as uploading encrypted files, much simpler. Additionally, SSH keys provide better logging, allowing you to keep track of who is accessing your systems and when. All in all, using SSH keys is a good practice for improving security and convenience in your authentication process.

  Documentation submission/data/uploading-files/inbox

• Whole Exome Sequencing in Alopecia Areata Identifies Rare Mutations in KRT82

  Alopecia areata (AA) is an autoimmune disorder, in which the body's own immune cells attack hair follicels resulting in hair loss. It is a complex genetic disease with 14 associated loci previously identified in our genome-wide association study (GWAS). In the study, we performed the whole-exome sequencing (WES) on 849 AA patients with the aim to identify the novel rare variants in genes contributing to AA susceptibility. We performed gene-collapsing analyses and identified keratin 82 (KRT82 Gene ID: 3888) as the highest associated AA gene in the three rare damaging collapsing models with p&lt;6.7E-07 in one of the models. The KRT82 gene is a hair-specific type II keratin expressed in the hair shaft cuticle during the anagen phase of the hair cycle. We reported the lower expression of KRT82 in AA hair follicles, compared to controls, and the KRT82 expression is absent from the bulb region, the site of AA immune attack. This study emphasizes the significance of using WES and gene collapsing analyses to identify rare variation in a functionally relevant risk gene using a moderate sample size.&nbsp;&nbsp;

  Study phs002632

• Genetic Underpinnings of Ethnic Disparities in Bone Toxicities Between Hispanic and Non-Hispanic Children Treated for Acute Lymphoblastic Leukemia

  Acute lymphoblastic leukemia (ALL) is the most common malignancy among children and young adolescents. More than 90% of children are cured with a combination of multiple chemotherapeutic drugs. Some may suffer from debilitating toxicities due to the cytotoxic drugs, necessitating pharmacogenetic research to search for genetic markers predictive of treatment toxicities, so that treatment can be better tailored based on patients' genetic makeup. Bone toxicities, including osteonecrosis and fractures, most often due to glucocorticoids, are the most common complications in ALL, which have long-lasting detrimental impact on the still developing skeletons in children. In DFCI ALL Consortium Protocol 05-001, a multi-center clinical trial for childhood ALL conducted in Canada and the US, we found that Hispanic children had less drug toxicities to their bones than non-Hispanic children. Although reasons for the observed ethnic differences are largely unknown, patients' inherited genetic background may be at play. This study seeks to perform a novel pharmacogenomic study based on multi-site clinical trials DFCI ALL Consortium Protocol 05-001 and 11-001 to identify genetic underpinnings of ethnic disparities in bone toxicities.

  Study phs002317

• Genetic coupling of enhancer activity and connectivity in gene expression control

  In this study, we investigated whether the genetic effects of eQTLs on enhancer activity and enhancer-promoter contacts (connectivity) are inherently coupled or distinct. To address this, we performed Capture Hi-C targeting ~1100 eQTL loci, along with ATAC-seq and RNA-seq, in monocytes from 34 healthy male donors. Using single-modality and multi-modality Bayesian statistical approaches, we demonstrated that eQTL effects are predominantly shared across enhancer activity, connectivity, and gene expression. However, we also identified a locus where an eQTL disrupts a CTCF binding site at a putative insulator, facilitating contacts between the PCK2 gene promoter and downstream enhancers. To validate these findings, we used 4C-seq to confirm the allelic effects of eQTLs on enhancer-promoter connectivity at the PCK2 locus and another eQTL region associated with THBS1 expression, both of which showed consistent eQTL effects across all three modalities. Additionally, we conducted Hi-C on monocytes from two donors to map reference TADs and performed ChIP-seq to confirm the allelic effects of the PCK2 eQTL on CTCF binding. Together, these data provide comprehensive insights into the genetic coupling of enhancer activity, connectivity, and gene expression.

  Study EGAS50000000756

• Reference Exome Data for a Northern Brazilian population

  Whole exome sequencing (WES) is a popular technology widely used in the diagnosis of rare genetic diseases and provides useful variant data for analysis of complex diseases. However, there is not always adequate population-specific reference data available to assist clinical geneticists in assigning a diagnostic variant to a specific clinical condition. Here we provide a catalogue of variants called after sequencing the exomes of 45 babies from the State of Rio Grande do Nord in Brazil. Sequence data were processed using an ‘intersect-then-combine’ (ITC) approach, using GATK and SAMtools to call variants. A total of 612,761 variants were identified in at least one individual in this Brazilian Cohort, including 559,448 single nucleotide variants (SNVs) and 53,313 insertion/deletions. Of these, 58,111 overlapped with nonsynonymous (nsSNVs) or splice site (ssSNVs) SNVs in dbNSFP. As an aid to clinical diagnosis of rare diseases, we used the American College of Medicine Genetics and Genomics (ACMG) guidelines to assign pathogenic/likely pathogenic status to 439 (0.755%) of the 58,111 nsSNVs and ssSNVs. Our data set provides a useful reference point for diagnosis of rare diseases in Brazil.

  Study EGAS00001004112

• Onco-exaptation of an Endogenous Retroviral LTR Drives IRF5 Expression in Hodgkin Lymphoma

  The transcription factor interferon regulatory factor 5 (IRF5) is upregulated in Hodgkin lymphoma (HL) and a key regulator of the aberrant transcriptome characteristic of this disease. Here we show that IRF5 upregulation in HL is driven by transcriptional activation of a normally dormant endogenous retroviral LOR1a long terminal repeat (LTR) upstream of IRF5. Specifically, through screening of RNA-seq libraries, we detected LTR-IRF5 chimeric transcripts in multiple HL cell lines but not in normal B-cell controls. In HL, the LTR was in an open and hypomethylated epigenetic state and we further show the LTR is the site of transcriptional initiation. Among HL cell lines, usage of the LTR promoter strongly correlates with overall levels of IRF5 mRNA and protein, indicating that LTR transcriptional awakening is a major contributor to IRF5 upregulation in HL. Taken together, oncogenic IRF5 overexpression in HL is the result of a specific LTR transcriptional activation. We propose that such LTR derepression is a distinct mechanism of oncogene activation (“onco-exaptation”) and that such a mechanism warrants further investigation in molecular and cancer research.

  Study EGAS00001001205

• Treatment-mediated selection of lethal prostate cancer clones defined by copy number architectures

  Despite initial responses to hormone treatment, metastatic prostate cancer invariably evolves to a lethal state. To characterize the intra-patient relationships of metastases that evade treatment, we performed genome- wide copy number profiling and bespoke approaches targeting the androgen receptor (AR) on 142 metastatic regions from 10 organs harvested post-mortem from nine men who died from prostate cancer. We identified diverse and patient-unique alterations clustering around the AR in metastases from every patient with evidence of independent acquisition of related genomic changes within an individual and, in some patients, the co-existence of AR-neutral clones. Using the genomic boundaries of pan-autosome copy number change, we confirmed a common clone of origin across metastases and diagnostic biopsies; and identified in individual patients, clusters of metastases occupied by dominant clones with diverged autosomal copy number alterations. Autosome-defined clusters were characterized by cluster-specific AR gene architectures that in two index cases were topologically more congruent than by chance (p-values 0.03, 3.07x10-8). Integration with anatomical site suggested patterns of spread and points of genomic divergence. Copy number boundaries identified treatment-selected clones with putatively distinct lethal trajectories.

  Study EGAS00001006598

• HLA-DR is absent in primitive macrophages through epigenetic silencing of master regulator CIITA

  The earliest macrophages are generated during embryonic development from erythro-myeloid progenitors (EMPs) via primitive haematopoiesis. This process is poorly understood in humans and in the mouse is thought to be spatially restricted to the yolk sac. Human fetal placental macrophages, Hofbauer cells (HBC), arise during the primitive haematopoietic wave at ~18 days post conception. Here we identify a population of placental erythro-myeloid progenitors (PEMPs) in the early human placenta that give rise to HBC. PEMP are fetal CD34+CD43+ progenitors found exclusively at early gestational timepoints. Transcriptomic analyses reveal that PEMP have conserved features of primitive yolk sac EMPs, including the lack of HLF expression. Using in vitro single-cell culture experiments we show that PEMP generate HBC-like cells that completely lack HLA-DR expression  and demonstrate that this is a conserved feature of all macrophages generated by primitive haematopoiesis in humans. Finally, we demonstrate that the class II transactivator, CTIIA, the master regulator HLA-DR expression, is epigenetically silenced during primitive haematopoiesis, leading to HLA-DRneg macrophages. These findings demonstrate that HBC are derived locally from PEMP and establish the human placenta as an additional site of primitive haematopoiesis.

  Study EGAS00001006981

• Defining Cutaneous Gene Expression Signatures in Juvenile Dermatomyositis

  Skin inflammation in juvenile dermatomyositis (JDM) can signal disease onset or flare up and prevent complete disease remission. Our objective was to evaluate JDM cutaneous expression signatures through tape stripping in both lesional and non-lesional skin and as compared to paired blood. A total of 28 JDM and 20 control (CTL) patients participated in the study. All patients had tape strip sampling of non-lesional (NL) skin at enrollment visit. Within our JDM cohort, paired lesional (L) skin was additionally sampled if rash was present. 16/28 of JDM patients had at least one additional skin sample associated with a follow-up (FU) visit (total number of follow-up samples included = 26 NL and 4 L). We identified a common innate immune signature in both lesional and non-lesional JDM skin. Using unsupervised hierarchical clustering of skin expression data, we distinguished a JDM molecular subgroup characterized by a mitochondrial dysfunction signature. In comparison to blood expression data, we determined that skin may more effectively highlight the JDM subgroup characterized by a mitochondrial dysfunction expression signature. Data available include bulk RNA-sequencing data from tape strips and blood of the JDM and control patients from this study.

  Study phs003884

• Pediatric Whole Genome Sequencing Diagnostic Utility

  The standard of care for first-tier clinical investigation of the etiology of congenital malformations and neurodevelopmental disorders is chromosome microarray analysis (CMA) for copy number variations (CNVs), often followed by gene(s)-specific sequencing searching for smaller insertion-deletions (indels) and single nucleotide variant (SNV) mutations. We compared diagnostic rate of WGS to CMA and targeted gene testing of 100 patients referred to The Hospital for Sick Children Genetics clinic in 2014. WGS identified genetic variants meeting clinical diagnostic criteria in 34% of cases, representing a 4-fold increase in diagnostic rate over CMA (8%) (p-value = 1.42e-05) alone and &gt;2-fold increase in CMA plus targeted gene sequencing (13%) (p-value = 0.0009). WGS identified all rare clinically significant CNVs that were detected by CMA. In an additional 26 patients, WGS revealed indel and missense mutations presenting in a dominant (63%) or a recessive (37%) manner. We found four subjects with mutations in at least two genes associated with distinct genetic disorders, including two cases harboring a pathogenic CNV and SNV. When considering medically actionable secondary findings in addition to primary WGS findings, 38% of patients would benefit from genetic counselling.

  Study EGAS00001001623

• PATL2 is a key actor of oocyte maturation whose invalidation causes infertility in women and mice

  The genetic causes of oocyte meiotic deficiency (OMD), a form of primary infertility characterised by the production of immature oocytes, remain largely unexplored. Using whole exome sequencing, we found that 26% of a cohort of 23 subjects with OMD harboured the same homozygous nonsense pathogenic mutation in PATL2, a gene encoding a putative RNA-binding protein. Using Patl2 knockout mice, we confirmed that PATL2 deficiency disturbs oocyte maturation, since oocytes and zygotes exhibit morphological and developmental defects respectively. PATL2's amphibian orthologue is involved in the regulation of oocyte mRNA as a partner of CPEB. However, Patl2's expression profile throughout oocyte development in mice, alongside colocalisation experiments with Cpeb1, Msy2 and Ddx6 (three oocyte RNA-regulators) suggest an original role for Patl2 in Mammals. Accordingly, transcriptomic analysis of oocytes from WT and Patl2-/- animals demonstrated that in the absence of Patl2, expression levels of a select number of highly relevant genes involved in oocyte maturation and early embryonic development are deregulated. In conclusion, PATL2 is a novel actor of mammalian oocyte maturation whose invalidation causes OMD in humans.

  Study EGAS00001002903

• 16S-based fecal microbiota composition

  The dataset reports the 16S rRNA gene sequencing of the fecal microbiota of donors from the Milieu Intérieur Cohort. The Milieu Intérieur cohort includes a total of 1,000 healthy individuals of western European ancestry, recruited in France as part of the Milieu Intérieur project. To assess their fecal microbiota composition, 16S rRNA profiles were generated from stool samples of 863 of the 1,000 donors. Human stool samples were produced at home no more than 24 hours before the scheduled medical visit and collected in a double-lined sealable bag maintaining strict anaerobic conditions. Upon reception at the clinical site, the fresh stool samples were aliquoted and stored immediately at -80°C. DNA was extracted from stool and barcoding PCR was carried out using indexed primers targeting the V3-V5 region of the 16S rRNA gene. Equal volumes of normalized PCR reaction were pooled and thoroughly mixed. The amplicon libraries were sequenced on Illumina MiSeq.

