Frequently Asked Questions (FAQ)
Services Provided
Eligibility
Application Process
General Issues
Data Use and Dissemination
Questions Specific to Resequencing
R1. Study Design
R2. Technical Issues
R3. Post Approval
R4. Data
Questions Specific to Genotyping
G1. Study Design
G2. Technical Issues
G3. Post Approval
G4. Data
Services Provided
What resequencing does the RS&G Service provide?
The RS&G Service will resequence DNA samples submitted by the investigator using next-generation technology. For a limited time, Sanger sequencing for a small candidate gene or region (typically less than 10kb) for hundreds of DNA samples will be considered. The majority of projects will be processed using next-generation sequencing.What genotyping does the RS&G Service provide?
The RS&G Service will provide high-throughput single nucleotide polymorphism (SNP) genotyping for fine mapping and association studies. Genotyping will be performed with custom panels on investigator-supplied DNA samples from user-defined SNP panels, (> 384 variants) or from fixed content chips such as the Immunochip. Genome wide arrays are not available through the RS&G service.Eligibility
Do you have to have an NHLBI grant to be eligible?
No, but you will need to provide evidence in your application that you have adequate resources to analyze and use the data generated by the service.Are researchers based outside of the U.S. eligible to apply?
Researchers from outside the United States should discuss their application with NHLBI program staff before applying. In addition to the standard criteria, these applications will be assessed for whether the project presents special opportunities for furthering the NHLBI's research programs through the use of unusual talent, resources, populations, or environmental conditions in other countries that are not readily available in the United States or that augment existing U.S. resources.Are researchers at other federal agencies eligible (e.g., FDA researchers doing blood-related research)?
Yes, a memorandum of understanding will need to be completed between the NHLBI and the agency.Are animal studies accepted?
No, this service is currently limited to human studies only, but we plan to expand our offerings soon; therefore, if you have a project for which you would like resequencing or genotyping on non-human samples, you should discuss your project with the ACC before applying.Are all health topics eligible?
No, the focus of this service is heart, lung, and blood disorders and diseases.If my project does not qualify are there other services available?
If your resequencing or genotyping project does not qualify for RS&G Service, you may be able to obtain genotyping through the NIH CIDR mechanism.Application Process
Can I submit documentation for my application in non-PDF format?
No, only a single PDF-formatted document should be submitted for review; otherwise, applications will be deemed incomplete and require resubmission by the applicant.How quickly will I be informed if my study has been approved?
You will receive an e-mail informing you that your application has been either approved or declined within 3 to 4 months after the application submission deadline.How long after approval can we start working with the laboratory?
Immediately following notification of approval, the RS&G Service Laboratory Center will contact you to schedule an introductory conference call and discuss project specifications and detailed logistics.Can I modify my project after my application is accepted?
The project is approved as defined in the application, contingent upon implementation of any reviewer recommendations. Minor technical modifications may be necessary. Any other changes in the project-such as alterations in scope, genes, target regions, or number of samples-will necessitate submission of a revised application subject to review during the next application cycle; i.e., in approximately 3 months.If rejected, will I be told why my application was not accepted?
Yes, written notification will include comments.If I am revising can I have a longer application?
Yes, you may add two pages to your application to respond to and clarify the issues from the previous request.How many times may I resubmit an application for the same project or request?
You may submit two revised applications.General Issues
How many samples do I need to have available to start a study?
As specified in the program requirements and stipulated in the application materials, you will need to ensure that all of the samples needed to carry out the project are readily available and have passed recent quality checks. Any delay in the delivery of samples to the laboratory center (within 3 weeks of first contact) and/or sample quality testing failures may lead to lower prioritization of the project or rejection of the project from the service.Does the RS&G Service include DNA controls?
For sequencing, controls are not included, but samples are initially fingerprinted using a standardized panel and genotypes for the panel compared to the resequencing results. For genotyping, a certain number of wells (typically two samples per 96-well plate) will be reserved for Coriell controls. These spots will be reserved in the DNA manifest.Do we need to provide internal controls for genotyping?
Yes, replicates should be provided. Most case-control studies include 5 percent replicated samples, and family-based studies can include replicated trios (parent-parent-child).How long will it take to process my samples?
