FDA Postdoctoral Fellowship in Microbiology
*Applications will be reviewed on a rolling-basis.
A research opportunity is currently available at the U.S. Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN), Office of Regulatory Science (ORS) located in College Park, Maryland.
Approximately 265,000 cases of foodborne illness per year can be attributed to Shiga toxin-producing Escherichia coli (STEC). Assessment of the potential pathogenicity of STECs requires characterization of the whole genome, particularly the presence of Shiga toxin (stx1 and/or stx2) and intimin (eae) genes, serotype, and other virulence factors (e.g. tir, esp, toxB). Current FDA reporting protocols for STECs require approximately two weeks of analysis time for detection by qPCR and microbiological techniques, including several rounds of selective plating to generate a single isolate, followed by classification by whole genome sequencing. We propose development and dissemination of a pipeline that would detect, quantify, and classify STECs in enriched irrigation water in less than half the time (3-4 days) by using a combined qPCR and metagenomic sequencing approach using the Oxford Nanopore MinION and Illumina MiSeq. This analysis pipeline (STEC precision metagenomics) will also have the potential to distinguish several different STECs present in the same tested sample. By determining the limits of detection of each technique (qPCR, Nanopore and MiSeq sequencing), we can effectively tailor our proposed pipeline to obtain a closed or fragmented STEC genome(s). We can use the current qPCR detection method to quantify the STECs in the enrichment and determine the expected level of sequencing coverage to be obtained. Determining the limit of detection will establish the necessary concentration for obtaining a complete or fragmented STEC genome, which would allow us to conduct an in silico analysis of the recovered STEC strain(s) and predict the risk of those STECs to human health. Our findings and the methods proposed herein will have wide implications for food safety, including decreased time to STEC identification during outbreaks, characterization of the microbial community, and the potential to use these methods to determine the limits for other foodborne pathogens.
Under the guidance of a mentor, the participant will be involved in the following activities:
- Testing the effectiveness of new MinION sequencing kits and flow cells using 8 genomes already been sequenced by PacBio or Sanger methods
- Data captured in reports and presentation in meetings
- Perform experiments as designed by PI
- Maintaining a detailed report and present to PI
- Maintaining the MiSeq and MinION laboratory, inventories of chemicals and reagents
- Collaborating with the PI in growing cultures, inoculating food products with bacterial cultures, and extracting DNA using various protocols
- Participating in writing of publications
- Evaluating the utility of PromethION nanopore sequencing for deep sequencing analysis of metagenomic samples and determine its detection limit
- Compare DNA extraction method to produce high quality DNA with focus on DNA integrity, ease of use, time, and cost
- Develop a pipeline to analyze the metagenomic data produced by the Nanopore MinION using the high-performance computing system here at FDA. Emphasis in extracting E. coli reads from total sequenced reads
- Test the probability of obtaining high quality bacterial closed genomes (that can be used for SNP analyses) by also using MiSeq data (hybrid assembly) from samples that were within STEC levels 1 and 2 from the nanopore detection limit experiment
- Validate these pipelines using new unknown samples. To improve accuracy, these samples will also be sequenced using the Illumina MiSeq.
- Transfer the use of this technology to other FDA laboratories that might find the use of this portable sequencer a cost-effective application for their procedures, for both the wet laboratory as well as Bioinformatic tools to support analyses.
If you have questions, send an email to ORISE.FDA.CFSAN@orau.org. Please include the reference code for this opportunity in your email.