1. Which molecular assays are already performed or planned on the samples and therefore would not be appropriate for RFA-RM-12-009?
2. Is the RNAseq being performed capturing microRNAs?
3. Can the funding be used to analyze other samples in addition to GTEx samples?
4. Which are the assays that you might be interested in?
5. How specific should the scientific question be? Is it preferable to focus on specific disease area?
6. Can we request GTEx samples prior to submission in order to test our molecular assay?
7. Would analyzing only a subset of the samples be perceived as a weakness? What “dimensions” of data generation are most important to the goals of this FOA? More tissues, individuals or assays?
8. Would focus on a single tissue, such as the brain collection, be attractive for GTEx, or would we need a wider purview of tissues?
9. What fraction of the samples should be studied to be considered responsive?
10. How much fresh-frozen brain tissue is available?
11. Is it possible for other fresh frozen tissue (in addition to brain regions currently listed) to be made available from future donors? Can we request fresh samples?
12. Will you allow the samples to be assembled into tissue arrays?
13. What is the success rate for the establishment of fibroblast and lymphoblastoid cell lines?
14. What type of medical history information is available for most samples?
15. 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?
16. Is there any cost associated with obtaining the samples?
17. What is the approximate range of the anticipated annual direct costs?
18. Will there be a mechanism to coordinate with other groups what tissues and individuals to analyze to maximize the utility of the data for the community?
19. Which data will be controlled access and which will be freely available?
20. Who will review these applications?
21. Is there an application process separate from the grant to obtain samples?
22. Do we need a partner from GTEx?
23. Would multiplexed IHC be considered an omics approach?
24. 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?
25. What are the deliverables for the project? Just the raw data or some preliminary analysis with respect to gene expression/genotype association?
26. Can we design a study to simply understand the baseline characteristics of the selected tissue/s or does the analysis have to specifically include a clinically-relevant question?
27. Are there limitations on the team working with the data beyond the scope described in the application?
28. Are you expecting a multi-PI team approach or more an R01-like effort?
29. 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?
30. Can foreign institutions submit applications to this FOA?
31. Would it be better for the application to be submitted by a US instead of a foreign institution?
32. How will the NIH staff be involved in the project?
33. What is the Common Fund and how does it operate?
34. Would fundamental data integration proposals be responsive? Is there funding to develop innovative analysis protocols, or is this FOA just for data production?
35. Do we know the ethnicity of the donors?
36. Is SNP typing done for blood only or separately on tissues?
37. Are there any special considerations for human subjects research for GTEx samples?
38. Which samples have exome sequences available? What depth does exome sequencing go to?
39. How many brain samples have already been processed? What limits the amounts of brain samples collected?
40. Would metabolomics approaches on blood samples be responsive?

Which molecular assays are already performed or planned on the samples and therefore would not be appropriate for RFA-RM-12-009?

The following assays have already been performed on GTEx samples:

• Whole-genome single nucleotide polymorphism (SNP) and copy number variation (CNV) genotyping are performed using high-density genotyping arrays (Illumina Omni5 and HumanExome BeadKit) on the peripheral blood DNA. In addition, some blood DNA samples are being piloted with whole-exome sequencing analysis. The blood DNA sample may eventually undergo whole genome sequencing to more comprehensively identify genetic variation, so this would not be appropriate for this RFA. There are no immediate plans for analysis of DNA from other tissues, and therefore proposals on this topic would be responsive.


• RNA-sequencing of all tissues of sufficient quality is performed using TruSeq library preparation on the Illumina Hiseq 2000 platform. This method involves a poly-A selection to analyze mRNA and is not strand-specific. The RNA­sequencing uses a 72 base paired-end protocol to approximately 50 million mapped reads. A limited number of biospecimens have also undergone gene expression array analysis using Affymetrix Gene 1.0ST or 1.5ST platform. No micro-dissection or laser capture is currently being performed on samples, but it may be done in the future so it would not be appropriate for this RFA.

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Is the RNAseq being performed capturing microRNAs?

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.

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Can the funding be used to analyze other samples in addition to GTEx samples?

Except for experimental controls, this FOA is to support analysis of GTEx samples only.

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Which are the assays that you might be interested in?

