Q301--Modification - Q & A for Advanced Genetic Sequencing 36C24E25Q0045

RFQ 36C24E25Q0045 Advanced Genetic Sequencing Solicitation Clarification and Question & Answer, PART 2 **Quote submission is extended to no later than 11:59AM EST, Thursday, June 26, 2025.** Quotes shall be submitted via email to millicent.covert@va.gov. Offerors must reference Solicitation 36C24E25Q0045 in the subject line of the email. No telephone requests for information will be considered. Incomplete packages will be considered nonresponsive. ___________________________________________________________ Response to Question 1: Scope, Library Prep, and Sequencing Capacity We appreciate your request for clarification and have refined the expectations to ensure alignment with the full Statement of Work (SOW). The following applies: 1. Workflow Scope Full Single-Cell RNA-seq Services Required The requirement includes complete execution of the Parse Biosciences single-cell RNA-seq pipeline, starting from fixed single-cell suspensions. This encompasses: - Sample intake and QC - Cell barcoding and library preparation using Parse WT or MEGA kits - Library quality control - High-throughput sequencing on NovaSeq X Plus PE150 (25B lanes) - Full bioinformatics pipeline (alignment, clustering, DEG, pathway analysis) Pre-made libraries are not in scope, and the contractor must have demonstrated experience handling Parse Biosciences workflows end-to-end, including immediate sequencing upon library completion without outsourcing. 2. Library QC and Kit Considerations Library QC is embedded within the full workflow and must be included in the price per library. The contractor should have: - Infrastructure and staffing to process up to 20 libraries/year in multiple independent batches (2 6 per shipment) - Access to and experience with Parse WT and MEGA kits, with the ability to scale per batch and adjust based on submission schedule without delays or combining batches 3. Sequencing Specifications and Lane Requirements Sequencing must reach 30,000 50,000 PE150 reads per cell in line with Parse s UMI-based design. The contractor should be able to independently: - Assess and balance sample pools - Perform high-throughput sequencing using NovaSeq X Plus - Deliver per-library and per-cell metrics, not only per-lane summaries Contractor must control both library prep and sequencing in-house, ensuring minimal sample handoff and continuity of sample metadata. Response to Question 2: Misalignment Between Pricing Table and Deliverables We acknowledge that the originally provided table structure may have introduced ambiguity. To ensure it accurately reflects the technical and operational deliverables, we recommend the pricing table be realigned to match the complete workflow. Recommended Table Format (Reflecting SOW) Line Item Qty Description 0001 Fixed Sample Receipt & Batch Intake 20 Intake, logging, and QC of methanol-fixed single-cell suspensions 0002 Parse Library Prep (WT or MEGA) + QC 20 Barcoding, cDNA synthesis, library prep, Bioanalyzer/Qubit QC 0003 Sequencing NovaSeq X Plus PE150 TBD Based on UMI complexity and required depth (30 50K reads per cell) 0004 Data Processing & Bioinformatics 20 UMI alignment, cell clustering, DEG, GSEA, UMAPs, raw & processed outputs Summary of Key Requirements To ensure scientific success, the vendor must: - Handle full Parse Evercode single-cell RNA-seq pipeline internally - Have proven experience with Parse kits (WT, MEGA) and NovaSeq X Plus - Support batch-level flexibility (2 6 samples), not pooled 20-sample prep - Deliver complete sequencing + bioinformatics per sample - Ensure end-to-end traceability and QC without third-party outsourcing __________________________________________________________________ RFQ 36C24E25Q0045 Advanced Genetic Sequencing Solicitation Clarification and Question & Answer, PART 2 **Quote submission is extended to no later than 11:59AM EST, Thursday, June 26, 2025.** Quotes shall be submitted via email to millicent.covert@va.gov. Offerors must reference Solicitation 36C24E25Q0045 in the subject line of the email. No telephone requests for information will be considered. Incomplete packages will be considered nonresponsive.