  Dataset EGAD00001004979

• DNA whole-exome sequencing data from patients with metastatic basal cell carcinoma

  Whole-exome sequencing data in fastq format of matched tumour and germline DNA from 8 patients with metastatic basal cell carcinoma. Samples are labeled as Primary, Local or Metastasis: Primary: Sample was obtained from primary tumour. Local: Sample was obtained from local recurrence of primary tumour. Metastasis: Sample was obtained from a metastatic site. Germline: Sample was obtained from normal adjacent tissue. DNA was isolated from FFPE tissue sections of the tumor biopsies using the AllPrep DNA/RNA FFPE Kit (Qiagen) and quality controls conducted using the Qubit fluorometer (Thermo Fisher Scientific). Library preparation was performed using the Agilent SureSelect Human All Exon v7 XTHS2 probes and sequenced on a NovaSeq 6000 S2 2x100bp

  Dataset EGAD00001008675

• Investigation of mutational signatures associated with DNMT3A deficiency (2019-04-03)

  Mutational signatures have been shown to be attributable to specific genetic contexts, such as mutations in DNA repair genes. DNMT3A is a DNA methyltransferase that helps maintain the DNA methylation pattern in a site-specific manner and may participate in DNA repair or the stress response. We have identified an adult individual who is a germline mosaic for a DNMT3A mutation. We have obtained clonal lymphoblastoid cells (LCLs) from the subject representing both WT and mutant lines grown in the same individual for &gt;50 years. These clones represent a unique opportunity to examine the mutational impact of the DNMT3A mutation in a well-controlled setting. Our goal is to perform WGS on whole blood, representing the pool, as well as several WT and several mutant clones, in order to investigate the contribution of DNMT3A to mutation rates and signatures. . This dataset contains all the data available for this study on 2019-04-03.

  Dataset EGAD00001004889

• Single cell targeted DNA-sequencing (and antibody sequencing) of high hyperdiploid B-ALL

  This dataset contains all sequencing data of the publication "Single-cell DNA and Surface Protein Characterization of High Hyperdiploid Acute Lymphoblastic Leukemia at Diagnosis and During Treatment". We provide both the raw fastq-files as well as the processed data in the form of .h5 files. There are 13 patients: XJ176, XH135, XJ180, XI145, XI148, XI167, XI150, XJ175, XJ178, XI162, XF98, XG111 and XG115. Of 9 patients, also follow-up samples during/after treatment were sequenced: XJ180, XI145, XI148, XI167, XI150, XJ175, XJ178, XI162 and XG111. To identify the cell types and distinguish between normal and leukemic cells, cell surface proteins were also captured for 6 samples: XF98, XG111 (at diagnosis and during treatment), XG115, XI162 (after treatment) and XJ175 (after treatment). All samples were processed on the MissionBio platform with a custom amplicon panel, and DaB-seq for the combined targeted DNA and protein sequencing.

  Dataset EGAD50000000829

• fragmentomic features of individuals with different cfDNA concentrations

  The uploaded data includes sequencing data of 862 individuals from the nasopharyngeal carcinoma (NPC) screening study. Samples from this cohort were sequenced using targeted sequencing methods for the Epstein Barr Virus (EBV) and selected autosomal DNA, but only ‘off-target’ reads were used for fragmentomic analyses. We have also performed genome-wide (non-targeted sequencing) for 1) Individuals with the highest and lowest cell-free DNA concentration (40 individuals); 2) A subsequent collection of the subjects with the highest and lowest DNA concentrations after six years (26 individuals); 3) 30 cases of pregnancy and 4) 20 patients with hepatocellular carcinoma. All sequencing data are of extracted plasma cfDNA from human subjects. The targeted sequencing samples from 862 individuals were sequenced on the NextSeq500 System (Illumina) and aligned to the EBV genome (AJ507799.2) and the human genome (hg19). The alignments were provided in bam format. The original fastq files of the remaining non-target sequencing samples were provided.

  Dataset EGAD50000000970

• IMCISION RNAseq

  RNA sequencing of 32 primary head and neck squamous cell carcinoma (HNSCC) samples prior to treatment with neoadjuvant anti-PD-1 (n=6) or anti-PD-1 + anti-CTLA-4 (n=26) immunotherapy, and 30 paired on-treatment HNSCC samples (i.e. after neoadjuvant immunotherapy). RNA quantity used: 10ng. Library Preparation Kit: SMART Stranded Total RNA Seq Kit (Takara). Sequencing parameters: NovaSeq 6000, 2x 100 bp. File type: fastQ

  Dataset EGAD00001008127

• single cell RNA-seq of peripheral blood from analysis in infants with MLL-rearranged Acute Lymphoblastic Leukemia

  This dataset contains two experiments. 1) Single cell RNA-seq of peripheral blood diagnostic samples from patients with MLL-rearranged infant ALL that underwent relapse or not (samples ending in R relapsed, samples ending in N did not), sequenced with SORT-seq (see cell systems, 2016, doi:10.1016/j.cels.2016.09.002). For some of the patients, multiple indipendent plates were produced (each plate is a sample). Barcode-well correspondence can be found here: https://bitbucket.org/princessmaximacenter/sharq/src/master/data/celseq2_bc384-v4.csv . 2) Single cell RNA-seq of peripheral blood diagnostic samples from patients with MLL-rearranged infant ALL that underwent relapse or not (samples ending in R relapsed, samples ending in N did not), sequenced with10x Genomics Version 2.

  Dataset EGAD00001007752

• Germline RUNX1 Variation and Predisposition to Childhood Acute Lymphoblastic

  Somatic mutations of RUNX1, which encodes the myeloid and lymphoid transcriptional factor RUNX1, are common in both B- and T- acute lymphoid leukemia (ALL) and are associated with poor prognosis of T-ALL. However, there has been no comprehensive investigation of the pattern or prevalence of RUNX1 germline mutation in both B- and T-ALL. Here we report germline RUNX1 variants in 1.23% of B-ALL and 2.11% of T-ALL, identifying 31 unique variants in 62 B-ALL and 18 unique variants in 26 T-ALL children. The majority of frameshift and nonsense variants affected RUNX1 function in transcriptional regulation, hematopoiesis, and cellular proliferation. We identified JAK3 as the most frequent somatic mutation in T-ALL with RUNX1 variants. These results not only identify RUNX1 as a leukemia predisposition gene but also further underline the importance of germline genetic variants to the development of ALL

  Dataset EGAD00001007793

• Metabolic reprogramming towards OXPHOS identifies a novel therapeutic target for mantle cell lymphoma

  Metabolic reprogramming is linked to cancer cell growth and proliferation, metastasis, and therapeutic resistance in a multitude of cancers. Targeting dysregulated metabolic pathways to overcome resistance, an urgent clinical need in all relapsed/refractory cancers, remains difficult. Through genomics analysis of clinical specimens, we show that metabolic reprogramming towards oxidative phosphorylation (OXPHOS) and glutaminolysis is associated with therapeutic resistance to the Bruton’s tyrosine kinase inhibitor ibrutinib in mantle cell lymphoma (MCL), an incurable B-cell lymphoma with poor clinical outcomes. Inhibition of OXPHOS with a novel, clinically applicable small molecule, IACS-010759, which targets complex I of the mitochondrial electron transport chain, results in significant growth inhibition in vitro and in vivo in ibrutinib-resistant patient-derived cancer models. This work suggests that targeting metabolic pathways to subvert therapeutic resistance is a clinically viable approach to treat highly refractory malignancies.

  Dataset EGAD00001004577

• How to request data

  Request Data Access If you're looking to access data from the EGA for your research, you are in the right documentation page! In this page you will find a step by step guide on how to request data archived at the EGA, as well as some frequently asked questions you might find useful. When requesting data, you'll need to provide information about your research project and intended use of the data. Keep in mind that your data access request may require additional approvals or agreements depending on the datasets and data providers involved. Step by step guide Register yourself as an EGA user. Validate your account. A validation link will be sent to your email to activate your EGA user account. Log into the EGA page. Search the dataset of interest from the dataset catalogue or search bar. Click on “Request access” Add a comment requesting these files and send your request Your request is successfully registered, and the DAC will receive a notification about your request In addition to submitting your request, you should also contact the DAC directly via e-mail. You can access the DAC's contact information from the relevant dataset page. Frequently Asked Questions (FAQ) Which is the role of the EGA in the data access process? The European Genome-phenome Archive (EGA) facilitates the secure distribution under controlled access of personally identifiable genetic and phenotypic data. Data Access Committee (DAC) is a body of one or more individuals who are responsible for data release to external requestors, based on participant consent and/or National Research Ethics terms. How do I request access to multiple datasets? To request access for multiple datasets managed by the same Data Access Committee (DAC), please start by visiting the DAC catalogue to locate the DAC of your choice. Once you've found the relevant DAC, proceed to its dedicated page where you'll find a "Request access" button. After logging in to the EGA website, you'll be presented with a comprehensive list of datasets under the chosen DAC. Select the datasets you're interested in, and then compose a message indicating your access request. Click the "Send" button to submit your request, which will include the chosen datasets. Please be sure to review the data usage policies for the requested datasets, as policies can vary among datasets managed by the same DAC. I am a co-applicant. Will I be given a joint access account? Each applicant needs to create an EGA account themselves, with unique login details. User log-in details are for individual use only and should never be shared, under any circumstances. EGA services are only provided to EGA account holders. Therefore, the co-applicant will send a formal request to the DAC. What happens if a co-applicant leaves or a new member needs to be added to an existing DAC application? The appropriate Data Access Committee (DAC) should be contacted and informed about the required changes so that the application can be updated. I have changed my place of work since my EGA account was created. Can I still have access to data? You must contact the relevant Data Access Committee (DAC). The DAC did not reply to my data access request. How should I proceed? There is no estimated time for DAC response. Please allow up to four weeks for a response. In the meantime, please send them a weekly reminder of the status of your request by emailing them directly.If the DAC becomes unresponsive, meaning that they fail to communicate with a requestor within the specified time frame, please contact the EGA Helpdesk team. I have an account with the EGA and would like access to further datasets. How should I proceed? You need to reapply to the relevant Data Access Committee (DAC). Once the DAC has approved your application your account will be updated and an email will be sent to notify you of a change made to your account. How do I download files of the datasets I have been granted access to? The preferred method for data download and decryption is the EGA Secure Download Client. Please carefully review the provided documentation. How do I download metadata? Metadata is retrieved from the EGA webpage; you will not need to use any download client. Be informed that you will only be able to download metadata of your approved datasets. For such download, you should first log in the EGA webpage with your credentials. Then browse the metadata's dataset you have gained access and you will find a metadata download button. You may also want to have a look our metadata schema. I have forgotten or misplaced my user account password; how do I get a new one? Click on “Log in”, located in the top right corner of the EGA homepage. Then click “Forgot password”, type in your registered institutional email address, and instructions for resetting your password will be sent to you. Please check both regular and junk/spam folders. I was sent a link to set a password for my download account, but it seems like the link has expired. What should I do? The procedure is the same as the one for a forgotten password, explained in the previous question.

  Documentation access/request-data/how-to-request-data

• RNA-seq of high grade serous ovarian tumours

  Somatic RNA for 40 samples matched to the WGS was extracted using the Qiagen Qiasymphony RNA protcol (cat no 931636). The tissue was initially homogenised using a Qiagen Bioruptor, followed by the manufacturers recommended protocol (including DNase digestion). The resulting RNA the underwent quality control as follows: firstly, A260 and A280nm were measured on a Denovix DS-11 Fx to qualitatively illustrate A260/280nm and A260/230nm ratios as measures of RNA purity. A260/280 had to be 2.0 and A260/230 had to be 2.0-2.2. Then RNA was quantified using LifeTechnologies Qubit RNA BR kit (cat no Q10210). RNAseq was carried out by the Edinburgh Clinical Research Facility on an Illumina NExtSeq500. Total RNA samples were assessed on the Agilent Bioanalyser (Agilent Technologies, #G2939AA) with the RNA 6000 Nano Kit (#5067-1512) for quality and integrity of total RNA, and then quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific Inc, #Q32866) and the Qubit RNA HS assay kit (#Q32855). Libraries were prepared from total-RNA sample using the NEBNext Ultra 2 Directional RNA library prep kit for Illumina (#E7760S) with the NEBNext rRNA Depletion kit (#E6310) according to the provided protocol. 400ng of totalRNA was then added to the ribosomal RNA (rRNA) depletion reaction using the NEBNext rRNA depletion kit (Human/mouse/rat) (#E6310). This step uses specific probes that bind to the rRNA in order to cleave it. rRNA-depleted RNA was then DNase treated and purified using Agencourt RNAClean XP beads (Beckman Coulter Inc, #66514). RNA was then fragmented using random primers before undergoing first strand and second strand synthesis to create cDNA. cDNA was end repaired before ligation of sequencing adapters, and libraries were enriched by PCR using the NEBNext Multiplex oligos for Illumina set 1 and 2 (#E7500). Final libraries had an average peak size of 271bp. Libraries were quantified by fluorometry using the Qubit dsDNA HS assay and assessed for quality and fragment size using the Agilent Bioanalyser with the DNA HS Kit (#5067-4626). Sequencing was performed using the NextSeq 500/550 High-Output v2 (150 cycle) Kit (# FC- 404-2002) on the NextSeq 550 platform (Illumina Inc, #SY-415-1002). Libraries were combined in an equimolar pool based on the library quantification results and run across 5 High-Output Flow Cell v2.5.

