V4V5

V4V5 refers to the combined variable regions 4 and 5 of the 16S ribosomal RNA gene, a common target in culture-independent microbiology for profiling bacterial communities. The 16S gene contains nine hypervariable regions (V1–V9); V4 and V5 together provide a balance between taxonomic resolution and amplicon length that is well suited for short-read sequencing platforms. The V4V5 segment typically spans roughly 400 to 450 base pairs across many bacteria, though exact length varies by species.

Amplification of V4V5 is typically achieved with polymerase chain reaction using primers such as 515F and 926R (including degenerate variants like 515F-Y) that flank the region. The resulting amplicons are sequenced on high-throughput platforms, most commonly Illumina MiSeq or HiSeq, using paired-end reads (often 2x250 bp) that are merged to cover the entire V4V5 region.

Data analysis involves quality control, chimera removal, and grouping sequences into operational taxonomic units (OTUs) or amplicon sequence variants (ASVs). taxonomic assignment is performed by comparing sequences to reference databases such as SILVA, Greengenes, or RDP. Outputs include relative abundance profiles and diversity metrics that enable comparisons across samples or treatments.

Advantages of the V4V5 approach include generally good taxonomic resolution for many bacterial groups and compatibility with established short-read workflows. Limitations include primer bias, region-dependent resolution that may underrepresent certain taxa, and challenges in cross-study comparability when different primer sets or regions are used. V4V5 remains a widely used option in environmental, clinical, and agricultural microbiome studies, particularly when moderate resolution and compatibility with common sequencing platforms are desired.