STATEMENT OF WORK (SOW) 1. Contract Title. Exome sequencing analysis collaboration for the identification of novel rare genetic determinants of early onset stroke. 2. Background. The overall goal of this Early Onset Ischemic Stroke Consortium funded VA grant is to detect genes associated with ischemic stroke and its subtypes in persons whose first stroke was before the age of 60. To complete the goals of the grant we will require an academic partnership with McMaster University s Drs. Michael Chong and Guillaume Paré, who will support exome sequencing at a highly competitive rate and co-lead the bioinformatic analysis with the Baltimore VAMC/UMD Stroke Genetics Research team. Drs. Chong and Paré are well-established stroke genetics researchers, directors of the state-of-the-art biobank and genomics testing facility Clinical Research Lab & Biobank - Genetic & Molecular Epidemiology Lab (CRLB-GMEL), in Hamilton, Ontario, Canada. They are long-time active collaborators with the Baltimore VAMC/UMD Stroke Genetics Research team on other projects (SiGN, CANVAS, etc.) dating back to 2016. The CRLB-GMEL is the central biobank for many large international studies (> 4M specimens collected from 85+ countries). As such, Drs. Chong and Paré have the necessary infrastructure, staffing, and experience to perform the exome sequencing and analyses to be performed as per the AIMS of the grant. Independent of this initiative, Drs. Chong and Paré have already sequenced hundreds of early stroke cases from their INTERSTROKE study, with an additional ~1K cases ready to sequence with these cases analyzed as part of the AIMS of this grant. Furthermore, recent large public databases (>100K) of control sequences are now available; Drs. Chong and Paré leveraged this to develop a new method to harmonize and compare the burden of rare variants among locally sequenced cases vs. public control sequences, "RV-EXCALIBER" (Nat Commun. 2021 Oct 6;12(1):5852. PMID: 34615865) As such, we will use RV-EXCALIBER to effectively boost the number of controls from the Genome Aggregation Database (gnomAD) to ~570K controls to be contrasted to a large number of cases (~10K) for analysis. Specifically, the CRLB-GMEL will sequence ~3-4K cases, supplemented by ~6K cases already sequenced by other initiatives (UKBB, TOPMED, Geisinger, INTERSTROKE). At the most recent ISGC workshop, Dr. Chong showcased promising results from an RV-EXCALIBER analysis of ~4000 older stroke cases vs. only ~30K gnomAD controls, which bodes well for our planned much larger analysis of ~10K early stroke cases vs. ~570K controls. 3. Scope. The Baltimore VAMC/UMD Stroke Genetics Research team will prepare, arrange and ship ~2-3K EOS case DNA samples to the CRLB-GMEL for exome sequencing, with the CRLB-GMEL supplying ~1K EOS cases of their own. As per protocol, ~500 ng of DNA per case sample will be sent; this allows some leftover material for repeat exome processing in the unlikely case sample processing fails the first time-around. These ~3-4K cases will be combined with ~6K additional cases already sequenced by other initiatives (UKBB, TOPMED, Geisinger, INTERSTROKE) for a combined analysis of ~10K early stroke cases vs. ~570K controls. Analysis will be a joint effort between the Sequencing Data Analysis Core (University of Maryland), led by Dr. Braxton Mitchell, and the CRLB-GMEL bioinformatics team (McMaster University), led by Dr. Chong. 4. Specific Tasks. 4.1 Task 1 - Supply DNA specimens from early-onset stroke patients Deliverables: Identification of ~1000 early stroke patients (< 60 yo) within the supplier s (CRLB-GMEL) database fitting the project Aims. 4.2 Task 2 Laboratory Testing Deliverables: Normalization and preparation of DNA specimens extracted from > 3,000 DNA specimens. 1000 samples to come from Supplier (CRLB-GMEL) directly. 2000+ samples to be shipped to Supplier as coordinated by Balt VA/U Maryland. Exome sequencing of > 3,000 DNA specimens @ ~100x mean sequencing depth. 4.3 Task 3 - Data Processing and Statistical Genetic Analyses Deliverables: Alignment of raw exome sequence reads to the human reference genome (hg38). Germline variant calling of SNPs and INDELs. Identification of rare protein-altering variants through in silico database filtering. Association testing to identify genes associated with early stroke using the RV-EXCALIBER method. 5. Performance Monitoring Monthly meetings will be held to discuss project planning, task progress, and projections. 6. Security Requirements All identifiers, associated participant information, and derived data products will be stored on secure, password- and firewall-protected servers. Visibility and access to the transferred specimens and any derived data products will be restricted exclusively to staff involved in providing the Services. Servers housing data will be backed up regularly. Data sent between institutions will be transferred via secure file transfer protocols. 7. Government-Furnished Equipment (GFE)/Government-Furnished Information (GFI). N/A 8. Other Pertinent Information or Special Considerations. CRLB-GMEL: Will supply their own large collection of early-onset stroke cases. Has the necessary experience to deliver exome sequencing of thousands of samples within the 18-month time frame Has the necessary statistical genetics expertise in conducting case-only rare variant association testing to successfully complete the AIMS of the grant. Identification of Possible Follow-on Work. Additional exome sequencing beyond 3K cases. Other types of next-generation sequencing - (e.g., clonal hematopoiesis of indeterminate potential events) Copy number and structural variant analysis Acceptance Criteria: 90% targets > 20x Genotypic concordance with array data > 95% (when available) Genetically determined sex and ancestry match reported information No evidence of contamination or excess heterozygosity 9. Risk Control N/A 10. Place of Performance. The work will be performed at the contractor s site 11. Period of Performance. Period of performance will be October 1, 2023 September 30, 2024. 12. Delivery Schedule. Task 1. Supply DNA specimens from early-onset stroke patients ~1000 DNA specimens from EOS cases will be identified and normalized for exome sequencing by December 31, 2023 Task 2. Laboratory testing Exome sequencing of 1st batch of 2,000 samples completed by March 31, 2024 Exome sequencing of 2nd batch of 1,000 samples completed by December 31, 2024 (to be completed under FY25 Purchase Order) Task 3. Data Processing and Statistical Genetic Analyses (to be completed under FY25 Purchase Order) Interim Analysis of the first 2,000 samples completed by December 31, 2024 Final Analysis of all 3,000 samples completed by September 30, 2025
