Table 1.

Main features of culture-independent detection methods of gut microbiota

Question	Target	Approach	Data generated	Can microbes be identified directly?	Main benefit	Main limitation
Which microbes are present in the GI tract?	Isolates	Cultivation	Phenotypic characterisation	Yes	Accurate species identification	Not representative
	16S rRNA gene	Cloning and sanger sequencing	Phylogenetic identification	Yes	Complete 16S rRNA gene sequence data	Cloning bias
	16S rRNA gene	High-throughput sequencing	Phylogenetic identification	Yes	High-throughput data generation	Short reads
	16S rRNA gene	Fingerpinting	Community profile	No	Fast comparison between communities	No direct link with phylogeny
	16S rRNA	FISH	Cell numbers	Yes	Accurate enumeration	Dependent on 16S rRNA databases
	16S rRNA gene	qPCR	16S rRNA gene abundances	Yes	Wide dynamic range	Dependent on 16S rRNA databases
	16S rRNA gene	Phylogenetic microarray	Phylogenetic identification	Yes	High-throughput phylogenetic profiling	Dependent on 16S rRNA databases
What microbial genes are present in the GI tract?	Community DNA	Sequence-based metagenomics	Gene sequences	Not always	High-throughput data generation	Function mainly based on predictions
	Community DNA	Function-based metagenomics	Functional properties encoded on DNA fragment	Not always	Functional properties linked to DNA sequences	Suitable cloning host/system and screening assays needed
What are GI tract microbes doing?	mRNA	Metatranscriptomics	Community gene expression	Not always	Direct information about microbial activity	Community RNA extraction challenging
	Proteins	Metaproteomics	Community protein production	Not always	Direct information about microbial activity	No uniform protocol for all cell fractions
	Metabonomics	Metabonomics/metabolomics	Community metabolity profiles	No	Microbiota activity representation	No link with microbes or its function
	Lactulose hydrogen breath test	Measuring GI tract gas production	Hydrogen and methane breath content	No	Unclear, simple test but not validated for diagnosing SIBO	May simply measure small intestinal transit time to caecum
	Glucose hydrogen breath test	Measuring GI tract gas production	Hydrogen breath content	No	Same as above	Poor sensitivity; misses distal SIBO

FISH, fluorescent in situ hybridisation; GI, gastrointestinal; qPCR, quantitative PCR; SIBO, small intestinal bacterial overgrowth.