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• Modifier Genes in 21-hydroxylase Deficiency

  CAH is a genetic steroidogenesis disorder. The most common form, 21OHD, leads to cortisol deficiency and, in turn, an excess of androgen, a hormone that promotes the development and maintenance of male sex characteristics. As a result of this androgen excess, prepubescent males and newborn, prepubescent, and grown females exhibit mature masculine characteristics. The symptoms and severity of 21OHD vary among individuals with the disease and in adults versus children. The reasons for these differences are not yet known. Current therapy for 21OHD consists of administration of glucocorticoids to replace cortisol and suppress excessive pituitary function. With more information about what genes or factors contribute to the severity of 21OHD, researchers may be able to better treat children and adults with the disease. This study will examine participants' DNA to determine what other genes may affect the severity of 21OHD and may make the disease milder in adults than in children This is a 3-day inpatient study. Once eligibility is confirmed through physical exam and blood analysis, eligible participants are admitted to the study site in the morning on the first study day. A blood sample is taken and participants receive one 10-mg pill of hydrocortisone. Heart rates and blood pressures are taken every 4 hours throughout the day. In the morning of Day 2, a blood sample will be obtained and a 24 hour urine collection will begin. On the morning of Day 3, another blood sample is obtained. Participants then receive intravenous cosyntropin, a synthetic form of a hormone that the body makes. About 1 hour after this, participants provide a final blood sample. Participants receive a pill of hydrocortisone prior to the end of the study.

  Study phs001313

• The Human Pancreas Analysis Program (HPAP)

  The past decade has seen a dramatic improvement in our ability to phenotype and molecularly profile human tissues with unprecedented resolution at the genomic, epigenomic, protein, and functional levels. The Human Pancreas Analysis Program (HPAP), part of the Human Islet Research Network and supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) through multiple NIH grants, is performing deep phenotyping of the human endocrine pancreas to better understand the cellular and molecular events that precede and lead to beta-cell loss and/or dysfunction in type 1 diabetes (T1D) and type 2 diabetes (T2D) as well as to accumulate, analyze, and distribute high-value data sets to the diabetes research community through the HPAP PANC-DB database (and additional details provided at that site). To this end, HPAP employs state-of-the-art technologies to perform comprehensive analyses of pancreas biology as it pertains to organ donors with T1D, autoantibody-positive donors without diabetes, donors with T2D, and control donors. Pancreas procurement and analyses take advantage of the expertise and extensive network of Organ Procurement Organizations (OPOs) and autoantibody screening centers established through the JDRF/The Lenona M. and Harry B. Helmsley Charitable Trust (HCT) – funded nPOD program. In contrast to nPOD, the major product provided by HPAP is not archived biomaterial subject to broad distribution, but rather, the delivery of extensive and high-quality molecular data sets to the diabetes research community in order to facilitate further discovery. Together, nPOD and HPAP are complementary programs that assist the diabetes community and afford the maximal opportunity for advancing knowledge about the pathogenesis of T1D and T2D.

  Study phs002465

• A Personalized Neoantigen Vaccine Generates Anti-Tumor Immunity in High-Risk Renal Cell Carcinoma

  Personalized cancer vaccines (PCVs) can generate circulating immune responses against predicted neoantigens. However, whether such responses can target cancer driver mutations, lead to immune recognition of a patient's tumor, and result in clinical activity are largely unknown, particularly in patients with lower mutation burden tumors. We conducted a phase I trial testing a neoantigen-targeting PCV in patients with high-risk, fully resected clear cell renal cell carcinoma (RCC; stage III or IV), with or without ipilimumab administered adjacent to the vaccine. At a median follow-up of 40.2 months from surgery, none of the 9 initial participants experienced a recurrence of RCC. No dose-limiting toxicities were observed. All patients generated T cell immune responses against PCV antigens, including RCC driver mutations in VHL, PBRM1, BAP1, KDM5C, and PIK3CA. Following vaccination, there was a durable expansion of peripheral T cell clones, and T cell reactivity against autologous tumor was detected in 7 of the 9 patients. Our results demonstrate that neoantigen-targeting PCVs in high-risk RCC are highly immunogenic, capable of targeting key driver mutations, and induce anti-tumor immunity. These observations, in conjunction with the absence of recurrence in all 9 vaccinated patients, highlights the promise of PCV as effective adjuvant therapy in RCC. We are submitting WES data from whole blood samples and tumor FFPE samples, and RNA-Seq data from tumor FFPE samples. We are also submitting RNA-Seq data from purified tumor samples. scRNA-seq data will be submitted for skin samples (vaccine site biopsies at Weeks 0, 4, and 13), tumor samples (including resected metastatic tumor samples for two patients), and normal kidney samples. Bulk TCR-seq data will be submitted for blood samples.

  Study phs003710

• CHARGE (Cohorts for Heart and Aging Research in Genomic Epidemiology) Consortium Summary Results from Genomic Studies

  GWAS have successfully identified genetic loci associated with a variety of conditions such as type 2 diabetes and coronary disease. The large number of statistical tests required in GWAS has posed a special challenge because few studies that have DNA and high-quality phenotype data are sufficiently large to provide adequate statistical power for detecting small to modest effect sizes. Even before the era of GWAS, the requirement for large sample sizes and the importance of replication have served as powerful incentives for collaboration. Meta-analyses combining summary data from multiple sources have improved the ability to detect new loci. The Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium was formed to facilitate GWAS meta-analyses and replication among multiple large and well-phenotyped cohort studies. The design of the CHARGE Consortium was formed initially from 5 prospective cohort studies from the United States and Europe: the Age, Gene/Environment Susceptibility (AGES) - Reykjavik Study, the Atherosclerosis Risk in Communities (ARIC) Study, the Cardiovascular Health Study (CHS), the Framingham Heart Study (FHS), and the Rotterdam Study (RS). Additional studies have expanded the CHARGE consortium based upon the phenotypes and willingness to share information across the research community. In order to facilitate investigators across the world to examine relationships between phenotypes and genetic markers within CHARGE published reports, an open site is made available on dbGaP that provides the rsID and the p-value for inspection. Access to detailed summary statistics (including minor allele frequency, odds ratio/effect size) requires approval of a Data Access Request (DAR).

  Study phs000930

• CPTAC Proteogenomic Study

  Recently, significant progress has been made in characterizing and sequencing the genomic alterations in statistically robust numbers of samples from several types of cancer. For example, The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC) and other similar efforts are identifying genomic alterations associated with specific cancers (e.g., copy number aberrations, rearrangements, point mutations, epigenomic changes, etc.) The availability of these multi-dimensional data to the scientific community sets the stage for the development of new molecularly targeted cancer interventions. Understanding the comprehensive functional changes in cancer proteomes arising from genomic alterations and other factors is the next logical step in the development of high-value candidate protein biomarkers. Hence, proteomics can greatly advance the understanding of molecular mechanisms of disease pathology via the analysis of changes in protein expression, their modifications and variations, as well as protein=protein interaction, signaling pathways and networks responsible for cellular functions such as apoptosis and oncogenesis. Realizing this great potential, the NCI launched the third phase of the CPTC initiative in September 2016. As the Clinical Proteomic Tumor Analysis Consortium, CPTAC continues to define cancer proteomes on genomically-characterized biospecimens. The purpose of this integrative approach was to provide the broad scientific community with knowledge that links genotype to proteotype and ultimately phenotype. In this third phase of CPTAC, the program aims to expand on CPTAC II and genomically and proteomically characterize over 2000 samples from 10 cancer types (Lung Adenocarcinoma, Pancreatic Ductal Adenocarcinoma, Glioblastoma Multiforme, Acute Myeloid Leukemia, Clear cell renal Carcinoma, Head and Neck Squamous Cell Carcinoma, Cutaneous Melanoma, Sarcoma, Lung Squamous Cell Carcinoma, Uterine Corpus Endometrial Carcinoma) .Germline DNA is obtained from blood and Normal control samples for proteomics varied by organ site. All cancer samples were derived from primary and untreated tumor.

  Study phs001287

• Prenatal lead exposure is associated with decreased cord blood DNA methylation of the glycoprotein VI gene involved in platelet activation and thrombus formation

  Early-life lead exposure impairs neurodevelopment and later exposure affects the cardiovascular system. Lead has been associated with reduced global 5-methylcytosine DNA methylation, suggesting that lead toxicity acts through epigenetic mechanisms. The objective of this study is to clarify how early life lead exposure alters DNA methylation of specific genes, using an epigenomic approach. We measured lead concentrations in urine (gestational week, GW, 8) and erythrocytes (GW 14), using ICP-MS, for 127 pregnant mothers recruited in the MINIMat food and supplementation cohort in rural Bangladesh. Cord blood DNA methylation was analyzed with the Infinium HumanMethylation450K BeadChip and top sites were validated by methylation-sensitive high-resolution melt curve analysis. Maternal urinary lead concentrations (divided into quartiles) showed significant (after adjustment for FDR) inverse associations with methylation at nine CpGs. Three of these sites were in the 5’ end, including the promoter, of glycoprotein IV (GP6); cg18355337 (q=0.029, β=-0.30), cg25818583 (q=0.041, β=-0.18) and cg23796967 (q=0.047, β=-0.17). The methylation in another CpG site in GP6 was close to significant (cg05374025, q=0.057, β=-0.23). The erythrocyte lead concentrations (divided into quartiles) were also inversely associated with CpG methylation in GP6, although this was not statistically significant after FDR-adjustments. Eight CpG sites in GP6 constituted a differentially methylated region in relation to urinary lead (p=0.005, q=0.48) and erythrocyte lead (p=0.007, q=0.46). In conclusion, we found that moderate prenatal lead exposure appear to epigenetically affect GP6, a key component of platelet aggregation and thrombus formation, suggesting a novel link between early lead exposure and cardiovascular disease later in life.

