DRAFT_Statement of Work_scRNA Sequencing Service.pdf

Statement of Work Title: scRNA Sequencing Service of Influenza Vaccine Study Samples

The period of performance of this contract is: 12 months

SECTION 1 – BACKGROUND

The Centers for Disease Control and Prevention (CDC) is charged with influenza pandemic preparedness and response, with one of the primary goals being developing and assessing effective influenza vaccines. The immune response elicited by a vaccine is characterized by the induction of protective immunological memory. Although the precise mechanisms underlying this response are not yet fully understood, modern techniques such as bulk RNA sequencing and single-cell RNA sequencing (scRNA-seq) have greatly enhanced our understanding of the immune response following vaccination.

Bulk RNA sequencing and scRNA-seq, both workflows using Next Generation Sequencing (NGS) technologies, offer complementary insights into the immune response post-vaccination. NGS technologies are advantageous due to their high throughput, sensitivity, accuracy, reproducibility, and the specialized bioinformatics tools available for data analysis.

Bulk RNA sequencing enables the identification of differentially expressed genes between vaccinated and control groups. This approach provides a global view of gene expression changes following vaccination, which can reveal important insights into the molecular processes that underlie the immune response.

Conversely, single-cell RNA sequencing (scRNA-seq) offers a higher-resolution view of immune cell populations and their heterogeneity. This method identifies distinct immune cell subsets, their activation states, and their functional roles in response to vaccination. By characterizing the cellular composition and transcriptional profiles of individual immune cells, scRNA-seq can uncover novel genes and pathways involved in the immune response. Further, monitoring different responses along the identified pathways to different vaccine candidates can help improve the overall quality of vaccines produced.

Combining the results of bulk RNA sequencing and scRNA-seq allows for identifying key genes, cell types, and molecular pathways involved in the protective immune response elicited by vaccines. In turn, these insights will inform the development of more effective vaccines and therapeutic strategies for combating influenza and other infectious diseases.

SUBSECTION A – Definitions

1. Sequencing: Sequencing is the process of determining the order of nucleotides (adenine, guanine, cytosine, and thymine) in a DNA or RNA molecule. DNA sequencing reveals the genetic information encoded in an organism's genome, while RNA sequencing (also known as transcriptome sequencing) provides insights into the genes that are actively expressed in a particular cell or tissue under specific conditions.

2. DNA (Deoxyribonucleic Acid): DNA is a double-stranded, helical nucleic acid molecule that carries genetic information. DNA molecules consist of two long chains of nucleotides (adenine, guanine, cytosine, and thymine) arranged in a specific sequence, often called a “sequence.”

3. RNA (Ribonucleic Acid): RNA is a single-stranded nucleic acid molecule that plays a central role in the coding, decoding, regulating, and expressing genes. Composed of a sequence of ribonucleotide units, RNA molecules are involved in various cellular processes, including the synthesis of proteins through transcription and translation, gene regulation, and the catalysis of certain biochemical reactions.

4. NGS (Next Generation Sequencing): NGS refers to a group of high-throughput DNA sequencing technologies that enable rapid and cost-effective sequencing of large portions of genomes or entire genomes.

These methods allow for the simultaneous sequencing of millions of DNA fragments, which has revolutionized genomics and transcriptomics research.

5. scRNA-seq (Single-cell RNA sequencing): scRNA-seq is a technique for analyzing gene expression patterns at the single-cell level. This method enables researchers to study the heterogeneity of cell populations, identify distinct cell types, and characterize their transcriptional profiles, providing a detailed understanding of cellular diversity and function in various biological contexts.

Author Is the POP flexible?

6. bp (base pair): A base pair is the complementary pairing of two nucleotides in a double-stranded DNA or RNA molecule.

7. Paired-end reads: Paired-end sequencing involves sequencing both ends of a DNA fragment, generating two reads per fragment. This approach provides more information about the fragment's position and orientation in the genome, which is helpful for accurate mapping, assembly, and detection of structural variations.

8. NGS library: A collection of DNA or RNA fragments that have been prepared for high-throughput sequencing using NGS platforms.

9. FASTQ: A widely used text-based file format in genomics and bioinformatics for storing raw DNA or RNA sequencing data. It is designed to contain both the nucleotide sequences (also known as sequence reads) and their corresponding quality scores.

9. CDC (Center for Disease Control and Prevention): The Government entity that needs work proposed.

10. Offeror: an entity that provides a proposal to CDC for listed work.

11. Contractor: entity, if any, awarded a contract to perform proposed work.

12. Fragments Per Kilobase of transcript per Million mapped reads (FPKM): FPKM is a statistical measure used to quantify the expression levels of genes in RNA sequencing experiments. This measure normalizes the number of fragments (reads) that are mapped to a gene, taking into account both the length of the gene and the total number of mapped reads in the sequencing experiment. The normalization for the length of the gene allows genes of different lengths to be compared, and the normalization for the total number of reads allows for comparison between samples with different total read counts.

