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• High-throughput telomere length measurement at nucleotide resolution using the PacBio high fidelity sequencing platform

  Telomeres are specialized nucleoprotein structures at the ends of linear chromosomes that are essential for the stability as well as the complete replication of the human genome. The progressive shortening of steady-state telomere length in normal human somatic cells is a promising biomarker for age-associated diseases. However, there remain substantial challenges in quantifying telomere length in clinics and research laboratories due to the lack of accurate and high-throughput telomere length measurement method. Here, we describe a novel workflow to capture and analyze telomeres accurately at the single-nucleotide resolution. By using newly designed telobaits that are complementary to the single-stranded G-rich 3’ telomeric overhangs, we can specifically and efficiently capture full-length telomeres and subject them to single-molecule real-time (SMRT) sequencing. We show that the telomere length can be measured at nucleotide resolution using the PacBio high fidelity (HiFi) sequencing platform, which provides the necessary accuracy and throughput to uncover the steady-state telomere length distribution in human culture cell lines as well as clinical patient samples. This new method is a valuable tool not only in population-wide epidemiology studies but also for the longitudinal assessment of telomere attrition in individuals. Our results also reveal the extreme heterogeneity of telomeric variant sequences (TVSs) that is dispersed throughout the telomere repeat region. Importantly, the unique distribution pattern and sequence of TVSs in individuals may be used as a fingerprint for sample identification. In addition, the presence of TVSs disrupts the continuity of the canonical (5’-TTAGGG-3’)n telomere repeats, which could affect the binding of shelterin complexes at the chromosomal ends. Therefore, TVSs may act as a form of genetic polymorphisms that impairs telomere protection. Together with the absolute telomere length in each individual, TVSs may have profound implications in human aging and diseases.

  Study EGAS00001006103

• High-throughput telomere length measurement at nucleotide resolution using the PacBio high fidelity sequencing platform, P49-P104 and run1 replicates

  Telomeres are specialized nucleoprotein structures at the ends of linear chromosomes that are essential for the stability as well as the complete replication of the human genome. The progressive shortening of steady-state telomere length in normal human somatic cells is a promising biomarker for age-associated diseases. However, there remain substantial challenges in quantifying telomere length in clinics and research laboratories due to the lack of accurate and high-throughput telomere length measurement method. Here, we describe a novel workflow to capture and analyze telomeres accurately at the single-nucleotide resolution. By using newly designed telobaits that are complementary to the single-stranded G-rich 3’ telomeric overhangs, we can specifically and efficiently capture full-length telomeres and subject them to single-molecule real-time (SMRT) sequencing. We show that the telomere length can be measured at nucleotide resolution using the PacBio high fidelity (HiFi) sequencing platform, which provides the necessary accuracy and throughput to uncover the steady-state telomere length distribution in human culture cell lines as well as clinical patient samples. This new method is a valuable tool not only in population-wide epidemiology studies but also for the longitudinal assessment of telomere attrition in individuals. Our results also reveal the extreme heterogeneity of telomeric variant sequences (TVSs) that is dispersed throughout the telomere repeat region. Importantly, the unique distribution pattern and sequence of TVSs in individuals may be used as a fingerprint for sample identification. In addition, the presence of TVSs disrupts the continuity of the canonical (5’-TTAGGG-3’)n telomere repeats, which could affect the binding of shelterin complexes at the chromosomal ends. Therefore, TVSs may act as a form of genetic polymorphisms that impairs telomere protection. Together with the absolute telomere length in each individual, TVSs may have profound implications in human aging and diseases.

  Study EGAS00001006595

• Resource for Genetic Epidemiology Research on Adult Health and Aging (GERA)