  Study EGAS00001001575

• Combining a Universal Telomerase Based Cancer Vaccine with Ipilimumab in Patients with Metastatic Melanoma - Five-year Follow up of a Phase I/IIa Trial

  Background Ipilimumab improves survival for patients with metastatic malignant melanoma. Combining a therapeutic cancer vaccine with ipilimumab may increase efficacy by providing enhanced anti-tumor immune responses. UV1 consists of three synthetic long peptides from human telomerase reverse transcriptase (hTERT). These peptides comprise epitopes recognized by T cells from cancer patients experiencing long-term survival following treatment with a first-generation hTERT vaccine, and generate long-lasting immune responses in cancer patients when used as monotherapy. The objective of this trial was to investigate the safety and efficacy of combining UV1 with ipilimumab in metastatic melanoma. Patients and methods In this phase I/IIa, single center trial [NCT02275416], patients with metastatic melanoma received repeated UV1 vaccinations, with GM-CSF as an adjuvant, in combination with ipilimumab. Patients were evaluated for safety, efficacy and immune response. Immune responses against vaccine peptides were monitored in peripheral blood by measuring antigen-specific proliferation and IFN-γ production. Results Twelve patients were recruited. Adverse events were mainly diarrhea, injection site reaction, pruritus, rash, nausea and fatigue. Ten patients showed a Th1 immune response to UV1 peptides, occurring early and after few vaccinations. Three patients obtained a partial response and one patient a complete response. Overall survival was 50% at 5 years. Conclusion Treatment was well tolerated. The rapid expansion of UV1-specific Th1 cells in the majority of patients indicates synergy between UV1 vaccine and CTLA-4 blockade. This may have translated into clinical benefit, encouraging the combination of UV1 vaccination with standard of care treatment regimes containing ipilimumab/CTLA-4 blocking antibodies.

  Study EGAS00001005253

• Genetic Risk for Subsequent Neoplasms among Long-term Survivors of Childhood Cancer in the St. Jude Lifetime Cohort

  While childhood cancer survivors are known to be at increased risk of subsequent neoplasms (SNs), the contribution of germline mutations in cancer predisposition genes to the risk of SN is largely unknown. Whole-genome sequencing (30X) was performed on 3,006 5+ year survivors of childhood cancer (median age, 35.8 [range: 7.1-69.8] years) from the St. Jude Lifetime Cohort. Survivors underwent a comprehensive clinical assessment. Germline mutations in 156 cancer predisposition genes (SJCPG156), including a subset of 60 (SJCPG60) autosomal dominant genes with moderate to high penetrance, were classified for their pathogenicity. Piecewise exponential regression, stratified by radiation exposure, was used to evaluate the relative rate (RR) and 95% confidence interval (95% CI) of SN occurrence by mutation status. Pathogenic or likely pathogenic mutations in SJCPG156 and SJCPG60 were identified in 11.7% and 5.8% of survivors, respectively. The most frequently mutated SJCPG60 genes included RB1 (n=43), NF1 (n=22), BRCA2 (n=14), BRCA1 (n=12) and TP53 (n=10). Mutations in SJCPG60 were associated with the rate of subsequent sarcoma (RR, 10.3; 95% CI, 4.2 to 25.6) and breast cancer (RR, 13.3; 95% CI, 5.8 to 30.5) among irradiated survivors, and the rate of developing any SN (RR, 4.8; 95% CI, 2.4 to 9.4) and breast cancer (RR, 7.8; 95% CI, 2.5 to 24.0) among non-irradiated survivors. The rate of developing ≥2 histologically distinct SNs was increased 17.8-fold (95% CI, 3.5 to 89.8) among SJCPG60 mutation carriers treated without radiotherapy. Similar but attenuated associations were observed for mutations in SJCPG156. Survivors who develop a SN without prior radiotherapy or who are diagnosed with a subsequent sarcoma or breast cancer within a site of prior radiotherapy should be referred to genetic counseling.

  Study EGAS00001002499

• HGSC WGS for the study Genomic consequences of aberrant DNA repair mechanisms stratify ovarian cancer histotypes

  HGSC cases in the OvCaRe and CRCHUM Tumour Banks were selected according to the following criteria: (i) were administered platinum taxane based therapy; (ii) relapsed within 12 months (365 days) or had at least longer than 4.5 years (1642.5 days) follow-up data; (iii) had at least 50% tumour content by H&E staining and expert pathology review. All cases were re-reviewed by expert pathologists to confirm the diagnosis of HGSC. Germline BRCA1 and BRCA2 was determined for all patients through hereditary cancer screening programs. The design of cases selection as a discovery cohort was engineered to amplify biological differences by selecting cases from the extremes of the outcome distribution. All HGSC tumours are primary tumour samples. Library construction and sequencing Frozen specimens with >50% tumour cellularity (based on initial slide review) were used for cryosectioning and subsequent nucleic acid extraction. Patient tumour and normal blood samples derived from primary, untreated fresh frozen tumour specimens harvested at diagnosis during standard of care debulking surgery. Germline DNA was provided from peripheral blood buffy coat on all specimens except 13 from Tokyo, where non-cancer frozen tissue was used as a germline source. DNA extraction from both matched normal (blood) and tumour samples (frozen tissue) were performed using the QIAamp Blood and Tissue DNA kit (Qiagen) and quantified using a Qbit fluorometer and reagents (high-sensitivity assay). Three lanes of Illumina HiSeq 2500 v4 chemistry for normal samples and five lanes for tumour samples were obtained. The PCR-free protocol was adopted to eliminate the PCR-induced bias and improve coverage across the genome.

  Dataset EGAD00001003268

• ENOC WGS for the study Genomic consequences of aberrant DNA repair mechanisms stratify ovarian cancer histotypes

  For ENOC cohorts, OvCaRe cases were reviewed, including frozen material, by at least two expert gynecopathologists prior to inclusion in the sequencing cohort who provided the confirmation on final selected cohort. Frozen H&E from Tokyo were also used for evaluation along with representative H&E photos and review done at the Jikei School of Medicine. For ENOC, DAH985 and DG1288 are recurrent and both were treated with chemotherapy after their first surgery. DAH123 is a untreated sample, metastasis from an primary endometrial tumour. All HGSC, GCT, CCOC and the rest ENOC tumours are primary tumour samples. Library construction and sequencing Frozen specimens with >50% tumour cellularity (based on initial slide review) were used for cryosectioning and subsequent nucleic acid extraction. Patient tumour and normal blood samples derived from primary, untreated fresh frozen tumour specimens harvested at diagnosis during standard of care debulking surgery. Germline DNA was provided from peripheral blood buffy coat on all specimens except 13 from Tokyo, where non-cancer frozen tissue was used as a germline source. DNA extraction from both matched normal (blood) and tumour samples (frozen tissue) were performed using the QIAamp Blood and Tissue DNA kit (Qiagen) and quantified using a Qbit fluorometer and reagents (high-sensitivity assay). Three lanes of Illumina HiSeq 2500 v4 chemistry for normal samples and five lanes for tumour samples were obtained. The PCR-free protocol was adopted to eliminate the PCR-induced bias and improve coverage across the genome.

  Dataset EGAD00001003266

• HTAN MCL Pre-Cancer Atlas Pilot Project - Targeted Sequencing Development Study

