Frequently Asked Questions (FAQ)
Eligibility
Application Process
Technical Issues
Questions Specific to Resequencing
Questions Specific to Genotyping
Data Use and Dissemination
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 U.S. 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 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 U.S. 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 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.
Are there additional
resequencing services available for new investigators?
The NHLBI also supports resequencing through the Program for
Genomic Applications. You may be able to
receive resequencing through the Seattle SNPs PGA or
the Berkeley PGA.
If my genotyping
project does not qualify are there other genotyping services available?
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 documention for my application in non-PDF format?
No, only PDF formated documents will be reviewed.
How quickly will I be
informed if my study has been accepted?
You will receive email informing you that your application
has been either accepted or declined within 3 to 4 months after the application
submission deadline.
How long after
acceptance can we start working with the laboratories?
Immediately following notification of acceptance, applicants
will be contacted by the Laboratory Center that will complete
the work.
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
and clarify the issues from the previous request.
How many times may I resubmit an application for the same project or request?
You may resubmit a revised application twice after your original submission.
Technical Issues
What services,
exactly, 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 Repository, and/or panels of
investigator-supplied DNA samples.
The RS&G Service also provides high-throughput genotyping of SNPs for fine mapping and association studies. Genotyping generally will be performed on an Illumina system with custom panels of user-selected SNPs (374, 758 or 1526 as justified in the request). Investigator-supplied DNAs are prechecked using a Taqman gender assay to identify any significant discrepancies with the pedigree or DNA manifests that are requested for each study.
Are there limits on the size of the study I can propose for resequencing or genotyping? How many genes and DNA samples are acceptable?
There are no limits on the size of the study proposed for resequencing or genotyping, but projects may be modified according to the capacity of the RS&G laboratories.
The genotyping lab processes approximately 100 96-well plates per year (50 96-well plates per application cycle). For genotyping, about 5 ug (35 ul @ 75-150 ng/ul)
is requested. For resequencing, 120 ng/kb of sequence is requested.
How many samples do I
need to have available to start a study?
Investigator-supplied DNA samples must be already collected,
available and ready for processing at the time of application.
Do you include DNA
controls?
Yes. For genotyping,
all plates processed have parent-parent-child CEPH trios and one replicate of
the child. These spots will be reserved
in the DNA manifest. For resequencing, a certain number of wells will be
reserved for Coriell DNA controls (positive and negative).
Do we need to provide
our own internal controls for genotyping?
Yes. Replicates
should be provided. Most case-control
studies include five 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 to process the samples depends on the
extent of the area to be resequenced or genotyped, in addition to the number of
samples submitted and the workload and capacity of the laboratory. Each laboratory will be able to discuss the
timeline with the researcher upon notification of acceptance to the program.The resequencing service timeline is
explained in further detail in the next FAQ section.
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 genotyping/resequencing laboratory 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 six months).
As specified in the program requirements and stipulated in the application materials, please ensure that the samples needed
to carry out this project are available and have passed recent quality checks. Any delay in the delivery of samples to the
genotyping/resequencing laboratory center and/or sample quality testing failures may lead to lower prioritization of the project.
Questions Specific to Resequencing
What is the average resequencing project size?
For resequencing the average project size (baseline reference sequence x number of samples) is approximately 30 Mb. This could 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., 576 samples across 52 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.
How is resequencing done?
We amplify the specified gene(s) or region(s) requested by the investigator using PCR.
Four types of coverage can be requested: a) Protein coding exons only, b) All exons (UTR and coding) only,
c) NHLBI Standard coverage which consists of exons, conserved non-coding sequences plus 2 kb upstream and
2 kb downstream of the gene(s) across a region(s), d) NHLBI Full (complete) coverage of the gene(s) or
across the region(s) including all coding and non-coding 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 their overall project.
For PCR:
PCR primers are Tm-matched and designed from a masked reference sequence to exclude design in problematic regions such as repeat motifs and high-GC content. PCR primers are designed to specifically target either standard or complete sequencing of the specified gene(s), or region(s). 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.
For Sequencing:
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 or regions, or failed PCR handled?
Approaches include the following:
- Computationally identifying 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.
- If a region fails two attempts with alternative conditions, that region is considered refractory to analysis. In general, 10 to 15% of targeted regions are refractory in this manner.
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 a 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.
What data is provided by the resequencing service?
We have a defined set of output data that will be provided
to approved RS&G investigators at the completion of a project. These include 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 and Haploview.
Do you provide trace files?
Our resequencing 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. The
investigators will be responsible for any additional analysis with these
traces.
What is the expected timeline for the resequencing service?
Following approval of a resequencing project, the investigator will be notified of
the resequencing center (UW or VI) assignment. The resequencing center PI
(Dr. Nickerson for UW or Dr. Levy for VI) will contact the investigator within two
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 and preparation, and the timeline for transferring samples. Shortly
following 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 standard instructions apply for the provision of resequencing service?
Investigator
The investigator should be prepared to:
- 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 (i.e., standard or full), 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 providing samples, the investigator should be prepared to:
- 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 "underscore" 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.
Resequencing Center
Following assignment of a project, the resequencing center will:
- 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 potentially 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.
The only DNA variations that the resequencing centers report are single nucleotide polymorphisms (SNPs) or small (<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 (CGH) to verify suspected CNVs.
Questions Specific to Genotyping
What is the average genotyping project size?
For genotyping the average project size (number of samples x number of SNPs) is approximately 1.5 Million genotypes. This could be obtained by genotyping fewer samples for more SNPs (e.g., ~1,000 samples for 1,536 SNPs) or by genotyping more samples for fewer SNPs (e.g., ~4,000 samples for 384 SNPs).