  The Resource for Genetic Epidemiology Research on Aging (GERA) Cohort was created by a RC2 "Grand Opportunity" grant that was awarded to the Kaiser Permanente Research Program on Genes, Environment, and Health (RPGEH) and the UCSF Institute for Human Genetics (AG036607; Schaefer/Risch, PIs). The RC2 project enabled genome-wide SNP genotyping (GWAS) to be conducted on a cohort of over 100,000 adults who are members of the Kaiser Permanente Medical Care Plan, Northern California Region (KPNC), and participating in its RPGEH. The purpose of the RPGEH is to facilitate research on the genetic and environmental factors that affect health and disease by linking together clinical data from electronic health records, survey data on demographic and behavioral factors, and environmental data from various sources, with genetic data derived from biospecimens collected from participants. At the time of the award of the RC2 project in late 2009, the RPGEH had established a cohort of about 140,000 individuals who had answered a detailed survey, provided saliva samples for extraction of DNA, and given broad consent for the use of their data in studies of health and disease. To maximize the diversity of the resulting sample, the GERA cohort was formed by including all racial and ethnic minority participants with saliva samples (N = 20,925; 19%); the remaining participants were drawn sequentially and randomly from white non-Hispanic participants (89,341; 81%). A total of 110,266 participant samples were included to ensure that at least 100,000 were successfully assayed. The resulting GERA cohort is 42% male, 58% female, and ranges in age from 18 to over 100 years old with an average age of 63 years at the time of the RPGEH survey (2007). The sample is ethnically diverse, generally well-educated with above average income. Approximately 69% of the participants are married or living with a partner. Length of membership in KPNC averages 23.5 years. UCSF and RPGEH investigators worked with the genomics company Affymetrix to design four custom microarrays for genotyping each of the four major race-ethnicity groups included in the GERA Cohort, described in detail in Hoffmann et al., 2011a and 2011b. Following genotyping and quality control procedures, and after removal of invalid, discordant, or withdrawn samples, about 103,000 participants were successfully genotyped. The resulting genotypic data were linked to survey data and data abstracted from the electronic medical records. As described below, all RPGEH participants were mailed new consent forms with explicit discussion of the placement of data in the NIH-maintained dbGaP. About 77% of participants returned completed consent forms, resulting in a final sample size of 78,486 participants in the GERA Cohort with data for deposit into dbGaP. Origins of the RPGEH GERA Cohort The goal in creating the RPGEH GERA cohort was to create a large, multiethnic, and comprehensive population-based resource for research into the genetic and environmental basis of common age-related diseases and their treatment, and factors influencing healthy aging and longevity. The GERA Cohort consists of a diverse cohort of more than 100,000 adults who are members of the Kaiser Permanente Medical Care Plan, Northern California Region (KPNC), and participating in its Research Program on Genes, Environment and Health (RPGEH). KPNC is an integrated health care delivery system with a population of about 3.3 million people in northern California. The membership of KPNC is representative of the general population in the 14 county area in which facilities are located, although the membership is underrepresented for the extremes of income at both ends of the spectrum. The RPGEH utilizes the longitudinal electronic health records (EHR) of KPNC to obtain clinical, laboratory, imaging and pharmacy information on all cohort members, to which personal demographic, behavioral and health characteristics have been added through member surveys. The GERA Cohort comprises a subsample of the RPGEH participant cohort, and was created through the RC2 award from the NIA, NIMH, and NIH Common Fund as described above. GERA Study Design The GERA Cohort is a subsample, as described above, of the longitudinal cohort enrolled in the Kaiser Permanente RPGEH. The RPGEH cohort includes about 400,000 survey participants of whom about 200,000 have provided broad consent and a sample of saliva or blood for use in studies of genetic and environmental factors in health and disease. The GERA Cohort was developed from a mailed survey sent to all adult members of KPNC who had been members for two years or more in 2007. All survey respondents were contacted and asked to complete a consent form; those who completed consent forms were asked to provide a saliva sample. Additional male participants were added to the RPGEH through inclusion of the Northern California sample of the California Men's Health Study (CMHS) cohort of about 40,000 men from KPNC, ages 45-69 years old at the time of the CMHS survey in 2002-2003. The CMHS participants contributed about 15,400 saliva samples to the RPGEH and were eligible for inclusion in the GERA Cohort. CMHS participants were included according to the same sampling design as for the RPGEH cohort as a whole. Specifically, all minority participants were selected for inclusion in order to maximize representation of minorities in the GERA Cohort, and Non-Hispanic White participants were selected at random to complete the sample of 110,266 GERA Cohort participants. GERA Genotypic Data High-density genotyping was conducted at UCSF using custom designed Affymetrix Axiom arrays, as described in Hoffmann et al. (2011a; 2011b). To maximize genome-wide coverage of common and less common variants, four specific arrays were designed for individuals of Non-Hispanic White (EUR), East Asian (EAS), African-American (AFR), and Latino (LAT) race/ethnicity. There was broad overlap among the SNPs on the arrays, which were designed using a hybrid greedy imputation algorithm (Hoffmann et al., 2011b) applied to genotype information validated by Affymetrix from the 1000 Genomes Project. However, in order to capture low frequency variants specific to particular race-ethnicity groups, SNP content varies between arrays. A more detailed description of the process of genotyping and results is included in Genotyping of DNA Samples. Description of the analyses of population structure and development of principal components for adjustment of population structure is included in Population Structure Analysis. GERA Phenotypic Data RPGEH and CMHS Survey Data. The sources of data on demographic and behavioral factors deposited in dbGaP for the GERA Cohort are the RPGEH and CMHS surveys. Data on common demographic factors such as gender, race/ethnicity, marital status, and education and on behavioral factors such as smoking, alcohol consumption, and body mass index, have been cleaned, edited, reconciled between the two surveys, and compiled into summary indices, where appropriate, for deposition into dbGaP. A more complete description of the survey variables is included in Survey Variables Documentation. Please note that the terms of use of the GERA Cohort Data, as specified in the Data Use Certification (DUC), prohibit the use of survey variables as outcomes in analyses. For example, a genome-wide association study (GWAS) of education or smoking is not permitted as specified by the DUC. Only health conditions can be used as outcome variables in analyses. Health Conditions derived from Kaiser Permanente Electronic Medical Records. Data on the occurrence of health conditions in participants in the GERA Cohort have been derived from summarizing ICD-9 coded diagnoses in Kaiser Permanente's electronic medical records. An algorithm that aggregates specific ICD-9 codes into appropriate diagnostic groups for selected conditions is applied to outpatient and inpatient databases; see Disease and Conditions Definitions Documentation for details. The criterion for including a condition as "present" for a participant is the occurrence of two or more diagnoses within a diagnostic category occurring on separate days. Two or more is used as the criterion in order to reduce false positives due to mistakes or rule-out diagnoses. When compared with validated disease registries, the criterion of 2+ diagnoses yields high specificity and good sensitivity. ICD-9 codes in the electronic records are specified in several ways. For outpatient visits occurring during the period 1995 to 2006, diagnoses were assigned by the treating physician who endorsed specific diagnoses on an optically scanned list that varied by specialty. Beginning in 2006 with the advent of an integrated, fully electronic medical record, outpatient diagnoses are made by physicians/ providers using a pull down menu. Discharge diagnoses from inpatient stays are specified by physicians and coded by specially trained coders. Databases of ICD-9 codes for diagnoses assigned at outpatient visits, or as one of the discharge diagnoses following inpatient stays, are complete and available for all KPNC members dating back to 1995. Although the average length of KPNC membership among GERA cohort members is 23.5 years in 2007, not all have been members since 1995, so the history for some conditions, such as those that are not chronic or recurrent, may not be complete for all cohort members. The year of first membership in KPNC is included as a variable in the list of survey variables, enabling investigators to estimate the number of years of observation of each Cohort member. RPGEH Access and Collaborations Website and Procedures The RPGEH maintains a web portal for inquiries and applications for collaboration and access to data. The url is: https://rpgehportal.kaiser.org/. RPGEH has an application process and an Access Review Committee that reviews applications for collaboration and use. For more details, please contact RPGEH through the website.

