Table 2 Technical factors affecting the microbiome
From: Tiny microbes, enormous impacts: what matters in gut microbiome studies?
| Covariate | References | Findings |
|---|---|---|
| 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. |