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

Hu, H.; Karwautz, C.; Duszka, K.; Karner, T.; Wagner, I.; Grander, C.; Grander, W.; Steinwidder, L.; Boito, L.; Velde, V. V. d.; Bauters, M.; Boeckx, P.; Seki, D.; Glasl, B.; Thiele, S.; Schmidt, H.; Seneca, J.; Wagner, M.; Pjevac, P.

2025-11-26 microbiology
10.1101/2025.11.26.690684 bioRxiv
Show 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 Chloroflexota 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.

Published in ISME Communications (predicted rank #2) · training set

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