Highlights

  • Quality of Whole Genome Sequencing (WGS)

  • Proficiency Testing for WGS

  • IAFP 2021 Symposium: WGS Quality and Quantity – Can You Have It All?

Quality of Whole Genome Sequencing (WGS)

Whole genome sequencing (WGS) has been recognized as the gold standard for bacterial fingerprinting in the US. Federal agencies such as FDA and CDC utilize this tool to investigate foodborne illness outbreaks. Food companies also have started applying WGS for internal contamination investigations. WGS is becoming a common tool for food microbiology because of its ability to generate an abundance of data and to deliver complete genomic information with the help of bioinformatics tools in a relatively short amount of time. But there are also concerns surrounding WGS such as data security, results interpretation, and the quality of the WGS data.  

The workflow of WGS consists of two distinct processes: the “wet” lab where samples are manipulated to be put on the sequencer and the dry lab portion where the output of sequencing data is analyzed. Both the quality of the raw data generated in the laboratory and the data analysis pipeline will affect the final results. Critical control points are important to monitor to ensure high quality results are generated from the samples that are processed. There are various contributing factors that may impact many quality parameters throughout the WGS process. Some of them are listed below:

Quality Parameters

  • Sample purity
  • DNA quality and quantity
  • GC-content
  • Read coverage

  Contributing Factors

  • Variations in genera
  • Sequencing platform
  • Library preparation
  • Bioinformatic pipeline

To this end, an ISO working group (ISO TC 34/SC 9/WG 25) has been developing a technical standard for laboratories to use as a framework for the entire WGS workflow. This standard would provide some sequencing quality control metrics throughout the WGS process from handling bacterial cultures, to genomic DNA isolation, library preparation, bioinformatics analysis, and validation of the end-to-end WGS workflow.

Proficiency Testing for WGS 

One way for laboratories to assess if the WGS method being performed is standardized and meeting quality parameters is to participate in proficiency testing (PT) schemes. There are a few PT schemes available for WGS. Some notable proficiency programs include:

  • Global Microbial Identifier (GMI) launched a questionnaire in 2011 to assess the harmonization and standardization of WGS data. A proficiency test was launched in 2015 and 2016 for both wet and dry lab portions of the WGS workflow. Results were returned from over 40 participants using different bioinformatic tools.
  • 2017-2018 US PulseNet and GenomeTrakr networks worked within the Genomics for Food Safety consortium to harmonize and coordinate sequencing among network laboratories. High reproducibility was demonstrated by the 21 labs that  participated in this PT.  

Given the wealth of information gleaned from the previously mentioned trials, Mérieux NutriSciences undertook an intracompany PT with its three WGS centers.  Each of the participants performed WGS on the same pool of strains using their routine WGS procedures and protocols. A cumulative analysis of the outputs from each of the participating laboratories was performed. Despite the differences in operators, reagents, and sequencing platforms, the overall results were almost completely identical between all three facilities. This PT not only proved the consistency and accuracy of the WGS results achieved by the three laboratories in varying environments but also identified opportunities to harmonize the workflows and quality metrics. Overall, confidence in WGS results can be enhanced by implementing uniform quality standards and assessing such performance with PT programs within any given network. 

IAFP 2021 Symposium: WGS Quality and Quantity – Can You Have It All?

If you are interested in more discussion about the quality of WGS, please join us on July 21st, 2021 for our virtual IAFP 2021 symposium S47 – “WGS Quality and Quantity – Can You Have It All?”. Professionals from public health agencies, industry and service laboratories will share their experiences and thoughts on this topic. The full results of our internal PT results will also be presented at IAFP 2021. Please visit poster P1-173 – Intracompany Proficiency Trial for Whole Genome Sequencing of Listeria monocytogenes and Salmonella enterica on July 19th, 2021. If you would like information about our WGS capabilities, feel free to reach out to our experts

 

References:

  1. Brinkmann, A., et al. (2019). “Proficiency Testing of Virus Diagnostics Based on Bioinformatics Analysis of Simulated In Silico High-Throughput Sequencing Data Sets.” J Clin Microbiol 57(8).
  2. Moran-Gilad, J., et al. (2015). “Proficiency testing for bacterial whole genome sequencing: an end-user survey of current capabilities, requirements and priorities.” BMC Infect Dis 15: 174.
  3. Timme, R. E., et al. (2020). “Gen-FS coordinated proficiency test data for genomic foodborne pathogen surveillance, 2017 and 2018 exercises.” Sci Data 7(1): 402.
  4. Timme, R. E., et al. (2018). “GenomeTrakr proficiency testing for foodborne pathogen surveillance: an exercise from 2015.” Microb Genom 4(7).

 

 

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