  Dataset EGAD00001009048

• RNA-seq of high grade serous ovarian tumours

  Somatic RNA for 37 samples was extracted using the Qiagen Qiasymphony RNA protcol (cat no 931636). The tissue was initially homogenised using a Qiagen Bioruptor, followed by the manufacturers recommended protocol (including DNase digestion). The resulting RNA the underwent quality control as follows: firstly, A260 and A280nm were measured on a Denovix DS-11 Fx to qualitatively illustrate A260/280nm and A260/230nm ratios as measures of RNA purity. A260/280 had to be 2.0 and A260/230 had to be 2.0-2.2. Then RNA was quantified using LifeTechnologies Qubit RNA BR kit (cat no Q10210). RNAseq was carried out by the Edinburgh Clinical Research Facility on an Illumina NExtSeq500. Total RNA samples were assessed on the Agilent Bioanalyser (Agilent Technologies, #G2939AA) with the RNA 6000 Nano Kit (#5067-1512) for quality and integrity of total RNA, and then quantified using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific Inc, #Q32866) and the Qubit RNA HS assay kit (#Q32855). Libraries were prepared from total-RNA sample using the NEBNext Ultra 2 Directional RNA library prep kit for Illumina (#E7760S) with the NEBNext rRNA Depletion kit (#E6310) according to the provided protocol. 400ng of totalRNA was then added to the ribosomal RNA (rRNA) depletion reaction using the NEBNext rRNA depletion kit (Human/mouse/rat) (#E6310). This step uses specific probes that bind to the rRNA in order to cleave it. rRNA-depleted RNA was then DNase treated and purified using Agencourt RNAClean XP beads (Beckman Coulter Inc, #66514). RNA was then fragmented using random primers before undergoing first strand and second strand synthesis to create cDNA. cDNA was end repaired before ligation of sequencing adapters, and libraries were enriched by PCR using the NEBNext Multiplex oligos for Illumina set 1 and 2 (#E7500). Final libraries had an average peak size of 271bp. Libraries were quantified by fluorometry using the Qubit dsDNA HS assay and assessed for quality and fragment size using the Agilent Bioanalyser with the DNA HS Kit (#5067-4626). Sequencing was performed using the NextSeq 500/550 High-Output v2 (150 cycle) Kit (# FC- 404-2002) on the NextSeq 550 platform (Illumina Inc, #SY-415-1002). Libraries were combined in an equimolar pool based on the library quantification results and run across 5 High-Output Flow Cell v2.5.

  Dataset EGAD00001010139

• Study of Women's Health Across the Nation (SWAN) Repository

  The Study of Womens Health Across the Nation (SWAN) is an active multi-site, multi-disciplinary, longitudinal study of women&#39;s health. Initially funded in 1994 by the National Institute on Aging (NIA), the National Institute of Nursing Research (NINR), and the Office of Research on Women&#39;s Health (ORWH), the overall goal of SWAN is to describe the natural history of the menopausal transition and the post menopause including biological, behavioral, and psychosocial characteristics. SWAN focuses on the impact of menopause on age-related chronic diseases, such as diabetes, cardiovascular disease, depression, bone loss and osteoporosis, as well as physical and cognitive functioning. SWAN has seven clinical study sites located in six states, two in California, and one each in Chicago, Boston, Detroit area, northern New Jersey and Pittsburgh. The SWAN cohort was recruited from these areas and consists of 3,302 African American, Caucasian, Chinese American, Hispanic and Japanese American women. SWAN participants were enrolled in 1996-1997 and have been seen annually or bi-annually for clinic visits, which include interviews, measurements, and the collection of blood and urine samples. SWAN participants have now been seen for the baseline and 15 follow-up visits.

  Study phs001470

• International Multi-Center ADHD Genetics Project

  The goal of the project is to complete a 600,000 tag SNP genome-wide association scan of 958 parent-child trios from the International Multisite ADHD Genetics (IMAGE) project, in order to assess the association of SNP markers with ADHD, analyze quantitative ADHD phenotypes, complete copy number analyses, assess parent of origin effects and season of birth effects, and test for epistasis among apparently uncorrelated genes. Acquiring DNA Samples All consent forms stipulate that the samples can only be used by researchers who have been approved by the National Institute of Mental Health (NIMH), National Institutes of Health. All consent forms, except those used at the Z&#252;rich site (N=141 subjects), explicitly indicate that the samples may be used by researchers from commercial enterprises seeking to benefit financially from the analysis of the samples. The Z&#252;rich consent does not prohibit such use. The Z&#252;rich consent form also included an &quot;opt out&quot; that allowed the subjects to indicate that they did not want their samples stored at the NIMH repository or used by researchers external to the project. No subjects enrolled in the project opted out. Consent groups and participant set ADHD (ADHD): 2758 (924 trios)

  Study phs000016

• Integrated Genomic Analysis of Periampullary Tumors at The Human Genome Sequencing Center, Baylor College of Medicine (HGSC-BCM)

  Adenocarcinomas arising in the complex environment of the ampulla of Vater constitute a histopathological heterogenous group, presumably originating from the different epithelial cellular constituents present at the site: pancreas, bile duct, and intestinal duodenum. These tumors have been described in many different ways: intra-ampullary, periampullary, intra-ampullary papillary-tubular neoplasm, ampullary-ductal, periampullary-duodenal, and ampullary-not otherwise specified. These varied classifications reflect the difficulty in classifying these tumors into specific groups. Only the tumors clearly localized in the bile duct or duodenum are identified as distal cholangiocarcinomas (CAC) or duodenal adenocarcinomas (DUOAC). The current classification is based on macroscopic features that may distinguish the epithelium of origin, microscopic features, clinicopathological criteria, histopathology and expression of differential markers. This classification is subjective and prone to inter-observer variability and significantly impacts on treatment selection and therapeutic development. In order to define subtypes of periampullary cancer with clinical relevance, we performed whole exome sequencing and copy number analysis of 160 cancers arising in the periampullary region, 62 of these clearly arising from either the bile duct (n = 44), or the duodenum (n = 18) and 98 periampullary cancers (AMPAC) where the epithelium of origin could not be clearly defined.

  Study phs000895

• Trisomy21: Risk Factors for Chromosome Nondisjunction (T21NDJ)

  The overall goal of the project is to identify genetic risk factors associated with chromosome 21 nondisjunction in the oocyte. The dataset derives from multi-site collection of live birth probands with Down syndrome due to standard trisomy 21 (T21) and their biological parents. The type of nondisjunction error (NDJ)(maternal or paternal) in all cases has been determined to be maternal in origin based on the chromosome 21 variants contributed from parent to proband. The Center of Inherited Disease with support from NICHD has conducted genome-wide genotyping using the Illuminia Human OmniExpress Plus Exome array on approximately 800 women who have been identified through their offspring with DS and have been characterized as having a maternal meiois I (MI) or meiosis II (MII) nondisjunction error. Genotypes from biological fathers of the offspring with DS can be used with the data on mothers to better define the type of nondisjunction error (MI or MII) and to refine the chromosome 21 recombination profile. We provide the type of nondisjunction error, knowing that this will be updated based on the new panel. We do not provide the recombination profile, as this can be best defined using the new comprehensive set of SNPs in the OmniExpress panel.

  Study phs000718

• Precision Medicine for Dilated Cardiomyopathy in European and African Ancestry

  The DCM Precision Medicine Study is a multi-site consortium-based cross-sectional study of families with an embedded open-label randomized controlled trial. The aims of the study are to test the hypothesis that dilated cardiomyopathy (DCM) has a substantial genetic basis and to evaluate the effectiveness of a family communication intervention in improving the uptake of preventive screening and surveillance in at-risk first-degree relatives. The Study goal was to recruit 1,300 individuals (600 non-Hispanic African ancestry, 600 non-Hispanic European ancestry, and 100 Hispanic; 50% female) meeting rigorous diagnostic criteria for idiopathic DCM (probands) and 2,600 of their relatives. The probands were inpatients or outpatients identified by heart failure and cardiac transplant cardiovascular physicians and clinical research personnel in heart failure/heart transplant programs at sites in the DCM Consortium. The DCM phenotype was selected for this study because of its prevalence and its prior inclusion in the development of virtually all pharmacotherapy for non-ischemic heart failure from reduced ejection fraction. Study procedures included obtaining family history, cardiac history, medical record review, surveys, and blood sample collection. Exome data from 1219 idiopathic DCM probands and 127 idiopathic DCM relatives are included in dbGaP.

  Study phs002641

• Joint-Specific TF Regulation in RA

  The mechanisms responsible for the distribution and severity of joint involvement in rheumatoid arthritis (RA) are not known. To explore whether site-specific fibroblast-like synoviocyte (FLS) biology might be associated with location-specific synovitis and explain the predilection for hand (wrist/metacarpal phalangeal joints) involvement in RA, we generated bulk, whole-genome transcriptomic and chromatin accessibility data from FLS. Samples were matched in age, sex, and were homogeneous in that all had significant joint damage with longstanding disease requiring surgical intervention. Analysis revealed joint-specific patterns of FLS phenotypes, with proliferative, migratory, proinflammatory, and matrix-degrading characteristics observed in resting FLS derived from the hand joints compared with hip or knee. TNF-stimulation amplified these differences, with greater enrichment of proinflammatory and proliferative genes in hand FLS compared with hip and knee FLS. Hand FLS also had the greatest expression of markers associated with an &#8216;activated' state relative to the &#8216;resting' state, with the greatest cytokine and MMP expression in TNF-stimulated hand FLS. Predicted differences in proliferation and migration were biologically validated with hand FLS exhibiting greater migration and cell growth than hip or knee FLS. Distinctive joint-specific FLS biology associated with a more aggressive inflammatory response might contribute to the distribution and severity of joint involvement in RA.

  Study phs003633

• NHLBI TOPMed - NHGRI CCDG: Pakistan Risk of Myocardial Infarction Study (PROMIS)

  The Pakistan Risk of Myocardial Infarction (PROMIS) study is a retrospective multicenter case-control cohort study of individuals with and without coronary heart disease from Pakistan. Multiple biological samples have been collected from the participants including DNA, plasma, serum, and whole blood. The goal of the study is to recruit 20,000 cases and 20,000 controls of Pakistani descent. Eligible cases were individuals aged 30-80 who presented to the emergency department of a participating recruitment center in Pakistan with clinical symptoms consistent with a myocardial infarction (MI), ECG changes consistent with MI, elevated troponin levels consistent with MI, and no prior history of cardiovascular disease. Controls were individuals of Pakistani descent who did not have a self-reported history of cardiovascular disease. This site hosts data generated via NHLBI's TOPMed program and NHGRI's Common Disease Genomics (CCDG) program. Both whole exome and whole genome data are presented here, namely 4,211 whole genomes from TOPMed, 3,859 whole genomes from NHLBI supplemental funds, 1,136 whole genomes and 16,855 whole exomes from CCDG. Please note that there is additional legacy whole exome data (7,298 subjects) also generated via NHGRI funds as part of the MIGen Exome Sequencing project that can be found in dbGaP at phs000917.

  Study phs001569

• Examination of Engineered LINE-1 Integration Events in HeLa Cells

  Long Interspsesed Element-1 (LINE1 or L1) encodes two proteins (ORF1p and ORF2p) required for its mobilization (i.e. retrotransposition) in the human genome. Human ORF2p, a 150kDa protein, has endonuclease (EN) and reverse transcriptase (RT) activities. L1 EN is classified as apurinic/apyrimidin-like endonuclease (APE) and is responsible for initiating target-site primer reverse transcription (TPRT) during the L1 retrotransposition cycle. During canonical TPRT, the L1 EN activity makes a single-strand endonucleolytic nick at a double-strand DNA target sequence in genomic DNA. The endonucleolytic nick is typically made at an L1 EN degnerate consensus cleavage sequence (5'-TTTT/A-3') exposing a free 3' hydroxy group that can be used as a primer by the ORF2p L1 RT activity to reverse transcribe the associated L1 RNA. Here, we characterized over 21,000 de novo engineered L1 retrotransposition events in HeLa cells to determine if L1 exhibits overt integration preferences in the human genome. Bru-seq of HeLa cells to determine actively transcribed regions of the genome was also performed. This Bru-seq data was used to determine if engineered L1 integration events preferentially integrate into transcribed regions of the genome, and to determine which strand during transcription may present itself as preferential during L1 retrotransposition.

  Study phs001669

• Rapid detection of expanded short tandem repeats in personal genomics using hybrid sequencing.

  Long expansions of short tandem repeats (STRs); i.e., DNA repeats of 2-6 nucleotides, are associated with some genetic diseases. Cost-efficient high throughput sequencing can quickly produce billions of short reads that would be useful for uncovering disease-associated STRs. However, enumerating STRs in short reads remains largely unexplored because of the difficulty in elucidating STRs much longer than 100 bp, the typical length of short reads. We propose ab initio procedures for sensing and locating long STRs promptly by utilizing the frequency distribution of all STRs and paired-end read information. We validated the reproducibility of this method using biological replicates and used it to locate an STR associated with a brain disease (SCA31). Subsequently, we sequenced this STR site in eleven SCA31 samples using SMRT? sequencing (Pacific Biosciences), determined 2.3-3.1 kb sequences at nucleotide resolution, and revealed that (TGGAA)- and (TAAAATAGAA)-repeat expansions determined the instability of the repeat expansions associated with SCA31. Our method could also identify common STRs, (AAAG)- and (AAAAG)-repeat expansions, which are remarkably expanded at four positions in an SCA31 sample. This is the first proposed method for rapidly finding disease-associated long STRs in personal genomes using hybrid sequencing of short and long reads.