This FOA does not focus on any particular assay or areas of biology, but rather is open to the most compelling ideas.

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How specific should the scientific question be? Is it preferable to focus on specific disease area?

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.

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Can we request GTEx samples prior to submission in order to test our molecular assay?

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.

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Would analyzing only a subset of the samples be perceived as a weakness? What “dimensions” of data generation are most important to the goals of this FOA? More tissues, individuals or assays?

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.

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Would focus on a single tissue, such as the brain collection, be attractive for GTEx, or would we need a wider purview of tissues?

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.

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What fraction of the samples should be studied to be considered responsive?

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.

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How much fresh-frozen brain tissue is available?

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).

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Is it possible for other fresh frozen tissue (in addition to brain regions currently listed) to be made available from future donors? Can we request fresh samples?

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.

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Will you allow the samples to be assembled into tissue arrays?

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 nhgrigtex@mail.nih.gov 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.

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What is the success rate for the establishment of fibroblast and lymphoblastoid cell lines?

Approximately 75% of donors will have fibroblasts and 50% EBV.

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What type of medical history information is available for most samples?

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.

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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 nhgrigtex@mail.nih.gov. 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.

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Is there any cost associated with obtaining the samples?

Applicants should not budget for shipping and preparation of samples; these costs will be covered by the CBR or LDACC.

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What is the approximate range of the anticipated annual direct costs?

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.

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Will there be a mechanism to coordinate with other groups what tissues and individuals to analyze to maximize the utility of the data for the community?

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.

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Which data will be controlled access and which will be freely available?

Openly available:

• Molecular data that do not provide direct genetic variation information (e.g., expression arrays, summary sequence-based gene expression estimates (rpkm – reads per kilobase of exon model- per million mapped reads)
• RNA Integrity Number (RIN) values
• Expression quantitative trait loci (eQTL) results data
• Standard Operating Procedures (SOPs)
• Histopathological interpretations
• Laboratory processing variables (e.g., cDNA library preparation methods, processing batch variables)
• Sex and age at death (in 5- or 10-year intervals) for each donor

Available only through controlled access (dbGaP):

• Medical and social history information - This information is collected from family members and/or the medical record, when available
• Molecular results that contain direct genetic variation information (e.g., SNP genotyping files, RNA-seq BAM files).

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Who will review these applications?

The Center for Scientific Review will organize this review.

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Is there an application process separate from the grant to obtain samples?

No, successful applicants will be provided with samples as part of the grant.

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Do we need a partner from GTEx?

No.

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Would multiplexed IHC be considered an omics approach?

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.

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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.

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What are the deliverables for the project? Just the raw data or some preliminary analysis with respect to gene expression/genotype association?

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.

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Can we design a study to simply understand the baseline characteristics of the selected tissue/s or does the analysis have to specifically include a clinically-relevant question?

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.

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Are there limitations on the team working with the data beyond the scope described in the application?

GTEx is designed as a public resource, so applicants and all other scientists are encouraged to download and analyze the data within GTEx.

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Are you expecting a multi-PI team approach or more an R01-like effort?

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.

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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.

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Can foreign institutions submit applications to this FOA?

Yes.

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Would it be better for the application to be submitted by a US instead of a foreign institution?

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.

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How will the NIH staff be involved in the project?

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.

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What is the Common Fund and how does it operate?

You can learn more about it at the following website http://commonfund.nih.gov/ and more specifically for GTEx at http://commonfund.nih.gov/GTEx.

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Would fundamental data integration proposals be responsive? Is there funding to develop innovative analysis protocols, or is this FOA just for data production?

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.

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Do we know the ethnicity of the donors?

Yes, race and ethnicity are collected for each donor, and frequency distributions can be viewed on the dbGaP website.

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Is SNP typing done for blood only or separately on tissues?

SNP typing is only done on blood samples.

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Are there any special considerations for human subjects research for GTEx 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.

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Which samples have exome sequences available? What depth does exome sequencing go to?

Only the blood DNA samples are being analyzed with whole-exome sequencing, to a depth of approximately 20X.

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How many brain samples have already been processed? What limits the amounts of brain samples collected?

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.

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Would metabolomics approaches on blood samples be responsive?

Metabolomics analyses are responsive, but we don’t currently collect serum or plasma samples.