  The HTAN-MCL Pre-Cancer Atlas Pilot Project (PCAPP) is the result of a collaboration between the seven members of the MCL consortium. Across four organ types, PCAPP's goal is to collect and profile pre-malignant lesions for gene expression, DNA mutations, single-cell gene expression and immune-environment. Most PML are small in size and only available come from formalin fixed paraffin embedded archived tissue. The primary goal of PCAPP is to 1) understand the logistical challenges of PML specimen collection, 2) document technical limitations of the assays that are specific to the PML and 3) overcome them to support the generation of a more comprehensive Pre-Cancer Atlas in the future. The current upload provides RNA and DNA sequencing from participants with DCIS who were studied at the University of San Diego and the University of Vermont. Description of the overall study: A. Background/Significance One of the critical barriers to developing new approaches for cancer detection and prevention is the lack of understanding of the key molecular and cellular changes that cause cancer initiation and progression. Unlike the extensive work that has been done profiling advanced stage tumors, few studies have comprehensively profiled the molecular alterations found in precancerous tissues. Premalignant lesions are currently characterized by histologic changes that precede the development of invasive carcinoma1,2.These lesions can often be identified in regions surrounding an invasive tumor or in biopsies taken from patients undergoing diagnostic evaluation for suspicion of cancer. Currently, limited metrics exist to identify lesions that will likely progress to carcinoma and require intervention from those that will naturally regress or remain stable3,4. Characterization of the molecular alterations in premalignant lesions and the corresponding changes in the microenvironment would hasten the development of biomarkers for early detection and risk stratification as well as suggest preventive interventions to reverse or delay the development of cancer. Our pilot study will establish the feasibility of transcriptomic, genomic and immune profiling of FFPE premalignant lesions from multiple organ sites, collected and profiled with uniform SOPs across multiple institutions within the MCL consortium. We will characterize the molecular alterations in precancerous lesions and the corresponding microenvironment in four major organ sites, in order to uncover the molecular and cellular determinants of premalignancy, and establish standardized sequencing and immunohistochemistry protocols on FFPE precancerous tissue. We will also evaluate the technical feasibility of single nuclei sequencing of small FFPE pre-cancer lesions. Successful completion of the proposed pilot study will set the stage for expansion and development of a comprehensive Pre-Cancer Atlas (PCA) as part of the NCI's moonshot.B. Specific Aims Aim 1: Collect premalignant lesions (PML) and their associated microenvironment via LCM from FFPE tissue across four organ sites (breast, lung, pancreas & prostate). Aim 2: Perform bulk RNA and DNA seq on premalignant FFPE samples (and flash frozen tissue where available) and compare the genomic/transcriptomic alterations within and across organ sites. C. ApproachAim 1: Collect premalignant lesions (PML) and their associated microenvironment via LCM from FFPE tissue across four organ sites (breast, lung, pancreas & prostate). MethodsI. Patient Population/Sample Collection: Overview of the sites collecting PML tissue from the respective organs is provided in Table 1 and a full description of the biospecimens to be obtained is described in detail for each organ type below. Table 1. Breakdown of cohort by tissue type and collection site.Organ siteBreastLungPancreasProstateType of PMLDCISAAH, Squamous Dysplasia/CISIPMNsPINCollection of PatientsUCSF/UCSDUVMBU*/UCLAVanderbilt/MoffittMDACC*JHUStanford*# of Patients201920 (10 of each type)20 (10 of each type)242020Total patients per Organ39402440Note: single nuclei/cell RNA-Seq will be performed on 4-5 FFPE samples from each of the organ types 1. DCIS lesions from breast tissue: DCIS lesions will be collected from 39 patients (20 from UCSF/UCSD & 19 from UVM) with primary low or high-grade DCIS diagnosed from a breast core biopsy. Subsequent resected lumpectomy or mastectomy tissues will be prospectively sampled in the vicinity of the prior biopsy site using multiple approaches: 1) Live cells (heterogeneous mix) will be obtained as a cell scrape slurry from the lesion surface or by fine needle aspirate (FNA); 2) For a subset of specimens where size is sufficient, a block of breast tissue with DCIS will be fresh-frozen; 3) The remainder of the specimen will be taken for routine formalin-fixation and paraffin-embedding (FFPE). The FFPE sample will be annotated to identify the matched FFPE tissue block adjacent to the fresh-frozen sample and will be sectioned for use in bulk and single nuclei sequencing . We will dissect DCIS, adjacent normal and when available, associated carcinoma. In addition, when possible, normal tissue will be collected from a tissue block lacking lesions as well as collection of blood. A subset of patients (n = 5 | FFPE, flash frozen and fresh) will be sent to the Broad Institute for single nuclei/cell sequencing.2. AAH and squamous dysplastic/CIS lesions from airway and lung tissue: For squamous cell lung cancer, we will collect endobronchial biopsies from abnormal airway regions identified on autofluroscence bronchoscopy or identify PMLs in the margins of resected lung tissue. We will study 20 patients (5 each from BU/UCLA/Vanderbilt/Moffitt) with pre-invasive squamous lesions (moderate-severe dysplasia or carcinoma in situ (CIS)) identified on pathologic examination. LCM of the premalignant region and adjacent normal epithelium will be performed as well as the invasive tumor for those collected from the resection margin (n=5 from UCLA). On a subset of lesions collected at bronchoscopy (n=5), we will collect additional biopsies that will be flash frozen and fresh for single nuclei and cell sequencing, respectively, performed at the Broad Institute. In parallel to the work at the Broad, BU will perform single cell RNA-seq on these freshly cell sorted tissues (n = 5). Blood will be collected on all patients for genomic studies. For lung adenocarcinoma, we will collect resected FFPE lung tissues from 20 patients (10 from UCLA and 10 from Vanderbilt/Moffitt) with early stage lung adenocarcinoma that harbor atypical adenomatous (AAH) premalignant lesions in the resection margin. We will LCM multiple AAH regions (3-5 per patient) as well as adjacent regions of normal epithelium and invasive adenocarcinoma. In addition, blood will be collected on all patients for genomic studies. 3. IPMNs from pancreatic tissue: For pancreatic cancer PML, we will collect low and high grade lesions from 24 patients representing macroscopic Intraductal Papillary Mucinous Neoplasms (IPMN) (n=24) from surgically resected specimens along with blood samples. Archival FFPE specimens of microscopic PanIN lesions, occurring multi-focally adjacent to invasive PDAC, and archival IPMN lesions (with or without associated invasive cancer), along with the adjacent normal tissue, will undergo LCM and utilized for bulk DNA and RNA sequencing. If matched frozen tissues are available for a subset of these FFPE samples, we will bank for comparison of profiles. Because IPMNs are macroscopic lesions, they provide an opportunity for obtaining the samples fresh and therefore can be used for single cell sequencing (in contrast to PanINs). Therefore, 5 freshly obtained IPMNs will be used for the single cell RNA sequencing studies performed at both the Broad Institute and MDACC, and the matched FFPE and/or frozen sections from these lesions (obtained from the adjacent PML) will be sent to Broad Institute as a pilot to assess "single nuclei" RNA sequencing.4. PINs from prostate tissue: For prostate cancer PML, there will be 40 samples of Prostatic Intraepithelial Neoplasia (PIN) collected between the Stanford and JHU sites (20 cases per site). At the Stanford site, 20 prostate specimens detected by PSA screening who have/will undergo surgery (radical prostatectomy) for clinically localized disease will make up the final cohort. The age range of the participants would be 40-75, and we anticipate that 18 will be Caucasian, 1 Asian and 1 Latino or African American based on the practice demographics practice at Stanford. Clinical and MRI data will also be collected for these samples. We will collect low grade (e.g. Gleason score of 6/Grade group 1; n=10) and high grade (Gleason score 4+3=7 or higher/Grade group 3 or higher; n=10) PINs from FFPE samples that have prostate carcinoma. In addition to obtaining LCM archival samples of low and high grade PIN, we will also obtain normal prostatic epithelial from the peripheral, central and transition zones as well as multiple samples of prostate carcinoma in order to obtain the spectrum of Gleason grades in the carcinoma as needed. LCM samples will be used for bulk DNA and RNA sequencing. In addition, single cells will be dissected from FFPE samples to prepare single cell RNA seq libraries using techniques developed at Stanford, and FFPE tissue will be sent to the Broad for single nuclei sequencing. When available, flash frozen and fresh samples from these prostates will be archived and prepared for single nuclei and cell sequencing, respectively, at the Broad Institute and at Stanford (single cell only). JHU will also capture 10 cases (5 grade group 1 and 5 grade group 2) of high grade PIN, normal and invasive adenocarcinoma using frozen sections from fresh frozen tissues. When possible these will be from the same patients as the FFPE samples. Since frozen sections can be quite challenging to morphologically determine high grade PIN from normal epithelium, for these samples we will perform a number of additional tissue-based characterizations. These will include a multicolor combined basal cells (p63 and CK903) and PIN/carcinoma markers (AMACR) referred to in the cocktail as "PIN4", c-MYC (referred to as MYC) protein5, by IHC and mRNA by in situ hybridization (AM De Marzo, Q Zheng unpublished observations), telomere length by in situ hybridization6 and the 5'ETS/45S rRNA7. For these slides, the whole slides will be scanned with a Hammamatsu Nanozoomer with a 40x objective and regions of interest will be annotated as a guide for LCM.II. Laser-capture microdissection (LCM): FFPE tissue blocks will be sectioned at 7μm thickness and serial sections will be stained with H&E. LCM will be performed utilizing standard LCM systems, such as Leica LMD7000 and ArcturusXT at each site. Regions of premalignancy will be dissected and RNA/DNA will be extracted from microdissected cells using the Qiagen All Prep DNA/RNA FFPE Kit. Aim 2: Perform bulk RNA and DNA seq on premalignant FFPE samples and compare the genomic/transcriptomic alterations within and across organ sites. Rationale: There have been limited studies characterizing the genomic and transcriptomic landscape of premalignant lesions associated with breast, pancreatic, lung or prostate cancers. Characterizing the molecular determinants of premalignant disease that are unique and shared across multiple organs will enable new candidate biomarkers for early detection and novel therapeutic strategies for early intervention. MethodsBulk RNA-seq of LCM FFPE tissue: All participating sites will perform bulk RNA-seq in accordance with SOPs developed at BU. In brief, total RNA will be isolated from LCM'd lesion and associated microenvironment tissue using the Qiagen All Prep DNA/RNA FFPE Kit and quality will be assessed with the Agilent Bioanalyzer. Libraries will be generated with the Illumina TruSeq Access kit (for FFPE samples). They will be sequenced on the Illumina HiSeq2500 with 75base-pair paired-end reads. Quality of FASTQ files will be assessed with FastQC. Reads will be aligned to the human genome with STAR and gene-level and isoform-level expression will be quantified with RSEM. Splice junction saturation, transcript integrity, and biotype distributions will be calculated for each sample with RSeQC. DESeq2 and EdgeR will be used to identify associations between gene expression profiles and clinical variables while controlling for confounding covariates. BU will serve as an RNA-seq Core to assess reproducibility of FFPE RNA-seq methods across sites. We will perform RNA-seq according to the SOP listed above on a subset of samples for each organ type (total n ~ 20). Bulk seq of DNA from FFPE tissue: All participating sites will perform targeted or whole exome-seq (WES) in accordance with SOPs. In brief, DNA from laser captured material will be isolated using the Qiagen All Prep DNA/RNA FFPE Kit and undergo stringent quality control to ensure high quality input material for genomic profiling. Purified DNA (ideally 100-200 ng) will be used for library preparation and amplification, followed by next generation sequencing using standard protocols distributed by CDMG. Exome-seq methods are considered standardized, thus we will not need a DNA-seq Core to assess reproducibility across sites. We anticipate local centers will use Illumina paired end reads, following the following general approach. 1) DNA library preparation: Paired-end libraries will be prepared following the manufacturer's protocols (Illumina and Agilent), fragmented to 150-200 bp 2) Capture of targeted exome: Whole exome capture will be carried out using the protocol for Agilent's SureSelect Human All Exon kit. Purified capture products will be amplified using the SureSelect GA PCR primers (Agilent) for 12 cycles. 3) Sequencing will be carried out for the captured libraries using at least 100 bp paired-end reads. To achieve high level sensitivity and accuracy for detecting all the mutations in the whole exome, each sample will be sequenced at 200X mean depth. 4) Read mapping and alignment and variant analysis: Sequence short reads will be aligned to a reference genome (NCBI human genome assembly build 38) using BWA-MEM. Local realignment of aligned reads will be performed using Genome Analysis Toolkit (GATK).Data QC: To ensure scientific rigor and consistency among sites in RNA and DNA processing we will include a preliminary analysis of steps in processing and analysis. Protocols for extraction of high quality RNA and DNA from formalin fixed paraffin embedded (FFPE) tissues, which will be used extensively in these studies continue to improve and may have variable implementation among the sites participating in this study. To evaluate consistency of preliminary steps in processing and downstream analyses, we will initially distribute slides from one large FFPE fixed cancer of origin from prostate, breast, lung and pancreatic cancer. Analysis of these samples will allow us to review the DNA and RNA characteristics (yield, purity and strand length) among sites. Downstream analysis of these same samples will also allow us to compare among sites the consistency of variant calls among centers. We will be able to identify if there are some times of calls (such as small insertion deletions) that are more variable among centers versus other types of calls (such as relative gene expression or single base pair substitutions) that we expect to be less variable and to characterize the reliability of findings across sites. We are also including a 5% blind duplicate analysis of RNA sequencing. Samples will be analysed by the participating genomics cores without knowledge of the phenotype. RNA seq and CNA analyses are normalized for batch effects. We will also compare the observed sex to the self-reported sex as based on RNA profiles and exome sequencing of X chromosome genes as another check for processing accuracy and sample management. D. References 1. Wacholder, S. Precursors in Cancer Epidemiology: Aligning Definition and Function. Cancer Epidemiol. Prev. Biomark. 22, 521-527 (2013). PMID: 23549395.2. Berman, J. J. Precancer: The Beginning and the End of Cancer. (Jones & Bartlett Learning, 2011).3. Nasiell, K., Nasiell, M. & Vaćlavinková, V. Behavior of moderate cervical dysplasia during long-term follow-up. Obstet. Gynecol. 61, 609-614 (1983). PMID: 6835614.4. Merrick, D. T. et al. Persistence of Bronchial Dysplasia Is Associated with Development of Invasive Squamous Cell Carcinoma. Cancer Prev. Res. (Phila. Pa.) 9, 96-104 (2016). PMID: 26542061.5. Gurel, B. et al. Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis. Mod. Pathol. Off. J. U. S. Can. Acad. Pathol. Inc 21, 1156-1167 (2008). PMID: 18567993.6. Meeker, A. K. et al. Telomere shortening is an early somatic DNA alteration in human prostate tumorigenesis. Cancer Res. 62, 6405-6409 (2002). PMID: 12438224.7. Guner, G. et al. Novel Assay to Detect RNA Polymerase I Activity In Vivo. Mol. Cancer Res. MCR 15, 577-584 (2017). PMID: 28119429.