  Study JGAS000002

• The Spatial Heterogeneity in Multiple Myeloma - from the Subclonal Architecture to the Immune Microenvironment (partly hipo_K08K)

  Multiple myeloma (MM) cells show pronounced heterogeneity not only within a given patient but also between spatially separated tumor loci in the bone marrow. Understanding this spatial heterogeneity is emerging as a critical challenge for the successful treatment of the disease. Yet, our understanding of spatial differences in the subclonal architecture, molecular signatures and interactions with the tumor microenvironment remains very limited. To address this shortcoming, we performed bulk and single-cell multi-region sequencing, including random bone marrow samples from the iliac crest and paired imaging-guided focal lesion specimens from 15 newly diagnosed MM patients. We found a median of 5 subclones per patient and unique subclones in focal lesions. Central features of spatial heterogeneity included a consistent down-regulation of the chemoattractant cytokines CXCL7 and CXCL12 in focal lesion tumor cells as well as a significant depletion of macrophages in the focal lesion microenvironment. In contrast, a site-specific expansion of T-cell clones was not detected in focal lesions. In conclusion, our results demonstrate the relevance of considering spatial heterogeneity with potential implications for immunotherapies and molecular studies analyzing the role of gene signatures and MM-microenvironment interactions in disease progression.

  Study EGAS00001006090

• Inhibiton of the GABPB1L-containing GABP tetramer is sufficient to reverse replicative immortality in TERT promoter mutant glioblastoma cells.

  Over 50 types of cancer acquire TERT promoter mutations. These single point mutations reactivate telomerase, allowing for indefinite maintenance of telomere length and enabling cellular immortalization. The transcription factor binding site created by the point mutations specifically recruit the ETS factor GABP, a multimeric transcription factor composed of the GABPα and GABPβ subunits. GABP can form two functionally independent transcription factor species – a dimer or a tetramer – depending on which of the structurally distinct GABPβ isoforms is incorporated into the complex. In this study, we show that genetic disruption of GABPβ1L, a tetramer forming isoform of GABPβ that is dispensable in normal development, results in TERT silencing in a TERT promoter mutation dependent manner. Failure to activate TERT expression by GABPβ1L culminates in telomere dysfunction, DNA damage, and mitotic cell death exclusively in TERT promoter mutant cells. Furthermore, exogenous expression of TERT is sufficient to prevent telomere degradation and loss of cell viability in GABPβ1L-reduced lines bearing the mutant TERT promoter. Orthotopic injection of tetramer-deficient mutant TERT promoter GBM cells rendered lower tumor burden and prolonged the overall survival of the tumor-bearing mice. These results highlight the potentially widespread role of GABPB1L in enabling replicative immortality of TERT promoter cancers.

  Study EGAS00001002582

• Mutational Patterns in Metastatic Cutaneous Squamous Cell Carcinoma

  Cutaneous squamous cell carcinoma (cSCC) from the head and neck typically metastasizes to the lymph nodes of the neck and parotid glands. When a primary is not identified, they are difficult to distinguish from metastases of mucosal origin and primary salivary gland SCC. Ultraviolet radiation causes a mutation pattern that predominantly features cytosine to thymine transitions at dipyrimidine sites and has been associated with cSCC. In this study, we used whole genome sequencing data from 15 cSCC metastases and show that a UV signature mutation is pervasive across the cohort and distinct from mucosal SCC. The mutational burden was exceptionally high and concentrated in some regions of the genome, especially insulator elements (mean 162 mutations / Mb). We therefore evaluated the likely impact of UV-induced mutations on the dipyrimidine rich binding site of the main human insulator protein, CCCTC-binding factor (CTCF), and the possible implications on CTCF function and the spatial organization of the genome. Our findings suggest that mutation signature analysis may be useful in determining the origin of metastases in the neck and the parotid gland. Furthermore, UV-induced DNA damage to insulator binding sites may play a role in the carcinogenesis and progression of cSCC.

  Study EGAS00001003370

• A somatic reference standard for cancer genome sequencing with COLO829

  Identification of somatic alterations in cancer has become feasible with the massive adoption of next generation sequencing. However, due to variability in sequencing and informatics pipelines, a common somatic reference is needed. We thus performed paired whole genome sequencing of a metastatic melanoma cell line (COLO829) and a matched lymphoblastoid line (COLO829BL) across three institutions (TGen, Illumina, Genome Sciences Centre at the British Columbia Cancer Agency). We performed a meta-analysis of all data, in combination with the originally reported analysis of these cell lines by Pleasance et al (PMID: 20016485), and report a somatic reference standard based on consensus events. DNA extractions, library preparation, sequencing, and analysis were separately performed at each site. Results were compiled with sequence data previously generated for the cell lines to identify true positive variants, collectively representing a somatic reference standard. Overall, common somatic events include point mutations, small insertion/deletions, and genes affected by concurrent copy number changes. We additionally show divergence of somatic mutations in COLO829 associated with differences in cell line lineage. We present this reference to the community as a standard for enabling interpretation and identification of somatic mutations across institutions and analytical pipelines.

  Study EGAS00001001385

• Quantitative analysis of a novel DNA hypermethylation panel for lung cancer diagnosis using bronchial specimens

  Non-diagnostic findings in Transbronchial Lung Biopsy (TBLB) and Endobronchial Ultrasound-guided Transbronchial Lung Biopsy (EBUS-TBLB) are not uncommon. A challenge is to improve lung cancer detection by TBLB/EBUS-TBLB. In order to improve the diagnostic yield of bronchoscopy, we used 850K methylation chip to identify methylation sites that distinguish malignant from benign lung nodules.We found that the combination of HOXA7, SHOX2,and SCT methylation analysis has the best diagnostic yield in 123 bronchial washing (sensitivity: 74·1%; AUC: 0·851) and 172 brushing samples (sensitivity: 86·1%; AUC: 0·915). We developed a kit comprising these three genes and validated the kit in 329 unique bronchial washing samples, 397 unique brushing samples, and 179 unique patients with both washing and brushing samples. The diagnostic accuracy of the panel alone in lung cancer diagnosis was 86·9%, 91·2% and 95% in bronchial washing, brushing, and washing+brushing samples, respectively. The panel's sensitivity in combination with cytology, rapid on-site evaluation (ROSE), and histology in lung cancer diagnosis was 90·8% and 95.8% in bronchial washing and brushing samples, respectively, and 100% in washing+brushing samples. Quantitative analysis of the three-gene panel improves lung cancer diagnosis by bronchoscopy.

  Study EGAS00001007089

• Platinum Genomes

  Improvement of variant calling in next-generation sequence data requires a comprehensive, genome-wide catalogue of high-confidence variants called in a set of genomes for use as a benchmark. We generated deep, whole-genome sequence data of seventeen individuals in a three-generation pedigree and called variants in each genome using a range of currently available algorithms. We used haplotype transmission information to create a phased "platinum" variant catalogue of 4.7 million single nucleotide variants (SNVs) plus 0.7 million small (1-50bp) insertions and deletions (indels) that are consistent with the pattern of inheritance in the parents and eleven children of this pedigree. Platinum genotypes are highly concordant with the current catalogue of the National Institute of Standards and Technology for both SNVs (&#62;99.99%) and indels (99.92%), and add a validated truth catalogue that has 26% more SNVs and 45% more indels. Analysis of 334,652 SNVs that were consistent between informatics pipelines yet inconsistent with haplotype transmission ("non-platinum") revealed that the majority of these variants are de novo and cell-line mutations or reside within previously unidentified duplications and deletions. The reference materials from this study are a resource for objective assessment of the accuracy of variant calls throughout genomes.

  Study phs001224

• A Chromatin Accessibility Atlas of the Developing Human Telencephalon

  Gene expression differs between cell types and regions within complex tissues such as the developing brain. To discover regulatory elements underlying this specificity, we generated genome-wide maps of chromatin accessibility in nine anatomically-defined regions of the developing human telencephalon. Additionally, we defined the histone modification landscape of the prefrontal cortex and generated chromatin accessibility maps of its upper and deep layers. We predicted a subset of open chromatin regions (18%) that are most likely to be active enhancers, many of which are dynamic with 26% differing between early and late mid-gestation and 28% present in only one brain region. These predicted regulatory elements (pREs) are enriched proximal to genes with expression differences across developmental stages, regions, and cortical laminae; they harbor distinct sequence motifs that suggest potential upstream regulators. We leveraged this atlas to predict and validate novel regulatory elements of genes that control cortex laminar identity and genes associated with autism spectrum disorder (ASD). These include enhancers proximal to FEZF2 and BCL11A that were validated in mouse, and an enhancer of ASD gene SLC6A1 containing two functional de novo mutations in individuals with ASD whose enhancer function we validated via CRISPRa. We also include data for transcription factor binding in mid-fetal cortex tissues, using CUT&amp;RUN methods.

  Study phs002033

• Single_Cell_Sequencing_of_Sperm__scSperm_

  The aim of this project is to genotype and sequence single spermatozoa from two men, one in his twenties and the other in his seventies. The resulting data is used to quantify the mutations that have arisen in the gametes of both individuals in order to better understand the effect of aging on mutation rates and modes.Project Outline. In order to quantify mutations, semen from two individuals are sequenced. 48 single sperm cells are isolated from each individual, and their DNA is extracted. The resulting genomes are amplified using PicoPlex, GenomiPhi MDA, Repli-G MDA, and MALBAC. QC step is applied to check the quality of WGA DNA using standard Sequenom plex (26 SNPs). A subset of 32 amplification products which pass the intiall QC, are genotyped using Affymetrix SNP6 chips. 12 of the genotyped amplification products are also sequenced. In addition, one multi-cell sample per individual is sequenced as a reference and for validation purposes.Altogether, 12 single cell sperm genomes and two multi-cell genomes are sequenced, coming to a total of 14 genomes. Of the single cell sperm genomes, 2 are sequenced to 50x coverage, and the other 10 to 25x coverage. Both multi-cell genomes are sequenced to 25x coverage.

  Study EGAS00001000935

• Pancreatic, Small-intestinal and Pulmonary Neuroendocrine Tumors

  Purpose: The primary origin of neuroendocrine tumor metastases can be difficult to determine by histopathology alone, but is critical for therapeutic decision making. DNA methylation-based profiling is now routinely used in the diagnostic workup of brain tumors. This has been enabled by the availability of cost-efficient array-based platforms. We have extended these efforts to augment histopathological diagnosis in neuroendocrine tumors. Experimental Design and Results: We compiled data of 69 small-intestinal, pulmonary, and pancreatic neuroendocrine tumors. These data were used to build a ridge regression calibrated random forest classification algorithm (NEN-ID) that predicts the origin of tumor samples with high accuracy (&amp;gt; 95%). The model was validated during 3x3 nested cross validation and tested in a local (n=26) and external (n=172) cohort. In addition, we show that our diagnostic approach is robust across a range of possible confounding experimental parameters such as tumor purity and array quality. A software infrastructure and online user interface was built to make the model available to the scientific community. Conclusions: This DNA methylation-based prediction model can be used in the workup for patients with neuroendocrine tumors of unknown primary. To facilitate validation and clinical implementation, we provide a user-friendly, publicly available web-based version of NEN-ID.

  Study EGAS00001004878

• Spatiotemporal Genomic Profiling of Intestinal Metaplasia Reveals Clonal Dynamics of Gastric Cancer Progression

  Intestinal metaplasia (IM) is a pre-malignant condition of the gastric mucosa associated with increased gastric cancer (GC) risk. We analyzed 1256 gastric samples (1152 IMs) from 692 subjects from a 10-year prospective study. We identified 26 IM driver genes in diverse pathways including chromatin regulation (ARID1A) and intestinal homeostasis (SOX9), largely occurring as subclonal events. Analysis of clonal dynamics between and within subjects, and also longitudinally across time, revealed that IM clones are likely transient but increase in size upon progression to dysplasia, with eventual transmission of genetic events to paired GCs. Single-cell and spatial profiling highlighted changes in tissue ecology and lineage heterogeneity in IM, including an intestinal stem-cell dominant cellular compartment linked to early malignancy. Expanded transcriptome profiling revealed expression-based molecular subtypes of IM, including a body-resident “pseudoantralized” subtype associated with incomplete histology, antral/intestinal cell types, ARID1A mutations, inflammation, and microbial communities normally associated with the healthy oral tract. We demonstrate that combined clinical-genomic models outperform clinical-only models in predicting IMs likely to progress. Our results raise opportunities for GC precision prevention and interception by highlighting strategies for accurately identifying IM patients at high GC risk and a role for microbial dysbiosis in IM progression.