13. Principal Component Analysis (PCA): PCA is a statistical technique used in data analysis and machine learning to reduce the dimensionality of large datasets while preserving as much variability as possible. This is achieved by transforming the original variables into a new set of variables called principal components. These components are ordered so that the first few retain most of the variation present in all of the original variables.

PCA is commonly used to simplify data, reduce noise, and identify patterns in data. It's particularly useful in genomics and transcriptomics for visualizing and analyzing the relationships among samples, such as comparing genetic expression data across different conditions or identifying genetic markers.

SECTION 2 – PURPOSE/OBJECTIVE

The overall objective is to provide the Influenza Division with library construction and NGS services for RNA sequencing.

The Vaccine Preparedness Team of the Virology, from the Virology Surveillance and Diagnosis Branch within the Influenza Division of the CDC, shall be the principal scientific and technical party representing the Government’s needs, working with an indetermined Contractor from selected Offerors.

Samples from various infected, vaccinated, and/or control cohorts will be provided to the Contractor for bulk RNA sequencing and scRNA-seq.

SECTION 3 – SCOPE OF WORK

Turnaround time (TAT) is defined as the time from when the Government sends samples to the contractor for library construction and sequencing to when the sequencing data are delivered to the Government.

The Contractor shall provide the following:

(a) Bulk RNA-seq: library construction, strand-specific transcriptome sequencing (20 million 150bp paired-end reads per sample), and analysis. TAT is given for each batch and starts after quality control is completed. Maximum TAT will be 35 business for every 48 samples.

(b) scRNA-seq: Government-made libraries for single-cell RNA-seq will be sequenced, and the Contractor will provide > 300 million 150bp paired-end reads per sample. TAT is given for each batch and starts after quality control is completed. The maximum TAT will be 14 business days for libraries prepared on flow cells with 16 lanes and 28 business days for 32 lanes.

(c) Bioinformatics analysis assures and displays the quality of sequencing reads, samples, and analysis, along with the gene expression data, both normalized and raw.

The tasks in section 4 outline how these can be specifically achieved but outline the general needs of the work.

SECTION 4 – TASKS TO BE PERFORMED

Task 1: Bulk RNA Sequencing

1. To be strand-specific transcriptome resequencing/directional RNA sequences

a. 450 samples is the maximum number of samples to be provided over multiple runs

b. Coverage depth minimum of 20 million reads, at least 150bp pair-end reads per sample.

Task 2: scRNA-seq

1. scRNA-seq of up to 225 samples

a. Coverage depth minimum of 300 million reads that are at least 150bp pair-end reads per sample

b. Samples to be batched in unknown quantities, most likely 8-48 samples at a time.

2. Quality control analysis of libraries is to be performed before the sequencing of each sample and provided to CDC so that authorization may be given to proceed with sequencing.

a. It is not anticipated that many, if any, samples will fail to meet the quality thresholds needed to provide minimum coverage depth. This provision can cause unexpected degradation due to shipping or other issues and prevent the costly expenditure of reagents on poor-quality samples.

Task 3: NovaSeq-X Scale Sequencing Operations

1. High-Throughput NovaSeq-X Sequencing

a. Utilize the NovaSeq X Series 10B for high-throughput sequencing operations.

b. Each sequencing run will be completed on the NovaSeq X platform or equivalent, capable of producing up to 1.25 billion reads per run over 300 cycles.

c. Sequencing runs are batched, accommodating full lane capacity per run.

2. Genomic DNA Quality Control

a. Perform genomic DNA screening using the Agilent TapeStation system or validated equivalent.

b. Ensure detailed quality control and pooling of samples prior to sequencing to maintain sample integrity.

3. Lane Preparation and Sequencing for NovaSeq X Series 10B

a. Preparation and handling of lanes using the NovaSeq X Series 10B setup, or their validated equivalent.

b. Each lane to yield approximately 1-1.25 billion reads and generate about 300 Gb of output over 300 cycles.

c. Ensure efficient lane management to maximize output and maintain sequencing quality.

Task 4: Bioinformatics Analysis for Transcriptomics

1. Provide comprehensive bioinformatics analysis on RNA samples.

2. Output summaries shall include at minimum:

a. Alignment

b. FPKM raw read count

c. Expression estimates,

d. Gene body coverage,

e. Splicing annotation

3. Post-sequencing analyses shall include:

a. Hierarchical clustering

b. PCA

c. Heatmap correlation to assess sample relationships and data quality.

SECTION 5 – GOVERNMENT-FURNISHED PROPERTY

The CDC will collect samples and extract RNA from them. This RNA will be provided to the Contractor for bulk RNA sequencing. The CDC will create scRNA libraries that will be provided to the Contractor for RNA sequencing of sufficient quality so that a coverage depth of 300 million 150bp pair-end reads can be obtained for each sample.

The CDC will furnish samples via standard express courier services at the completion of batches and at its discretion during the performance period.

SECTION 6 – PLACE OF PERFORMANCE AND CONTRACT SUPPORT HOURS

The contractor’s facility will be the place of performance, and the contractor will determine the support hours needed to complete all tasks under this contract.