  Study phs000674

• The landscape of Usher syndrome-associated transcript isoforms in the human neural retina

  Usher syndrome is an autosomal recessively inherited disorder characterized by the combination of progressive loss of visual function due to retinitis pigmentosa, sensorineural hearing loss and vestibular dysfunction in a subset of cases. Usher syndrome is clinically and genetically heterogeneous. To date, eleven genes have been identified that are associated with Usher syndrome. It is known that these genes express multiple isoforms, however, for most of the known isoforms their relative importance in the human retina is unknown. To fill this gap, using PacBio long-read mRNA sequencing we aimed to provide an overview of the Usher syndrome transcripts in the human retina.

  Study EGAS50000000504

• 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

• Transcriptome sequencing of Gingivo-buccal Cancer : ICGC-India Project_YR03

  Transcriptome Sequencing of Gingivo-buccal Cancer: ICGC-India ProjectAs a part of the ICGC, India has undertaken genomic studies on gingivobuccal cancer of the oral cavity, which is the most prevalent form of cancer among men in India. There are various known environmental (life-style) correlates of this cancer, the most important of which are tobacco chewing and HPV infection. Transcriptome sequencing of paired RNA samples – isolated from the tumor and adjacent normal tissues of 12 patients – have been performed. Detailed clinical characterization of the patients, collection of data on demographic and environmental exposures, and isolation of DNA samples from adjacent normal and tumor tissues collected from each patient are being done at the Advanced Centre for Research, Treatment and Education on Cancer, Mumbai. Sequencing is being performed at the National Institute of Biomedical Genomics, Kalyani.

