Frequently Asked Questions about RFA-RM-12-009
The following assays have already been performed on GTEx samples:
No, the current RNA-Seq does not capture microRNAs. According to the manufacture, the RNA extraction protocol preserves miRNA but this has not been validated on GTEx samples.
Except for experimental controls, this FOA is to support analysis of GTEx samples only.
This FOA does not focus on any particular assay or areas of biology, but rather is open to the most compelling ideas.
The FOA is general, as is the resource, and there are no pre-conceived ideas about the specificity of the scientific question. Applications will be reviewed by a Center for Scientific Review Special Emphasis Panel set up for this FOA, so the most important step is to have well supported aims and to score well.
No, for this FOA, GTEx tissue cannot be made available prior to a funding decision. We encourage applicants to test their assays by collecting enough human or mammalian tissues processed with PAXgene Tissue or other relevant GTEx collection methods (e.g., flash frozen) to show technical suitability.
We are more concerned with at least one dimension being deep than which dimension(s) is(are) chosen. While there is not a minimum number of samples or assays, applicants are encouraged to submit applications that go deeply in at least one dimension, and to have scientifically justified means of selecting samples that take advantage of some of the unique aspects of GTEx.
Depending on the scientific question and assay requirements, applications focusing on one or a small number of tissues may be appropriate (e.g. assays that require frozen samples, for example, will be necessarily restricted to the brain). As long as applications that have a well-supported rationale for focusing on one or a few tissues score well in peer review and otherwise meet FOA requirements, they will be considered responsive and could be attractive.
There are no pre-set thresholds for the fraction of samples that must be studied to be considered responsive. The cost of the assay(s) and study question addressed will determine the proportion of samples that can be analyzed.
The cerebral cortex and cerebellum are sampled immediately, and the remaining whole brain is shipped to a brain bank, where 9 additional regions are sampled and analyzed with RNA-Seq. The remaining brain is sectioned and stored frozen, so the availability of tissue will be limited only by the size of the anatomic region being studied. For a more detailed description of the brain bank protocol, see the GTEx portal (http://www.broadinstitute.org/software/gtex/?q=sample_collection).
The possibility of collecting and freezing more organs is being evaluated, but given the uncertainty of whether and when they will be available, they are not relevant for this FOA. Fresh tissue specimens are not currently available nor are they expected to be during the course of the GTEx project.
We cannot guarantee that tissue microarrays (TMAs) can be assembled for this RFA. However, since the GTEx Comprehensive Biospecimen Resource (CBR) has the capability to create them, it might be considered depending on the complexity of the TMA and whether its composition will be of relatively general interest to the scientific community. Applicants interested in analysis of paraffin block samples assembled into TMAs are strongly encouraged to send detailed requests to Dr. Simona Volpi at email@example.com as early as possible, and no later than February 8th. Depending on the results of NIH’s assessment of our ability to provide the requested TMAs, an applicant may need to describe how they would design their study with and without TMAs and the associated cost implications.
Approximately 75% of donors will have fibroblasts and 50% EBV.
You can view the history questions and frequency distributions of the responses on the dbGaP website. For example, of the first 100 donors, approximately 20% have a history of type 2 diabetes. An authorized user of dbGaP can access and download the individual level data here.
How can we select individuals/tissue based on whether a particular organ was collected, a particular sample has RNA-Seq data, etc., as well as information about age and ethnicity of individuals? What is the letter of sample availability from the PO?
There is a GTEx sample inventory search (http://www.broadinstitute.org/software/gtex/?q=samples) to perform simple searches. To perform more complex searches and to obtain a letter of sample availability documenting that the requested samples are available, please contact Dr Volpi at least 3 weeks before the application due date at firstname.lastname@example.org. You should describe your donor and sample selection criteria and the amount and type of material required, and Dr Volpi will contact you if she needs more information.
Applicants should not budget for shipping and preparation of samples; these costs will be covered by the CBR or LDACC.
The requested direct costs should reflect the work proposed. Using a range of 2 - 10 awards and a 3-year total cost (direct and indirect) of $10 million, the rough annual direct cost amount would be between $200,000 and $1,000,000.
