What is the appropriate approach to ordering a microbiome test, specifically a 16S (16S ribosomal ribonucleic acid) rRNA (ribonucleic acid) gene sequencing test, for an adult patient with gastrointestinal symptoms?

How to Order Microbiome Testing for Gastrointestinal Symptoms

Microbiome testing using 16S rRNA gene sequencing is not currently recommended for routine clinical decision-making in patients with gastrointestinal symptoms, as the current state of knowledge and technology does not provide sufficient value for clinical decisions. 1

Current Clinical Utility

• Direct-to-consumer and clinical microbiome tests do not yet have established clinical utility for diagnosing or managing gastrointestinal conditions, despite growing commercial availability 1
• The field lacks universally accepted standards for conducting clinical microbiome studies, making interpretation of results problematic 2
• Considerable research remains before microbiome diagnostics can be reliably incorporated into individualized clinical care 1

When Microbiome Testing May Be Considered

If you determine that microbiome analysis is necessary for research purposes or in specialized clinical contexts, follow this structured approach:

Step 1: Determine Sample Type Based on Clinical Question

• For general gut microbiome assessment: Use stool samples, which provide the highest microbial biomass (up to 75% of fecal mass is bacterial) and lowest host DNA contamination (<1%) 3
• For small intestinal bacterial overgrowth (SIBO) concerns: Use breath testing (combined hydrogen and methane) rather than microbiome sequencing, as this is the validated diagnostic approach 4
• Tissue biopsies contain 50-80% host DNA, making them less suitable for standard microbiome analysis 3

Step 2: Sample Collection Protocol

• Immediate freezing at -80°C is the gold standard for preserving microbial community composition 3
• If immediate freezing is not feasible, use stabilization buffers:
  • 95% ethanol or RNALater are comparable to immediate freezing for DNA preservation 3
  • DNA Genotek's OmniGene commercial transport kit induces less change than typical inter-individual variation 3
  • Avoid 70% ethanol or room temperature storage, as these cause substantial changes in microbial profiles 3
• Homogenize samples immediately upon collection when using stabilization buffers 3

Step 3: Specify Laboratory Requirements

Critical quality control measures that must be included 3:

• Sampling blanks: Unused collection materials (swabs, containers, transport fluids) to assess contamination during collection 3
• DNA extraction blanks: Process alongside samples to assess contaminant DNA from extraction kits (the "kitome") 3
• No-template amplification blanks: Assess contamination from PCR reagents 3
• Positive controls: Mock communities or synthetic DNA controls processed with each batch 3
• Place controls asymmetrically in purification plates to verify proper sample tracking 3

Step 4: Choose Sequencing Method

• 16S rRNA gene sequencing (V3-V4 or V4 region) is appropriate for:

  • Low biomass samples (skin, tissue biopsies) 3
  • Initial taxonomic profiling studies 5
  • Budget-constrained projects 6

• Shotgun metagenomic sequencing provides more information but at higher cost:

  • Identifies functional genes and metabolic pathways, not just taxonomy 3
  • Better suited for high biomass samples like stool 3
  • Requires higher DNA concentrations for reliable results 7

Step 5: Specify Analysis Requirements

The laboratory report must include 3:

• Taxonomic reference database used (SILVA, Ribosomal Database Project, or GreenGenes) and version number 3
• Sequencing depth: Minimum read numbers per sample 3
• Diversity metrics: Both alpha-diversity (within-sample) and beta-diversity (between-sample) measures 3
• Batch effect assessment: Documentation that batch effects do not overlap with clinical variables of interest 3
• Contaminant mitigation methods: How contamination was identified and addressed 3

Critical Pitfalls to Avoid

• Low biomass samples are particularly vulnerable to contamination: DNA extraction kits contain bacterial DNA that can dominate samples with low microbial content 3
• The inverse relationship between sample DNA concentration and contaminant DNA means technical variation is highest in low biomass samples 7
• Different DNA extraction kit lot numbers can cause samples to cluster by kit rather than by biology 3
• Laboratory personnel can introduce human oral microbiome contaminants 3
• Without proper controls, you cannot distinguish true microbial signal from contamination in your results 3

Alternative Validated Testing for GI Symptoms

For clinical decision-making in patients with gastrointestinal symptoms, use these established diagnostic approaches instead:

• Combined hydrogen and methane breath testing for suspected SIBO or intestinal methanogen overgrowth 4
• Qualitative small bowel aspiration during endoscopy when breath testing is unavailable 4
• Standard stool studies for pathogens, inflammatory markers, and malabsorption 3

Documentation Requirements

When ordering microbiome testing, document 3:

• Patient antibiotic use in the preceding 3 months (minimum) 3
• Other medications affecting the microbiome (proton pump inhibitors, laxatives, probiotics) 3
• Recent dietary changes or restrictions 3
• Age, sex, and relevant comorbidities as these significantly influence microbiome composition 3