The length of time it takes to process the samples depends on the extent of the area to be resequenced or genotyped, the number of samples submitted, and the current workload and capacity of the RS&G Service Laboratory Center. We will discuss the timeline with you upon notification of approval to use the RS&G Service. The resequencing and genotyping timelines are explained in further detail in the appropriate FAQs sections.Data Use and Dissemination
What will happen to the data after the work is completed (i.e., will they be published on the web site or will they go into the public domain)?
The RS&G Service`s Data Release Policies indicate that data will be delivered to the applicant, and then the applicant is expected to submit the genotype and related phenotype data to the appropriate databases, (e.g., dbSNP and/or other NCBI databases such as the database of genotype and phenotype, dbGaP) no later than 3 months after receiving the data, if appropriate. The plan for sharing data, as well as any request and justification for an exception to this policy, should be included in the application. The RS&G Service Laboratory Center can assist in submitting these data.Do I have to acknowledge the RS&G Service in my publications that utilized data obtained through this program?
Yes. We ask that any publications resulting from the data you receive from the RS&G Service contain an acknowledgment as follows: (Resequencing or Genotyping) services were provided through the RS&G Service by the Northwest Genomics Center at the University of Washington, Department of Genome Sciences, under U.S. Federal Government contract number HHSN268201100037C from the National Heart, Lung, and Blood Institute. If you are unable to make this acknowledgment, you must provide the declination reason as part of your application for the service. It is not necessary to include the principal investigators of the RS&G Service Laboratory Center as authors on any publication.Questions Specific to Resequencing
R1. Study Design Issues
Are there limits on the size of the study I can propose for resequencing?
Resequencing projects are calculated as "baseline target reference sequence x number of samples." "Baseline target reference sequence" is defined as the sum of the lengths of the candidate genes and/or genomic regions to be targeted in each sample. The sample size should be between 96 and 200-300 samples, but projects with a minimum of 24 also will be considered. Investigators must contact the NHLBI program staff before submitting a project with a total output >2 Gb.What is the average resequencing project size?
The average project size for resequencing is approximately 0.3 - 1 Gb (baseline reference sequence x number of samples). This amount can be obtained by resequencing fewer samples at longer genomic coverage (e.g., 48 samples across 625 kilobases of baseline sequence) or by resequencing more samples at shorter coverage (e.g., 300 samples across 100 kilobases of baseline sequence). Baseline reference sequence is defined as the sum of the lengths of the candidate genes and/or genomic regions to be targeted in each sample.What coverage can be requested for resequencing?
An investigator can request any group of genes or genomic regions. All regions to be targeted must be explicitly defined using the February 2009 human reference sequence (hg19, GRCh47) coordinates. Regions submitted for custom capture design may need to be modified. All changes to the proposed regions will be discussed prior to project initiation.R2. Technical Issues
How much DNA do I need per sample?
All custom capture resequencing projects require 4 ug of genomic DNA regardless of the amount of targeted sequence. We discourage the use of whole genome amplified DNA samples but if they are used, we may require larger amounts and they will be subjected to more rigorous quality control. For Sanger resequencing projects the amount of DNA per sample is 120 ng multiplied by the target size (in kb) (i.e., 120 ng X 10 kb equates to 1200 ng or 1.2 ug of DNA per sample).How is resequencing done?
Resequencing is typically done using second-generation sequencing on the Illumina platform. A custom capture probe set is designed and ordered. DNA is fragmented and libraries constructed to be compatible with second-generation sequencing. Libraries are hybridized to oligo sets specific for the targeted regions. Enriched libraries are loaded and multiplexed to produce deep sequencing data for each sample at a minimum depth of 20x across >80% of the targeted region. Typically the average coverage is >60x across a region.R3. Post Approval
What is the expected timeline for the resequencing service?
Following approval of a resequencing project, the RS&G Service Laboratory Center will contact the investigator within 2 weeks of assignment to discuss the scope and size of the project and any logistical issues related to sample processing (e.g., a Material Transfer Agreement and/or Human Subjects/Institutional Review Board approval). Shortly after contact with the investigator, the RS&G Service Laboratory Center will provide custom bar-coded plastic ware for the samples as well as an estimated timeline for the project and details on the standard data files and formats the center will deliver.What specifically will the RS&G Service need from me after my project is approved?