  Study phs002225

• Type 2 Diabetes Genetic Exploration by Next-generation sequencing in multi-Ethnic Samples (T2D-GENES)

  The T2D-GENES/GoT2D 13K exome sequencing study includes ~13,000 samples, half T2D cases and half T2D controls, from five ancestries (~5K Europeans, ~2K each of African-American, East-Asian, South-Asian, and Hispanic). Samples underwent deep exome sequencing, with SNVs and INDEls called according to GATK best practices; variant sites were then filtered according to the GATK best practices, and then samples and variants underwent further filtering based on aggregate genotype quality as described in Fuchsberger et al. (e.g. low call rate, excess heterozygosity for samples, low call rate or coverage for variants). Please note that one of the samples in the T2D-GENES vcf does not have phenotype data.

  Dataset EGAD00001002246

• Use of Deep Sequencing to Dectect Clonal Mutations In Sun Exposed Skin Epidermis PART2

  This study aims to define the landscape of somatic mutations in sun exposed human skin by deep sequencing, analyse their frequency and use the data to infer the effect of mutations on proliferating cell behaviour. The frequency of each mutation will reflect the size of the clone of cells in the tissue sample. By analyzing small samples, clones with as few as 100 cells will be detectable. Allele frequency distributions for each mutation will be used to infer cell fate using published methods (Klein et al. 2010). This study will shed unprecedented light on the early clonal events that lead to the emergence of cancer.

  Dataset EGAD00001000825

• Use of Deep Sequencing to Dectect Clonal Mutations In Sun Exposed Skin Epidermis

  This study aims to define the landscape of somatic mutations in sun exposed human skin by deep sequencing, analyse their frequency and use the data to infer the effect of mutations on proliferating cell behaviour. The frequency of each mutation will reflect the size of the clone of cells in the tissue sample. By analyzing small samples, clones with as few as 100 cells will be detectable. Allele frequency distributions for each mutation will be used to infer cell fate using published methods (Klein et al. 2010). This study will shed unprecedented light on the early clonal events that lead to the emergence of cancer.

  Dataset EGAD00001001090

• GoT2D: Genetics of Type 2 Diabetes, a study of the the genetic architecture of type 2 diabetes

  The GoT2D study includes ~2800 samples, half T2D cases and half T2D controls, of Northern European ancestry sequenced over 3 three technologies: deep whole exome sequencing, low-pass (4x) whole genome sequencing, and OMNI 2.5M genotyping. Samples were ascertained to be phenotypically "extreme" (e.g. leaner, younger cases and older, more obese controls). Genotypes (SNVs, INDELs, and SVs) were called separately for each technology and then integrated via genotype refinement into a single phased reference panel; samples and variants were then excluded based on QC procedures described in Fuchsberger et al. Please note that 2 of the samples in the GoT2D vcf do not have phenotype data.

  Dataset EGAD00001002247

• DPY30 ChIP-seq

  H9 human embryonic stem cells (hESCs) were cultured in feeder-free chemically-defined conditions in medium containing 10ng/ml Activin A and 12ng/ml FGF2 (Vallier L. 2011, Methods in Molecular Biology, 690: 57-66). Chromatin immunoprecipitation was performed as described in Brown S. et al. 2011. Stem Cells 29: 1176-85 by using 5ug of anti-DPY30 antibody (Sigma, cat. number HPA043761). This protocol was performed in control hESCs (expressing a scrambled shRNA) and in hESCs stably expressing an shRNA against DPY30 (Sigma, clone n. TRCN0000131112).This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Dataset EGAD00001001268

• Cross-site Concordance Evaluation of Tumor DNA and RNA Sequencing Platforms of CIMAC-CIDC Network

  The Cancer Immune Monitoring and Analysis Centers (CIMACs) and the Cancer Immunologic Data Commons (CIDC)  has engaged in efforts to harmonize Whole-exome (WES) and RNA-sequencing (RNA-seq) data from three different experimental platforms (MD Anderson, NCI MoCha lab, and Broad Institute) and are using a series of developed pipelines to process the early trial samples. To evaluate the consistency of tumor WES and RNA-seq profiling platforms across different centers, the CIMACs-CIDC conducted a systematic harmonization study. DNA and RNA were centrally extracted from fresh frozen (FF) and formalin-fixed paraffin-embedded (FFPE) non-small cell lung carcinoma (NSCLC) tumors and distributed to three centers for WES and RNA-seq profiling

  Study phs002295

• Test dataset: Sequence and variant data from public 1000 Genomes Project

  This is a test dataset derived from public data of the 1000 Genomes Project. Its purpose is not to allow for any inference about cohort data or results, but to aid bioinformaticians in the technical development and testing of tools, as well as data consumers in learning how to access information. This dataset consists of 2508 samples from the 1000 Genomes Project (https://www.nature.com/articles/nature15393). Samples' (e.g. NA18534) data can be accessed through the IGSR portal (e.g. https://www.internationalgenome.org/data-portal/sample/NA18534) or their corresponding folder at the 1000 Genomes' FTP site (e.g. http://ftp.1000genomes.ebi.ac.uk/vol1/ftp/data_collections/1000_genomes_project/data/CHB/NA18534/exome_alignment/). There are several different types of data this dataset encompasses: Variant Calling Format (VCF, or its binary counterparts BCF) files, both joint (e.g. ALL_chr22_20130502_2504Individuals.vcf.gz) and split (HG01775.chrY.vcf.gz); exome sequencing CRAM files (e.g. NA18534.GRCh38DH.exome.cram); whole genome sequencing CRAM/BAM files (e.g. NA19239.cram). Additionally, there are multiple files that were sliced to create shorter files, which allows for a quick download, formated as "{FILE-INFO}__{NUMBER-OF-READS}r__{CHR}.{START-COORDINATE}-{END-COORDINATE}.{FILETYPE}" (e.g. "HG01500.GRCh38DH__90r__3.10000-10500__4.10000-10500.cram"). These files can be downloaded directly through the EGA-download-client PyEGA3 (https://github.com/EGA-archive/ega-download-client).

