BMott-SoW-2.9.2023-Vers02.1final.docx

Statement of Work (SOW) Project: NextGenSeq-Amplicon 16S-18S-ITS Sequencing

GENERAL INFORMATION

1.0 Scope of Work: Describe honey bee-associated microbial diversity and disease with microbial amplicon sequencing.

2.0 Background: The Anderson lab at USDA-ARS-PWA-CHBRC requires external DNA sequencing facilities utilizing specialized Next-Generation-Sequencing technologies to perform PCR Amplicon Metagenomic sequencing with 16S, 18S or ITS rRNA operon primers. The primers are universal to major microbial clades (bacteria, fungi, etc.) and allow for microbial community characterization and research. We are seeking a multi-year contract with fixed pricing (per sample or per 96-well plate) to control costs and guarantee turn-around times (TAT). This contract is critical for research objectives in both our CRIS and our multi-year NIFA grant focused on leveraging next-gen seq ‘big data’ to study brood disease.

CONTRACTOR REQUIREMENTS

3.0 Technical Requirements/Tasks:

3.1 General Requirements

3.1.1 The Contractor, as an independent contractor and not an agent of the United States Department of Agriculture, Agricultural Research Service, Carl Hayden Bee Research Center shall supply the necessary personnel, reagents, and laboratory equipment needed to perform: initial sample QC, PCR amplification, amplicon library preparation, library sequencing, post-run demultiplexing and quality control filtering of data, and data transfer to contracting lab (Anderson lab at USDA-ARS-CHBRC, henceforth CHBRC).

3.2 Specific requirements

3.2.1 For each sub-project CHBRC will:

1) CHBRC will: Provide frozen-liquid DNA templates extracted and purified via commercial silica-column kits using modified SOPs. These will be labeled and supplied per contractor’s instructions (e.g., in 96-well PCR plates or 1.5ml snap-cap microcentrifuge tubes). CHBRC will ship samples overnight on dry ice to contractor’s receiving facility located within the continental U.S.

2) CHBRC will submit batches of samples for each experiment (e.g., 192, 384, or 768 samples per batch).

3) CHBRC will complete a sample-submission-form per contractor’s instructions, including sample IDs (for demultiplexing) and PCR primers to be used for sequencing.

3.2.2 For each sub-project contractor will:

1) Contractor will: Receive, process and invoice each batch of CHBRC-submitted samples separately.

2) Contractor will: Receive, handle and store the provided samples following best practices to ensure sample quality. Contractor will perform initial quality checks on all received samples and provide CHBRC with a report on sample DNA quantity and quality. If any CHBRC-provided samples fail this QC check, contractor will provide reasonable options for CHBRC to either replace, remedy, or exclude failed samples from downstream processing. Any additional work related to these samples (e.g., attempted re-purification) will be quoted at reasonable rates, and samples excluded from downstream processing will only be invoiced a reasonable fee for initial QC checks.

3) Contractor will: After QC checks, all approved samples will proceed through specified sequencing workflow and contractor guarantees the specified quantities of high-quality amplicon-sequencing data (i.e., DNA sequence reads) within the specified TAT (see Table 2 below).

a. (High quality sequencing data is defined as, for each sample, ≥75% of reads with ≥Q30 PHRED score).

4) Contractor will: provide the appropriate molecular biology laboratory equipment and all consumable supplies and reagents required to process the template DNA provided by CHBRC and produce the specified quantities of high-quality amplicon-sequencing data (i.e., DNA sequence reads, see table below).

a. Sequencing platform should be Illumina MiSeq, NovaSeq, or equivalent platform capable of providing the specified quantities of high-quality amplicon-sequencing data for the specified amplification primers.

b. Appropriate equipment, supplies, kits, and reagents (ESKR) are defined by standard operating procedures (SOPs) and guidelines from the sequencing-machine-manufacturer (e.g., Illumina) and other reputable molecular biology vendors (e.g., NEB, ThermoFisher, Bio-Rad, etc.) that produce ESKR with demonstrated quality and compatibility with the proposed platform and workflow (e.g., Illumina MiSeq v2-PE250).

c. Appropriate reagents include a selection of well-studied 16S-, 18S-, and ITS- rRNA-operon sequencing primers previously used in peer-reviewed published academic research. These primers must be suitably universal for, and specified to, a target clade (eubacteria, fungi, etc., see Table 3 below). Primers must provide sufficient amplicon length that is well-suited for the proposed sequencing platform (e.g., Illumina MiSeq v2-PE250) and allows for microbiome studies with good taxonomic resolution (e.g., 300-500bp).

5) Contractor will: have all knowledge, expertise, tools and other items needed to perform the stated work and deliver the digital results to CHBRC within the guaranteed timeframe (see table below).

