SOW 143021240296 redacted.docx

STATEMENT OF WORK

For Next-Generation Sequencing of long- and short-read DNA and RNA for draft genome assembly and annotation May 17, 2024

1.0 Background. The National Oceanic and Atmospheric Administration (NOAA), National Marine Fisheries Service (NMFS), Northeast Fisheries Science Center (NEFSC) is the research arm of NOAA Fisheries in the region. The Center plans, develops, and manages a multidisciplinary program of basic and applied research. Wild harvest of Atlantic sea scallops Placopecten magellanicus is the most lucrative commercial bivalve fishery in the US ($488 million USD annually), however sea scallop landings are projected to decline by 50% at the end of the century in part from ocean acidification (OA; elevated ρCO2). Adaptive genetic change can occur rapidly and offset consequences of environmental change yet depends on variation maintained within an existing population. Thus, population genetics are essential to improve accuracy of risk assessment models by examining the capacity for sensitive species to evolve and counteract long-term ecosystem-scale change. We collectively experience the prolific research initiatives enabled by publicized reference genomes for commercial bivalves (i.e. Crassostrea and Mytilus sp.). To date, there is no reference genome for sea scallops P. magellanicus; however, the few existing studies postulate that there is high genetic differentiation between sea scallops in the Mid-Atlantic bight (MAB) and the Gulf of Maine (GOM). Expansion of this thread is key, as warming will press populations poleward and shift wild harvesters to cooler, more-acidified systems such as the GOM. An assembled and annotated genome will be an invaluable product for end-users and will catalyze research initiatives; such work is dependent on the rigor of assembly and annotation. A continuous genome and gene annotation will be generated using whole-genome sequencing of short-read DNA (Illumina Hi-C Library) and long-read DNA platforms (PacBio Sequencing Revio) and RNA sequencing, each with a tissue sample from the same individual scallop. Because of the need for a reliable assembly, subsequent services are planned for quality assurance via re-sequencing (i.e. low-coverage whole genome sequencing), DNA assembly (i.e. PacBio Assembly with misjoin detection/correction), haplotyping analysis (phasing services), and gene calling. Ultimately, an assembled and annotated Atlantic sea scallop genome will enable cost effective –omics approaches to sustain a viable blue economy in the North Atlantic. End-users of the reference genome include evolutionary biologists and fisheries management/modelers and will permit exploration into possible solutions, such as assisted gene flow, sanctuaries, and rotated fishing grounds.

2.0 General Requirements

2.1 Scope of Work. Non-Personal Service. Contractor shall provide all plant, trained labor, equipment, materials, supervision, transportation, permits, insurance, and quality control necessary to provide Next-Generation Sequencing of prepared tissues for Ilumina and PacBio Whole Genome Sequencing and RNA sequencing for the NMFS/NEFSC (Milford Lab). Contractor shall provide haplotype analysis, resequencing, assembly, and annotation/gene calling services. Performance under this contract known as “Work”. Contractor shall be fully knowledgeable of all contract requirements and shall ensure Work is accomplished in accordance with the terms and conditions of the contract in a manner that will promote and maintain a safe environment.

We require one contact task in the base year, encompassing different sequencing platforms and bioinformatic services to accommodate chromosome-scale assembly and annotation from the vendor. Pacbio and Hi-C technologies are required for these sequencing approaches to keep with industry standards for shellfish genome assembly and annotation. They are as follows:

1. Task 1: DNA and RNA extractions, library preparation, and sequencing and subsequent genome assembly and annotation:

a. Must be able to receive tissue samples on dry ice, with or without fixative added (ethanol, DNA/RNA shield, or RNAlater). Government shall submitAround 6-10 tissue samples from a single individual, which will be stored in sterile 2-mL tubes and submitted, sufficient for multiple extraction methods and repeat attempts if necessary.

b. Must use the same individual scallop for task 1 (both library preparations within task 1)

c. HMW-DNA extraction and QC. Extraction using PacBio NanoBind PanDNA kit (PacBio, 103-260-000), DNA QC using Qubit, and fragmentation (if applicable) using Megaruptor 3. Evaluation of fragment sizes with Agilent TapeStation.

d. PacBio library preparation and sequencing. Use PacBio SMRTbell® prep kit 3.0 with the SMRTbell® barcoded adapter plate 3.02 for library preparation and sequence the library on PacBio Revio

e. Hi-C library preparation and sequencing. Use Proximo Hi-C 4.0 Kit and sequence the Hi-C library ~100M read-pairs on Illumina NovaSeqX plus PE150

f. Low coverage DNA sequencing for library quality assurance (<1M paired end Illumina reads). Prior to production sequencing, determine library performance by low-pass sequencing. Generate ~200,000 read pairs of sequencing data on an Illumina iSeq and map to the reference assembly

g. PacBio genome assembly with misjoin detection and correction, using PacBio Hifi long reads

h. Phasing services. Use the Hi-C data to phase the assemblies producing a draft for each haplotype.