  Study EGAS00001002851

• Transcriptome sequencing of Gingivo-buccal Cancer : ICGC-India Project_Batch05

  Transcriptome Sequencing of Gingivo-buccal Cancer: ICGC-India ProjectAs a part of the ICGC, India has undertaken genomic studies on gingivobuccal cancer of the oral cavity, which is the most prevalent form of cancer among men in India. There are various known environmental (life-style) correlates of this cancer, the most important of which are tobacco chewing and HPV infection. Transcriptome sequencing of paired RNA samples – isolated from the tumor and adjacent normal tissues of 28 patients – have been performed. Detailed clinical characterization of the patients, collection of data on demographic and environmental exposures, and isolation of DNA samples from adjacent normal and tumor tissues collected from each patient are being done at the Advanced Centre for Research, Treatment and Education on Cancer, Mumbai. Sequencing is being performed at the National Institute of Biomedical Genomics, Kalyani.

  Study EGAS00001003285

• Age related Macular Degeneration (AMD)-- Michigan, Mayo, AREDS, Pennsylvania (MMAP) Cohort Study: A Joint Genome Wide Association Study

  Age-related Macular Degeneration (AMD) is a leading cause of incurable blindness in people over the age of 65. AMD is a late-onset multi-factorial neurodegenerative disease and its pathogenesis involves interaction of genetic and environmental factors. Several chromosomal regions have been associated with AMD susceptibility through linkage analysis (Swaroop et al., 2009). More recent studies provide strong evidence that variants within the CFH gene cluster on chromosome 1 and at/near LOC387715/ARMS2 on chromosome 10 are strongly associated with disease. Variants at other genes including C2/BF, C3, CFI and APOE4, also contribute to AMD susceptibility. Our primary goals are to identify genetic variants and haplotypes that are associated with AMD. The underlying hypothesis is that DNA variation(s) in multiple genetic susceptibility loci will predispose individuals to AMD pathogenesis, and comparison of DNA of cases and controls should identify these susceptibility variants. Our studies are focused on the genetic analysis of advanced AMD and should provide novel insights into disease diagnosis, progression and pathology. We have assembled a collaborative group of researchers from the University of Michigan, Mayo Clinic, University of Pennsylvania, and the AREDS group including National Eye Institute intramural investigators, who collected clinical data and DNA from a large number of patients affected with AMD and from unaffected controls. The primary source of funding was National Eye Institute. Through this collaborative effort, we submitted and obtained usable genotyping data on 2184 patients and 1155 controls from the Center for Inherited Disease Research (CIDR).

  Study phs000182

• National Institute on Aging (NIA) Late-Onset Alzheimer's Disease Genetics Initiative: The Multiplex Family Study

  Multiplex Family StudyThe purpose of the NIA Genetics Initiative: Multiplex Family Study is to identify families with multiple members diagnosed with late-onset Alzheimer&#39;s Disease. Families will be characterized clinically and blood samples will be collected to establish cell lines. If a blood sample is not available, autopsy samples will be collected for DNA extraction and storage. Our goal is to recruit 1,000 families over the course of the study. Clinical and demographic data from these families will be collected at the local site and coded data, without identifiers, will be sent and included in a national database of families with Alzheimer&#39;s Disease. This database, along with the biological samples, will be housed at the National Cell Repository for Alzheimer&#39;s Disease (NCRAD) at Indiana University. The Center or provider and the Cell Repository must sign a Material Transfer Agreement for shipment of biological samples and phenotypic data to NCRAD. The biological samples and data from these families will be available to qualified researchers, who must sign a Material Transfer Agreement before receiving DNA and data. An oversight committee known as the Cell Bank Advisory Committee (CBAC) will review and monitor the process of family identification and enrollment, data collection, the establishment of cell lines and access to samples. LOAD CIDR ProjectThe first 362 families of the Multiplex Family Study (including 2,105 family members) were included in a 6K SNP genome-wide scan at the NIH-supported, Center for Inherited Disease Research (CIDR). An additional 297 unrelated, healthy controls were also genotyped at CIDR. The average age of onset of AD in the genotyped sample is 74 years and 62% of the sample is women. While primarily Caucasian, 3% are African-American, and 3% are of Hispanic ancestry. 72% of the families had at least 2 affected siblings, whereas 21% had 3 or more sampled affected individuals and 7% had 4 or more genotyped, affected family members.

  Study phs000160

• Tumor-resident T-cell regulate responses to checkpoint blockade immunotherapies

  In cancer models, CD103+CD8 TRM cells are implicated in the response to anti-PD-1 immunotherapy, and a subset of CD4 TRM cells expressing the integrin CD49a is required for response to anti-CTLA-4 immunotherapy. We performed single-cell RNA sequencing on tumour-infiltrating T lymphocytes (TILs) isolated from colon carcinoma et unsupervised analyses reveal several TRM populations.

  Study EGAS50000000826