6) Contractor will: follow all appropriate manufacturer SOPs, guidelines and best practices for each step of the library preparation, sequencing, and post-run processing. Contractor will state clearly what manufacturer ESKRs and SOPs will be utilized so CHBRC has complete information about how the samples are processed and can be assured that resulting data are high-quality and reproducible with other contractors or researchers utilizing equivalent ESKRs and SOPs.

Table 1: Amplicon Sequencing (16S / 18S / ITS) services and quantities per year

(1) Amplicon Metagenomics Sequencing, (30,000 raw reads per sample)

Table 2: Turn-Around-Times for specified batch size

Table 3: Example primers and target regions

4.0 Government Furnished: As described above, government (CHBRC) will provide extracted and purified template DNA samples to sequencing facility, plus the necessary labeling information so samples can be received, processed, their relevant data returned to CHBRC in an orderly manner.

5.0 Deliverables / Schedule: Contractor will deliver the final product, DNA sequence results data for each sample, to CHBRC via an encrypted web-server. Delivery times will depend on the quantity of samples CHBRC submits in a batch, but should not exceed the guaranteed turn-around-times (TAT) as specified in Table 2 above.

6.0 Travel: n/a

7.0 Contractor’s Key Personnel: n/a

8.0 Security Requirements: n/a

9.0 Data Rights: All sequence data produced by the contractor shall be the sole property of USDA and will not be shared with any other party, except for purposes of encrypted transfer to CHBRC via secure web-server (e.g., data may be uploaded to Illumina BaseSpace for secure transfer to CHBRC). Per Results turn-around-times specified above, data for each sample-batch will released to CHBRC promptly after completion of sequencing and post-run processing. Contractor will retain data for sufficient time to ensure successful transfer to CHBRC (minimum 60 days).

10.0 Section 508 – Electronic and Information Technology Standards: n/a

11.0 Recommended Sources, Competitors, and Attached market research:

11.1 Recommended Source:

Novogene Company Address:

8801 Folsom Blvd, Suite 290, Sacramento, CA 95826 VAT number:

GB 273975163

Phone:

916-252-0068(toll free) Fax: 916-252-0068 Novogene Representative:

Samantha Jurecki: samantha.jurecki@novogeneusa.com Technical Support:

Yao Xue Novogene: xue.yao@novogeneusa.com

11.2 Competitors found via Market Research:

IDGenomics SPC www.idgenomics.com Address: 13751 Lake City Way NE, Suite 350 Seattle, WA 98125 Phone: +1(425)5496100

ASU Genomics Facility https://cores.research.asu.edu/genomics Genomics Facility The Biodesign Institute Arizona State University 1001 S. McAllister Ave.

Tempe, AZ 85287-6401 Lab Phone: 480-965-4998 Email: genomicshelp@asu.edu Joy Blain, Facility manager, Email: joy.blain@asu.edu

UA Genetics Core https://uagc.arl.arizona.edu/services University of Arizona Genetics Core 1657 E Helen St Rm 106 Tucson, AZ 85721 Phone: (520) 621-9791 Fax: (520) 621-9247 Email: uagc@arizona.edu(link sends e-mail) Manager Genomic Research Services: Barbara Fransway, PSM, ASCPCM

MrDNA https://www.mrdnalab.com/contact.html MR DNA (Molecular Research LP) 503 Clovis Rd Shallowater, TX 79363 Telephone: +1 806 789 7984

FAX: +1 866 429 7407

Email:info@mrdnalab.com

UC Davis Genome Centre https://dnatech.genomecenter.ucdavis.edu/ Inquiries to

EMAIL: DNATECH@UCDAVIS.EDU

LAB PHONE: 530-754-9143

FAX: 530-754-9658

Attachments:

NovoGeneSampleQuote.pdf IDG_quote-Aug2021.pdf UAGC_quotes_Dec2022.zip EzBiomeBid-Sep2022.pdf RTLgenomicsBid-Sep2022.pdf

Pricing for ASU Genomics Facility, MrDNA, and UC Davis Genome Center was all publically available on their websites as of March 2022. Brendon Mott can provide this upon request.

12.0 Evaluation factors:

12.1 Best value will be considered for awarding the contract with these factors

1) Lowest price (30%)

2) Best guaranteed turn-around-Time (20%)

3) Best guaranteed data quality (20%)

4) Contractor’s ability to provide clear and complete information on sequencing workflow and ESKR (equipment, supplies, kits and reagents) to be used so CHBRC can be confident in the quality and reproducibility of results. (10%)

5) Past performance and other indications of technical capability (e.g., quality of ESKR and QC steps in process). (20%)