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Shotgun metagenome analysis

When both microbiota community composition and function are of interest, all of the genetic information has to be evaluated. Genes are identified by comparing the sequencing reads with databases, and the genes encoded in the metagenome allow conclusions to be drawn on the function of the microbiota.

In contrast to 16S metagenomics, shotgun metagenomics is not targeted, is independent of primer binding sites, and is not biased by PCR during sample preparation. This gives the advantage that a wide variety of different sequences can be detected and no selection takes place. Therefore, in addition to the taxonomic information received from 16S metagenomics, the presence of non-bacterial microorganisms can also be discovered and functional analysis can be performed.

The microbiota can influence its host in positive and negative ways. One example of the interaction between bacteria and host is in mammalian digestion. Some substances cannot be absorbed and used as an energy source by humans. Bacteria colonizing the gastrointestinal tract can pre-digest these substances, and transform them into nutrients the human body can process. Changes of the microbiota in a way that stops pre-digestion can negatively influence human health.

DNA is isolated from high quality samples. The DNA is sheared into fragments of a defined length and barcode and linker sequences are attached. No amplification or selection of certain DNA sequences is performed. The order of the bases is determined by next-generation-sequencing. Comparison of the detected sequences with databases allows assignment of taxonomy or functions to the microbiota.

Applications

Compared to healthy controls, the intestinal microbiome of Type 2 Diabetes patients contains less Butyrate producing bacteria, more opertunistic pathogens and an accumulation of genes necessary for sulfate reduction and resistance against oxidative stress. (Qin et al. (2012) Nature)

Despite variations of the community structure between individuals, the metabolic pathways encoded are stable. (The Human Microbiome Project Consortium (2012) Nature)

Method

Upon receipt of a sample, the first step is quality control. It is important to verify that the samples were sufficiently cooled and not damaged during transport. Project-specific sample requirements are also checked. DNA is isolated using an established extraction protocol. After validation of DNA quality, it is prepared for sequencing with the Nextera XT DNA Sample Prep Kit, according to manufacturer’s instructions. DNA is sequenced using the Illumina HiSeq2500 and after data is demultiplexed the paired-end reads are merged. PrinSeq is used to perform quality control of the approximately 10 million reads per sample and the reads are then compared to the reference sequences available in specialized databases (NCBI Taxonomy for identification of microorganisms and SEED, KEGG and COG Classification for categorizing functional genes). After taxonomic and functional binning, a report is created.