  Study EGAS00001007067

• RNA sequencing on colonic biopsies from inflammatory bowel disease patients treated with vehicle control, MEK inhibitor, or infliximab

  This dataset consists of RNA sequencing data (FASTQs) from intestinal mucosal biopsies from 9 IBD patients. All patients endoscopically active disease and were not receiving immunosuppressive or biologic therapies. All biopsies (6 per donor) were collected from a single inflamed site. Biopsies were cultured for 18 hours at an air-liquid interface in media containing either DMSO (vehicle control), PD-0325901 (0.5uM) or infliximab (10ug/ml; MSD) - two biopsies per condition. Sequencing was performed on a NovaSeq 6000 (100bp, PE reads). After 18 hours, biopsies were harvested and snap frozen. After lysis, RNA was extracted using an AllPrep DNA/RNA Mini Kit (Qiagen). Sequencing libraries were prepared from 10ng RNA using the SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara) following the manufacturer’s instructions. The quality and molarity of all libraries was assessed using a BioAnalyzer 2100 and the libraries were sequenced on a NovaSeq 6000 (100bp, PE reads).

  Dataset EGAD00001011333

• Effects of Metformin on Transcriptomic and Metabolomic Profiles in Breast Cancer Survivors Enrolled in the Randomized Placebo-Controlled MetBreCS Trial

  RNA from snap-frozen breast tissue biopsies were purified after lysing by tissuelyser (Qiagen) and using RNA Purification Plus Kit (Norgen biotek CORP, 47700) with additional on-column DNase-I treatment (Qiagen, 79254) at 27 °C. RNA purity and integrity (RIN) were quantified using RNA 6000 Nano kit (Agilent Technologies, 5067-1511) on the 4200 TapeStation (Agilent, Santa Clara, USA). Library preparation and 2x75bp paired-end of 160 ng total RNA input was performed using Illumina Stranded Total RNA Prep Ligation kit and Illumina HiSeq4000 system (Illumina, Sand Diego, CA, USA). RNA sequencing data from HiSeq4000 were quality checked and aligned to GRCh38 (GCA_000001405.15) reference genome using HISAT2 2.0.5 and submitted to subread v.1.5.2 for feature counts calculation. Finally, 36 paired biopsies samples (metformin n=26 and placebo n=12) were sequenced and included in the final analyses.

  Dataset EGAD50000001274

• National Cancer Institute (NCI) Adding Hispanics to Ongoing GWAS in Colorectal Cancer

  Genome-wide association studies (GWAS) of colorectal cancer (CRC) have been instrumental in identifying a number of common susceptibility loci in Non Hispanic (NH)-White populations, and a NCI priority is to extend GWAS findings to other populations to address racial/ethnic disparities in cancer susceptibility. Currently, GWA studies of CRC in NH-Whites, Japanese and African-Americans are ongoing. We propose a complementary study to address this critical research area in Hispanics. Hispanics represent the fastest growing ethnic population in the U.S. and have been largely understudied in terms of genetic susceptibility to cancer. There are noted differences in incidence, survival and mortality in CRC by ethnic/racial groups. Hispanics often present with CRC at a younger age and have a significantly greater incidence of stage IV tumors or metastatic disease compared to NH-Whites. We propose to conduct a large, cost-efficient, population-based GWAS in Hispanics by building upon existing NIH-funded resources, the Colon Cancer Family Registry (Colon CFR) and the Multiethnic Cohort Study (MEC). We plan to recruit 2,500 Hispanic men and women diagnosed with CRC between 01/2008 to present using cancer registries in California, physican referrals and familial referrals. Risk factor/diet questionnaires, pathology reports, Oragene saliva samples (for genotyping), optional blood samples (for genotyping and biometric analysis) and tumor blocks (for MSI testing) will be collected using methodologies developed in the Colon CFR/MEC. Cases of CRC in the MEC (currently 473; anticipated 600 at end) will also be included. Population-based Hispanic individuals without a diagnosis of CRC participating in other GWA studies in the MEC (n=3,900, U01HG004726, Haiman) will be used as controls. We will genotype all 3,100 cases using the Illumina 1M array and use available genotype and epidemiologic data collected on 3,900 controls. Our statistical analyses will include: single-SNP and haplotype effects, gene-environment interactions and heterogeneity by MSI, tumor subtype and family history of CRC. We will replicate findings in a second-stage using CRC cases and controls from Mexico (1,000 cases and 1,000 controls, EU FP7 funding, CHIBCHA, Carvajal-Carmona/Tomlinson). We will also examine heterogeneity of the risk estimates by ethnicity/race by leveraging GWA data on NH-Whites (2,142 cases, 1,909 controls, U01 CA122839, Casey), (4,000 cases, 6,000 NH-White controls, UK-CHIBCHA, Tomlinson), Colombians (2,000 cases and 2,000 controls, CHIBCHA), Japanese (1,000 cases and 1,000 controls) and African-Americans (1,500 cases and 1,500 controls, R01CA126895, Le Marchand). We will genotype replicated significant SNPs in our main and combined analysis in several Hispanic populations (note: studies funded by EU or NIH for data collection but not GWAS), including 800 Puerto Ricans, 2,000 Brazilians, 2,000 Argentineans and 3,000 Spanish/Portuguese, to assess generalizability of findings. We will examine the differences in inflammatory gene transcription dynamics in leukocytes (from blood sample collection) by fatigue level (as assessed from study questionnaire data). This study will have a high impact by addressing the key question of racial/ethnic disparities related to genetic susceptibility to CRC, will provide translational guidelines on biological mechanisms during the cancer survivorship period to increase quality of life among cancer survivors, and will enable further growth and investment into research among Hispanics by providing a resource of genetic data and biospecimens, which is lacking.

  Study phs001193

• 3/7 Psychiatric Genomics Consortium: Finding Actionable Variation

  The goal of this study was to generate new therapeutic hypotheses by targeted sequencing of 161 genes most likely to harbor schizophrenia (SCZ) causal variants in multi-ancestry schizophrenia case-control cohorts. The gene panel was intended to contain putative SCZ risk genes, from within which, the majority of new discoveries from additional WES/WGS would come. To this end, we integrated current knowledge of the genetic architecture of SCZ (results from The Schizophrenia Exome Sequencing Meta-Analysis Consortium, or SCHEMA) and gene membership in gene sets that have been implicated in SCZ using a Bayesian framework. A total of 23,352 individuals selected to be non-overlapping with SCHEMA, as well as other previous and ongoing sequencing efforts in the field, were identified from over 20 individual study sites. Sequencing of the exonic regions of 161 prioritized genes was performed on the Ion Torrent platform in between June 2018 and April 2019. Sequencing plates were matched with respect to ancestry and case/control composition whenever possible. The average sequencing depth across all samples was 224x. Single sample calling was performed using Torrent Variant Caller v5.8.0, which is specially optimized to exploit the underlying flow signal information generated by the Ion Torrent sequencing. The schizophrenia case-control status was obtained from each original study site.

  Study phs003138

• Ischemic Stroke Genetics Study (ISGS)

  The third leading cause of death in the United States, stroke is an acute neurological event leading to death of neural tissues. Although the majority of strokes are ischemic strokes, meaning there is oxygen deprivation to the brain, almost 20% of strokes are hemorrhagic, resulting from bleeding into the brain. Stroke is a complex disorder and likely multigenic in nature, resulting from a combination of genetic and environmental factors. These well characterized risk factors that contribute to the incidence of stroke include hypertension, cardiac disease, sickle cell disease, hyperhomocysteinemia, family history of stroke and smoking. ISGS aim is to perform a prospective genetic association study of ischemic stroke focusing on the hemostatic system. ISGS is a 5-center case-control study of first-ever ischemic stroke cases and concurrent controls individually matched for age, sex and recruitment site. This data includes that from subjects both banked in the NINDS repository with biologicals publicly available, and those whose samples are not banked/not available. Important links to apply for individual-level data Data Use Certification Requirements (DUC) Apply here for controlled access to individual level data Participant Protection Policy FAQ --> This study utilized the NINDS Repository Cerebrovascular/Stroke Study, and neurologically normal controls from the sample population which are banked in the National Institute of Neurological Disorders and Stroke (NINDS Repository) collection for a first stage whole genome analysis.

  Study phs000102

• The Gene Partnership (TGP) - eMERGE Data

  The Gene Partnership (TGP) is a prospective longitudinal registry at Boston Children&#39;s Hospital (BCH) to study the genetic and environmental contributions to childhood health and disease, collect genetic information on a large number of children who have been phenotyped, and implement the Informed Cohort and the Informed Cohort Oversight Board (ICOB). The term "The Gene Partnership" reflects a partnership between researchers and participants. Children seen at BCH are offered enrollment, as are their parents and siblings. DNA is collected on all enrollees. BCH has a comprehensive EMR system, and virtually all inpatient and outpatient data are captured electronically. Clinical data in the BCH EMR is loaded in the i2b2 data warehouse which is available to investigators. Cases, phenotypes, and covariates are ascertained using the i2b2 database. Participants at BCH in TGP have consented to receive any research result and/or incidental finding that arises from studies using TGP that is approved by the Informed Cohort Oversight Board (ICOB) and is in accordance with the participants&apos; preferences; results are returned through the Personally Controlled Health Record (PCHR). BCH and Cincinnati Children&#39;s Hospital Medical Center (CCHMC) have partnered as the Pediatric Alliance for Genomic and Electronic Medical Record (EMR) Research (PAGER) site for the eMERGE Phase II network for pediatric institutions, and the cohort for eMERGE at BCH is TGP.

  Study phs000495

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

  The National Institute on Aging (NIA) Alzheimer&#39;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&#39;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&#39;s Disease (NCRAD). DNA was prepared by NCRAD for genotyping and sent to the genotyping site at Children&#39;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&#39;s disease. Nature Genetics 43, 436-441 (2011). doi:10.1038/ng.801. PMID: 21460841.]

  Study phs000372

• Whole genome sequencing of matched primary and metastatic acral melanomas

  Next generation sequencing has enabled systematic discovery of mutational spectra in cancer samples. Here we used whole genome sequencing to characterise somatic mutations and structural variation in a primary acral melanoma and its lymph node metastasis. Our data show that the somatic mutational rates in this acral melanoma sample pair was more comparable to the rate reported in cancer genomes not associated with mutagenic exposure than in the genome of a melanoma cell line or the transcriptome of melanoma short-term cultures. Despite the perception that acral skin is sun-protected, the dominant mutational signature in these samples is compatible with damage due to ultraviolet light exposure. A nonsense mutation in ERCC5 discovered in both the primary and metastatic tumours, could also have contributed to the mutational signature through accumulation of unrepaired dipyrimidine lesions. However, evidence of transcription-coupled repair was suggested by the lower mutational rate in the transcribed regions and expressed genes. The primary and the metastasis are highly similar at the level of global gene copy number alterations, loss of heterozygosity and single nucleotide variation (SNV). Furthermore, the majority of the SNVs in the primary tumour were propagated in the metastasis and one non-synonymous coding SNV and one splice site mutation appeared to arise de novo in the metastatic lesion.

  Study EGAS00001000169

• Analysis_of_genomic_integrity_of_disease_corrected_human_induced_pluripotent_stem_cells_by_exome_sequencing

  Induced pluripotent stem (iPS) cells hold great promise for autologous cell transplantation. In order to apply this therapy to monogenic disorder, disease-causing mutations must be corrected prior to transplantation. We generated iPS cells from patients with alpha-1 antitrypsin deficiency, which is caused by homozygous non-synonymous mutation in the SERPINA1 gene. In vitro differentiation of patient-derived iPS cells to hepatocytes showed recapitulation of the disease phenotype. We corrected the mutations by gene targeting using zinc finger nuclease (ZFN) and piggyBac transposon technologies. Our mutation correction is highly accurate. There is no exogenous sequence left behind at the targeted site. The corrected iPS cell-derived hepatocytes were indistinguishable from healthy hepatocyte, suggesting that the corrected gene was fully functional. The genomic integrity was also analyzed by CGH and SNP array, showing no genomic alterations. These are important points to ensure clinical safety as well as functional restoration of the corrected gene. One remaining question is if these corrected iPS cells are clinically safe at the single base-pair level. Recent publications suggested that cancer-related mutations are significantly accumulated during iPS cell propagation. We aim to assess the clinical safety of the corrected iPS cells by using exome sequencing. Outcome from this project would bring iPS cell technology one step closer to clinical applications.

  Study EGAS00001000055

• Structural variant analysis of homologous recombination-deficient genomes

  Homologous recombination (HR) deficiency causes DNA breaks and cytogenetic aberrations. Paradoxically, the types of DNA rearrangements specifically associated with HR-deficient cancers only minimally impact chromosomal structure. Addressing this, we combined a genome graph analysis of short-read whole genome sequencing (WGS) profiles across thousands of tumors with deep linked-read (LR) WGS of 46 BRCA1 or BRCA2 mutant breast cancers to discover a distinct class of HR deficiency-enriched rearrangements called reciprocal pairs. LR WGS showed that reciprocal pairs with identical rearrangement orientations gave rise to one of two distinct chromosomal outcomes, distinguishable only with long molecule data. While one (cis) outcome corresponded to the copy and pasting of a small segment to a distant site, a second (trans) outcome was a quasi-balanced translocation or multi-megabase inversion with substantial (10kb) duplications at each junction. The full spectrum of reciprocal pair outcomes could be explained by an HR-independent replication restart repair mechanism. LR WGS additionally identified single-strand annealing (SSA) as a BRCA2-deficiency specific repair pathway in human cancers. Replication restart- and SSA-associated SVs improved BRCA1- vs. BRCA2- deficiency classification and identified metastatic cancer cases with favorable chemotherapy responses. Our data reveal classes of BRCA1- and BRCA2-deficiency specific rearrangements as drivers of cytogenetic aberrations in HR deficient cells.