The investigator should be prepared to do the following:- Confirm the list of candidate genes and/or genomic regions to be targeted (as approved by the NHLBI).
- Provide data about any gene or region the investigator knows to be potentially difficult to sequence; e.g., duplicated regions.
- Confirm the approved level of sequence coverage for each gene or region and/or whether custom resequencing was approved.
- Discuss the logistics of the resequencing project and the investigator's expectations for project output data files and formats.
- Normalize the concentration and volume of all of the samples. (Concentration and volume of samples will be determined based on the size of the project.)
- Present plans for performing whole genome amplification of primary DNA samples if sufficient genomic DNA is not available. Specifics on the amplification method should also be provided.
- Ship samples in plastic ware provided, labeled as specified and packed on dry ice. Use overnight delivery scheduled to arrive Tuesday through Thursday.
- Provide an electronic sample manifest (using the supplied template) listing the plate location for each sample. Sample identifiers will need to conform to the provided specifications.
- Provide documentation of Human Subjects/Institutional Review Board approval for genetic testing of the samples and finalize a Material Transfer Agreement.
What will the RS&G Service Laboratory Center do after my project is approved?
Following assignment of a project, the RS&G Service Laboratory Center will do the following:- Confirm the list of candidate genes and/or genomic regions to be targeted (as approved by the NHLBI).
- Acquire information about any gene or genomic region in the project that could be difficult to amplify or sequence.
- Determine the project size based on approved coverage.
- Hold a teleconference with the investigator to discuss project logistics, the project timeline, and the output data files and formats to be delivered.
- Provide plastic ware, DNA concentration and volume requirements and reiterate the shipping requirements.
R4. Data
How are single nucleotide polymorphisms (SNPs) identified?
Sequences are base-called and assembled on the reference sequence, and then computational tools are applied to identify variants. The specifics of the algorithm(s) used to identify variants will be discussed at project initiation.Does the RS&G Service detect and report copy number variations (CNVs)?
The only DNA variations that will be reported are single nucleotide variants or small insertion/deletion variants. While CNVs in the gene or region being sequenced may manifest as hemizygous or null (missing data) genotype data, it is not possible to assess such data as CNVs with confidence. In addition to using online databases such as the Structural Variation track at the UCSC genome browser (genome.ucsc.edu), investigators should consider other methods or comparative genomic hybridization to verify suspected CNVs.What data are provided by the RS&G Service?
We have a defined set of output data that will be provided to RS&G Service investigators at the completion of a project. The output includes the sequence files in FASTA format for the regions sequenced, the genotype and frequency of each allele for each single nucleotide and insertion/deletion variant according to position mapping in the human genome, mapping in the context of gene structure, and if requested, input files for programs like PLINK. These data files are provided as plain text.Do you provide all the data files?
The RS&G Service includes the data analysis and quality assurance steps necessary to identify single nucleotide and small insertion/deletion variants, alleviating the need for the applicant to perform these tasks. However, we will provide BAMs or copies of the Consed-compatible trace files at the completion of the study through FTP access. Please note that these are extremely large data sets and require extensive disk storage and computational resources. The investigators will be responsible for any additional analysis with these data.Questions Specific to Genotyping
G1. Study Design Issues
Are there limits on the size of the study I can propose for genotyping? How many SNPs and DNA samples are acceptable?
Genotyping projects are calculated as "number of samples x number of variants." Examples of size calculations for projects are as follows: 384 variants X 1880 samples for 720 K genotypes or 30,000 variants X 188 samples for 5.6 M genotypes. Previously funded proposals have requested between 0.5 M to 10 M genotypes. Contact the NHLBI program staff if your project exceeds 6 M genotypes.What SNP information do I need to include in the application?
For custom genotyping applications, you must provide an overview of variant selection with the application. Describe the selection rationale such as function, density, frequency, heterozygosity, relevance for the ethnic/racial background of the study, etc. The overview also should specify how your team intends to deal with analysis problems such as population stratification, missing data, etc. Remember not every variant will work with the system, so small changes may need to be made after application submission. A final variant list should be completed within 3 weeks of approval notification. For fixed content, such as the Immunochip, you must provide a rationale for the selected fixed content chip and clearly justify why this larger content chip is required compared to a custom genotyping approach.G2. Technical Issues
How much DNA do I need per sample?