There is no formal pre-application mechanism for groups to coordinate which tissues and individuals are analyzed by different groups. However, NIH program staff will work with applicants, and eventually grantees, to avoid duplication and maximize the utility of the work that can be funded in this RFA.
The Center for Scientific Review will organize this review.
No, successful applicants will be provided with samples as part of the grant.
We realize that some technologies such as IHC do not lend themselves to whole genome or proteomic approaches. Rounds of multiplexed IHC might be considered appropriate for this FOA. Applications will have to make a compelling case that the proposed targets to be studied are of interest of a wider scientific community or are adding value to the GTEx resource; applications that focus only on very small number of biomarkers are not considered responsive.
In regards to the description in the FOA about limiting the complexity of the analysis, is there a recommendation on what this is (in other words, what is "too complex") or is that up to the collaboration team?
It is up to the applicant to propose the level of complexity of analysis of the data generated, within the general considerations mentioned in the FOA. The intent is for grants to focus primarily on data generation and basic statistical analysis, rather than on complex statistical analysis of a smaller set of newly generated data.
Both raw data and preliminary analysis are deliverables for this project. The nature of the newly generated data will determine the kind of preliminary analysis that might be appropriate.
Analysis of the data generated under this FOA could focus on baseline characteristics. While the primary questions don’t need to be clinically oriented, given the nature of the resource, we expect that most studies could inform questions of clinical relevance.
GTEx is designed as a public resource, so applicants and all other scientists are encouraged to download and analyze the data within GTEx.
This FOA uses the U01 Research Project (Cooperative Agreement) mechanism and it is up to the PI to decide the size and composition of the team.
Is there a need for our own data-sharing mechanism or would we leverage the existing GTEx infrastructure? Would immunohistochemistry images be part of the deliverables and would the awarded institution have to make a basic viewer publicly accessible?
All controlled access data generated from GTEx samples are expected to be deposited into dbGaP (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gap) or other NIH Trusted Partners databases. All data without privacy concerns, such as IHC images, are expected to be returned to the GTEx portal (www.broadinstitute.org/gtex) and-or other appropriate repositories with open access. The goal is to have grantees work closely with NCBI and the Laboratory, Data Analysis and Coordinating Center (LDACC) at the Broad Institute (or other appropriate repositories) whenever possible to integrate and share data as widely as possible. Therefore, grantees are not expected to develop their own data sharing mechanism except in unusual circumstances. If there are no readily available existing repositories or means of sharing the data, grantees will work with the NIH Project Scientist to come up with a solution.
Not necessarily. The scientific peer review of applications is the same whether it is a U.S. or foreign institution. However, there are some additional considerations in award decisions for a foreign institution; details are at http://grants.nih.gov/grants/foreign/index.htm.
The involvement of the Project Scientist from NIH is described in the FOA. Since these grants will be U01s (Cooperative Agreements), it is anticipated that NIH staff will have substantial programmatic involvement that is above and beyond the normal stewardship role in awards. See Section VI. Award Administration Information, 2. Cooperative Agreement Terms and Conditions of Award in the FOA.
We are hoping to fund applications which strike a balance between data generation and data analysis. Since the sizes of the awards are limited, we a hope to fund proposals that generate a significant amount of new molecular data. However, we realize that substantial statistical analysis may be needed to interpret and integrate the newly generated data. GTEx is a community resource and we would like as much data generated as possible to maximize its long term utility.
Yes, race and ethnicity are collected for each donor, and frequency distributions can be viewed on the dbGaP website.
SNP typing is only done on blood samples.
GTEx is not considered human research because all donors are deceased. Authorization for research use is gained from donor Next of Kin. The relatively small number of GTEx samples collected from living surgical patients is not part of this RFA.
Only the blood DNA samples are being analyzed with whole-exome sequencing, to a depth of approximately 20X.
See RFA-RM-12-009 for a table of current and projected numbers. Approximately 25-30% of donors have brain tissue collected thus far. We project to have ~260 donors from whom brain is collected by the end of project. Brains are collected only from donors who have not been on ventilator for 24 hours before death. Since many GTEx subjects are organ donors who are on a ventilator at the time or organ recovery, this limits the number eligible for brain collection.
Metabolomics analyses are responsive, but we don’t currently collect serum or plasma samples.