SUBSECTION A – PLACE OF PERFORMANCE

Work will be conducted entirely at the Contractor’s facility.

SECTION 7 – TRAVEL

No travel is anticipated for this award.

SECTION 8 – DELIVERABLES/REPORTING SCHEDULE

A. Reporting Schedule

(a) Quality control results for samples will be provided to the CDC within seven (7) days after receipt of samples.

(b) All sequencing data delivered under this agreement shall contain the following minimum information:

(a) QC report

(b) FASTQ files

Task Deliverable Quantity/Format Due Date Deliver To

1.1 Bulk RNA

Sequencing Data

450 results, digital: FASTQ at minimum

35 business days after receipt of samples.

SME

2.2 Quality control

report of sample libraries

225 reports, digital: PDF 7 days after receipt of samples.

SME

4.2 scRNA Sequencing Data

225 results, digital: FASTQ at minimum

17 business days for libraries prepared with flow cells of 16 lanes or less;

28 business days for libraries prepared with 32 lanes.

SME

4.3 Post-

sequencing Bioinformatics Report

450 reports, digital, PDF and supporting data files

7 days after completion of sequencing, deliverable task 4.2

SME

SECTION 9 – REFERENCE MATERIALS

None.

SECTION 10 – MINIMUM VENDOR QUALIFICATIONS

1. Proven capacity to produce library construction, strand-specific transcriptome sequencing, and analysis for 24 samples with a TAT of 20 business days.

2. Proven capacity to produce sequencing of 16 scRNA-seq premade libraries with > 300 million 150bp paired-end reads, each with a TAT of 14 business days.

SECTION 11 – ADDITIONAL REQUIREMENTS

Section 508 Compliance

Electronic and Information Technology Accessibility

(a) Section 508 of the Rehabilitation Act of 1973 (29 U.S.C. 794d), as amended by the Workforce Investment Act of 1998 and the Architectural and Transportation Barriers Compliance Board Electronic and Information (EIT) Accessibility Standards (36 CFR part 1194), require that when Federal agencies develop, procure, maintain, or use electronic and information technology, Federal employees with disabilities have access to and use of information and data that is comparable to the access and use by Federal employees who are not individuals with disabilities, unless an undue burden would be imposed on the agency. Section 508 also requires that individuals with disabilities, who are members of the public seeking information or services from a Federal agency, have access to and use of information and data that is comparable to that provided to the public who are not individuals with disabilities, unless an undue burden would be imposed on the agency.

(b) Accordingly, any offeror responding to this solicitation must comply with established HHS EIT accessibility standards. Information about Section 508 is available at http://www.hhs.gov/web/508. The complete text of the Section 508 Final Provisions can be accessed at http://www.access-aboard.gov/sec508/standards.htm.

(c) The Section 508 accessibility standards applicable to this contract are: 1194.

205 WCAG 2.0 Level A & AA Success Criteria 302 Functional Performance Criteria 502 Inoperability with Assistive Technology 504 Authoring Tools 602 Support Documentation 603 Support Services

In order to facilitate the Government's determination whether proposed EIT supplies meet applicable Section 508 accessibility standards, offerors must submit an HHS Section 508 Product Assessment Template, in accordance with its completion instructions. The purpose of the template is to assist HHS acquisition and program officials in determining whether proposed EIT supplies conform to applicable Section 508 accessibility standards. The template allows offerors or developers to self-evaluate their supplies and documentation detail - whether they conform to a specific Section 508 accessibility standard, and any underway remediation efforts addressing conformance issues. Instructions for preparing the HHS Section 508 Evaluation Template are available under Section 508 policy on the HHS Web site http://hhs.gov/web/508.

In order to facilitate the Government's determination whether proposed EIT services meet applicable Section 508 accessibility standards, offerors must provide enough information to assist the Government in determining that the EIT services conform to Section 508 accessibility standards, including any underway remediation efforts addressing conformance issues.

(d) Respondents to this solicitation must identify any exception to Section 508 requirements. If a offeror claims its supplies or services meet applicable Section 508 accessibility standards, and it is later determined by the Government, i.e., after award of a contract or order, that supplies or services delivered do not conform to the accessibility standards, remediation of the supplies or services to the level of conformance specified in the contract will be the responsibility of the Contractor at its expense.

http://www.hhs.gov/web/508 http://www.access-aboard.gov/sec508/standards.htm http://www.access-aboard.gov/sec508/standards.htm http://hhs.gov/web/508

(e) Electronic content must be accessible to HHS acceptance criteria. Checklist for various formats are available at http://508.hhs.gov/, or from the Section 508 Coordinator listed at https://www.hhs.gov/web/section-508/additional-resources/section-508-contacts/index.html. Materials that are final items for delivery should be accompanied by the appropriate checklist, except upon approval of the Contracting Officer or Representative.

http://508.hhs.gov/ https://www.hhs.gov/web/section-508/additional-resources/section-508-contacts/index.html