RFQ 36C24E25Q0045 Advanced Genetic Sequencing Solicitation Clarification and Question & Answer **Quote submission is extended from Wednesday, June 11, 2025, to no later than 11:59 EST, Monday, June 16, 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. _______________________________________________________________________ 1. For the "spatially segregated mouse brain and lung" samples, please clarify their processing prior to shipment. Specifically, will they be fresh-frozen (snap-frozen) or fixed-frozen (e.g., briefly fixed and then frozen)? The spatially segregated phrase refers to dissection of brain and lung regions prior to dissociation. No spatial transcriptomic techniques will be performed. Tissues are enzymatically dissociated into single-cell suspensions, then fixed and frozen in cryovials using Parse Biosciences methanol-based fixation protocol. 2. If able, can you also comment on: a) Tissue Details: Expected section thickness, mounting method, and clarity on section orientation (e.g., anatomical landmarks, images). Not applicable, no sectioning or mounting will be involved. Only single-cell suspensions will be used. b) Quality & Shipping: Anticipated RNA Integrity Number (RIN), detailed shipping conditions (e.g., dry ice amount, packaging), and frequency of shipments. Cells are fixed at our facility prior to shipment using Parse Evercode protocols. Shipping: Frozen in cryovials on dry ice using validated shippers. Shipments are expected 2 3 times per year, based on project progress. c) Histology: Will accompanying H&E stained slides or images be provided for morphological context? N/A Morphological context (e.g., H&E slides or images) may be provided separately for selected samples, but is not required for scRNA-seq processing. 3. For the single-cell suspensions, please provide details on: Single-Cell Suspensions a) Cell State & Handling: Pre-fixation viability: 85% Target cell concentration: 1 million cells/mL Total cells per sample: 50,000 100,000 Suspension buffer: PBS + 0.04% BSA b) Fixation: Will cells be provided as fresh suspensions (requiring our lab to fix them upon receipt) or will they be pre-fixed by the VA? If pre-fixed, please specify the fixation protocol/reagents. Cells will be pre-fixed by the VA using Parse Biosciences recommended Evercode protocol (methanol-based). c) Shipping: Specific shipping conditions (e.g., frozen in cryovials on dry ice). Cryovials shipped on dry ice; each vial contains fixed single-cell suspensions. 4. Finally, to plan the project effectively: a) Metadata: What specific metadata will accompany each sample? Sample ID, tissue type (brain/lung), treatment group, sex, genotype, age, fixation date, cell counts, and batch number. b) Bioinformatics: Beyond raw and processed sequencing data, are specific downstream analyses or visualizations expected (e.g., cell clustering, differential gene expression)? Required analyses include: Demultiplexing and UMI deduplication Alignment (mouse genome mm10) Unsupervised clustering (Seurat/Scanpy) Differential gene expression analysis Pathway analysis (GO, KEGG, GSEA) Visualizations: UMAP, heatmaps, volcano plots c) Timeline: What is the needed turnaround time for processing and data delivery once samples are received? 6 8 weeks from receipt of samples to delivery of final data and reports. 5) How many cells the VA wants processed along with how many reads per cells. The SOW mentions 100M read per samples, but with single-cell seq, reads are on a per cell level instead of a per sample level. Cell and Read Depth Targets: Targeting 50,000 100,000 cells per sample 30,000 50,000 reads per cell, as recommended for high-quality gene expression profiling using the Parse platform. __________________________________________ Section 3 states: Contractor shall provide start-to-finish advanced genetic sequencing and quantitation services tailored to the analysis of single-cells. Start-to-finish includes the comprehensive workflow required for single-cell RNA sequencing using the PARSE platform, to include RNA tagging, library construction, sequencing, and bioinformatics. However, Section 8 (Report/Deliverables section) Statement of Work notes pre-made libraries and only requires pricing for sequencing (a portion of the workflow): Pre-made libraries lane sequencing (NovaSeq X Plus 25B PE150) .        Thus, we respectfully ask: Is the scope to provide the entire single cell workflow (i.e. receipt of frozen mouse tissue from VA and the start to finish Parse single cell workflow), or will we receive pre-made libraries requiring sequencing only? The scope includes the entire Parse single-cell workflow from receipt of fixed single-cell suspensions, through barcoding, library prep, sequencing, and bioinformatics. Pre-made libraries will not be provided. Pre-made libraries will not be supplied; all samples will be delivered as fixed single-cell suspensions, and the contractor is expected to perform library preparation. What is the sequencing depth required per cell? Sequencing Depth per Cell: 30,000 50,000 paired-end reads (150 bp) per cell on the NovaSeq X Plus or equivalent. How many samples (or pre-made libraries) will be provided per batch (since the contract states 4-20 samples per year? Sample Throughput: 4 20 samples per year, sent in batches of 2 6 samples depending on study progress. For the Parse kit does the VA want to use the Parse WT or MEGA kit? We need to know how many cells per sample are needed and if they will be shared across a WT (100K kit) or 1M cells (MEGA kit). Kit Selection: Parse WT kit (up to 100K cells) will be used for most studies. The MEGA kit (up to 1M cells) may be used when pooling samples or scaling up. The number of cells per kit will be provided prior to shipment. Please define the bioinformatics needed. Required bioinformatics deliverables include: Demultiplexing and UMI deduplication Alignment to reference genome (mouse: mm10) Quality control metrics Dimensionality reduction (e.g., PCA, UMAP) Clustering of cell populations Differential gene expression analysis between experimental groups Pathway enrichment analysis (GO, KEGG, GSEA) Visualizations: UMAP plots, heatmaps, and volcano plots Exported count matrices and metadata for downstream analysis All data must be provided in formats compatible with Seurat and/or Scanpy pipelines. The specification notes single cell sequencing (scRNA).  If we cannot recover enough live/healthy cells from the frozen mouse tissue provided by the VA, should be process the tissue for single nuclei (snRNA) instead?  Yes, if cell viability is compromised or cell recovery is insufficient, processing for single-nucleus RNA sequencing (snRNA-seq) using Parse-compatible protocols is an acceptable contingency. This should be discussed and approved by the VA before proceeding with snRNA processing. _____________________________________________ 1. This SOW mentioned using Parse technology for single-cell RNAseq. Are you sending in plates? if so, how many samples in each plate? Based on the plates, number of samples, cell count, the type of Parse kit will vary and sequencing strategies. Additional Clarifications Scope: The scope includes the entire Parse single-cell workflow from receipt of fixed single-cell suspensions, through barcoding, library prep, sequencing, and bioinformatics. Pre-made libraries will not be provided. Sequencing Depth per Cell: 30,000 50,000 paired-end reads (150 bp) per cell on the NovaSeq X Plus or equivalent. Sample Throughput: 4 20 samples per year, sent in batches of 2 6 samples depending on study progress. Kit Selection: Parse WT kit (up to 100K cells) will be used for most studies. The MEGA kit (up to 1M cells) may be used when pooling samples or scaling up. The number of cells per kit will be provided prior to shipment. snRNA Contingency: If viable single-cell recovery from frozen tissues is inadequate, tissue will be reprocessed for single-nucleus RNA-seq (snRNA) using Parse-compatible protocols, with prior discussion. Plates: No plates will be sent. Samples will be shipped as fixed suspensions in cryovials only. **See Updated SOW Attachment 1** __________________________________________________________ RFQ 36C24E25Q0045 Advanced Genetic Sequencing Solicitation Clarification and Question & Answer **Quote submission is extended from Wednesday, June 11, 2025, to no later than 11:59 EST, Monday, June 16, 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.
