Table 4.

Specific considerations for designing a microbiome assessment study in upper gastrointestinal disease.

Workflow		Considerations
Study design	Prospective case-control	Cross-sectional observational studies to determine microbial–disease associations Longitudinal studies: premalignant conditions to identify causative factors for diagnostic purposes, therapeutic response, prognostication
	Patient factors influencing the microbiome	Neoadjuvant treatment naive Use of proton pump inhibitors and/or histamine-2 antagonists Use of antibiotics (consider time interval between use and microbiome assessment) Immunosuppressive states Synchronous cancer Smoking status Previous gastrointestinal surgery
	Geographical location	Genetic and lifestyle factors such as diet and exercise
	Matching groups	Matched age and gender as a minimum
	Sample size calculation
	Sub-group analysis	E.g., tumour stage, ethnicity, geographical location and its association with therapeutic response and prognostication—homogeneity will allow for a more accurate microbiome assessment.
Sampling process	Sample weight	Endoscopic tissue biopsies can be of variable size. Establish a minimum weight of tissue for adequate analysis.
	Positive control	Consider adjacent healthy tissue.
	Negative control	Consider storing an empty tube and/or storage medium at the same time as the sample.
	Replicates	(where possible)
	Minimise freeze–thaw cycles
Laboratory techniques		This is comprehensively covered by the STORMS reporting checklist [110].