RFQ 36C24E25Q0045 Advanced Genetic Sequencing Solicitation Clarification and Question & Answer, PART 3 **Quote submission is extended to no later than 11:59AM EST, Wednesday, August 20, 2025.** Quotes shall be submitted via email to millicent.covert@va.gov. Offerors must reference Solicitation 36C24E25Q0045 in the subject line of the email. No telephone requests for information will be considered. Incomplete packages will be considered non-responsive. ______________________________________________________________________ This project requires high-throughput, high-quality single-cell RNA sequencing (scRNA-seq) services compatible with Parse Biosciences library preparation technology for murine brain tissue samples. The sequencing provider must meet specific technical requirements necessary for data quality, compatibility, and compliance with VA standards. These include the ability to accommodate Parse's unique chemistry, ensure precise read configurations, and deliver data in a timely and secure manner. Our specifications are designed to eliminate ambiguity, ensure an equitable vendor comparison, and reflect scientifically justified parameters aligned with our research goals. Technical Requirements 1. Specialized Sequencing Compatibility Parse Biosciences uses a non-standard read configuration: Read 1: 28 bp (cell barcode + UMI) Read 2: 91 100 bp (transcript) Index i7: 8 bp (sample index) The sequencing provider must demonstrate prior experience or validated capability with Parse Biosciences libraries and must support these custom read configurations on Illumina platforms. 2. High-Throughput Capacity with Fast Turnaround For each sample consisting of 10,000 nuclei sequenced at 75,000 reads per cell, ~750 million paired-end reads are required. The provider must offer access to NovaSeq S1 or S2 lanes and the ability to sequence multiple samples per run. Turnaround time must be no more than 10 15 business days from receipt of libraries. 3. Cost Efficiency with Fixed Project Design To enable precise cost estimates and avoid ambiguity: Number of Samples: 20 Batch Size: Fixed at 4 samples per batch (5 total batches) Sequencing Depth: Fixed at 75,000 paired-end 150 bp reads per cell Target Cells per Sample: 10,000 nuclei Library Format: Parse Biosciences Evercode WT v2 Midi kits (supports 4 8 samples) This fixed structure balances scientific rigor with cost-effective use of reagents and sequencing lanes, and eliminates the variability introduced by a range of read depths or batch sizes. 4. Validated Quality Control and Data Delivery Infrastructure The provider must deliver raw FASTQ files and associated QC metrics compatible with Parse's cloud-based or command-line pipelines. Services must include demultiplexing, base calling, and sequencing documentation suitable for regulatory compliance and publication. 5. Data Security and Regulatory Compliance Sequencing services must be performed within the United States. Data transfer must occur through secure, encrypted platforms. No biological samples or raw data may be transferred internationally, in alignment with VA requirements. Scientific and Operational Requirements Summary Requirement Specification Compatibility with Parse Biosciences Required; including support for custom read layout Sequencing platform Illumina NovaSeq 6000 Number of samples 20 Batch size 4 samples per batch, 5 total batches Required read depth 75,000 reads per cell Target cell number per sample 10,000 nuclei Read configuration PE150 (R1: 28 bp, R2: 91 100 bp, Index: 8 bp) Turnaround time 10 15 business days Bioinformatics compatibility FASTQ format compatible with Parse/Seurat pipelines Data security U.S.-based, secure upload/download infrastructure The above technical requirements reflect essential capabilities needed to meet the scientific aims of our VA-funded research project. The ability to support Parse Biosciences chemistry, deliver high-throughput sequencing at fixed and justifiable specifications, ensure domestic sample handling, and provide compatible bioinformatics output are critical. These parameters have been established to ensure technical quality, cost transparency, and regulatory compliance. Any vendor responding must fully meet these defined specifications. ______________________________________________________________________ RFQ 36C24E25Q0045 Advanced Genetic Sequencing Solicitation Clarification and Question & Answer, PART 3 **Quote submission is extended to no later than 11:59AM EST, Wednesday, August 20, 2025.** Quotes shall be submitted via email to millicent.covert@va.gov. Offerors must reference Solicitation 36C24E25Q0045 in the subject line of the email. No telephone requests for information will be considered. Incomplete packages will be considered non-responsive. **Quote Submission Instructions** Quotes shall be sent electronically via e-mail. All quotes shall include one (1) separate copy of each of the items listed below. Submission of a quote that does not contain all items requested below may result in elimination from consideration of award. Offerors must submit each item of quote in a separate electronic attachment clearly identified as described below. No price information shall be included in the technical or past performance section. Offerors are responsible for including sufficient details to permit a complete and accurate evaluation of each quote. First Attachment - Volume 1 1. Completed Schedule of Supplies and Services (Attachment 2 to this solicitation). Completed CLIN price information in the Schedule of Supplies/Services. *Offerors must use the CLINs specified in the Schedule of Supplies/Services. It is not permissible for offerors to use offeror created CLINs or SUBCLINs. 