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7  Metagenomic sequencing

First step is to design an experimental design

Metagenomes is the study of whole DNA of a biological communities.

Metagenomic of a single gene (16S, ITS, etc.) is also called metataxonomic (amplicon sequencing). In contrast all the genes/ genomes in the sample is actually metagenomics.

Some differences :

Metagenomic | Metataxonomic expensive | cheap more samples| less specific regions | random regions

See the paper from Torsten Seeman, Gilbert, Meyer - A guide from sampling data analysis

7.1 Designing the experiment

Stablish the basic experimental unit and the appropriate replicates, control, randomization !

It is important to have metadata of the experiment in a plant microbial community is common to have pH, geo-reference, temperature, etc.

In metataxonomic we analyses hypervariable regions (V3-V4) and compare relative abundance to identify which is the most informative (abundance, richness)

7.2 DNA extraction and Sequencing (Illumina)

Followed common sequencing is also important to make quality check of the data

7.3 Denoising

There are several way to define OTUs (97-99 % of similarity) for instance :

• It is defined operational unit of species or species groups (be careful to assign a sequence as an species)

• Taxonomic level of sampling selected in the study… individual, population, species,…

Pipelines for OTUs or ASVs are Quimme and Silva and deNBI

7.4 Chimera

Reads that result from combinations in the sequencing

7.5 Taxonomic annotation

Using Silva or deNBI