i. RNA extraction and purification. QC quantification using Qubit RNA HS and fragment analysis with Agilent Bioanalyzer RNA 600 pico

j. RNAseq library preparation. cDNA synthesis with NEBNext® Ultra II Directional Library Prep Kit for Illumina. QC quantification using Qubit RNA HS and fragment analysis with Agilent Bioanalyzer RNA 600 pico

k. RNA-seq on Illumina NovaSeqX plus, PE150

l. Proximo assembly for medium genome

m. Gene calling and annotation. This service includes alignment of RNA-seq reads to the genome assembly validated by >one method (such as STAR and RepeatModeler, using the NCBI), masking repeats in the assembly using RepeatMasker. Apply MAKER to use repeat families generated from RepeatModeler, RNA-seq alignments from STAR, and the UniProt functional protein sequence database as evidence for gene annotation. Include a second round of annotation for ab initio gene prediction using AUGUSTUS. Finalize functional annotation with MAKER using the UniProt functional protein sequence database. Assess final scaffolds using BUSCO.

n. List all specific needs / details here

DNA sequencing on both platforms will be conducted according to the following specifications:

· Must provide all extraction (HMW-DNA and RNA extraction) and library preparation services (RNAseq, PacBio, and Hi-C)

· Must provide an Illumina Hi-C Library of genome-wide chromatin interactions for de novo assembly; ~100,000,000 read pair data

· Must provide PacBio Revio sequencing for long-read data on a single sample for de novo assembly. In addition to sequencing, must provide demultiplexing, quality control, and adapter trimming performed on the PacBio Revio instrument via Lima3

· Must provide RNAseq on Illumina NovaSeqX plus, PE150. Metrics on output and numbers of reads for Illumina sequencing should conform to Q30 greater than 80% (Q30 > 80%)

· Must provide low-coverage Illumina iSeq and sequencing for library quality assurance to map to the reference assembly; <1 million paired end illumina reads on a single sample

· Must be able to provide assembly services for PacBio Revio long-read data

· Must be able to provide a fast turnaround time for raw data (< 8 weeks from sample submission)

· Must be able to provide a fast turnaround time for QC and assembly (< 12 weeks from sample submission)

· Must provide a quality report and FASTQ files in an electronic format

· Must provide a FASTA file containing chromosome-scale assembly with a minimum Contig N50 of >5Mb and BUSCO score >85-90%

· Must provide sequencing statistics from each platform genome statistics.

· Must provide a GFF3 or GTF gene annotation file

2.1.1 Contractor shall be a fully trained authorized service provider of Next Generation Sequencing on PacBio Revio and Illumina platforms HiSeq and NovaSeq S4. Contractor shall at all times ensure Work is accomplished in accordance with the terms and conditions of the contract in a safe and professional manner.

2.1.2 Special Qualifications and Permits. Contractor shall ensure all required skill sets, and permits are obtained and maintained for performance under this contract.

2.1.3 Quality Control. Quality Control is the responsibility of the Contractor

2.2 Place of Performance. At Vendor's Location.

2.3 Period of Performance/Delivery: (1) Base year with no option years. Contractor shall deliver sequences to NEFSC within 4 months of sample submission and no later than August 31, 2025 for all CLINS. for each period of performance (See table below for dates).

3.0 Security Requirements. Security Clearances are not required.

3.1 Contractor employees and associated subcontractors performing under this contract MUST be U.S. citizens or MUST have established and maintain legal residence in the U.S., and be authorized by the US Government to work in the United States (i.e. Green card, worker authorization, etc.).

4.0 Safety, Security, Fire Protection, Environmental Controls, Conservation of Utilities, and Compliance with Laws and Regulations.

4.1 Safety Requirements. Contractor shall take all necessary precautions to meet proper safety standards, and regulations of all local, state and federal codes and regulations.

4.2 Security. There are no specific security requirements for performance under this contract.

5.0 Government Roles.

5.1 The Contracting Officer (CO) is the only individual with the authority to authorize changes within this contract that will have an effect on the scope, and monetary values (increase or decrease) of contract.

5.2 The Technical Point of Contact (TPOC) is the individual designated by the CO at contract award to perform administrative functions on the contract. The key role of the TPOC is to monitor the performance of the contract. The TPOC will observe, document, and communicate Contractor performance to the CO. TPOC and alternate TPOC (if applicable) will be responsible for the inspection and acceptance of performance and any deliverables under the contract. The TPOC does NOT have authority to change the terms and conditions of the contract. When in the opinion of the Contractor, the TPOC requests efforts outside the existing scope of the contract, the Contractor shall promptly notify the CO in writing. Contractor under such direction shall take no action until the CO has resolved the issue or has otherwise issued a modification to the contract.