  Dataset EGAD00001003338

• Uncovering the Genetic Architecture of Colorectal Cancer with Focus of Rare and Less Frequent Variants

  The Genetics and Epidemiology of Colorectal Cancer Consortium (GECCO) is a collaborative effort comprised of a coordinating center and scientific researchers from well-characterized cohort and case-control studies conducted in North America and Europe. This international consortium aims to accelerate the discovery of common and rare genetic risk variants for colorectal cancer by conducting large-scale meta-analyses of existing and newly generated genome-wide association study (GWAS) data, replicating and fine-mapping of GWAS discoveries, and investigating how genetic risk variants are modified by environmental risk factors. To expand these efforts, we assembled case-control sets or nested case-control sets from 20 different North American or European studies. Summary descriptions and study participant inclusions/exclusion criteria for each of these studies are detailed below. The Black Women's Health Study (BWHS): Is the largest follow-up study of the health of African-American women (Cozier et al., 2004; Rosenberg et al., 1995) [PMID: 15018884; PMID: 7722208]. The purpose is to identify and evaluate causes and preventives of cancers and other serious illnesses in African-American women. Among the diseases being studied are breast cancer, colorectal cancer, type 2 diabetes, uterine fibroids, systemic lupus erythematosus, and cardiovascular disease. The study began in 1995, when 59,000 black women from all parts of the United States enrolled through postal questionnaires. The women provided demographic and health data on the 1995 baseline questionnaire, including information on weight, height, smoking, drinking, contraceptive use, use of other selected medications, illnesses, reproductive history, physical activity, diet, use of health care, and other factors. The participants are followed through biennial questionnaires to determine the occurrence of cancers and other illnesses and to update information on risk factors. Self-reports of cancer are confirmed through medical records and state cancer registry records. Mouthwash-swish samples, as a source of DNA, were obtained from ~26,000 BWHS participants in 2002-2007. DNA was isolated from the mouthwash-swish samples at the Boston University Molecular Core Genetics Laboratory using the QIAAMP DNA Mini Kit (Qiagen). All incident colorectal cancer cases with a DNA sample were included in the present analysis. Two controls per case, selected from among BWHS participants free of colorectal cancer at end of follow-up, were matched to cases on year of birth (+/- 2 years) and geographical region of residence (Northeast, South, Midwest, and West). A total 209 colorectal cancer cases and 423 controls were sent for genotyping. Campaign Against Cancer and Heart Disease (CLUE II): The Campaign Against Cancer and Heart Disease, is a prospective cohort designed to identify biomarkers and other factors associated with risk of cancer, heart disease, and other conditions (Kakourou et al., 2015) [PMID: 26220152]. 32,894 participants were recruited from May through October 1989 from Washington County, Maryland and surrounding communities. Colorectal cancer cases (n = 297) and matched controls (n = 296) were identified between 1989 and 2000 among participants in the CLUE II cohort of Washington County, Maryland. Colorectal Cancer Study of Austria (CORSA): In the ongoing colorectal cancer study of Austria (CORSA), more than 13,000 Caucasian participants have been recruited within the province-wide screening project "Burgenland Prevention Trial of Colorectal Disease with Immunological Testing" (B-PREDICT) since 2003 (Hofer et al., 2011) [PMID: 21422235]. All inhabitants of the Austrian province Burgenland aged between 40 and 80 years are annually invited to participate in fecal immunochemical testing and haemoccult positive screening participants are invited for colonoscopy. CORSA includes genomic DNA and plasma of colorectal cancer cases, low-risk and high-risk adenomas, and colonoscopy-negative controls. Controls received a complete colonoscopy and were free of colorectal cancer or polyps. CORSA participants have been recruited in the four KRAGES hospitals in Burgenland, Austria, and additionally, at the Medical University of Vienna (Department of Surgery), the Viennese hospitals "Rudolfstiftung" and the "Sozialmedizinisches Zentrum Sud", and at the Medical University of Graz (Department of Internal Medicine). 1403 colorectal cancer and advanced colorectal adenoma cases, and 1404 matched controls were selected for the study. Distribution of factors sex and age (5 year strata) were evenly matched between cases and controls. Cancer Prevention Study II (CPS II): The CPS II Nutrition cohort is a prospective study of cancer incidence and mortality in the United States, established in 1992 and described in detail elsewhere (Calle et al., 2002; Campbell et al., 2014) [PMID: 12015775; PMID: 25472679]. At enrollment, participants completed a mailed self-administered questionnaire including information on demographic, medical, diet, and lifestyle factors. Follow-up questionnaires to update exposure information and to ascertain newly diagnosed cancers were sent biennially starting in 1997. Reported cancers were verified through medical records, state cancer registry linkage, or death certificates. The Emory University Institutional Review Board approves all aspects of the CPS II Nutrition Cohort. A total of 360 cases and 359 controls were selected for this study. Czech Republic Colorectal Cancer Study (Czech Republic CCS): Cases with positive colonoscopy results for malignancy, confirmed by histology as colon or rectal carcinomas, were recruited between September 2003 and May 2012 in several oncological departments in the Czech Republic (Prague, Pilsen, Benesov, Brno, Liberec, Ples, Pribram, Usti and Labem, and Zlin). Two control groups, sampled at the same time of cases recruitment, were included in the study. The first group consisted of hospital-based individuals with a negative colonoscopy result for malignancy or idiopathic bowel diseases. The reasons for the colonoscopy were: i) positive fecal occult blood test, ii) hemorrhoids, iii) abdominal pain of unknown origin, and iv) macroscopic bleeding. The second control group consisted of healthy blood donor volunteers from a blood donor center in Prague. All individuals were subjected to standard examinations to verify the health status for blood donation and were cancer-free at the time of the sampling. Details of CRC cases and controls have been reported previously (Vymetalkova et al., 2014; Naccarati et al., 2016; Vymetalkova et al., 2016) [PMID: 24755277; PMID: 26735576; PMID: 27803053]. All subjects were informed and provided written consent to participate in the study. They approved the use of their biological samples for genetic analyses, according to the Declaration of Helsinki. The design of the study was approved by the Ethics Committee of the Institute of Experimental Medicine, Prague, Czech Republic. All subjects included in the study were Caucasians and comprised 1792 cases and 1764 matched controls. Controls were matched to CRC cases as 1:1 ratio. Matching was done on age and sex. Age was matched on +-5 years, whereas sex was matched exactly. For the cases without matched controls, matching was done only on sex. Early Detection Research Network (EDRN): The aim of the EDRN initiative is to develop and sustain a biorepository for support of translational research (Amin et al., 2010) [PMID: 21031013]. High-quality biospecimens were accrued and annotated with pertinent clinical, epidemiologic, molecular and genomic information. A user-friendly annotation tool and query tool was developed for this purpose. The various components of this annotation tool include: CDEs are developed from the College of American Pathologists (CAP) Cancer Checklists and North American Association of Central Cancer Registries (NAACR) standards. The CDEs provides semantic and syntactic interoperability of the data sets by describing them in the form of metadata or data descriptor. A total of 352 colorectal case samples and 399 controls were selected for this study. Controls were matched to CRC cases based on age and sex. The EPICOLON Consortium (EPICOLON): The EPICOLON Consortium comprises a prospective, multicentre and population-based epidemiology survey of the incidence and features of CRC in the Spanish population (Fernandez-Rozadilla et al., 2013) [PMID: 23350875]. Cases were selected as patients with de novo histologically confirmed diagnosis of colorectal adenocarcinoma. Patients with familial adenomatous polyposis, Lynch syndrome or inflammatory bowel disease-related CRC, and cases where patients or family refused to participate in the study were excluded. Hospital-based controls were recruited through the blood collection unit of each hospital, together with cases. All of the controls were confirmed to have no history of cancer or other neoplasm and no reported family history of CRC. Controls were randomly selected and matched with cases for hospital, sex and age (+- 5 years). A total of 370 cases and 370 controls were selected for genotyping. Hawaii Adenoma Study: For this adenoma study, two flexible-sigmoidoscopy screening clinics were first used to recruit participants on Oahu, Hawaii. Adenoma cases were identified either from the baseline examination at the Hawaii site of the Prostate Lung Colorectal and Ovarian cancer screening trial during 1996-2000 or at the Kaiser Permanente Hawaii's Gastroenterology Screening Clinic during 1995-2007. In addition, starting in 2002 and up to 2007, we also approached for recruitment all eligible patients who underwent a colonoscopy in the Kaiser Permanente Hawaii Gastroenterology Department. Cases were patients with histologically confirmed first-time adenoma(s) of the colorectum and were of Japanese, Caucasian or Hawaiian race/ethnicity. Controls were selected among patients with a normal colorectum and were individually matched to the cases on age at exam, sex, race/ethnicity, screening date (+-3 months) and clinic and type of examination (colonoscopy or flexible sigmoidoscopy). We recruited 1016 adenoma cases (67.8% of all eligible) and 1355 controls (69.2% of all eligible); 889 cases and 1169 controls agreed to give a blood and 29 cases and 34 controls, a mouthwash sample. A total of 989 cases and 1185 controls were genotyped for this study. Columbus-area HNPCC Study (HNPCC, OSUMC): Patients with colorectal adenocarcinoma diagnosed at six participating hospitals were eligible for this study, regardless of age at diagnosis or family history of cancer. Patients with a clinical diagnosis of familial adenomatous polyposis were not eligible for this study. These six hospitals perform the vast majority of all operations for CRC in the Columbus metropolitan area (population 1.7 million). The institutional review board at all participating hospitals approved the research protocol and consent form in accordance with assurances filed with and approved by the United States Department of Health and Human Services. Briefly, during the period of January 1999 through August 2004, 1,566 eligible patients with CRC were accrued to the study (Hampel et al., 2008) [PMID 18809606]. A total of 1472 colorectal cancer samples had enough blood DNA remaining to be sent for genotyping. Control samples were provided by the Ohio State University Medical Center%#39;s (OSUMC) Human Genetics Sample Bank. The Columbus Area Controls Sample Bank is a collection of control samples for use in human genetics research that includes both donors' anonymized biological specimens and linked phenotypic data. The data and samples are collected under the protocol "Collection and Storage of Controls for Genetics Research Studies", which is approved by the Biomedical Sciences Institutional Review Board at OSUMC. Recruitment takes place in OSUMC primary care and internal medicine clinics. If individuals agree to participate, they provide written informed consent, complete a questionnaire that includes demographic, medical and family history information, and donate a blood sample. 4-7 ml of blood is drawn into each of 3 ACD Solution A tubes and is used for genomic DNA extraction and the establishment of an EBV-transformed lymphoblastoid cell culture, cell pellet in Trizol, and plasma. Controls were matched to CRC cases as 1:1. Matching was done on age at reference time (age_ref), race, and sex. Age_ref was matched on +-5 years. Sex and race were matched exactly. For the cases without matched controls, matching was done only on sex and race with 1:1 ratio. Since controls are fewer than cases, one control is matched on 2 cases at most. Health Professionals Follow-up Study (HPFS): A parallel prospective study to the NHS (Nurses' Health Study). The HPFS cohort comprised 51,529 men aged 40-75 who, in 1986, responded to a mailed questionnaire (Rimm et al., 1990) [PMID: 2090285]. Participants provided information on health related exposures, including current and past smoking history, age, weight, height, diet, physical activity, aspirin use, and family history of colorectal cancer. Colorectal cancer and other outcomes were reported by participants or next-of-kin and were followed up through review of the medical and pathology record by physicians. Overall, more than 97% of self-reported colorectal cancers were confirmed by medical record review. Information was abstracted on histology and primary location. Incident cases were defined as those occurring after the subject provided the blood sample. Prevalent cases were defined as those occurring after enrollment in the study but before the subject provided the blood sample. Follow-up evaluation has been excellent, with 94% of the men responding to date. Colorectal cancer cases were ascertained through January 1, 2008. In 1993-1995, 18,825 men in the HPFS mailed blood samples by overnight courier, which were aliquoted into buffy coat and stored in liquid nitrogen. In 2001-2004, 13,956 men in the HPFS who had not provided a blood sample previously mailed in a swish-and-spit sample of buccal cells. Incident cases were defined as those occurring after the subject provided a blood or buccal sample. Prevalent cases were defined as those occurring after enrollment in the study in 1986, but before the subject provided either a blood or buccal sample. After excluding participants with histories of cancer (except nonmelanoma skin cancer), ulcerative colitis, or familial polyposis, case-control sets were previously constructed. In addition to colorectal cancer cases and controls, a set of adenoma cases and matched controls with available DNA from buffy coat were selected for genotyping. Over the follow-up period, data were collected on endoscopic screening practices and, if individuals had been diagnosed with a polyp, the polyps were confirmed to be adenomatous by medical record review. Adenoma cases were ascertained through January 1, 2008. A separate case-control set was constructed of participants diagnosed with advanced adenoma matched to control participants who underwent a lower endoscopy in the same time period and did not have an adenoma. Advanced adenoma was defined as an adenoma 1 cm or larger in diameter and/or with tubulovillous, villous, or highgrade dysplasia/carcinoma-in-situ histology. Matching criteria included year of birth (within 1 year) and month/ year of blood sampling (within 6 months), the reason for their lower endoscopy (screening, family history, or symptoms), and the time period of any prior endoscopy (within 2 years). Controls matched to cases with a distal adenoma either had a negative sigmoidoscopy or colonoscopy examination, and controls matched to cases with proximal adenoma all had a negative colonoscopy. In total, 159 advanced adenoma cases and 109 controls were selected for genotyping. Leeds Colorectal Cancer Study (LCCS): Following local ethical approval, colorectal cancer cases were recruited from 1997 until 2012 in Leeds, UK through surgical clinics. Initially, funding was provided by the UK Ministry of Agriculture, Farming and Fisheries (subsequently the Food Standards Agency) and Imperial Cancer Research Fund (subsequently Cancer Research UK). Recruitment also occurred similarly in Dundee, Perth and York between the periods of 1997 and 2001 using the same protocol and the data and samples were combined. Pathologically confirmed cases were consented at outpatient clinics, providing information on known and postulated risk factors for colorectal cancer (diet, lifestyle and family history) as well as providing a blood sample for DNA. Exclusion criteria included pre-existing diverticular disease and an inability to complete the questionnaire. The General Practitioners of cases (all UK residents have a nominated General Practitioner to whom to refer