Table 1

Overview of sequencing-based methods to characterise microbiota composition and function

	Full-length 16S rRNA gene sequencing		Targeted 16S rRNA gene amplicon sequencing	Metagenome sequencing	Metatranscriptomics	Single-cell analysis
Technique	Clade-specific amplification of 16S rRNA gene, vector-based cloning and sequencing	Long-read-based amplification and sequencing of large 16S rRNA gene fragments	PCR-based amplification of target followed by sequencing	Sequencing of entire DNA extracted from samples	Sequencing of entire RNA extracted from samples	Cultivation-based isolation of single bacterial clones	Emulsion or droplet-based single-cell amplification
Target	Entire 16S rRNA gene	Entire 16S rRNA gene	Variable regions, eg, V1-V2; V3-V4; V4; V6	All DNA molecules	All transcribed RNA molecules	Multiple complete genomes	Single bacterial cells
Potential bias	Gene copy-number bias	Amplification bias	Primer/region-specific amplification bias; gene copy-number bias		Transcriptionally more active bacteria are over-represented	Anaerobic and hard-to-culture bacteria are under-represented	More abundant taxa over-represented
Sequencing technology	Sanger	PacBio or Oxford Nanopore	MiSeq	HiSeq/NextSeq	HiSeq/NextSeq	Any NGS technology	HiSeq/NextSeq
Advantages	Species-level resolution	High throughput; species-level resolution	Low cost; high throughput	Information on encoded functional repertoire; high-resolution taxonomic assignment; captures also viruses, fungi and archaea	Information on actively expressed functional content	Reconstruction of multiple complete genomes from complex communities	Reconstruction of multiple complete genomes from complex communities
Disadvantages	No information on functional repertoire; low throughput; much hands-on time	No information on functional repertoire; high error rates; higher cost per base than MiSeq	No information on functional repertoire; low resolution on species level	High cost; high computational burden; large amounts of unannotated data	High cost; high computational burden; large amounts of unannotated data Removal of 16S rRNA required	Tedious culturing and selection approaches (eg, media, oxygen); high cost	High cost

NGS, next generation sequencing.