Table 2 Technical factors affecting the microbiome

Sample storage

[76–79]

The gold standard for storage is −80 °C. Long-term storage at room temperature or multiple freeze-thaw cycles alter community stability. Room temperature preservation methods improve stability but may alter microbial community structure.

Primers and sequencing method

[32, 34, 82, 83]

Primer selection and hypervariable region influence the observed microbial community. Resolution is better with longer reads and the V2 and V4 regions of the 16S rRNA.

Extraction kit and kit lot

[80, 81, 90, 91]

Extraction kit alters the observed community by increasing the probability that certain bacteria will be observed. In low-biomass samples, reagent contamination in the extraction kit can have a larger effect on the observed community than the biological effect of interest.

Bioinformatics

[22, 61, 74, 84, 85, 88]

Clustering method, choice of reference, chimera removal, or de-noising method and quality filtering influence results and taxonomic assignments. Additionally, the choice of statistical analysis and data visualization can lead to conflicting conclusions with similar data.