DNA Resequencing and Genotyping Service

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 samples from defined populations in candidate genes or specified DNA regions. The variation discovery will be performed using panels of DNA samples such as those available from the Coriell Cell Repository, Coriell Institute for Medical Research, and/or panels of investigator-supplied DNA samples.

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 of user-selected SNPs in investigator-supplied DNA samples.

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 the RS&G Service 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 limited to human studies only.

Are all health topics eligible?
No, the focus of this service is heart, lung, blood, and sleep disorders and diseases.

If my project does not qualify are there other services available?
The NHLBI has been supporting limited resequencing efforts through the Seattle SNPs Program for Genomic Applications (PGA) and the Berkeley PGA, but these programs are no longer accepting requests. If your 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 laboratories?
Immediately following notification of approval, applicants will be contacted by the Laboratory Center that will complete the work.

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 to facilitate the Resequencing/Genotyping Center implementation. 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 available and have passed recent quality checks. Any delay in the delivery of samples to the laboratory center and/or sample quality testing failures may lead to lower prioritization of the project. If insufficient DNA is available, the investigator should consider the use of a DNA amplification service such as that provided by the Coriell Institute for Medical Research or Qiagen.

Does the RS&G Service include DNA controls?
Yes. For resequencing, a certain number of wells (typically two samples per 96-well plate) will be reserved for Coriell DNA controls. For genotyping, all plates processed have a parent-parent-child HapMap trio and one member of another HapMap trio, so three total sets create four parent-parent-child trios. Most trios are placed on more than one plate to create interplate duplicates. 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 laboratory assigned the project. Each laboratory will discuss the timeline with the investigator 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 results once the work is completed (i.e., will they be published on the website 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 a month later, data will be released to public databases. Typically, there is an additional delay of 3 months or more before publication in dbSNP. Applicants requesting exceptions to the standard policy must submit their justification as part of the application. The Resequencing Centers will submit allele frequency data for each project along with the number of samples and broad sample identifiers; e.g., continent of origin. The Genotyping Center will submit SNP allele frequency information to dbSNP under the JHU Genetic Resource Core Facility handle within 30 days of release of data

Do I have to acknowledge the RS&G Service in my publications that utilized data obtained through this program?
Yes. If you receive services, we ask that any publications resulting from the data you receive contain an acknowledgment of the Resequencing or Genotyping Center that provided the service. If you are unable to make this acknowledgment, you must provide the declination reason as part of your application for the service. Full details can be found in the Application Template relevant to the service being requested. It is not necessary to include the principal investigator(s) of the Resequencing or Genotyping Center(s) as author(s) 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 reference sequence x number of samples." "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. The sample size should be between 94 and 564, but projects with a minimum of 47 also will be considered. Investigators must contact the NHLBI before submitting a project larger than 60 Mb.

What is the average resequencing project size?
The average project size for resequencing is approximately 30 Mb (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., 564 samples across 53 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. Examples of completed project sizes can be found here.

What coverage can be requested?
Four types of coverage can be requested: (1) Protein coding exons only; (2) all exons (UTR and coding) only; (3) NHLBI standard coverage, which consists of exons, conserved noncoding sequences plus 2 kb upstream and 2 kb downstream of the gene(s) across a region(s); and (4) NHLBI full (complete) coverage of the gene(s) or across the region(s) including all coding and noncoding sequences, plus 2 kb upstream and 2 kb downstream of the gene(s) or region(s). In addition, only regions refractory to PCR amplification or sequencing (e.g., low-complexity repeats) are excluded. For these four options, precomputed estimates for the sequences have been identified and calculated to aid the investigator in defining the size of the overall project.

What if I want resequencing of a nonstandard gene isoform?
Coverage estimates for all reference gene isoforms are available using a precomputed estimates table. An investigator may submit a FASTA file of the cDNA or a set of genomic coordinates on the current NCBI human genome sequence of the region to be targeted if it is nonstandard. This should be noted in the RS&G application and discussed further with the Resequencing Center upon project approval. Following these discussions with the Resequencing Center, the targeted gene isoform cannot be altered.

R2. Technical Issues

How much DNA do I need per sample?
The amount of DNA required per sample depends on the amount of sequencing requested. The formula to use is 120 ng of DNA per kb of sequence requested.

