Design	Recommendations	Further reading
Sample size	Consult a statistician or utilize tools such as powmic or Micropower to estimate sample size before beginning study	(Chen, 2020; Debelius et al., 2016; Kelly et al., 2015)
Sample collection method	Stool samples: fresh sample	(Claesson et al., 2017; Liang et al., 2020)
	Tissue samples: whole biopsies rather than mucosal scrapes are preferable
Sample storage	Store samples immediately at −80°C or if study design requires RT storage, store in 95% ethanol	(Marotz et al., 2021; Pollock et al., 2018)
DNA extraction method	Use a mechanical lysis method and try to ensure samples are processed with the same kit	(Gerasimidis et al., 2016; Nearing et al., 2021)
Controls	Prudent use of negative and positive controls. We recommend at least one extraction control per batch and additional water controls during library preparation and sequencing	(Bedarf et al., 2021; de Goffaude et al., 2018)
Sequencer	Illumina MiSeq/HiSeq™ machines are appropriate for most 16S studies	(Caporaso et al., 2012; Pollock et al., 2018)
Hypervariable region	V1-V2/V3, V4, and V3-V4 are all commonly used and suitable for animal or human studies	(Abellan-Schneyder et al., 2021)