STATEMENT OF WORK – Next-Generation Amplicon Sequencing Services

GENERAL INFORMATION

A. INTRODUCTION

The USGS Upper Midwest Water Science Center’s (UMid-WSC) Michigan Bacteriological Research Laboratory (MI-BaRL) conducts state-of-the-art microbiology research in association with the Great Lakes Restoration Initiative (GLRI) and USGS Programs such as the Environmental Health Program and the Cooperative Water Program. The MI-BaRL needs an experienced next generation sequencing provider to continue to produce consistent, quality, cutting-edge science.

B. PROJECT BACKGROUND

The MI-BaRL is involved in microbial research on a variety of topics including bacterial pathogens, harmful algal blooms (HABs), microbial ecology of contaminated environments, antibiotic resistance, and microbial source tracking. Several of MI-BaRL’s research studies incorporate next-generation sequencing methods to better understand the microbial community and its response in contaminated environments and with harmful bloom formation. USGS microbiologists are using amplicon sequencing (i.e. 16S rRNA amplicon sequencing) to not only characterize “who’s there?” in regard to bacteria species, but also determine how the host’s or system’s microbiome changes in response to ecosystem changes including environmental modification (i.e. biological or chemical contamination). Other USGS microbiology studies are incorporating metagenomic profiling (shotgun metagenomic sequencing) to address not only “who’s there?” but also what these bacteria may be doing (metabolic capabilities and function). Additionally, MI-BaRL will be able to screen data for other genes of interest such as those involved in nutrient cycling, pathogenicity, and antibiotic resistance.

C. SCOPE

Services, non-personal, to provide all equipment, labor, travel, and supplies and materials (unless otherwise provided herein) necessary to provide next generation amplicon sequencing services.

3.0 PROJECT SPECIFICATIONS AND ASSOCIATED COSTS

The MI-BaRL staff will be responsible for extracting DNA from all samples, quantifying DNA concentrations, and submitting all administrative paperwork and samples to the contractor (next generation sequencing provider). Additionally, MI-BaRL does prepare sequencing libraries for targeted gene sequencing, such as antimicrobial resistance genes.

The MI-BaRL has several ongoing projects that require next generation sequencing services.

These projects vary in regard to sample size, number of sequencing runs needed, and type of sequencing technology required (Illumina MiSeq or AVITI). Several of these projects are multi-year projects and to ensure consistency and data quality on these projects, analyses shall be performed by the same contractor.

TECHNICAL REQUIREMENTS

The contractor shall:

1. Complete library preparation (PCR amplification including barcoding (tagging) as required to run multiple samples together on an Illumina MiSeq or AVITI sequencing run, normalization, quantitation, and pooling of product) according to documented lab quality control (QC) guidelines.

2. The contractor shall have access to and use of 515F and 806R 16S rRNA primers for 16S rRNA amplicon sequencing.

3. Contractor shall allow the use of other primers as deemed necessary by the USGS Technical Liaison.

4.Contractor shall provide QC and sequencing services of user-prepared library or pool.

5. Perform quality control checks on genomic DNA following library prep and prior to sequencing analysis. The USGS may decide to proceed with sequencing these samples after understanding and acknowledging that these samples have reduced quality.

USGS should be notified of quality control issues no later than 30 business days after receipt of samples.

6. Should provide a positive control that is run on each sequencing run.

7. Analyze samples and provide sequencing data using the Illumina MiSeq or AVITI instrument using the Illumina MiSeq (v2) kit or Cloudbreak Freestyle 600 cycle reagent kit (AVITI), 2 X 150 bp PE and 2 X 250 bp PE read format, or 2 X 300 bp PE (AVITI only), with chemistry targeting the V4 and V3/V4 regions respectively of the 16S rRNA. Samples shall be sequenced on a standard flow cell for Illumina Sequencing and either a half flow cell or full flow cell for AVITI sequencing.

8. Illumina sequencing results must meet the minimum of 3.6-4.5 Gbp or 12-15 (M) read pairs per 2 X 150 bp PE run and 6.0-7.5 Gbp or 12-15 (M) read pairs per 2 X 250 bp PE run. AVITI amplicon sequencing results must meet the minimum of 35 (M) read pairs for a medium flow cell or 100 (M) read pairs for a high flow cell run.

9. Provide demultiplex and raw sequencing data for all data.

10. Notify USGS regarding any samples that do not pass quality control checks following library prep, prior to sequencing, or after sequencing analysis.

11. Deliver data no later than 60 business days following receipt of samples for library prep. Data should be accompanied by a spreadsheet listing sample identifiers, sample barcodes, number of reads, read quality scores, % of read quality scores over a score of 30, and yield (Mbp). The overall % of quality scores over a score of 30 should be 75% per sequencing run. The contractor shall provide the USGS with access to an FTP server or other secure server for downloading sequencing data. All corresponding quality control information including barcodes (tags), library prep kit information, and primers (as appropriate) should be accompanied with data delivery whether through secure server or external hard drive. Provide access to data for downloading for at minimum 60 days following receipt of sequencing data. Archive data for retrieval for at least 1 year post sequencing.

12. Provide a written document describing the methods used for all processes including from library preparation through sequencing and provide all details on reagents, kits, and equipment used. This document is to be provided with data no later than 60 days following receipt of samples for library prep.

GENERAL REQUIRMENTS

The contractor shall:

1. Provide consultation services when planning experiments including guidance for selecting the appropriate sequencing technology and experimental design to best meet research objectives. Consultation services include but are limited to providing information on available sequencing technology and technical requirements and providing answers to questions regarding sequencing services.

2. Provide answers to technical questions regarding sequencing methods used and about technical issues with sequencing data.

3. Provide a Web-based LIMs system that allows the USGS to upload sample information for sequencing.

4. Provide the use of sequencing facility’s stock primers, including but not limited to use of 515F/806R.

5. Provide access to a secure server for downloading sequencing data or provide data on an external hard drive.

SUPPORTING INFORMATION

Period of Performance:

The contractor shall deliver data no later than 60 days following receipt of samples.

Samples will be delivered to the contractor between date of award and September 30, 2031.

USGS QUALITY ASSURANCE SURVEILLENCE PLAN

The USGS Technical Liaison (TL) will assure that the contractor delivers the quality products and services described. TL will monitor all aspects of the services performed by the contractor. Substandard Services, services that fall short of the described standards, shall be re-done by the contractor at no additional cost to the Government within 30 days of identified issue.

The Government may seek consideration for more than 2 sequencing failures per year.

SUPPORTING INFORMATION