How is resequencing done?
We amplify the specified gene(s) or region(s) requested by the investigator using PCR. The PCR primers are designed to specifically target the gene(s) or region(s) requested. They are Tm matched and designed from a masked reference sequence to exclude design in problematic regions such as repeat motifs and high GC content. Each primer pair is "tailed" with a universal M13 forward and reverse sequencing primer to enable subsequent sequencing. Optimal PCR conditions are determined empirically or computationally and confirmed by multiple quality assurance steps. Once the regions are amplified, each PCR product is sequenced from the forward and reverse direction to provide double-stranded coverage. Sequencing is carried out with Sanger Big-Dye Terminator sequencing, and detection with capillary-based sequencing machines.

How are difficult sequences/regions or failed PCR handled?
Approaches include the following:

Computationally identified problematic regions in the initial reference sequence such as repeat motifs and high-GC content sequences. These regions are masked to exclude these sequences in the design of PCR primers;
Designing multiple primer pairs to target difficult regions;
Attempting alternative PCR chemistries and thermocycling parameters for the failed region; and
If a region fails two attempts with alternative conditions, that region is considered refractory to analysis. In general, 10 to 15 percent of targeted regions are refractory in this manner.

R3. Post Approval

What is the expected timeline for the resequencing service?
Following approval of a resequencing project, the investigator will be notified of the Resequencing Center assignment (UW or VI). The PI of the appropriate center (Dr. Nickerson for UW or Dr. Levy for VI) 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. If the project involves patient samples, the investigator should be prepared to discuss issues including sample availability, preparation, and the timeline for transferring samples. Shortly after contact with the investigator, the Resequencing Center will provide an estimated timeline for the project and details on the standard data files and formats the center will deliver.

What specifically will the Resequencing 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 to the assigned Resequencing Center about any gene or region the investigator knows to be potentially difficult to amplify or 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.

If samples will be sent, the investigator should be prepared to do the following:

Normalize the concentration and volume of all of the samples as directed by the Resequencing Center. (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 (120 ng/kb).
Ship samples in plastic ware provided by the Resequencing Center, labeled as specified by the Resequencing Center and packed on dry ice-via overnight delivery to arrive Tuesday through Thursday.
Provide an electronic sample manifest listing the plate location for each sample. (The Resequencing Center will provide a template.) Sample identifiers will need to conform to the Resequencing Center's specifications. (For VI, letters, numbers, and underscoring are the only acceptable characters.)
Provide documentation of Human Subjects/Institutional Review Board approval for genetic testing of the samples and finalize a Material Transfer Agreement.

What will the Resequencing Center do after my project is approved?
Following assignment of a project, the Resequencing 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.
In the case of investigator-supplied samples, specify the amount of DNA per donor necessary to complete the project, provide plastic ware, and reiterate shipping requirements.

R4. Data

How are single nucleotide polymorphisms (SNPs) identified?
The sequencing traces are base-called and assembled on the reference sequence, and then computational tools are applied to resolve mixed (heterozygous) sequences in the trace (the signature of an SNP/variant). The SNPs/variants are identified by confirmation on both the forward and reverse strands and, if required, with manual review by specialized data analysts.

Does the resequencing service detect and report copy number variations (CNVs)?
The only DNA variations that the Resequencing Centers will report are SNPs or small (less than 20 bp) insertion/deletion polymorphisms. 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 SNP genotyping methods or comparative genomic hybridization to verify suspected CNVs.

What data is provided by the resequencing 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 SNP/variant and insertion-deletion polymorphism detected according to position mapping in the human genome, mapping of these SNPs/variants in the context of gene structure, and if requested, input files for programs like PHASEII Haploview and PLINK. These data files are provided as plain text and, as appropriate, may be imported into programs like Microsoft Excel for further analysis.

Do you provide trace files?
The RS&G Service includes the data analysis and quality assurance steps necessary to identify variants directly from the sequence traces, alleviating the need for the applicant to perform these tasks. However, if requested, we do provide copies of the Consed-compatible trace files at the completion of the study through FTP access or disk copy. Please note that these are extremely large data sets and require extensive disk storage and computational resources. Proficiency in working with Consed-compatible chromatograms is necessary. The investigators will be responsible for any additional analysis with these traces.

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 SNPs." The system uses 96-well plates, so sample estimates should use multiples of 90. The Genotyping Center can process approximately 200 plates per year or 50 plates per application cycle for the RS&G Service. The most commonly used genotyping platforms process 96, 384, 768, or 1536 SNPs per sample. A higher SNP-density platform also is available. Investigators must contact the NHLBI before submitting a project larger than 4 million genotypes.

