Amendment - RFQ 36C24E25Q0045_0001.docx

5. PROJECT NUMBER (if applicable)

CODE

7. ADMINISTERED BY

2. AMENDMENT/MODIFICATION NUMBER

CODE

6. ISSUED BY

8. NAME AND ADDRESS OF CONTRACTOR

4. REQUISITION/PURCHASE REQ. NUMBER

3. EFFECTIVE DATE

9A. AMENDMENT OF SOLICITATION NUMBER

9B. DATED

PAGE OF PAGES

10A. MODIFICATION OF CONTRACT/ORDER NUMBER

10B. DATED

BPA NO.

1. CONTRACT ID CODE

FACILITY CODE

CODE

Offers must acknowledge receipt of this amendment prior to the hour and date specified in the solicitation or as amended, by one of the following methods:

The above numbered solicitation is amended as set forth in Item 14. The hour and date specified for receipt of Offers

E. IMPORTANT:

is extended,

(a) By completing Items 8 and 15, and returning __________ copies of the amendment; (b) By acknowledging receipt of this amendment on each copy of the offer submitted; or (c) By separate letter or electronic communication which includes a reference to the solicitation and amendment numbers. FAILURE OF YOUR ACKNOWLEDGMENT TO BE RECEIVED AT THE PLACE DESIGNATED FOR THE RECEIPT OF OFFERS PRIOR TO THE HOUR AND DATE SPECIFIED MAY is not extended.

12. ACCOUNTING AND APPROPRIATION DATA

(REV. 11/2016)

is required to sign this document and return ___________ copies to the issuing office.

is not, A. THIS CHANGE ORDER IS ISSUED PURSUANT TO:

Specify authority)

THE CHANGES SET FORTH IN ITEM 14 ARE MADE IN THE CONTRACT ORDER NO. IN ITEM 10A.

15C. DATE SIGNED

B. THE ABOVE NUMBERED CONTRACT/ORDER IS MODIFIED TO REFLECT THE ADMINISTRATIVE CHANGES SET FORTH IN ITEM 14, PURSUANT TO THE AUTHORITY OF FAR 43.103(b).

RESULT IN REJECTION OF YOUR OFFER. If by virtue of this amendment you desire to change an offer already submitted, such change may be made by letter or electronic communication, provided each letter or electronic communication makes reference to the solicitation and this amendment, and is received prior to the opening hour and date specified.

C. THIS SUPPLEMENTAL AGREEMENT IS ENTERED INTO PURSUANT TO AUTHORITY OF:

D. OTHER

Contractor

16C. DATE SIGNED

14. DESCRIPTION OF AMENDMENT/MODIFICATION

16B. UNITED STATES OF AMERICA

Except as provided herein, all terms and conditions of the document referenced in Item 9A or 10A, as heretofore changed, remains unchanged and in full force and effect.

15A. NAME AND TITLE OF SIGNER

16A. NAME AND TITLE OF CONTRACTING OFFICER

15B. CONTRACTOR/OFFEROR

STANDARD FORM 30

PREVIOUS EDITION NOT USABLE

Prescribed by GSA - FAR (48 CFR) 53.243 (Type or print) (Type or print) (Organized by UCF section headings, including solicitation/contract subject matter where feasible.)

(Number, street, county, State and ZIP Code) (If other than Item 6) (Specify type of modification and authority) (such as changes in paying office, appropriation date, etc.)

(If required)

(SEE ITEM 11)

(SEE ITEM 13)

(X)

CHECK

ONE

13. THIS ITEM APPLIES ONLY TO MODIFICATIONS OF CONTRACTS/ORDERS,

IT MODIFIES THE CONTRACT/ORDER NO. AS DESCRIBED IN ITEM 14.

11. THIS ITEM ONLY APPLIES TO AMENDMENTS OF SOLICITATIONS

AMENDMENT OF SOLICITATION/MODIFICATION OF CONTRACT

(Signature of person authorized to sign) (Signature of Contracting Officer) 240E 06-11-2025 00240E Veterans Health Administration Regional Procurement Office East Building 32 Suite 1A1111 1010 Delafield Road Pittsburgh PA 15215-1802 00240E Veterans Health Administration Regional Procurement Office East Building 32 Suite 1A1111 1010 Delafield Road Pittsburgh PA 15215-1802 To all Offerors/Bidders

36C24E25Q0045

X X X ** HOUR & DATE for Receipt of Offers is EXTENDED to: 06-16-2025 11:59 EDT X -01- The purpose of this modification is to provide responses for questions asked in preparation of submissions and to extend the solicitation response date from no later than 11:59a, Wednesday, 6/11/2025, to no later than 11:59a, Monday, 6/16/2025.

All other terms and conditions of the solicitation remain unchanged.

Millicent Covert Contracting Officer

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:

1. 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.

1. 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.

1. 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.

1. 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.

1. 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.

1. 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.

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.

See attached document: Attachment 1 - Statement of Work_updated.