Revised 16S rRNA V4 hypervariable region targeting primers enhance detection of Patescibacteria and other lineages across diverse environments

Abstract Primer bias in 16S rRNA gene amplicon sequencing can distort microbial diversity estimates by underrepresenting key taxa. We introduce a modified primer pair (V4-EXT) targeting the hypervariable V4 region of bacterial and archaeal 16S rRNA genes, with improved in silico taxonomic inclusivity. To benchmark performance, we analyzed 938 samples from terrestrial, aquatic, and host-associated habitats, comparing microbial community profiles derived with V4-EXT and the currently most widely used V4-targeted primers. V4-EXT substantially improved the detection of Patescibacteria and other underrepresented lineages, such as Chloroflexi and Iainarchaeota, while enhancing recovery of novel amplicon sequence variants across sample types. Overall, V4-EXT provides broader taxonomic coverage and more inclusive microbial community profiles, particularly in high-diversity ecosystems such as groundwater and soils. We propose V4-EXT as a robust successor for comprehensive microbial community analysis across diverse habitats.