initial medical queries) and these GPs were asked to send letters to other persons on their patient list of the same gender and born within 5 years of the case. Subsequently to enhance the number of controls, we systematically invited patients from selected GP practices. Diet was assessed in cases and controls using an extensive dietary and lifestyle questionnaire modified by that produced by the European Prospective Investigation in Cancer (EPIC). The frequency that each specific food items were eaten was recorded and we also obtained average fruit and vegetable consumption as a cross-check. In total, 1591 cases and 739 controls provided a DNA sample. The North Carolina Colon Cancer Studies (NCCCS I/II): The North Carolina Colon Cancer Studies (NCCCS I- colon and NCCCS II-rectal) were population-based case-control studies conducted in 33 counties of North Carolina. Cases were identified using the rapid case ascertainment system of the North Carolina Central Cancer Registry. Patients with a first diagnosis of histologically confirmed invasive adenocarcinoma of the colon (cecum through sigmoid colon) between October 1996 and September 2000 were classified as potential cases in the NCCCS I. The NCCCS II included patients with a first diagnosis of histologically confirmed invasive adenocarcinoma of the sigmoid colon, rectosigmoid, or rectum (hereafter collectively referred to as rectal cancer) between May 2001 and September 2006. Additional eligibility requirements were: aged 40-80 years, residence in one of the 33 counties, ability to give informed consent and complete an interview, had a driver's license or identification card issued by the North Carolina Department of Motor Vehicles (if under the age of 65), and had no objections from the primary physician in regards to contacting the individual. Controls, identified and sampled during the respective study dates, were selected from two sources. Potential controls under the age of 65 were identified using the North Carolina Department of Motor Vehicles records. For those 65 years and older, records from the Center for Medicare and Medicaid Services were used. Controls were matched to cases using randomized recruitment strategies. Recruitment probabilities were done using strata of 5-year age, sex, and race groups. Dietary information was collected using a modified version of the semiquantitative food frequency questionnaire developed at the National Cancer Institute. In addition, participants were asked about vitamin and mineral supplementation, special diets, restaurant eating, sodium use, and fats used in cooking. In NCCCS I, 515 colorectal cases and 687 matched controls were sent for genotyping. In NCCCS II, 796 colorectal cases and 823 controls were sent from the NCCCS II for genotyping. Controls were matched to CRC cases as 1:1 ratio. Matching was done on age, race, and sex. Age was matched on +-5 years. Race and sex was matched exactly. For the cases without matched controls, matching was done only on sex and race. Nurses Health Study (NHS): The NHS cohort began in 1976 when 121,700 married female registered nurses age 30-55 years returned the initial questionnaire that ascertained a variety of important health-related exposures (Belanger et al., 1978) [PMID: 248266]. Since 1976, follow-up questionnaires have been mailed every 2 years. Colorectal cancer and other outcomes were reported by participants or next-of-kin and followed up through review of the medical and pathology record by physicians. Overall, more than 97% of self-reported colorectal cancers were confirmed by medical-record review. Information was abstracted on histology and primary location. The rate of follow-up evaluation has been high: as a proportion of the total possible follow-up time, follow-up evaluation has been more than 92%. Colorectal cancer cases were ascertained through June 1, 2008. In 1989 -1990, 32,826 women in NHS I mailed blood samples by overnight courier, which were aliquoted into buffy coat and stored in liquid nitrogen. In 2001-2004, 29,684 women in NHS I who did not previously provide a blood sample mailed a swish-and-spit sample of buccal cells. Incident cases were defined as those occurring after the subject provided a blood or buccal sample. Prevalent cases were defined as those occurring after enrollment in the study in 1976 but before the subject provided either a blood or buccal sample. After excluding participants with histories of cancer (except nonmelanoma skin cancer), ulcerative colitis, or familial polyposis, case-control sets were previously constructed from which DNA was isolated from either buffy coat or buccal cells for genotyping. In addition to colorectal cancer cases and controls, a set of advanced adenoma cases and matched controls with available DNA from buffy coat were selected for genotyping. Over the follow-up period, data were collected on endoscopic screening practices and, if individuals had been diagnosed with a polyp, the polyps were confirmed to be adenomatous by medical record review. Adenoma cases were ascertained through June 1, 2011. A separate case-control set was constructed of participants diagnosed with advanced adenoma matched to control participants who underwent a lower endoscopy in the same time period and did not have an adenoma. Advanced adenoma was defined as an adenoma more than 1 cm in diameter and/or with tubulovillous, villous, or high-grade dysplasia/carcinoma-in-situ histology. Matching criteria included year of birth (within 1 year) and month/year of blood sampling (within 6 months), the reason for their lower endoscopy (screening, family history, or symptoms), and the time period of any prior endoscopy (within 2 years). Controls matched to cases with a distal adenoma either had a negative sigmoidoscopy or colonoscopy examination, and controls matched to cases with proximal adenoma all had a negative colonoscopy. A total of 272 cases and 236 matched controls were sent to CIDR for the advanced adenoma case-control set. Northern Swedish Health and Disease Study (NSHDS): Comprises over 110,000 participants, including approximately one third with repeated sampling occasions, from three population-based cohorts (Dahlin et al., 2010; Myte et al., 2016) [PMID: 20197478; PMID: 27367522]. The largest is the ongoing Vasterbotten Intervention Programme, in which all residents of Vasterbotten County are invited to a health examination upon turning 30 (some years), 40, 50 and 60 years of age. Extensive measured and self-reported health and lifestyle data, as well as blood samples for central biobanking in Umea, Sweden, are collected at the health exam. Leucocyte DNA samples for 1:1-matched CRC case-control sets from the NSHDS, of which 878 samples are included in this study, have been selected for genotyping. This is in addition to 354 samples from the NSHDS previously analyzed as part of the multicenter EPIC cohort. Cancer-specific and overall survival data are available for all patients. For at least 425 patients, archival tumor tissue has been analyzed for the BRAF V600E mutation and by sequencing codon 12 and 13 for KRAS mutations, as well as for MSI screening status by immunohistochemistry and for an eight-gene CIMP panel using quantitative real-time PCR (MethyLight). Ohio Colorectal Cancer Prevention Initiative (OCCPI, OSUMC): OCCPI (ClinicalTrials.gov identifier: NCT01850654) is a population-based study of colorectal cancer patients diagnosed in one of 51 hospitals throughout the state of Ohio from January 1, 2013 through December 31, 2016. The OCCPI was created to decrease CRC incidence in Ohio by identifying patients with hereditary predisposition (statewide universal tumor screening for newly diagnosed CRC patients), increase colonoscopy compliance for first-degree relatives of CRC patients, and encourage future research through the creation of a biorepository. The 51 Ohio hospitals participating in the OCCPI were selected to represent a cross-section of clinical centers in the state based on high reported volume of CRC patients, affiliation with a high volume hospital, or interest in participation. Institutional Review Board (IRB) approval was obtained by the individual hospitals, Community Oncology Programs, or by ceding review to the OSU IRB. Written informed consent was obtained. A total of 2139 colorectal cases were genotyped. Patients were considered eligible for this study if they were age 18 or older at the time of enrollment, if they had a surgical resection (or biopsy if unresectable) in the state of Ohio demonstrating an adenocarcinoma of the colorectum from 1/1/13 - 12/31/16. Matched control samples were selected from the Ohio State University Medical Center's (OSUMC) Human Genetics Sample Bank in an identical way to the selection for the Columbus-area HNPCC Study (please refer to the description for the Columbus-area HNPCC Study). Prostate, Lung, Colorectal and Ovarian Cancer Screening Trail (PLCO): PLCO enrolled 154,934 participants (men and women, aged between 55 and 74 years) at ten centers into a large, randomized, two-arm trial to determine the effectiveness of screening to reduce cancer mortality. Sequential blood samples were collected from participants assigned to the screening arm. Participation was 93% at the baseline blood draw. In the observational (control) arm, buccal cells were collected via mail using the "swish-and-spit" protocol and participation rate was 65%. Details of this study have been previously described (Huang et al., 2016) [PMID: 27673363] and are available online (http://dcp.cancer.gov/plco). For this study 1651 advanced adenoma cases and 1392 controls were selected for genotyping. Selenium and Vitamin E Prevention Trial (SELECT): The Selenium and Vitamin E Cancer Prevention Trial (SELECT) was a double-blind, placebo controlled clinical trial which explored using selenium and vitamin E alone and in combination to prevent prostate cancer in healthy men (Lippman et al., 2009) [PMID: 19066370]. Secondary endpoints included the prevention of colorectal and lung cancers. SELECT was conducted at 427 sites and centers in the United States, Canada and Puerto Rico; 35,533 men 55 years and older (50 or older if African American) were randomized beginning August 22, 2001. Supplementation was discontinued on October 23, 2008 due to futility. 308 colorectal cancer cases and 308 matched controls were selected from the SELECT population and sent for genotyping. Screening Markers For Colorectal Disease Study and Colonoscopy and Health Study (SMS-REACH): Details on this study population were previously reported (Burnett-Hartman et al., 2014) [PMID: 24875374]. Participants were enrollees in an integrated health-care delivery system in western Washington State (Group Health Cooperative, Seattle, Washington) aged 24-79 years who underwent an index colonoscopy for any indication between 1998 and 2007 and donated a buccal-cell or blood sample for genotyping analysis. Study recruitment took place in 2 phases, with phase 1 occurring in 1998-2003 and phase 2 occurring in 2004-2007. Persons who had undergone a colonoscopy less than 1 year prior to the index colonoscopy, persons with inadequate bowel preparation for the index colonoscopy, and persons with a prior or new diagnosis of colorectal cancer, a familial colorectal cancer syndrome (such as familial adenomatous polyposis), or another colorectal disease were ineligible. Patients diagnosed with adenomas or serrated polyps and persons who were polyp-free at the index colonoscopy (controls) were systematically recruited during both phases of recruitment. Approximately 75% agreed to participate and provided written informed consent. Based on medical records, persons who agreed to participate and those who refused study participation were similar with respect to age, sex, and colorectal polyp status. Study protocols were approved by the institutional review boards of the Group Health Cooperative and the Fred Hutchinson Cancer Research Center (Seattle, Washington). A total of 575 cases and 508 matched were selected for the study. Controls were matched to CRC cases as 1:1 ratio. Matching was done on age_ref, race, and sex. Age_ref was matched on +-5 years. The Women's Health Initiative (WHI): WHI is a long-term national health study that has focused on strategies for preventing heart disease, breast and colorectal cancer, and osteoporotic fractures in postmenopausal women. The original WHI study included 161,808 postmenopausal women enrolled between 1993 and 1998. The Fred Hutchinson Cancer Research Center in Seattle, WA serves as the WHI Clinical Coordinating Center for data collection, management, and analysis of the WHI. The WHI has two major parts: a partial factorial randomized Clinical Trial (CT) and an Observational Study (OS); both were conducted at 40 Clinical Centers nationwide. The CT enrolled 68,132 postmenopausal women between the ages of 50-79 into trials testing three prevention strategies. If eligible, women could choose to enroll in one, two, or all three of the trial components. The components are: Hormone Therapy Trials (HT): This double-blind component examined the effects of combined hormones or estrogen alone on the prevention of coronary heart disease and osteoporotic fractures, and associated risk for breast cancer. Women participating in this component with an intact uterus were randomized to estrogen plus progestin (conjugated equine estrogens [CEE], 0.625 mg/d plus medroxyprogesterone acetate [MPA] 2.5 mg/d] or a matching placebo. Women with prior hysterectomy were randomized to CEE or placebo. Both trials were stopped early, in July 2002 and March 2004, respectively, based on adverse effects. All HT participants continued to be followed without intervention until close-out. Dietary Modification Trial (DM): The Dietary Modification component evaluated the effect of a low-fat and high fruit, vegetable and grain diet on the prevention of breast and colorectal cancers and coronary heart disease. Study participants were randomized to either their usual eating pattern or a low-fat dietary pattern. Calcium/Vitamin D Trial (CaD): This double-blind component began 1 to 2 years after a woman joined one or both of the other clinical trial components. It evaluated the effect of calcium and vitamin D supplementation on the prevention of osteoporotic fractures and colorectal cancer. Women in this component were randomized to calcium (1000 mg/d) and vitamin D (400 IU/d) supplements or a matching placebo. The Observational Study (OS)examines the relationship between lifestyle, environmental, medical and molecular risk factors and specific measures of health or disease outcomes. This component involves tracking the medical history and health habits of 93,676 women not participating in the CT. Recruitment for the observational study was completed in 1998 and participants were followed annually for 8 to 12 years. All centrally confirmed cases of invasive colorectal cancers, or deaths from colorectal cancer were selected as potential cases from September 30, 2015 database. Controls were participants free of colorectal cancer (invasive or in situ) as of September 30, 2015. Potential cases and controls were excluded if they (1) were non-White; (2) had history of colorectal cancers at baseline; (3) lost to follow-up after enrollment; (4) DbGAP ineligible; (5) had <1.25ug of DNA; (6) selected for WHI study M26 Phase I or II; (7) selected for WHI study AS224 and also included in the imputation project. A total of 578 cases and 104,429 controls met the eligibility criteria. Each case was matched with 1 control (1:1) that exactly met the following matching criteria: age (+-5 years), 40 randomization centers (exact), WHI date (+-3 years), CaD date (+-3 years), OS flag (exact), HRT assignments (exact), DM assignments (exact), and CaD assignments (exact). Control selection was done in a time-forward manner, selecting one control for each case from the risk set at the time of the case's event. The matching algorithm was allowed to select the closest match based on a criteria to minimize an overall distance measure (Bergstralh EJ, Kosanke JL. Computerized matching of cases to controls. Technical Report #56, Department of Health Sciences Research, Mayo Clinic, Rochester MN. April 1995). Each matching factor was given the same weight. When exact matches could not be found, the matching criteria were gradually relaxed among unmatched cases and controls until all cases had found matched controls. Using the matching criteria specified above, 559 of the 578 eligible cases found exact matches. The matching criteria was then relaxed to : Age+-5, randomization centers, WHI date +- 3 years, CaD date +- 3 years, OS flag, HRT flag, DM flag, CaD flag. 17 of the remaining 19 unmatched cases found matched controls. By matching on Age+-5, randomization centers, WHI date +- 3 years, CaD date +- 3 years, OS flag, HRT flag, the remaining 2 unmatched cases found their matches.

