**2. Requirements for a gut microbiota test**

The general requirements for a gut microbiota test is (i) simple sample collection, (ii) comprehensive, (iii) reproducible, (iv) high-throughput and (v) affordable. In addition, the test must deliver results that can be interpreted and be relevant to disease.

There are still major challenges with all these requirements. Most methods developed for research are actually not well documented, and promises far more than the methods in reality can deliver. A current example of this is the emergence of the next generation sequencing approaches. For instance, it is now being realized that these techniques inflate diversity measures (Kunin et al 2010). New software tools have been developed to reduce

The first step in 16S rRNA gene microbiota analyses is to purify DNA from all bacteria present in a sample without introducing bias due to e.g. differential lysis or recovery. Subsequent to DNA purification, all bacterial 16S rRNA genes in the samples are amplified using primers targeting generally conserved regions in the 16S rRNA gene. The ratio between 16S rRNA gene copies for different bacteria is (in theory) conserved during the

Currently the most widely used approach to analyze 16S rRNA gene diversity is through next generation sequencing (Kunin et al 2010). The depth of the information that can be obtained form such sequencing efforts are dependent on the number of reads that are being

An alternative approach is the use of high-density microarrays based on 16S rRNA sequence variations. These approaches are explorative, and intended for discovery rather than diagnostics. A major challenge with traditional 16S rRNA gene microarrays is probe specificity, and cross-reactivity between closely related species (Cox et al 2010). For microarrays, this challenge has recently been addressed by tilling probes covering the variable region of the 16S rRNA gene (Rajilic-Stojanovic et al 2009). The principle by tilling is that a large number of overlapping probes cover the region of interest, with the combined probe signals providing a relatively good signal-to-noise ratio. Both for next generation sequencing and microarray analysis, a comprehensive data analysis of the information is

A more direct approach for characterizing the gut microbiota is the use of highly specific single nucleotide primer extension (SNuPE) probes (Eggesbo et al 2011).The high specificity of the SNuPE assay is obtained by DNA polymerase based incorporation of a labeled dideoxynucleotide (Syvanen et al 1990). The SNuPE probes are constructed so that the probes hybridize adjacent to discriminative gene positions. If the target bacterium is present,

A technology platform have been developed that can readily be applied to analyze the gut microbiota based on the SNuPE principle. The method – called GA-map™ (Genetic Analysis' Microbiota Analysis Platform) - has already demonstrated its usefulness in applications for assessing various disease states based on analysis of the composition of the

Here, we discuss the possibility of using a novel comprehensive targeted approach for

The general requirements for a gut microbiota test is (i) simple sample collection, (ii) comprehensive, (iii) reproducible, (iv) high-throughput and (v) affordable. In addition, the

There are still major challenges with all these requirements. Most methods developed for research are actually not well documented, and promises far more than the methods in reality can deliver. A current example of this is the emergence of the next generation sequencing approaches. For instance, it is now being realized that these techniques inflate diversity measures (Kunin et al 2010). New software tools have been developed to reduce

quantifying the inhabitants of the human gut microbiota in disease diagnostics.

test must deliver results that can be interpreted and be relevant to disease.

PCR amplification process.

required in order to obtain interpretable results.

**2. Requirements for a gut microbiota test** 

a labeled dideoxynucleotide is incorporated by the polymerase.

done.

gut microbiota.

this inflation of diversity, but it is still uncertain if the number of different Operational Taxonomic Units (OTUs) obtained by next generation sequencing techniques gives a correct representation of the actual number of different bacteria present in the sample.

Perhaps the largest challenge in gut microbiota diagnostics is the establishment of the correlation between microbiota patterns and disease. Since the characterization of the gut microbiota is in its infancy, there are not many diseases that have actually been characterized with respect to specific dysbiosis of the gut microbiota. Furthermore, since the analysis represent patterns rather than single bacteria, new diagnostic principles must also be implemented.

Diagnostic approaches related to dysbiosis of the gut microbiota have until now been dominated by culture dependent techniques which only detect a minor portion of the true diversity. Doctors used to such techniques may find it challenging to adapt knowledge about non-cultivable bacteria in their diagnostics because that would include bacteria that they are not familiar with.

We have for instance found that the clostridal family Lachnospiraceae is the most stable and dominant phylogroup in the human gut (Sekelja et al., 2011).This phylogroup has been overlooked by the traditional cultivation dependent techniques, probably because it is strictly anaerobic and that it has special growth requirements that are difficult to simulate on plates.