Examples of completed project sizes can be found here.
What platforms are supported by the Genotyping Laboratory?
Currently the RS&G program uses the Illumina system. We are, however, constantly exploring new technology and may update the platform.
What SNP information do I need to have before I apply?
Investigators applying for genotyping on the Illumina platform should specify the number of SNPs requested. (Please note that choices are 96, 384, 768, 1536, or multiples of 1536.) Describe the rationale for SNP selection (density, frequency, heterozygosity, level of validation, relevance for the ethnic/racial background of the study, etc.). SNPs can be in one or multiple chromosomal regions or within candidate genes. You must provide an overview of SNP selection and also submit the final SNP list with the application. The overview should specify how the investigator 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. A poster describing our experience with Illumina Golden Gate chemistry SNP selection is available here. The Genotyping Lab staff is also available for advice. We suggest that you provide a final SNP list, including the Illumina design scores with your application. To obtain the design scores, you may submit a list of rs numbers, a list of gene names, a region list or a sequence list to Illumina technical support (techsupport-ilmn@illumina.com) and they will return a SNP design score file. Instructions for use of the Illumina Assay Design Tool as well as templates for submission of these lists can be found here. Illumina's scores can then be combined with other criteria to select your final set of requested SNPs.
If a final SNP list is not submitted with this application, or if small changes need to be made after application submission, a final SNP list should be completed within 1 month of approval notification.
How do I select SNPs?
a. What sources can be used to specify SNPs for genotyping?
For the Illumina platform, SNPs can be specified from any source available to the investigator. Generally, the investigator
supplies a list of genes and/or regions to the Genotyping Laboratory. We then work with Illumina to generate a
list of possible SNPs from which the final list for genotyping can be selected by the PI. Investigators may submit
private SNPs and SNPs from other databases as well. For information on how to submit these SNPs, please contact
the Genotyping Laboratory.
b. Do I need to mine the SNPs from dbSNP myself?
For those investigators who wish to use dbSNP, we will work with Illumina to prepare a list of all SNPs in a gene
or region along with calculated design scores. You need only provide a chromosomal region or the HUGO approved
gene name or NM_ identifier along with the number of flanking bases on either side of the gene. A list of SNPs
will be returned to you for your selection.
c. What issues should be considered in selecting SNPs?
For the Illumina platform, SNPs that are not validated (dbSNP category 0) should be avoided. NCBI designated "double hit"
or "Submitter validated" SNPs are more likely to succeed. Avoid SNPs with missing heterozygosity values. It is
advisable to use "Illumina validated" SNPs from dbSNP. View the poster
describing SNP selection criteria.
If you want to use "tag" SNPs you must keep in mind that there is not a definitive list of such markers at this time. Also, "tags" will differ depending on population, etc. There are several web-sites which can help with tag SNP selection. Each site has its own pluses and minuses, and care should be taken to familiarize oneself with these before committing to using a particular site. The Genotyping Laboratory is available to consult with you regarding our experience with various sites if desired.
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.
d. How do I know which SNPs will work the best?
For the Illumina platform, the design scores described above are a good indicator of likely success. In our 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.
e. Can the Genotyping Laboratory help me select SNPs?
The Genotyping Laboratory can help with SNP selection. We can act as consultants at any stage in the process or do the
majority of the work for you.
How are the data returned to the investigator upon completion of genotyping?
Data are supplied as CD or DVD-ROMs depending on the size of the files in a format that is readily accepted by most
genetic analysis packages. Accompanying information is supplied with QC values, project notes, etc. We can work with
the analyst for a given project to provide data in specific formats and we can assist investigators in understanding
the data through conference calls or Webex sessions.
What is the expected timeline for the genotyping service?
Project Initiation: After your project has been approved you will receive an email from the Genotyping
Laboratory 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 Laboratory; the date of this e-mail serves as the
project initiation date.
Various files must be prepared and submitted by the Investigator within 1 month of the project initiation date. If this is not feasible you must notify the Genotyping Laboratory. Please submit:
- 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 which are available for use (RS sequence list, region list, gene list etc.). The Genotyping Lab will assist in the preparation of this file and submit it to Illumina to obtain design scores for each of the SNPs. Once this list is finalized the custom SNP oligo pool order will be placed. This reagent takes 6-8 weeks for delivery.
- A copy of your current IRB approval for this project must be on file with the Administrative Coordinating Center [and should have been included with your application].
The Genotyping Laboratory 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 Laboratory within 2 months of project initiation. If your sample shipment will be delayed you must notify the Genotyping Laboratory.
Sample pretesting: A gender test will be performed to determine if there are any major plating or file errors. If the results indicate a major problem, the investigator will be given the chance to address it prior to the initiation of SNP genotyping. Gender discrepancies or sample performance problems may also be reported to the investigator at the individual sample level prior to the initiation of SNP genotyping.
Custom SNP Genotyping: Samples which perform poorly in the initial attempt at genotyping will be identified, cherry-picked and the assay will be repeated one time. At the conclusion of generation of laboratory data, the project data (both at the sample and SNP level) is carefully reviewed for data quality. All data for each SNP locus is manually reviewed and cluster definitions are edited if necessary. Genotyping results will be returned as a single batch for each project. The data will be presented in multiple formats on a CD or DVD along with various data summary and explanatory documents.
The Genotyping Laboratory will submit SNP allele frequency information to dbSNP under the JHU Genetic Resource Core Facility handle within 30 days of release of data.
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.
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 Laboratory Center that provided the service.
If you are unable to make this acknowledgement 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.