|1. Current microbiota sequencing methods rely heavily on 16S rRNA amplicon-based methodology which detects up to genus level with minimal capability for species-level detection.||• Investigators are encouraged to utilize other methodology, such as shotgun metagenomics for its expanded taxonomic range, strain-level resolution, and identification of other microorganisms (i.e., archaea, viruses, and fungi, etc.).|
• Omic techniques, such as metatranscriptomics, metaproteomics, and metabolomics, should be integrated with compositional data to provide a ‘functional’ readout of the microbiome providing data on the metabolic interplay between the host, diet, exercise, and the gut microbiota.
• For targeted genomic approaches specific primers for quantitative PCR can be effectively used.
|2. Primer selection and hypervariable regions of 16S rRNA (i.e., V1-V9) influence the observed microbial community.||• When using 16S rRNA amplicon-based sequencing it may be better to sequence a single variable region (V4 is mostly used in human gut microbiome studies) with reads that (almost) entirely overlap to reduce errors .|
|3. Several studies report less stringent inclusion/exclusion criteria and did not fully account/control for confounding variables.||• When outlining participant criteria, investigators should pay particular attention to important factors that may confound results such as medication use (e.g., antibiotics, bile acid sequestrants, metformin, etc.), dietary supplement use (e.g., prebiotics and probiotics), age, sex, ethnicity, geographical location, etc.|
• To control for confounding effects investigators should collect more detailed data on such factors as dietary intake, training history, level of physical fitness, gastrointestinal function (e.g., gastrointestinal symptoms rating scale ), fecal pH, fecal form (i.e., Bristol stool scale, ), etc.
|4. The majority of studies in the present review did not report power and sample size calculations.||• Many studies in this field are likely underpowered. Therefore, investigators are encouraged calculate and report sample size estimations when appropriate.|
|5. Several studies in the present review did not provide adequate detail on sample collection and processing. Both of these factors can significantly affect the analysis and ultimately the results of the study.||• While there is no one method, investigators should provide detail on these procedures and ensure these methods are the same across samples.|
• Time that samples are held at room temperature should be greatly minimized (i.e., < 24 h without preservative) and kept consistent across samples [238, 239].
• Homogenization of the whole stool sample may provide a more uniform sample. This method has been shown to reduce the variation in both the amount of DNA extracted and the relative abundance of bacterial taxa .
|6. Similar to issue #5, several reports did not provide adequate detail in regard to sample transportation and storage.||• Investigators should provide detail on these procedures and ensure these methods are the same across samples.|
• The gold standard for microbial storage is freezing samples at − 80 °C .
• Investigators are encouraged to keep freeze-thaw cycles minimal as they affect reproducibility .
|7. While not a weakness per se, feces is the only sample material used in the analyses of the gut microbiota from the studies included in this review.||• Eventually, more in-depth mapping of microbial community structure and function along the length of the GI tract and across gradients (i.e., from lumen to mucosa) should be considered .|
|8. The variation in microbial analysis across different studies can make comparing/contrasting study findings difficult.||• Variation in profiling techniques (e.g., sequencing strategies, platforms, variable regions, sequencing depth, etc.) may act as a confounding variable that can lead to significant differences due entirely to laboratory techniques rather than treatment. While a limitation of the current review, future, more specific reviews should provide deeper discussions on results in the frame of their methodological approaches.|