Optimal results are obtained with high molecular weight genomic DNA. We require a minimum of 15 ul of DNA at a concentration of 100 ng/ul (i.e., 1.5 ug/sample). We discourage the use of whole genome amplified DNA samples but if they are used, a higher concentration range (200-400ng/ul; 3 - 6 ug/sample) is required. Investigator-supplied DNA samples are pre-checked using a TaqMan gender assay to identify any significant discrepancies with the DNA manifests that are submitted for each study. Investigators will be notified immediately about any discrepancies.What types of genotyping formats are available?
Investigators can choose custom variant genotyping with either 384 - 3 k (in increments of 96, GoldenGate), or 30 k - 50 k (Infinium) variants per sample, or use selected customized formats up to 200K variants, (i.e. Immunochip).What SNP information do I need to have before I apply?
Investigators applying for genotyping should specify the number of SNPs requested. The most common format sizes available are 384 - 3k in increments of 96 (GoldenGate). These can be located in one or multiple chromosomal regions or within candidate genes. As evidence of your being ready to have the genotyping performed, you should provide a final or near final variant list, including the Illumina design scores (techsupport-ilmn@illumina.com) with your application.What sources can be used to specify SNPs for genotyping?
SNPs can be specified from any source available to the investigator. Generally, the investigator supplies a list of rs numbers and gene names, but investigators may submit private SNPs and SNPs from other databases as well.What issues should be considered in selecting SNPs?
NCBI-designated "double-hit" or "submitter-validated" SNPs are more likely to succeed. Avoid SNPs that are not validated (dbSNP category 0) or are missing heterozygosity values. If you want to use "tag" SNPs, you must keep in mind that tags will differ depending on population, etc. Additional issues to consider when picking SNPs are whether the SNP is polymorphic in your population, whether it is in a repeated and polymorphic region of the genome, whether there are other known SNPs near the target SNP, and the Illumina design scores.What does the design score tell me about how a SNP will work?
A design score file can be obtained by submitting the list of rs numbers, the list of gene names, a region list, or a sequence list to Illumina technical support (techsupport-ilmn@illumina.com). Instructions for use of the Illumina Assay Design Tool as well as templates for submission of these lists can be found at https://icom.illumina.com/custom/index. The design scores are a good indicator of likely success. In the Genotyping Center's experience, SNPs with a score above 0.8 have the greatest odds of success (both in providing data and being polymorphic). SNPs with a score of 1.1 have been used successfully on the Illumina platform in previous studies, and these SNPs should be selected whenever possible as long as they have useful minor allele frequencies for your study. SNPs with scores below 0.4 should be removed from the panel as they could affect overall performance of the assay. Previously reported heterozygosity information is also provided and should be considered when picking optimal SNPs.G3. Post Approval
What is the expected timeline for the genotyping service?
Following approval of a genotyping project, the RS&G Service Laboratory Center will contact the investigator within 2 weeks of assignment to discuss the scope and size of the project and any logistical issues related to sample processing (e.g., a Material Transfer Agreement and/or Human Subjects/Institutional Review Board approval). Shortly after contact with the investigator, the RS&G Service Laboratory Center will provide custom bar-coded plastic ware for the samples as well as an estimated timeline for the project and details on the standard data files and formats the center will deliver.What specifically will the RS&G Service need from me after my project is approved?
Various files will need to be submitted by investigators within 3 weeks of the project initiation date unless other arrangements are made with the RS&G Service Laboratory Center. Please plan to submit the following:- A sample information file.
- A duplicate samples file. Two blind duplicate samples per 90 experimental samples are required to be submitted by the investigator.
- A list of desired SNPs. There are a number of different file formats that are available for use (RS sequence list, region list, gene list, etc.). Once this list is finalized, the custom SNP oligo pool order will be placed. This reagent takes 6 to 8 weeks for delivery.
- A copy of the current IRB approval for this project and a Material Transfer Agreement.