The oversight of microbes

The knowledge and the monitoring of microbial flora are important steps in the quality and R&D processes of companies in health and food sectors.
The exploration of microbial world, for example to detect contaminants, is traditionally performed with Pasteur methods. These methods involve cultivating microorganisms on selective culture media, where only a few species grows. However this strategy has three main problems:

• The ability of identification is relatively low (usually it is possible to only determine the family or the genus of a microorganism);
• The detection capacity is limited: only the cultivable species can be detected;
• An a priori approach: only a preemptive spectrum is scanned with no warranty that the target species are causing the problem.

Moreover, microbes can exchanges some genetic material by several ways (transformation, transfection, conjugation). They are therefore able to quickly acquire new features, some of which are used by Pasteur methods to identify an organism. These transfers of genetic functions, called lateral transfers, may cause false positive through these techniques.

PCR and First Generation Sequencing.

To solve the problem of the ability of identification, it is possible to combine Pasteur methods with modern molecular biology methods. In this case, the DNA of the target microorganism is analysed. A universal barcode (usually the 16/18s ribosomal RNA gene) is amplified and sequenced to more accurately determine the exact species. These methods include PCR techniques (Polymerase Chain Reaction), quantitative or not, and first generation sequencing (Sanger method). Since part of the genome is only explored (500-1500 bp of 5 to 10 million bp), this is called micro-sequencing. Nevertheless, the latter two problems are not resolved: the ability to detect non-cultivable species and the possibility to explore an entire microbial flora.

NGS - Next-Generation Sequencing

Advances in DNA sequencing allowed the emergence of high-throughput methods: the next generation sequencers or NGS. With these machines, it is possible to sequence several million of DNA sequences in parallel, instead of one sequence at a time with the Sanger method. Three new paradigms appeared with NGS:

• Genomics: complete exploration of a genome (all the genes of a living being);
• Transcriptomics: exploration of all the genes used by a cell;
• Metagenomics: exploration of all the genomes of an environment.

Since DNA is a universal molecule of the living (in cellular organisms, the statement is false in viruses that also use RNA for storing their genetic information), metagenomics allows detecting which species are present in an environment. It also helps to know the biological functions encountered in an environment (eg. heavy metal detoxification, iron assimilation, photosynthesis, pathogenicity, etc.). But compared to genomics and transcriptomics, metagenomics is a very greedy research activity in bioinformatics resources. Indeed, we must analyse and interpret genetic information from hundreds or thousands of living species.

Metagenetics

To identify living species, it is not necessary to analyse all the genetic information present in an environment, but only the famous barcodes that are used in micro-sequencing. In this context, the term of metagenomics is not proper, and scientists chose metagenetics or partial metagenomics to describe this approach. According to the sequencing depth (amount of analysed sequences), a precise monitoring of microbial populations (Archaea, Bacteria or Eukaryota) can be undertaken.

Examples of applications :

• Studies of microbiota imbalances in humans (chronic disease, obesity, cancer, rare diseases, etc.);
• Analysis of the quality of water, air or environment;
• Effects of products on microbiota (effect of a food product on the gut microbiota, effect of a fertilizer on the soil microorganisms, choice of materials, etc.)

Biomanda turnkey solutions

Biomanda provides a customized solution allowing to any company to benefit of these technological advances:

• Advices on scientific strategy (statistical validation, qualification of the issue);
• Molecular biology techniques (NGS, PCR, DNA extraction, etc.);
• Bioinformatics analyses;
• Interpretations of results.