Shotgun metagenomic sequencing

 

Image: By Krista M. Ruppert, Richard J. Kline and Md Saydur Rahman - CC BY-SA 4.0. Source: https://commons.wikimedia.org/w/index.php?curid=99493087

The strangely named “shotgun metagenomic sequencing” involves sequencing all genes present in a clinical sample thus allowing identification of pathogens, and (as a bonus) their associated resistance genes and pathogenicity factors.

 

Shotgun metagenomics, is the untargeted ('shotgun') sequencing of all ('meta-') microbial genomes 'genomics' present in a sample. Shotgun sequencing can be used to profile taxonomic composition and functional potential of microbial communities and to recover whole genome sequences.

 

It sounds relatively straightforward, but there are certain challenges that one needs to be aware of.

• Sensitivity:- Unlike PCR, the process does not involve an amplification step, therefore it may not be as sensitive as other currently existing methodology. In addition, if the sample contains a lot of human DNA, or DNA from non-pathogens, then the pathogen can be “drowned out” in the testing process. (stoichiometric ratios). Methods to enhance pathogen and suppress non-pathogen/host nucleic acid during the sequencing process are in development to mitigate this issue, but it is a work in progress.

 

 

• Turnaround Time:- Traditional sequencing can take 1-2 weeks when you add the time for the various steps; extraction, library preparation, sequencing and bioinformatic analysis. This is still slow compared to current culture-based and PCR methodology. Newer Real-time sequencing techniques such as Oxford Nanopore can potentially reduce this turnaround down to a couple of days.
• Cost:- Cost is coming down, and depending on what sequencing platform you use, can be anything from a hundred dollars to a few hundred dollars per sample. The cost will almost certainly come down further but we are still some way from the cost of a couple of agar plates.
• Bioinformatic analysis and validation thereof:- The bioinformatic analysis of genetic sequences remains somewhat foreign to most microbiologists. Slowly but surely automated bio-informatic pipelines are being developed which automates this step for an increasing number of pathogens. However, validation of these pipelines is laborious and difficult and requires the input of specialist bioinformaticians.

There are now metagenomic assays commercially available in several areas, the most promising possibly being metagenomic analysis of CSF samples for infective causes of meningo-encephalitis. But it is still only a small niche area of the market, and it has a long way to go before becoming mainstream.

The above post is an edited etract from the blog Microbiology Matters!

Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)