  Study phs001415

• A Case-Controlled Study for Genotype-Phenotype Associations in Multiple Sclerosis (MS)

  This is a multi-centre, case-controlled study to develop a dataset containing 1000 MS cases and 1000 matched controls and to associate DNA sequence (allelic) variations with MS phenotypes. Study subjects were enrolled through a prospective effort initiated in 2003. Three MS clinical centres were involved in subject recruitment and biological specimen collection using identical inclusion/exclusion criteria, two in Europe (Vrije Universiteit Medical Center, Amsterdam; and University Hospital Basel) and one in the US (University of California San Francisco). This study recruited subjects of northern-European ancestry with a diagnosis of MS (McDonald et al., 2001), with dissemination in time and space. Patients with Clinically Isolated Syndromes (CIS) were also included if they fulfilled 3 of the 4 Barkhof criteria for dissemination in space as per application of the McDonald criteria (McDonald et al., 2001). While recruitment predominantly included subjects with a relapsing onset of MS, individuals with all clinical subtypes of the disease participated, including clinically isolated syndrome (CIS), relapsing remitting MS (RRMS), secondary progressive MS (SPMS), primary progressive MS (PPMS), and progressive relapsing MS (PRMS). The control group consisted of unrelated individuals, primarily spouses/partners, friends, and other volunteers. Control subjects were of northern-European ancestry and matched as a group, proportionally with cases according to age (±5 years) and gender. A familial history or current diagnosis of MS as well as a relation to another case or control subject were considered exclusionary for this group. Protocols were approved by the Committees on Human Research at all Institutions and informed consent was obtained from all participants prior to participation in the study. Primary Study Objective:To identify DNA sequence variations (genotype) and flanking sequences that are associated with clinical factors (phenotype) which differ between study subjects with and without MS. Secondary Study Objectives: To develop a clinical dataset including quantitative measures of 1000 well-characterized cases with MS, and 1000 ethnically matched controls. To identify other genotype-phenotype associations in MS study subjects such as magnetic resonance imaging (MRI) measures of disease burden and/or severity. To identify or confirm candidate surrogate markers of neurodegeneration using a variety of techniques including biochemical assays, blood transcriptome analysis, plasma proteomics and MRI*. GenotypingGenotyping of the complete dataset was performed at the Illumina facilities using the Sentrix® HumanHap550 BeadChip. *MRI results are not available on dbGaP.