What is the average genotyping project size?
For genotyping, the average project size (number of samples x number of SNPs) is approximately 2.0 million genotypes. This amount could be obtained by genotyping fewer samples for more SNPs (e.g., approximately 1,300 samples for 1536 SNPs) or by genotyping more samples for fewer SNPs; e.g., approximately 5,200 samples for 384 SNPs. Examples of completed project sizes can be found here.

What SNP information do I need to include in the application?
You must provide an overview of SNP selection with the application. Describe the rationale for SNP selection (density, frequency, heterozygosity, level of validation, relevance for the ethnic/racial background of the study, etc.). The overview also should specify how your team intends to deal with problems such as population stratification, small genes, regions of low heterozygosity, limited number of tagging SNPs, and/or regions with few ideal SNPs. Not every SNP will work with the Illumina system, so small changes may need to be made after application submission. A final SNP list should be completed within 1 month of approval notification.

G2. Technical Issues

How much DNA do I need per sample?
The amount of DNA required per sample depends on the amount of genotyping requested. About 2.5 ug DNA (25 ul @ 75-150 ng/ul) is needed per sample. If it is necessary to send samples amplified by WGA, we request 5 to 10 ug DNA (25 uL @ 200-400 ng/uL). Investigator-supplied DNA samples are prechecked using a Taqman gender assay to identify any significant discrepancies with the pedigree or DNA manifests that are requested for each study.

What platforms are supported by the Genotyping Center?
Currently, the RS&G Service uses mainly the Illumina systems. We are, however, constantly exploring new technology.

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 SNP sizes have been 384, 768, or 1536 per sample. 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 SNP list, including the Illumina design scores with your application. The Illumina scores should be combined with other criteria to select your final set of requested SNPs.

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. For information on how to submit these SNPs, please contact the Genotyping Center.

What issues should be considered in selecting SNPs?
NCBI-designated "double-hit" or "submitter-validated" SNPs are more likely to succeed. It is advisable to use "Illumina-validated" SNPs from dbSNP and to 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 a definitive list of such markers is not available at this time. Also, tags will differ depending on population, etc. There are several Web sites that can help with tag SNP selection. As each site has pluses and minuses, you will want to familiarize yourself with these before deciding on a particular site. 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, etc. A poster describing the genotyping center's experience with Illumina Golden Gate chemistry SNP selection is available here.

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 here. 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 0.2 should be avoided, and SNPs between 0.2 and 0.8 should be used only if necessary. 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?
After your project has been approved for access to the RS&G Service, you will receive an e-mail from the Genotyping Center with detailed instructions and the name of a contact person who will manage your project. You must select a person in your group who will, likewise, be the liaison for future contacts. An e-mail acknowledging receipt of the detailed instructions should be sent back to the Genotyping Center; the date of this e-mail serves as the project initiation date. Shortly after contact with the investigator, the Genotyping Center will provide an estimated timeline for the project and details on the standard data files and formats the center will deliver.

What specifically will the Genotyping RS&G Service need from me after my project is approved?
Various files will need to be submitted by investigators within 1 month of the project initiation date unless other arrangements are made with the Genotyping 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 on file with the Administrative Coordinating Center (included with your application).

How do I send my samples?
The Genotyping Center will send you barcode-labeled 96-tube latched racks, shipping supplies, and instructions for sample preparation and placement. Samples must be sent to the Genotyping Center within 2 months of project initiation. If your sample shipment will be delayed, you must notify the Genotyping Center.

Are samples pre-tested?
A gender test will be performed to determine if there are any major plating or file errors. Gender discrepancies or sample performance problems may be reported to the investigator at the individual sample level. If the results indicate a major problem, the investigator will be given the chance to address it prior to the initiation of SNP genotyping.

What happens if a sample still performs poorly?
Samples that perform poorly in the initial attempt at genotyping will be identified and cherry-picked, and the assay will be repeated one time.

G4. Data

How are data generated?
At the conclusion of generation of genotyping data, the project data (both at the sample and SNP level) are carefully reviewed for data quality. All data for each SNP locus are manually reviewed, and cluster definitions are edited if necessary.

How are the data returned to the investigator upon completion of genotyping?
Genotyping results will be returned as a single batch for each project. Data are supplied as CDs or DVD-ROMs, depending on the size of the files. The data will be presented in multiple formats readily accepted by most genetic analysis packages along with various data summary and explanatory documents such as QC values, project notes, etc. We can work with the analyst for a given project to provide data in specific formats if needed, and we can assist investigators in understanding the data through conference calls or Webex sessions.