Moderated by Paul W O’Toole and Joël Doré (@dorejoel)
Moderators aimed to make technical differences between the two categories of microbial community analysis, to help understand methods involved in gut microbiota analysis and to answer clinical questions involving analysis of the microbiota.
The human gut microbiota is the collection of bacteria, archea, virus and fungi. Paul O’Toole indicated that microbiome is a collection of genes inside microbiota and metagenomics is the method that consists in analyzing all genes of microbiota. Paul O’Toole summarized molecular tools available to investigate gut microbiota. There are two major approaches: studying phylogenetic marker using 16 rRNA genes amplification by pyrosequencing (called 16S barcodes profiling) or studying all DNA using metagenomics shotgun sequencing.
The 16S rRNA gene is a fundamental molecule of the bacterial machinery that is highly conserved to answer the question “who’s there?” thanks to the 16 barcoding.
Pyrosequencing technique is usually used to obtain 40000 reads per sample, then you can clusters reads into OTU (Operational taxonomic Units) which help to identify species and their respective abundance. Species detected abundance is based on reads count.
Bacteria can be classified using rank from phylum to genus and even clustered into species. In gut microbiota there is 4 majors phylum: Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria. They represented more than 95% of microbial mass in the gut.
With shotgun metagenomics, DNA is totally sequenced then reads are assembled and annotated to access the functional repertoire. Reads abundance can be summarized to gene to obtain functional profile after normalization. Quantitative metagenomics permit to obtain gene abundance profile so sample could be compared. Metagenomics finally answer these two questions: who’s there and what are they doing?
Metagenomics generate a huge amount of data, which makes an analysis with a simple laptop hard to complete. However, a terminal could be linked to a server and to a cluster, offering tools and the capacity to handle it.
Concerning technology, various sequencers exist in the market and biological question should determine the technical solution. Price matters, especially the cost per sequenced base. Nevertheless, sequencing is getting cheaper and finally the most important cost in terms of time comes from bioinformatics analysis. Depending on the question, for phylogenetic analysis, number of 16S rRNA gene reads is important but depending on the subject, it has to be appropriately adjusted. For babies, known to have a low diversity in microbiomes, a low number of reads is usually recommended. For shotgun metagenomics 4 to 5 Gigabases per sample become a standard. However, below certain abundance, shotgun metagenomics could fail to detect some species which could have a huge impact on health (ex.: Shigella). For such question, deeper or targeted sequencing is recommended.
Joël Doré started a presentation aiming to answer questions from the audience concerning standards operating procedure, a key step point for sample collection and alternative metagenomics approaches like those describing crosstalk between microbial community and human cells.
Joël Doré insisted that there is no established standard. He recommended to prepare sample aliquot within short delay and store it at -80°C. If there is any shipping process involved at room temperature, oxygen removal is essential to preserve anaerobic microbial community over 4 hours. It is absolutely necessary to avoid freezing/thawing cycle because this affect the recovering of microbial diversity.
Joël Doré talked about the my.microbes initiative, which aims to collect stool samples from participants all over the world. He detailed a procedure and a sampling kit which was notably developed for this project. The kit instruction is available on my.microbes.eu website and will be also available on www.gutmicrobiotaforhealth.com.
Concerning DNA extraction from stools or from biopsy, no standard exists yet, but an international consortium (called IHMS) aims to compare and standardize different procedures from many different labs across the world. DNA extraction really matters because it could lead to differences between studies. Some reagents like “RNA later”work quite well to preserve DNA but not so much with RNA. However, until now, there is no fair test study for the moment.
Beyond metagenomics, Joël Doré introduced microbiomics. Microbiomics is the field that makes the association of “omics” technique like 16S rRNA analysis, metagenomics, metatranscriptomics and metaproteomics. It was observed that at each level of “omics” data, a species core, a functions core and a proteins core were observed, leading together to a microbiome core.
Metagenomics approach is not only direct sequencing, it is also based on screening called functional metagenomics. A study showed that functional metagenomics permitted to isolate NF-KB pathway immunomodulatory metagenomics clones while transposon technique helped to identify genes which modulates NF-KB responders cells.