  Study phs000171

• High hyperdiploid ALL single cell whole genome sequencing

  This dataset was collected from viable bone marrow cells obtained at diagnosis from nine patients with high hyperdiploid ALL and one normal bone marrow sample. All samples were subjected to low pass single cell whole genome sequencing with the median sequencing coverage of 0.02x. Single nuclei in G0/G1 phase were isolated using a fluorescence-activated cell sorting (FACS) cytometer. DNA libraries were constructed and associated next-generation sequencing was carried out by European Research Institute for the Biology of Ageing (ERIBA), University of Groningen, University Medical Center Groningen, Groningen, The Netherlands. Further details regarding the DNA libraries construction are available by Bos et. al., 2019 (https://link.springer.com/protocol/10.1007/978-1-4939-8931-7_15).

  Dataset EGAD00001008988

• scRNA-seq and snRNA-seq of trophoblast stem cells (TSCs) differentiation into extravillous trophoblast organoids (EVTs)

  This dataset is part of a study that aims to compare in vivo human trophoblast differentiation into EVTs to different in vitro trophoblast organoids using single-cell and single-nuclei RNA sequencing. This specific dataset includes scRNA-seq and snRNA-seq data from trophoblast stem cells (TSCs). Trophoblast stem cell (TSC) lines BTS5 and BTS11 derived by Okae and colleagues were grown as described previously (Okae et al. 2018) together with EVT differentiation media. This study shows that the main regulatory programs mediating EVT invasion in vivo are preserved in in vitro models of EVT differentiation from primary trophoblast organoids and trophoblast stem cells. Data for primary trophoblast organoids is available under E-MTAB-12650.

  Dataset EGAD00001010017

• Longitudinal study of whole blood gene expression in Kenyan children exposed to malaria

  We have collected RNA samples from whole blood of Kenyan children exposed to malaria in the Kilifi region of Kenya. Collections were performed each year from 2015 until 2018. This is a follow-up study to that described in Bediako et al. (in preparation). The SIMS consortium is seeking to identifying the underlying reasons why some children are more susceptible to malaria than others. In this study we hope to track changes in children’s immune systems over time which relate to the number of malaria episodes they experience. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/

  Dataset EGAD00001015405

• An integrated single-cell reference atlas of the human endometrium

  This dataset includes both the anchor scRNA-seq and snRNA-seq datasets used to build the Human Endometrial Cell Atlas (HECA). HECA provides a comprehensive definition of endometrial cell types and states throughout the menstrual cycle in donor with and without endometriosis. It identifies consensus cell types across datasets generated by teams worldwide, as well as previously unreported cell types, all of which are validated and mapped in situ using spatial transcriptomics. Processed data is available from ArrayExpress with accession number E-MTAB-14039. See Marečková, M., Garcia-Alonso, L., Moullet, M. et al. An integrated single-cell reference atlas of the human endometrium. Nat Genet 56, 1925–1937 (2024) https://doi.org/10.1038/s41588-024-01873-w for more information.

  Dataset EGAD00001015446

• How to upload GPG files

  Uploading files Users that holds 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. In order to manage the available resources, we enforce a limit of 10Tb per submission account at any one time. Please do not exceed this limit. INBOX FTP The FTP is only compatible with files encrypted using the EGACryptor tool Before uploading Once your submission files have been prepared using the EGAryptor, the resulting encrypted files and associated md5sum files can be uploaded to your submission account using Aspera or FTP. The EGA is a shared, public service with limited resources. In order to manage the available resources, EGA submission boxes should not exceed 8Tb in size, and cannot exceed 12Tb. If you are approaching this limit please contact contact EGA Helpdesk so that we can advise on how to register the associated metadata and trigger the archiving of files, so that you can continue with your submission. If we note that your submission account increases above 10Tb on a consistent base your password will be changed until metadata is associated. Aspera Download Aspera Using Aspera FTP FTP windows FTP Linux / Unix FTP client (Filezilla) FTP and TLS Troubleshooting Troubleshooting Aspera Download Aspera is a commercial file transfer protocol that may provide faster transfer speeds than ftp especially over longer distances. The Aspera ascp command line client. Please select Aspera Connect. The ascp command line client is distributed as part of the aspera connect highperformance transfer browser plugin and is free to use, without registration. The minimum required version of the IBM Aspera Cli is V4. Further instructions. Using the Aspera ascp command line program The location of the ascp program in the filesystem: Mac: on the desktop go cd /Applications/Aspera\ Connect.app/Contents/Resources/ there you'll see the command line utilities where you're going to use ascp. Windows: the downloaded files are a bit hidden. For instance, in Windows 7 the ascp.exe is located in the users home directory in: AppData\Local\Programs\Aspera\Aspera Connect\bin\ascp.exe Linux: should be in your user's home directory, cd /home/username/.aspera/connect/bin/ there you'll see the command line utilities where you're going to use ascp. Your command should look similar to this: ascp -P33001 -O33001 -QT -l300M -L- /path/file ega-box-N@fasp.ega.ebi.ac.uk:/path If you wish to upload several files without being requested the password, please use the below command : ASPERA_SCP_PASS=ega-box-password ascp -P33001 -O33001 -QT -l300M /path/file ega-box-N@fasp.ega.ebi.ac.uk:/path/ Explanation of parameters l300M option sets the upload speed limit to 30MB/s. You may wish to lower this value to increase the reliability of the transfer. L option is for printing logs out while transferring files to upload can be a file mask (e.g. '/homes/submitter/*.srf) or a list of files. ega-box-N is your submission account login. Add k2 switch for transfer restarts Check the command line transfer usage for more configuration details. Using FTP to upload your prepared files Use your preferred ftp client. For example, lftp is a popular choice for Linux and Mac users. Use binary mode for file transfers. Use ftp.ega.ebi.ac.uk as the target host. Login with your ega-box username and password. Upload files to your private ega-box upload area. Depending on your network setting you might wish to start FTP in passive or active mode. Using default FTP command line client in Windows Start the command line interpreter: press WinR, type cmd, hit enter Enter ftp ftp.ega.ebi.ac.uk Enter your submission username Enter your submission password Type binary to enter binary mode for transfer To see a list of available ftp commands type help. Type ls command to check the content of your submission account. Type prompt to switch off confirmation for each file uploaded. Use mput command to upload files: mput *.bam* Use bye command to exit the ftp client. Use exit command to exit the command line interpreter. Using default FTP command line client in Linux / Unix Open a terminal and type ftp ftp.ega.ebi.ac.uk Enter your submission username Enter your submission password Type binary to enter binary mode for transfer To see a list of available ftp commands type help. Type ls command to check the content of your submission account. Type prompt to switch off confirmation for each file uploaded. Use mput command to upload files: mput *.bam* Use bye command to exit the ftp client. Using FTP client FileZilla We recommend the use of FileZilla, a free FTP client . FileZilla is open source software distributed free of charge under the terms of the GNU General Public License. Use the following connection details (File - Site Manager) and add yoursubmission account username and password: Using FTP client FileZilla Select the files you wish to upload and then select upload: Using FTP client Filezilla Using LFTP with TLS We recommend the following to force the use of a secure connection. lftp > set ftp:ssl-force yes We also recommend setting the following for not encrypting the bulk data itself for performance reasons (theauthentication will still be encrypted): lftp > ftp:ssl-protect-data no In order to verify the certificate,the recommended way would be to use the CA certificates from your machine. To do that use this command in lftp adjusting the path to your ca-certificates location. lftp > ssl:ca-file "/etc/ssl/certs/ca-certificates.crt" If that is not possible or certificates are old and you can't update them, you can download the certificates needed from Quo Vadis Digital Repository The two certificates to download in PEM format are: QuoVadis Root CA2 G3 QuoVadis EV SSL ICA G3 Then you can concatenate them in a file one after the other and save it as lftp-certificates.pem Once this is done you have to point the ssl:ca-file variable to the path lftp > set ssl:ca-file "/path/to/lftp-certificates.pem" Also note that you can save this configuration at ~/.lftp/rc Another option is to download the certificates and add them to the ca-certificates of your machine. For example: In RHEL7 and cenots and others box the process to add the certificates globally is: Download the two certificates (in PEM or DER format, doesn't matter) and save them to "/etc/pki/ca-trust/source/anchors/" Run "update-ca-trust extract" Another less secure option is to turn off certificate verification with the following command: lftp > set ssl:verify-certificate false Troubleshooting If you are having problems with Aspera connection timeouts, it can be down to either one of the following. Transfers cannot start the connection fails instantly. Ensure that TCP traffic on port 33001 is allowed (open) for outbound connections through your computer's firewall and network's firewall. The connection is made, transfers are started, but 0 bytes (0%) are uploaded for each file. Ensure that UDP traffic on port 33001 is allowed (open) for outbound connections through your computer's firewall and network's firewal

  Documentation submission/data/uploading-files/ftp