TABLE 2.

Commonly used methods for characterization of the intestinal microbiota

Method	Purpose	Description	Advantages	Disadvantages
Fluorescence in situ hybridization (FISH)	identification, quantification, visualization of bacterial cells in tissue	fluorescent dye‐labeled oligonucleotide probes are hybridized to ribosomal RNA sequence in bacterial cells	useful method for quantifying bacteria, allows localization of bacteria in tissue	labor intense, FISH probes need to be developed for each group of interest
Quantitative real‐time PCR	quantification of bacterial taxa	target organisms are quantified using fluorescent dye‐labeled primers and/or probes	rapid, reproducible, inexpensive, quantitative, RIs can be established	primer and probes need to be designed for each group of interest
16S rRNA sequencing	identification of bacteria in a sample, measures relative abundance	bacteria are amplified using universal primers targeting the 16S rRNA gene, PCR amplicons are separated and sequenced using a high‐throughput sequencer	high throughput, relative inexpensive, allows identification of bacteria, semi‐quantitative, allows to describe changes within a community	requires advanced bioinformatics, changes in taxonomic databases and bioinformatics pipelines make comparing results difficult across studies, does not allow to detect changes in total abundance of bacteria
Metagenomics (shotgun sequencing of genomic DNA)	identification of microbial genes present in sample	genomic DNA is fragmented and then randomly sequenced (without PCR amplification) on a high‐throughput sequencer	provides not only phylogenetic information but also what functional genes are present in sample	expensive, requires advanced bioinformatics, does not allow to detect changes in the total abundance of bacteria