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Brian Raphael, PhD, has been a research microbiologist at CDC since 2005 and currently leads the agency’ s Legionella activities, with a major focus on advanced molecular detection in the Pneumonia Response and Surveillance Laboratory in the Respiratory Diseases Branch. Before this, he led research involving molecular subtyping of botulinum toxin-producing clostridia at CDC’ s Enteric Diseases Laboratory Branch. Raphael graduated from the University of Arizona with a PhD in microbiology and immunology and completed a postdoctoral fellowship at Washington State University, studying mechanisms of Campylobacter jejuni pathogenesis. Here he discusses current and future applications of whole genome sequencing in the microbiology laboratory.

As a research microbiologist for the past decade or so, you have been at the forefront of next-generation sequencing( NGS) in public health. What are the benefits of this technology?

Before whole genome sequencing( WGS), we used various molecular methods to subtype bacteria, including PCR, PFGE and microarray technologies. But we were examining hundreds of targets instead of the entire genome, with millions of bases with potential diversity. With WGS, we’ re looking at the nucleotide variation all at once and we can begin to use comparative genomics to learn how strains differ from one another. Already, we are gaining an unprecedented understanding of the incredible diversity of microorganisms. For Legionella, some of the most important questions WGS can help us answer are: How can we confidently attribute an environmental source to isolates from patients? How common or rare is a particular strain? And how dangerous is it?

CDC maintains extensive culture collections. By sequencing these strains we can actually place the differences we detect among strains isolated from an outbreak within the context of the greater diversity that we now know exists. This information can help us understand if an outbreak strain is something new or unusual. One of the huge benefits of sequencing is that all of the data can be entered into public databases and queried by people all over the world. This greatly improves the value of this information by expanding the knowledge base. Bottom line: WGS gives us information very rapidly that would have taken weeks or months to learn previously.

You have experience using various bioinformatics tools and certifications in Linux, R programming and Python. Is access to bioinformatics expertise necessary to gain the full value of NGS?

Yes, it is absolutely necessary! As a microbiologist, I had to learn about bioinformatics tools to keep up with the changes that NGS brings. I am constantly evaluating new tools to determine if they are quicker, more streamlined and likely to perform better. Many of these tools have to be used with a command line interface. I think a lot of microbiologists may be intimidated by the command line. I say,“ Don’ t fear the command line.” I think it’ s great. It opens up the prospect of all kinds of new scientific software, most of which is open source, so anyone can use it.

What three pieces of advice would you offer to PHL scientists just now unpacking their laboratory’ s first Ion Torrent or MiSeq NGS platform?

The very first thing on the agenda: You should actually speak with local representatives of the instrument manufacturer and get a copy of the manual before your instrument arrives. This will probably save a lot of troubleshooting later.

The second thing: Make a plan right now to deal with data, including how to store, share and back up. Also, come up with a plan to systematically name your files. If you don’ t think about this now, you’ re going to have a mountain of data and important data may be hard to retrieve later.