6.0 Government Responsibilities:

6.1 Government shall inspect the Work performed under this contract. Any corrective action required because of the inspection will be accomplished prior to final monthly billing. Government will document instances of deficiencies.

6.2 Contractor may be notified of deficiencies in performance in writing by the CO. Oral deficiency notifications will be confirmed in writing by the CO. Contractor shall take prompt corrective action upon notice of deficiency. Prompt is defined as within 24 hours of notification.

6.3 Government Furnished Property, Materials, Equipment and Services (GFP/M/E/S). N/A

7.0 Contractor Responsibilities.

7.1 Contractor Furnished Property, Materials, Equipment and Services (GFP/M/E/S). Contractor shall provide all plant, trained labor, equipment, materials, supervision, transportation, permits, insurance, and quality control necessary to achieve the quality performance standards of the Work in this contract.

7.1.1 Contractor shall immediately notify the CO if discrepancies are discovered between the existing property conditions and those noted on the SOW.

7.1.2. Contractor shall notify the CO immediately when it is discovered that the deliverable schedule will not be met to work together to revise the schedule as necessary. Contractor shall communicate with the CO and revise the schedule as necessary. Contractor shall not deviate from schedule without prior approval from the CO.

7.1.3 Contractor shall be fully knowledgeable of all requirements of the contract documents and shall make themselves aware of all job site conditions that will affect their performance.

7.1.4 Contractor shall maintain an adequate workforce trained to safely and satisfactorily perform under this contract.

7.1.5 Contractor shall ensure all employees and subcontractors are fully trained and have a full understanding of the statement of work task requirements; government will not provide instruction or oversight.

7.1.6 Contractor shall ensure all Contractor employees, and all associated subcontractor(s) are experienced in the type of work involved and familiar with the specifications of this contract. Contractor is responsible for damage to any Government equipment and supplies caused by Contractor personnel.

7.1.7 Contractor shall comply with all applicable interstate, local, state, and federal codes and laws.

7.1.8 Contractor shall perform in accordance with professional industry standards.

7.1.9 Contractor shall accomplish all tasks to meet the requirements of the SOW.

8.0 Specific Tasks. Contractor and subcontractors working under this contract shall be fully knowledgeable and skilled in the scientific disciplines required to successfully fulfill this requirement. Contractor shall be fully knowledgeable of all contract requirements. Contractor shall at all times ensure Work is accomplished in accordance with the terms and conditions of the contract in a safe and professional manner. Contractor shall perform the following tasks (as detailed in section 2.1):

CLIN 0001: RNA extraction services CLIN 0002: RNA Library preparation CLIN 0003: Sequencing services for RNA sequences CLIN 0004: HMW-DNA extraction services CLIN 0005: PacBio Library Preparation CLIN 0006: Hi-C Library Preparation CLIN 0007: Low coverage sequencing for library QC (<1M paired end Illumina reads) CLIN 0008: Illumina Sequencing of Hi-C Library (~100M Read-Pairs) CLIN 0009: PacBio sequencing Revio CLIN 0010: PacBio Assembly with misjoin detection and correction (Genome Haplotyping Analysis) CLIN 0011: Phasing Service CLIN 0012: Proximo assembly for a medium genome CLIN 0013: Gene calling and annotation

9.0 Deliverables. Contractor shall provide the identified deliverables in the manner and time frame identified.

Deliverable #1: (for CLIN 0001 - 0013) A FASTA containing the chromosome-scale genome assembly and csv file containing genome sequencing stats. An annotation file containing gene calls and annotation. Required no later than four months from the time of sample submission, and no later than August 31, 2025 for all CLINs. .

10.0 IT Security. Not applicable. Requirements of 48 CFR 1352.239-73 do not apply and a Security Accreditation Package is not required.

11.0 Section 508. Not applicable.

12.0 Invoicing. Contractor shall provide “proper” invoices in arrears of tasks performed (and expensed) with the last invoice being submitted within 30 days of contract completion. Invoices shall be submitted (via electronic copy) no later than by the 10th day of the billing month.

1. Payments will be based on receipt of a proper invoice and satisfactory contract performance and guidelines in FAR 32.9, Prompt Payment.

2. To constitute a proper invoice, the Contractor’s invoice shall be prepared in accordance with, and contain the elements specified in FAR 52.212-4(g), “Invoices”.

3. The Government will return invoices that do not comply with FAR 52.212-4(g), “Invoices” within 7 days after the date the invoice was received along with a statement as to the reasons why it is not a proper invoice.

4. The Contractor will send invoices in accordance with the terms and conditions of this contract to the following address (electronic submission of invoices is preferred) Please reference the Contract/Order Number in your email subject line when submitting invoices.