Choosing which stretch of the 16S rRNA gene to sequence can change the picture researchers get of the vaginal microbiota during pregnancy, according to a benchmarking study published in BMC Microbiology. The analysis compared ten commonly used sub-regions of the gene against the full-length V1-V9 sequence and found that no single short region performed best across every measure.
The study drew on full-length 16S rRNA sequencing data from a Chinese pregnancy cohort. Using an in silico approach, the researchers extracted ten sub-regions from the full-length data: V1-V2, V1-V3, V3, V4, V3-V4, V3-V5, V4-V5, V4-V6, V6-V8, and V6-V9, and compared each with the complete V1-V9 sequence as an internal benchmark.
The main region-comparison analysis was conducted in a randomly selected sub-cohort of 104 pregnant women. A secondary case-cohort analysis of 176 women then examined whether the choice of region altered observed associations between the microbiota and preterm birth.
Across the comparisons, sub-regions differed from the full-length sequence in alpha diversity, beta diversity, community state types, taxonomic assignment, species co-occurrence, and predicted functional profiles. The extent of these differences varied by region and by the metric being assessed.
Some regions stood out for particular endpoints. V6-V9 came closest to the full-length sequence for richness metrics, while V1-V3 was most consistent for evenness metrics and for Bray-Curtis structure. V6-V8 and V6-V9 performed best for weighted and unweighted UniFrac distances, respectively, and V6-V9 showed the highest concordance for predicted pathway profiles.
V1-V3 showed high agreement with the full-length sequence for major community state types and vaginal types, suggesting that this region may be adequate when the goal is to classify samples into broad community categories.
In the secondary analysis of preterm birth, the species S_Dialister micraerophilus was associated with preterm birth in the full-length V1-V9 data, and the direction of this association was consistent across the sub-regions tested. This indicates that at least some taxonomic associations with clinical outcomes may be robust to the choice of sequenced region.
The authors conclude that because no single short region exhibits the highest consistency with the full-length sequence in all respects, region selection should be aligned with the intended analytical endpoint in vaginal microbiota studies. Researchers planning sequencing projects may therefore need to weigh which measurements matter most for their questions before committing to a particular variable region.
The results provide a reference for interpreting how profiles derived from different sub-regions compare with those obtained from full-length sequences in a pregnancy cohort.
The study was published as a peer-reviewed, accepted article shared ahead of the final Version of Record, with a permanent DOI, and is open access.
Subject of Research: Comparison of 16S rRNA gene sub-regions with full-length sequencing for vaginal microbiota profiling in pregnancy
Article Title: Benchmarking 16S rRNA gene sub-regions against full-length sequencing in vaginal microbiota profiling
Article References: Han, B., Liu, H., Cao, Y., Xu, Y., Cao, M., Qian, Z., Dong, Y., Pei, Y., Liu, J., Wu, F., Pan, L., Tan, Y., Li, Z., Li, Q., Hu, A., & Zheng, Y. (2026). Benchmarking 16S rRNA gene sub-regions against full-length sequencing in vaginal microbiota profiling. BMC Microbiology. https://doi.org/10.1186/s12866-026-05797-9
Image Credits: AI Generated
DOI: 10.1186/s12866-026-05797-9
Keywords: 16S rRNA, vaginal microbiota, pregnancy, full-length sequencing, preterm birth, community state types, alpha diversity, beta diversity, BMC Microbiology, sequencing methods, Benchmarking, rRNA
News Source: Morgan Morrow. (October 28, 2026). No single short 16S region matches full-length vaginal microbiota sequencing. Scienmag.