  Study EGAS00001007186

• Neoadjuvant immune checkpoint blockade in mismatch-repair proficient colon cancers

  Immune checkpoint blockade has led to paradigm shifts in the treatment of various tumour types, yet limited efficacy has been observed in patients with metastatic mismatch-repair proficient (pMMR) colorectal cancer. Here we report an in-depth analysis of patients with early-stage pMMR colon cancer from the phase II NICHE study (ClinicalTrials.gov: NCT03026140). A total of 31 patients received neoadjuvant treatment of nivolumab plus ipilimumab followed by surgery. The response rate was 26% and included six patients with a major pathological response (≤10% residual viable tumour). One patient with an ongoing clinical complete response did not undergo surgery. Responses were observed despite a low tumour mutational burden in all tumours, while chromosomal genomic instability scores were significantly higher in responders compared to non-responders. Responding tumours were more likely to be TP53 mutant, KRASG12 wildtype and had significantly higher baseline expression of proliferation signatures and TCF1. Imaging mass cytometry also revealed a higher percentage of Ki-67+ cancer and Ki-67+ CD8+ T cells in responders compared to non-responders. Meanwhile, in non-responders, we observed an enrichment for an inflamed-fibrotic microenvironment associated with TGF-β expression, hinting towards possible resistance mechanisms. These results provide a comprehensive analysis of response to neoadjuvant ICB in early-stage pMMR colon cancers and identify potential biomarkers for patient selection.

  Study EGAS50000000856

• JMML targeted sequencing (2013)

  Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic myeloproliferative neoplasm of early childhood initiated by germline or somatic RAS-activating mutations (Chang, Dvorak et al. 2014). Clinical evolution is heterogeneous, with acute myeloid leukemia (AML) transformation in 1/3 of cases and frequent relapses after hematopoietic-stem-cell transplantation (HSCT), although some patients experience spontaneous remission (Locatelli, Nollke et al. 2005; Matsuda, Shimada et al. 2007; Takagi, Piao et al. 2013; Locatelli and Niemeyer 2015). The initiating lesion incompletely predicts outcome, suggesting the involvement of additional mutations. Genetic profiling of a large JMML cohort (n=118), including whole-exome sequencing (WES) in 30 cases, uncovered additional genetic abnormalities in 56 cases (47%). Somatic events were rare (0.38/case/Mb) and restricted to sporadic (47/78; 60%) or NF1-associated (8/8; 100%) JMML. Multiple concomitant genetic hits were identified targeting the RAS pathway in 13/78 (17%), RAC2D63V, which activates the PI3K/PDK1/AKT and the mTORC2 pathways, and a novel pathway for JMML, the polycomb repressive complex 2 (PRC2), in 26/78 (33%) of the sporadic JMML cases. Interestingly, PRC2 imbalance was recently shown to synergize with RAS activation (De Raedt, Beert et al. 2014). JMML outcome was associated to the mutational profile, suggesting a dose-dependent effect for RAS-pathway activation, distinguishing very aggressive JMML rapidly progressing to AML.

  Study EGAS00001001324

• Extracellular transcriptome in seminal plasma for non-invasive prostate cancer diagnosis

  A diagnostic non-invasive biomarker test for prostate cancer at an early stage, with high sensitivity and specificity, would improve diagnostic decision making. Extracellular RNAs present in seminal plasma might contain biomarker potential for the accurate detection of clinically significant prostate cancer. So far, the extracellular messenger RNA (mRNA) profile of seminal plasma has not been interrogated for its biomarker potential in the context of prostate cancer. Here, we investigate the mRNA transcriptome in seminal plasma samples obtained from prostate cancer patients (n=25), patients with benign prostate hyperplasia (n=26) and individuals without prostatic disease (n=6). Seminal plasma harbors a complex mRNA repertoire that reflects prostate as its tissue of origin. The endogenous RNA content is higher in the prostate cancer samples compared to the control samples. Prostate cancer antigen 3 (PCA3), a long non-coding RNA with prostate cancer-specific overexpression, and ATP-binding cassette transporter 1 (ABCA1), known to be involved in the prostate cancer pathogenesis, were more abundant in the prostate cancer group. In addition, twelve high confidence fusion transcripts could be detected in prostate cancer samples, including the bona-fide prostate cancer fusion transcript TMPRSS2-ERG. Our findings provide proof-of-principle that the extracellular transcriptome of seminal plasma can reveal information of an underlying prostate cancer.

  Study EGAS00001005277

• Unraveling the transcriptomic profile of utero-tubal lavage fluid of ovarian cancer patients

  The majority of ovarian cancers are diagnosed at an advanced stage and have a high mortality rate. Current screening strategies fail to improve prognosis because markers of early stage disease are lacking. This medical need justifies the search for biomarkers enabling early detection of ovarian cancer. In this study, we explore the extracellular transcriptome of utero-tubal lavage fluid obtained from 26 ovarian cancer patients and 48 controls using messenger RNA (mRNA) capture and small RNA sequencing. Because of its contact with the fallopian tube and the ovaries, collection of utero-tubal lavage might be the preferred non-invasive method to identify localized lesions compared with analyses based on peripheral blood samples. We observed an enrichment of ovarian and fallopian tube specific messenger RNAs in utero-tubal lavage fluid compared to other human biofluids. Over 300 mRNAs and 41 miRNAs were upregulated in ovarian cancer samples compared with controls. Upregulated genes were enriched for genes involved in cell cycle activation and proliferation, hinting at a tumor derived signal. In conclusion, we provide proof-of-principle that mRNA capture sequencing of utero-tubal lavage fluid is technically feasible, and that the extracellular transcriptome of utero-tubal lavage should be further explored in larger cohorts to assess the diagnostic value of the biomarkers identified in this study.

  Study EGAS00001005498

• Cell-free DNA fragmentation patterns and personalized sequencing reveal circulating tumor DNA in urine and plasma of glioma patients

  Glioma-derived cell-free DNA (cfDNA) is challenging to detect using liquid biopsy as levels in body fluids are low. We determined the glioma-derived DNA fractions in tumor biopsies, in cerebrospinal fluid (CSF), plasma and urine samples, using deep sequencing of personalized capture panels. By sequencing cfDNA across thousands of mutations identified individually in each patient’s matched tumor we detected tumor-derived DNA in plasma (10/12) and urine samples (8/11). The median tumor fraction was 6.4x10-3 in CSF, 3.1x10-5 in plasma and 4.7x10-5 in urine. We identified a shift in the size distribution for mutant cfDNA fragments in these body fluids. Next, we analyzed cfDNA fragment sizes with paired-end shallow whole genome sequencing (WGS) in urine samples from 35 patients with gliomas, 8 individuals with non-malignant brain disorders, and 26 controls (n=69 individuals, 96 samples). cfDNA in urine of glioma patients was significantly more fragmented compared to urine from patients with non-malignant brain disorders (t-test, p=1.7x10-2) and compared to urine of controls (t-test, p=5.2x10-9). The proportion of DNA fragments &lt;60 bp was higher in glioma patients urine and could be used for classification (AUC=0.93). Machine learning models integrating fragment lengths could identify urine samples from glioma patients (AUC=0.97 in cross-validation).

  Study EGAS00001004355

• Single cell sequencing of a post-PD-1 inhibitor metastatic melanoma mass

  Three technical replicates of FACS-sorted T cells (CD45+CD3+) and one replicate of FACS-sorted tumor cells (MCSP+) were loaded to a targeted 10,000 cells per lane on the 10X Genomics Chromium Controler with the single cell 5’ Immune Repertoire and Gene Expression profiling kit. In total, we loaded ~30,000 individual tumor infiltrating lymphocytes (TILs) and ~10,000 melanoma cells on the 10X platform (10X Genomics, CA, USA). Reverse transcription, TCR enrichment, and library preparations were performed according to the 10X Genomics 5’ V(D)J protocol revision C. Transcriptome libraries were pooled and sequenced on the Illumina NovaSeq 6000 S2 flow cell with 26 R1, 8 i7, and 91 R2 cycles respectively. The TCR libraries were pooled and sequenced on the Illumina MiSeq V2 150 cycles paired-end. Single cell transcriptomic and TCR data was processed with the 10X Genomics Cell Ranger Pipeline version 2.2.0 with the software-provided GRCh38 reference transcriptomes. After quality control, there was RNAseq profile data available from 6267 immune and 4303 melanoma cells. Downstream processing and visualization was encompassed through Seurat and tSNE plots.

  Dataset EGAD00001006013

• Paired-WGS Sequencing of Primary lymphomas of the central nervous system (PCNSL)

  26 Tumor/Control pairs of WGS data of PCNSL tumors, sequenced on either Illumina HiSeq2000/2500 instruments or HiSeq X Ten. The controls are blood or buffy coat samples in most cases.

  Dataset EGAD00001007806

• Characterization of mutational signatures in human cancer cell lines reveals episodic APOBEC mutagenesis

  Multiple signatures of somatic mutations have been identified in human cancer genomes. To investigate whether mutational signatures continue to be generated, and if so their temporal patterns of activity, subsets of cell lines were cultured in vitro for extended periods and subjected to single cell cloning and whole genome or exome sequencing or directly to single cell whole genome sequencing. As expected, signatures of past exogenous exposures, such as tobacco smoke and ultraviolet light, were not generated in vitro. In contrast, signatures of normal and defective DNA repair and replication continued to be generated at essentially constant mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing activity exhibited a distinctive pattern with substantial fluctuations in mutation rate over time and episodic bursts of mutations. The initiating factors for these bursts are unclear although retrotransposon mobilisation may play a role. This cell line set now constitutes a comprehensive resource of live experimental models of mutational processes of both known and unknown aetiologies potentially retaining the patterns of activity and regulatory influences operative in human cells in vivo.

  Dataset EGAD00001004202

• TRanscriptomic ANalySis of left ventriCulaR gene Expression (TRANSCRibE)

  Myocardial ischemia occurs when there is a mismatch between coronary oxygen delivery and metabolic requirements of the myocardium, which may be clinically manifested during angina, coronary angioplasty or cardiopulmonary bypass (CPB). Myocardial ischemia may lead to a spectrum of myocardial stunning, hibernating myocardium, and ultimately cell death if the ischemic insult is severe. In the human heart, irreversible damage begins after approximately 20 to 40 minutes of oxygen deprivation. Observed molecular and cellular changes of myocardial ischemia are characteristic of an inflammatory response, but the exact mechanisms that underlie this pathological process are unclear and may not be full emulated by animal models of ischemia or infarction. Thus, we felt it valuable to investigate a human ischemia model. During cardiac surgery, CPB with aortic cross-clamping (AoXC) and cardioplegic arrest is associated with excellent clinical outcomes and suitable operative conditions. However, despite the use of cardioprotective strategies, AoXc during CPB is accompanied by a variable, yet obligate ischemic period lasting 1 to 3 hours, resulting in hypoxia, metabolic substrate depletion, reperfusion injury, apoptosis, and necrosis. Cardiac specific biomarkers of ischemia and infarction, including troponin, are elevated even after routine coronary artery bypass graft surgery and correlate with the duration of ischemia from AoXc.This process of CPB provides us with the ability to examine the transcriptional profile before and after an expected, consistent, and reproducible human ischemic event, albeit induced by cold cardioplegic arrest and not coronary occlusion. In addition, the absence of reperfusion in this time period allows us to examine the transcriptomic response to intermittent ischemia, without having to account for the perturbations of reperfusion injury. Although various animal models have been used to examine the effects of ischemia on cardiac function, no human data exist which examine the early transcriptomic response to a left ventricular (LV) ischemic insult. We therefore characterized the effect of cold cardioplegia induced acute ischemia on the transcriptional profile of the LV by performing whole transcriptome next-generation RNA-sequencing (RNA-seq) in patients undergoing cardiac surgery by sampling human LV tissue prior to, and after, the obligate ischemia during AoXC. We hypothesized that the cold cardioplegia induced ischemic injury will dramatically alter transcription in the human myocardium, and that we would identify genes and pathways, which will identify interventional targets for pharmacological therapy. Methods:We have collected left ventricle tissue samples and blood sample from patients undergoing heart surgery. We obtained punch biopsies (~3-5&#956;g total RNA content) from the site of a routinely placed surgical vent in the anterolateral apical left ventricular wall of patients undergoing elective aortic valve replacement surgery with cardiopulmonary bypass. After an average of 79 minutes of aortic cross-clamping with intermittent cold blood cardioplegia for myocardial protection every 20 minutes, a second biopsy was obtained in the same manner. Tissue samples were immediately placed in RNAlater&#174; (Ambion, ThermoFisher Scientific, Waltham, MA), and after 48 hours at +4&#176;C were stored at -80&#176;C until RNA extraction. Total RNA was isolated with Trizol and RNA quality was assessed using the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA). Libraries were prepared by poly(A) mRNA isolation and reverse transcription Polymerase Chain Reaction (RT-PCR), then sequenced on the Illumina HiSeq2000 or HiSeq2500 (Illumina, San Diego, CA). As samples were analyzed at different times, different read lengths were employed, initially using single-end reads (n=20) and then transitioning to paired end reads (n=216), ranging from 36 - 100 base pairs. Raw sequencing files were processed using Sickle, Skewer, and STAR software, and aligned to GrCh37 or UCSC Hg19. DNA was isolated from whole blood using standard methods. SNP genotyping was performed using the Illumina Omni2.5Exome-8 BeadChip array with additional exome content (Illumina, San Diego, CA) chip, version 1.1. We first phased and imputed 93 subjects using a phasing tool called SHAPEIT and an imputation tool called MINIMAC, with 1000 Genomes phase 1 version 3 for the reference panel. We then phased and imputed 26 more subjects using SHAPEIT, an imputation tool called IMPUTE2, and 1000 Genomes phase 3 version 5.