2. Completed certifications and representations located at FAR 52.212-3, or an indication that said certifications and representations are current and located in the SAM database. 3. Certifications that may be included as Addenda to FAR 52.212-1. 4. Signed acknowledgements of amendments (SF30). Applicable only if any amendments are issued against this solicitation. Amendments 001 and 002 shall be acknowledged. Second Attachment - Volume 2 Offerors shall provide a narrative (including any descriptive literature and/or certifications) which demonstrates the offeror s ability to meet the technical credentials and capability stated in the Statement of Work (SOW). The narrative shall not exceed ten (10) pages and address the requirements within Item 3 Major Requirements and Item 10 under A. General Information in the SOW. Third Attachment - Volume 3 Past Performance Record: Vendors shall provide information on Past Performance Record. This information is used in determining the level of confidence the Government has for a successful performance based upon the vendor s actions under previously awarded and/or relevant contracts. The information provided in Volume 3 will be utilized to evaluate the Vendor s Past Performance. To be considered part of the Past Performance record, we ask to provide two (2) or more relevant contracts in the last five (5) years. If a vendor or its major subcontractor determines that they have no relevant past performance data, they shall provide the following statement in lieu of the information provided below: (Insert vendor or major subcontractor s name) has determined that they possess no relevant past performance information in accordance with the RFQ. To be compliant with this factor, vendors shall provide the following information for both the vendor and proposed major subcontractors: (1) A list of Previous Contract Data for the vendor. Please provide two (2) PRIME contracts and relevant subcontracts that the vendor is currently performing or has completed during the last 5 years. The list shall include: - Contractor name - Name of customer/agency - Contract/Purchase Order Number - Customer points of contact (minimum of two; provide name, telephone number, fax number and e-mail addresses) - Length of contract - Period of Performance (dates) - Initial Contract Price - Description of product/service provided (include sufficient detail to demonstrate similarities/dissimilarities to the requirements of this solicitation) (2) A list of Previous Contract Data for all minor subcontractors containing the information listed in (1) above as it relates to relevant Prime and Subcontracts that the major subcontractor is currently performing or has completed during the last 5 years. (3) The Government may utilize the Contractor Performance Assessment Reporting System (CPARS), responses from the Performance Risk Assessment Questionnaire, data submitted by the vendor, and any other information available to determine the quality and relevance of the vendor s past performance. Fourth Attachment Volume 4 Contractors are required to include a copy of the FAR provision 52.212-3 Offeror Representations and Certifications Commercial Items with its proposal or may indicate completion of the representations and certifications on the internet at http://sam.gov. Failure to include the certifications along with the price proposal or complete certifications on the internet may result in elimination from consideration for award. To facilitate the award process, all quotes must include a statement regarding the terms and conditions herein as follows: "The terms and conditions in the solicitation are acceptable to be included in the award document without modification, deletion, or addition." OR "The terms and conditions in the solicitation are acceptable to be included in the award document with the exception, deletion, or addition of the following:" Quoters shall list exception(s) and rationale for the exception(s), if any. ______________________________________________________________________ RFQ 36C24E25Q0045 Advanced Genetic Sequencing Solicitation Clarification and Question & Answer, PART 3 **Quote submission is extended to no later than 11:59AM EST, Wednesday, August 20, 2025.** Quotes shall be submitted via email to millicent.covert@va.gov. Offerors must reference Solicitation 36C24E25Q0045 in the subject line of the email. No telephone requests for information will be considered. Incomplete packages will be considered non-responsive.

Please be advised that Solicitation 36C24E25Q0045 is hereby cancelled. Requirement may be reposted at a later date.