Requirements Document.pdf

Statement of Work

Title:

DNA isolation and16s rRNA Sequencing for microbiome analysis of murine fecal samples.

Period of Performance:

Date of Award through 12 months

Background:

The host microbiome plays a significant role in the pathogenesis of multiple gut infections.

Pathogens such as C. difficile and Vancomycin resistant Enterococcus (VRE) are typically only able to colonize the intestinal tract of humans following disruption of the hose microbiota by antibiotic therapy. The laboratory of Dr. Paul Carlson at CBER is examining the role of the microbiome in each of these infections. For C. difficile, we are examining the role of specific components of the immune system to shape the microbiome into a resistant/susceptible state and the manufacturing of Fecal Microbiota Transplantation (FMT) for treatment of C. difficile. For VRE, we are examining potential changes in the microbiome induced by phage therapy compared to standard antibiotic therapies. These studies will advance our understanding of how the microbiome develops, the role of the immune system in driving this development, the pathogenesis of C. difficile infection, and the effect of phage therapy on the gut microbiota.

Purpose/Objective:

Under this contract, the vendor will isolate bacterial DNA from mouse fecal samples. The vendor will then generate 16S rRNA libraries and run subsequent sequencing from each sample. The vendor will perform all necessary troubleshooting and quality control steps throughout the process. Samples will be sent as frozen fecal pellets in MoBio PowerMag Microbiome 96-well plates and shipped on dry ice.

Justification

The vendor will be required to isolate bacterial DNA from murine fecal samples. The vendor will be required to amplify bacterial DNA and generate libraries from each sample for 16s rRNA sequencing. The vendor will be required to multiplex up to 380 samples for one sequencing run.

The vendor shall have the capacity for high throughput, reproducible DNA isolation from fecal samples, preferably through automated methods to eliminate human error in sample preparation and processing. The vendor shall have experience preparing DNA libraries and performing 16s rRNA sequencing for microbiome analysis. The vendor must perform all steps, including troubleshooting and quality control measures, starting with murine fecal samples and ending with high quality sequence data to be returned to FDA.

Technical Requirements

The vendor must have demonstrated ability to isolate bacterial DNA from fecal samples that is suitable for 16S rRNA sequencing and subsequent microbiome analysis.

The vendor must have demonstrated ability to prepare 16S rRNA libraries from murine fecal samples that are suitable for sequencing. Including expertise to troubleshoot problems that arise at any point during the isolation, library generation, or sequencing process.

The vendor must have demonstrated the ability to perform quality control measures to ensure high quality DNA libraries. These measures should include qPCR and/or Bioanalyzer or similar methods. In addition, the vendor must be able to perform the necessary library normalization.

The vendor must have demonstrated ability to perform Illumina MiSeq sequencing of bacterial 16S rRNA.

The vendor must have the capability to multiplex 380 samples in a single MiSeq lane.

The vendor is NOT required to perform analysis of the resulting sequence data.

Deliverables:

1. Completion of Task 1: 16S rRNA sequencing of stool from various FMT specific experiments including efforts to understand the impact of manufacturing methods on product viability and to identify biomarkers of potency in these products.

Isolation of DNA from mouse fecal pellets 1520 samples spanning multiple treatments and time courses for these mice, preparation of 16S rRNA libraries for each sample, and sequencing to achieve sufficient coverage for microbiome analysis. These samples will be sent in batches of 380 samples each (in four 96-well plates). Following isolation and sequencing the vendor will return purified DNA from each sample as well as sequencing to the Carlson laboratory. The Carlson lab will pay for shipping of the DNA samples and the sequencing data will be downloaded from the vendor into the HIVE system using appropriate security procedures (this will be handled by Luis Santana-Quintero – HIVE). Samples will be shipped on dry ice overnight via UPS. Expected turn-around time for each group of 380 samples is approximately 4-6 weeks.

2. Completion of Task 2: 16S rRNA sequencing, WT mice with phage treatment vs antibiotic therapy. Isolation of DNA from mouse fecal pellets 1520 samples spanning multiple treatments and time courses for these mice, preparation of 16S rRNA libraries for each sample, and sequencing to achieve sufficient coverage for microbiome analysis. These samples will be sent in batches of 380 samples each. Following isolation and sequencing the vendor will return purified DNA from each sample as well as sequencing to the Carlson laboratory. The Carlson lab will pay for shipping of the DNA samples and the sequencing data will be downloaded from the vendor into the HIVE system using appropriate security procedures (this will be handled by Luis Santana-Quintero – HIVE). Samples will be shipped on dry ice overnight via UPS. Expected turn-around time for each group of 380 samples is approximately 4-6 weeks.

Anticipated timeline:

The first set of 380 samples will be ready to ship to the vendor immediately upon awarding of the contract. Subsequently, we anticipate having sets of 380 samples ready to ship to the vendor every 3 months.

Payment Schedule:

Payment will be made upon completion of each sequencing run (380 samples) described and receipt of invoice.

Special Requirements:

We are planning to sequence a large number of samples, representing a large financial commitment for both running the experiments and contracting out the sequencing. Because of this, the vendor must have experience in high throughput DNA isolation from fecal samples, library generation for 16S rRNA sequencing, and sequencing.

Project Officer

Paul Carlson FDA White Oak Campus 10903 New Hampshire Avenue Silver Spring, MD 20993 Bldg. 52/72 CBER Room 3306 240-402-4090 Yolanda Jamison FDA White Oak Campus 10903 New Hampshire Avenue Silver Spring, MD 20993 Bldg. 71 CBER Room 3309B 240-402-7142

Period of Performance The period of performance is twelve (12) months after award