  Study phs001679

• Whole exome sequencing of an invasive diffuse intrinsic pontine glioma sampled from different sites

  The lateral ventricle subventricular zone (SVZ) is a frequent site of high-grade glioma (HGG) spread and tumor invasion of the SVZ is a predictor of a worse clinical prognosis. A range of HGG types invade the stem cell niche of the SVZ, including both adult glioblastoma and pediatric high-grade gliomas such as diffuse intrinsic pontine glioma (DIPG). The cellular and molecular mechanisms mediating this frequent invasion of the SVZ are poorly understood. Here we demonstrate that neural precursor cell (NPC):glioma cell communication underpins the propensity of glioma to colonize the SVZ. SVZ NPCs secrete chemoattractant signals toward which glioma cells home. Biochemical and proteomic analyses of factors secreted by SVZ NPCs revealed a discrete list of candidate proteins. Necessity and sufficiency testing implicated the neurite outgrowth-promoting factor pleiotrophin, along with three required binding partners (secreted protein acidic and rich in cysteine (SPARC), SPARC-like protein 1, and heat shock protein 90B), as key mediators of this chemoattractant effect. Pleiotrophin protein expression is strongly enriched in the SVZ, and knockdown of pleiotrophin expression starkly reduced glioma invasion of the SVZ in the adult murine brain. Pleiotrophin, in complex with the three binding partners, activated the Rho/ROCK pathway in DIPG cells, and inhibition of ROCK resulted in decreased DIPG invasion toward SVZ neural precursor cell-secreted factors. These findings demonstrate a pathogenic role for neural precursor cell – glioma interactions and potential therapeutic targets to limit glioma invasion.

  Study EGAS00001002326

• Evolutionary origin and methylation status of human intronic CpG islands that are not present in mouse

  Imprinting of the human RB1 gene is due to the presence of a differentially methylated CpG island (CGI) in intron 2, which is part of a retrogene derived from the PPP1R26 gene on chromosome 9. The murine Rb1 gene does not have this retrogene and is not imprinted. We have investigated whether the RB1/Rb1 locus is unique with respect to these differences. For this we have compared the CGIs from human and mouse by in silico analyses and found out that there are at least 2033 human intronic CGIs that are not present in the mouse. Among these CGIs, 104 show sequences similarities elsewhere in the human genome, which suggests that they arose by retrotransposition. We could narrow down the time points when most of these CGIs appeared during evolution. Their methylation status was analysed in two monocyte methylome datasets from whole genome bisulfite sequencing and in 18 published methylomes. Five CGIs appear to be differentially methylated, which suggests that they are subject of genomic imprinting. They are located in introns of the following genes: RB1, ASRGL1, PARP11, PDXDC1 and MYO1D. Four of these CGIs are parts of retrogenes. Our study supports the notion that imprinting builds on host defence mechanisms by which the genome protects itself against retrotransposed and foreign DNA and that the epigenetic fate of the retrotransposed DNA depends on selective forces at the integration site.

  Study EGAS00001000719

• Integration of genomics and histology reveals diagnosis and effective therapy of refractory cancer of unknown primary with PDL1 amplification (H021)

  Identification of the tissue of origin in cancer of unknown primary (CUP) poses a diagnostic challenge and is critical for directing site-specific therapy. Currently, clinical decision making in patients with CUP primarily relies on histopathology and clinical features. Comprehensive molecular profiling has the potential to contribute to diagnostic categorization and, most importantly, guide CUP therapy through identification of actionable lesions. We here report the case of an advanced-stage malignancy initially mimicking poorly differentiated soft-tissue sarcoma that did not respond to multi-agent chemotherapy. Molecular profiling within a clinical whole-exome and transcriptome sequencing program revealed a heterozygous, highly amplified KRAS G12S mutation, compound-heterozygous TP53 mutation/deletion, high mutational load, and focal amplification of chromosomes 9p (including PDL1 [CD274] and JAK2) and 10p (including GATA3). Integrated analysis of molecular data and conventional histopathology suggested a diagnosis of triple-negative breast cancer (TNBC) and provided a rationale for immune checkpoint inhibitor therapy with pembrolizumab, which resulted in rapid clinical improvement and a lasting partial remission. Analysis of 157 TNBC samples from The Cancer Genome Atlas revealed focal, high-level PDL1 amplification coinciding with excessive PDL1 mRNA expression in 24% of cases. Collectively, these results illustrate the diagnostic utility of multidimensional tumor profiling in cases with non-descript histology and immune phenotype, demonstrate the predictive power of genomic PDL1 amplification for immune checkpoint inhibition, and suggest a targeted therapeutic strategy in chromosome 9p24.1/PDL1-amplified cancers.H021

  Study EGAS00001001846

• Copy number profiling of putative cancer stem from pleural effusion aspirates from breast cancer patients

  A common symptom during late stage breast cancer disease is pleural effusion, which is related to poor prognosis. In pleural effusions malignant cells can be detected, which indicates a metastatic spread from the primary tumor site. Beside the usefulness for investigation of the metastatic process, it has been a source to isolate cells with putative cancer stem cell (CSC) properties. For this study, pleural effusion aspirate from 25 metastatic breast cancer patients were processed to establish in vitro culture. Patient-derived aspirates were cultured under sphere forming conditions and isolated primary cultures were sorted for cancer stem cell populations ALDH1+ and CD44+CD24-/low and sphere forming efficiency of CSC and non-CSC subpopulation was determined. Unsorted cells had a higher potential to form spheres compared to CSC subpopulations. Subsequently, DNA was isolated from pleural effusions before and after cell sorting, to compare their copy number profiles generated by low-coverage whole genome sequencing to DNA from FFPE primary tumors or bone metastasis. Unbalanced profiles could be detected in four pleural samples before sorting, in two ALDH1+ sorted samples, in one CD44+CD24-/low and in all control samples. Additionally, spheroid culture from one pleural sample could be passaged on and was characterized. Therefore, pleural effusions are an appropriate media for enrichment of putative CSC. However, low-coverage sequencing is difficult due to low quantities of abnormal cells compared to normal cells.

  Study EGAS00001002343

• Defining the metastasome in colorectal cancer: Implications for hypotheses on metastasis evolution and personalized therapy (HIPO-032)

  Personalized cancer therapy aims at individual genetic changes characterizing primary cancers. Still, however, metastasis is the major clinically unmet problem, also due to an incomplete understanding of its molecular evolution.This study is the first to define the metastasis-specific whole genome landscape of human colorectal primary vs. matched metastatic lesions.Protein coding, ncRNA genes and 3’UTRs harbored about a third each of single nucleotide variations (SNVs)/indels, 19% of them being metastasis-specific. We found novel mutational hills among ncRNAs and 3’UTRs, copy number changes able to explain changes in microRNA expression in metastases, and novel metastasis-specific mutations in the 3’UTRs on important cancer signalling genes. Some metastatic lesions showed targetable mutations not detected in the primaries. Analysis of mutual exclusivity patterns supported a model that has progressive alterations in important colorectal cancer genes and revealed new partners. Metastatic lesions were enriched in SNVs, specific structural variations, and alterations in genes affecting cell adhesion and extracellular matrix interaction.Furthermore, a hepatic stellate activation cascade was enriched in metastases, suggesting genetic programs for site-specific colonization. Allele frequency distribution and mutational signature analysis suggests a common ancestor clone to both the primary tumor and the metastasis, with additional mutagenesis ongoing in both sites independently. Taken together, our results significantly add to hypothesis generation on metastasis evolution, and suggest novel metastasis-specific genomic changes which would be missed by current personalized therapy concepts. (HIPO-032)

  Study EGAS00001002717

• Conserved features of TERT promoter duplications reveal an activation mechanism that mimics hotspot mutations in cancer

  Mutations in the TERT promoter are the single most common non-coding mutation in cancer and represent the genetic underpinnings of tumor cell immortality. Beyond the two most common point mutations, G228A and G250A, which selectively recruit the ETS factor GABP to activate TERT, the significance of other variants in the TERT promoter are unknown. We identified duplications of wildtype sequence within the core promoter region of TERT in 7 different cancer types that have strikingly similar features including size, insertion position, and inclusion of one of the native ETS motifs. Each duplication activates the TERT promoter to a similar level as G228A and G250A and is critically dependent on the insertion site. The GABP tetramer binds to the TERT duplicated promoter sequence by virtue of the native ETS motif and its duplicated version with precise spacing, and it is necessary for the transcriptional activation by all duplications tested. Spatiotemporal analysis in a multifocal glioblastoma shows the duplication is clonal and its activation of TERT is readily detectable at the single cell level and in bulk tumor tissue. We conclude that recurrent TERT promoter duplications of the native ETS sequence are functionally and mechanistically equivalent to the hotspot mutations that confer tumor cell immortality. The shared mechanism of these divergent somatic genetic alterations suggests a strong selective pressure for recruitment of the GABP tetramer to activate TERT.

  Study EGAS00001006118

• Whole Exome Sequencing of Chronic Lymphocytic Leukemia

  The somatic genetic basis of chronic lymphocytic leukemia (CLL), a common and clinically heterogenous adult leukemia, remains poorly understood. Massively parallel sequencing technology now provides a method for systematic discovery of genetic alterations that underlie disease, and for uncovering new therapeutic targets and biomarkers. In study version 2 we presented a dataset consisting of DNA sequencing from 169 CLL samples (with matched germline controls). Samples were collected from patients displaying a wide range of characteristics representing the broad clinical spectrum of CLL. Understanding the mutational landscape of CLL provides a starting point for systematic analyses to address fundamental questions in CLL, including how mutated genes alter cellular networks and phenotypes, and thereby contribute to disease heterogeneity. Intratumoral heterogeneity plays a critical role in tumor evolution. To define the contribution of DNA methylation to heterogeneity within tumors, we performed genome-scale bisulfite sequencing of &#62;100 primary chronic lymphocytic leukemias (CLLs; data presented in study version 3). Compared with 26 normal B cell samples, CLLs consistently displayed higher intrasample variability of DNA methylation patterns across the genome, which appears to arise from stochastically disordered methylation in malignant cells. Transcriptome analysis of bulk and single CLL cells revealed that methylationdisorder was linked to low-level expression. Disordered methylation was further associated with adverse clinical outcome. We therefore propose that disordered methylation plays a similar role to that of genetic instability, enhancing the ability of cancer cells to search for superior evolutionary trajectories.

  Study phs000435

• Genetics of 24 hour urine composition

  Genetic and environmental factors play an important role in the etiology of nephrolithiasis. This project will build on and extend our previous efforts (examining environmental risk factors for stone formation) by allowing us to study the risk of stone formation associated with specific genes and gene-environment interactions. We will take advantage of previously collected data in three large cohort studies: Nurses&#39; Health Study I (n=121,000 women), Nurses&#39; Health Study II (n=116,000 women), and Health Professionals Follow-up Study (n=51,000 men). Over a period of 17 to 26 years, information has been collected prospectively on important exposures including diet, family history, body size measures, past medical history, and medications. We have confirmed over 2000 incident cases of kidney stones in each cohort (DK59583, PI Curhan). Further, we have collected 24-hour urine samples from over 4100 stone formers and non-stone formers; the majority of participants have performed two collections. The primary objective of this project is to examine the association between single nucleotide polymorphisms and the 24-hour urinary excretion of stone promoters (calcium and oxalate) and a stone inhibitor (citrate). The secondary objective is to explore the impact of interactions between the genetic factors and dietary factors on 24-hour urinary excretion of relevant lithogenic factors. These findings should provide new insight into regulation of these important factors and also into novel approaches for prevention of stone formation.

  Study phs000460

• Evaluation Study of Congestive Heart Failure and Pulmonary Artery Catheterization Effectiveness (ESCAPE-BioLINCC)

  Accessing Data Please refer to &#8220;Authorized Access&#8221; below regarding accessing data through the BioData Catalyst ecosystem. The data from this accession is not available for download through dbGaP. Objectives To test whether pulmonary artery catheter use was safe and could improve clinical outcomes in participants hospitalized with recurrent heart failure. Background Pulmonary artery catheters have been used to guide adjustment of therapy in multiple settings, but recent studies have raised concern that pulmonary artery catheters may lead to increased mortality in hospitalized participants. Participants A total of 433 participants at 26 sites were enrolled, and randomly assigned to receive therapy guided by clinical assessment and the pulmonary artery catheter or clinical assessment alone. Patients with acute decompensation in which the attending heart failure physician considered pulmonary artery catheterization (PAC) was required or likely to be required within the next 24 hours were entered into a PAC registry. A total of 439 patients were added to the registry. Conclusions Therapy to reduce volume overload during hospitalization for heart failure led to marked improvement in signs and symptoms of elevated filling pressures, with or without the pulmonary artery catheter. Addition of the pulmonary artery catheter to careful clinical assessment did not impact overall mortality and hospitalization. Future trials should test noninvasive assessments with specific treatment strategies that could be used to better tailor therapy for both survival time and survival quality as valued by participants. (Binanay, C. et al., JAMA, 2005)

  Study phs003782

• Neoadjuvant combination PD-L1 plus CTLA-4 blockade in patients with cisplatin-ineligible operable urothelial carcinoma

  Immune checkpoint therapy (ICT) is being tested in the neoadjuvant setting for patients with localized urothelial carcinoma (UC), with one study reporting data in cisplatin-ineligible patients who received anti PD-L1 monotherapy. The study reported that patients with bulky tumors, a known high-risk feature defined as greater than clinical T2 disease, had fewer responses, with pathologic complete response (pCR) rate of 17%. Here, we report on the first pilot combination neoadjuvant trial (NCT02812420) with anti-PD-L1 (durvalumab) plus anti-CTLA-4 (tremelimumab) in cisplatin-ineligible patients, with all tumors identified as having high risk features (N=28). Primary endpoint was safety and we observed 6 of 28 patients (21%) with grade ≥3 immune-related adverse events, consisting of asymptomatic laboratory abnormalities (N=4), hepatitis and colitis (N=2). We also observed pCR of 37.5% and downstaging to pT1 or less in 58% of patients who completed surgery (N=24). In summary, we provide initial safety, efficacy and biomarker data with neoadjuvant combination anti-PD-L1 plus anti-CTLA-4, which warrants further development for patients with localized UC, especially cisplatin-ineligible patients with high-risk features who do not currently have an established standard-of-care neoadjuvant treatment. Publication: Gao et al. Neoadjuvant PD-L1 plus CTLA-4 blockade in patients with cisplatin-ineligible operable high-risk urothelial carcinoma. Nature Medicine volume 26, pages1845–1851(2020)

  Study EGAS00001004074

• Non-coding mutations reveal cancer driver cistromes in luminal breast cancer

  Whole-genome sequencing of primary breast tumors enabled the identification of cancer driver genes and non-coding cancer driver plexuses from somatic mutations. However, differentiating between driver and passenger events among non-coding genetic variants remains a challenge to understand the etiology of cancer and inform the delivery of personalized cancer medicine. Herein, we reveal enrichment of non-coding mutations in cis-regulatory elements that cover a subset of transcription factors linked to tumor progression in luminal breast cancers. Using a cohort of 26 primary luminal ER+PR+ breast tumors, we compiled a catalogue of ~100,000 unique cis-regulatory elements from ATAC-seq data. Integrating this catalogue with somatic mutations from 350 publicly available breast tumor whole genomes, we identified four recurrently mutated individual cis-regulatory elements. By then partitioning the non-coding genome into cistromes, defined as the sum of binding sites for a transcription factor, we uncovered cancer driver cistromes for ten transcription factors, namely CTCF, ELF1, ESR1, FOSL2, FOXA1, FOXM1 GATA3, JUND, TFAP2A, and TFAP2C in luminal breast cancer. Nine of these ten transcription factors were shown to be essential for growth in breast cancer, with four exclusive to the luminal subtype. Collectively, we present a strategy to find cancer driver cistromes relying on quantifying the enrichment of non-coding mutations over cis-regulatory elements concatenated into a functional unit drawn from an accessible chromatin catalogue derived from primary cancer tissues.

  Study EGAS00001005235

• RNA-sequencing from duodenal bipsies of Celiac disease patients

  Samples, in a form of PAXgene fixed and paraffin-embedded biopsies, were collected from the multi-site, double-blind, randomized, placebo-controlled trial, aimed at dose-finding and assessing the efficacy and tolerability of a 6-week treatment with ZED1227 capsules vs. placebo in subjects with well-controlled celiac disease undergoing gluten challenge. Total RNA was extracted from the PaxFPE biopsy specimens (n = 116) using additional cuttings from the samples on which histomorphometry was previously assessed. For the extraction, an RNeasy Kit (Qiagen, Hilden, Germany) was used according to the manufacturer’s instructions. Library preparation and next-generation sequencing (NGS) were performed by the Qiagen NGS Service. A total of 10 ng of purified RNA was converted into cDNA NGS libraries. Library preparation was quality controlled using capillary electrophoresis. Based on the quality of the inserts and the concentration measurements, the libraries were pooled in equimolar ratios and then sequenced on a NextSeq (Illumina Inc., San Diego, USA) sequencing instrument according to the manufacturer’s instructions, with 100 bp read length for read 1 and 27bp for read 2. The raw data were de-multiplexed, and FASTQ files for each sample were generated using bcl2fastq2 software (Illumina Inc., San Diego, USA).

  Dataset EGAD50000000491

• Partner independent fusion gene detection by multiplexed CRISPR Cas9 enrichment and long read Nanopore sequencing (FUDGE)

  Targeted long-read nanopore sequencing. Abstract: Fusion genes are hallmarks of various cancer types and important determinants for diagnosis, prognosis and treatment. Fusion gene partner choice and breakpoint-position promiscuity restricts diagnostic detection, even for known and recurrent configurations. To accurately and impartially identify fusions, we developed FUDGE: FUsion Detection from Gene Enrichment. FUDGE couples target-selected and strand-specific CRISPR/Cas9 activity for fusion gene driver enrichment - without prior knowledge of fusion partner or breakpoint-location – to long-read Nanopore sequencing with the bioinformatics pipeline NanoFG. FUDGE has flexible target-loci choices and enables multiplexed enrichment for simultaneous analysis of several genes in multiple samples in one sequencing run. We observe on-average 665 fold breakpoint-site enrichment and identify nucleotide resolution fusion breakpoints - within two days. The assay identifies cancer cell line and tumor sample fusions irrespective of partner gene or breakpoint-position. FUDGE is a rapid and versatile fusion detection assay, providing unparalleled opportunity for diagnostic pan-cancer fusion detection.

  Dataset EGAD00001006111

• Cell-free DNA Methylome Analysis Enables Early Preeclampsia Prediction

  This dataset contains in solution target-enrichment bisulfite sequencing of placental tissue, buffy coat and plasma DNA from pregnant women. Blood samples were taken for cell-free DNA (cfDNA) DNA extraction from 64 women at the time of early-onset preeclampsia (PE) diagnosis, or from 38 controls (uncomplicated pregnancies) at a similar gestational age that did not develop preeclampsia subsequently. Among these subjects, plasma samples from 7 PE patients and 6 controls were also subjected to oxidative bisulfite sequencing. Placental tissues from 11 PE and 26 control subjects after delivery, and buffy coat from 16 PE and 16 control subjects at the same time of cfDNA sampling were profiled. A discovery cohort for early PE assessment in the first trimester was collected. In this cohort, cfDNA from 75 pregnancies that went on to develop early-onset PE and from 124 matched controls were collected and methylome sequencing were carried out. An independent validation cohort to validate early PE assessment with methylome profiling was collected as well. This validation cohort includes cfDNA samples from 61 PE and 136 control pregnancies.

  Dataset EGAD00001010159

• Single Cell Sequencing of Sperm (scSperm)

  The aim of this project is to genotype and sequence single spermatozoa from two men, one in his twenties and the other in his seventies. The resulting data is used to quantify the mutations that have arisen in the gametes of both individuals in order to better understand the effect of aging on mutation rates and modes.Project Outline. In order to quantify mutations, semen from two individuals are sequenced. 48 single sperm cells are isolated from each individual, and their DNA is extracted. The resulting genomes are amplified using PicoPlex, GenomiPhi MDA, Repli-G MDA, and MALBAC. QC step is applied to check the quality of WGA DNA using standard Sequenom plex (26 SNPs). A subset of 32 amplification products which pass the intiall QC, are genotyped using Affymetrix SNP6 chips. 12 of the genotyped amplification products are also sequenced. In addition, one multi-cell sample per individual is sequenced as a reference and for validation purposes.Altogether, 12 single cell sperm genomes and two multi-cell genomes are sequenced, coming to a total of 14 genomes. Of the single cell sperm genomes, 2 are sequenced to 50x coverage, and the other 10 to 25x coverage. Both multi-cell genomes are sequenced to 25x coverage.

  Dataset EGAD00001001216

• A panel of reference haplotypes for imputing complement component 4 (C4) gene structural variation

  Neuropsychiatric and autoimmune disorders have substantial epidemiological correlations (Benros et al., JAMA Psychiatry 2013, PMID: 23760347) and broad, genome-wide patterns of shared genetic risk (Pouget et al., Hum Mol Genet, PMID: 31211845; Tylee et al., Am J Med Genet B Neuropsychiatr Genet 2018, PMID: 30325587). Some cases of psychosis involve autoantibodies against the NMDA receptor, AMPA receptor, or other synaptic proteins (reviewed in Dalmau et al, Lancet Neurol 2011, PMID: 21163445). The related autoimmune conditions lupus and Sjogren&#39;s Syndrome also involve the development of autoantibodies. The possibility that neuropsychiatric disorders can have molecular mechanisms in common with autoimmune disorders - for example, that psychosis involves an inflammatory or autoimmune component in some patients, or that immune molecules are re-used in the brain to underlie other important biological activities (Stevens et al., Cell 2007, PMID: 18083105) - could open novel therapeutic possibilities for neuropsychiatric disorders. At a genetic level, the strongest genetic associations of schizophrenia, lupus, and Sjogren&#39;s Syndrome to common genetic variation involve associations to genetic markers in the Major Histocompatibility Complex (MHC) locus. Bipolar disorder in some studies also associates with variation in or near the MHC locus, though less strongly than schizophrenia does. Intriguingly, the same specific SNPs appear to associate strongly with schizophrenia, lupus, and Sjogren&#39;s; these strongly associating SNPs span a genomic segment that includes the HLA class II genes (which have an important role in antibody production) and the complement component 4 (C4) genes. The specific genes and alleles responsible for these associations need to be completely defined, and the extent to which they represent shared or distinct genetic influences in neuropsychiatric and autoimmune illnesses needs to be clarified. The complement component 4 (C4A and C4B) genes are present in the MHC locus, between the class I and class II HLA genes. C4A and C4B commonly vary in genomic copy number and encode complement proteins with distinct affinities for molecular targets. The complex genetic variation at C4 - arising from many alleles with different numbers of C4A and C4B genes - has been challenging to analyze in large cohorts. We recently developed an approach to this problem based on imputation: people share long haplotypes with the same combinations of SNP and C4 alleles, such that C4A and C4B gene copy numbers can be imputed from SNP data (Sekar et al., Nature 2016, PMID: 26814963). In the current work, to analyze C4 in large cohorts, we developed a way to identify C4 alleles from whole-genome sequence (WGS) data, then analyzed WGS data from 1,234 individuals to create a large multi-ancestry panel of 2,530 reference haplotypes of MHC SNPs and C4 alleles that can then be imputed into still-larger cohorts for which SNP data are available. With this dbGaP submission, we make this reference panel available for other studies. Protocols and software for imputing C4 alleles into genome-wide SNP data, and for performing molecular analyses on the C4 genes (such as direct measurement of copy number from genomic DNA), can be found on the McCarroll Lab web site (http://mccarrolllab.org/resources). We are also working to create additional reference panels for imputation of C4 alleles that will be based on still-larger and more diverse population samples; links to these will also be available on the McCarroll Lab web site as we create and validate them.

  Study phs001992

• Genetics of Mood Disorders: Aging and Emotion Regulation Brain Circuitry in Bipolar

  This single-site study is designed to investigate age-related changes in structure and function of a ventral prefrontal cortex (VPFC) neural system, that subserves emotion processing and regulation, during mid to later adulthood in individuals with bipolar disorder (BD), compared to healthy controls (HC). It is a 5-year study of age-related changes in mood, emotion regulation and related symptoms and behaviors using multimodality imaging measures [structural magnetic resonance imaging (MRI) of gray matter, diffusion tensor imaging (DTI) of white matter, and functional MRI measures of functional connectivity and of brain responses during implicit and explicit emotion regulation] comparing BD and HC groups ages 40-79 years, 50% female; a subgroup will be reassessed and rescanned after 2 years to study brain and associated behavioral changes over time. We will explore relationships between brain aging with risk factors implicated in progression. This will include genes, as well as adverse outcomes of progression such as suicide ideation and behavior. As there has been little study of effects of aging of the VPFC emotional circuitry in healthy individuals, findings could provide new insights into brain circuitry and emotional regulation in older adults. In addition to the importance of contributing to understanding BD, as BD is unique in having both elevated and depressive episodes, its study can provide a model for understanding progressive emotion dysregulation to both intervention and prevention that can be better targeted specifically for older individuals with BD. A de-identified blood sample will be sent to the Repository and Genomics Resource.

  Study phs001631