V3-V4 vs V4 vs V1-V3: Choosing a 16S Region

The best 16S region is the one your sequencing run can cover and that matches the studies you want to compare with. V3-V4 (341F/805R) is the most common Illumina target and needs 2×300 bp reads. V4 (515F/806R) is shorter, merges reliably with 2×250 bp reads and is the Earth Microbiome Project standard. V1-V3 (27F/534R) suits skin microbiome work but needs long, high-quality reads. Whatever you choose, use the same region and primers for every sample in a study.

Why does the 16S region matter?

The 16S rRNA gene has nine hypervariable regions (V1–V9) separated by conserved stretches where primers bind. Short-read sequencers cannot read the whole 1,500 bp gene, so a study amplifies one or two neighbouring regions. That choice affects three things:

• Which taxa you detect. Every primer pair has mismatches to some groups, so some taxa are under-amplified (Klindworth et al., 2013).
• How finely you can tell taxa apart. Longer, more variable regions separate more closely related genera and species.
• Whether your reads can be merged. Paired reads must overlap across the amplicon, which sets a minimum read length.

How do the common 16S regions compare?

Insert length is the sequence between the primers after they are removed. The combined length is what the trimmed forward and reverse reads must reach together for a 20 bp overlap.

How does read length limit your choice?

DADA2 merges each forward read with its reverse read. To do that, the two reads must overlap, so after primers are removed and low-quality ends are truncated, R1 + R2 must be at least the insert length plus the overlap.

• 2×150 bp reads (MiSeq v2 300-cycle, NextSeq, NovaSeq) are too short for V3-V4, V1-V2 or V1-V3. They can cover V4 only when the reads start after the PCR primer, as in the Earth Microbiome Project protocol; when the primers are part of the reads, trimmed 2×150 bp pairs fall just short.
• 2×250 bp reads (MiSeq v2 500-cycle) handle V4 and V1-V2 comfortably, and V3-V4 only if read ends keep good quality.
• 2×300 bp reads (MiSeq v3) cover every region in the table, though V1-V3 leaves little margin when R2 quality drops.

If the reads cannot overlap, most read pairs are discarded at the merge step, a result that can look like a successful run with surprisingly few reads. BioAnalysis.ca expects the primers at the start of the reads, so it can confirm the region and remove them, and it checks merge length before denoising, stopping with an explanation if the reads cannot merge.

Which region should you use for your samples?

• Human gut: V3-V4 or V4 both work well. Use V4 if you want to compare against large public datasets built on the Earth Microbiome Project protocol, and V3-V4 if your sequencing provider runs Illumina's 16S Metagenomic Sequencing Library Preparation protocol.
• Soil, water and other environments: V4 with the updated 515F (Parada et al., 2016) and 806R (Apprill et al., 2015) primers, which fixed the original primers' bias against marine SAR11 and Thaumarchaeota.
• Skin: V1-V3, which captures Cutibacterium and separates Staphylococcus better than V4 (Meisel et al., 2016).
• Samples with a lot of host or plant DNA: check how well your primer pair avoids chloroplast and mitochondrial 16S; V4 and V3-V4 primers amplify chloroplasts in plant samples.

Some Canadian facilities use other pairs. The Integrated Microbiome Resource at Dalhousie University, for example, uses V4-V5 (515FB/926R) primers (Walters et al., 2016). On BioAnalysis.ca you can analyze any pair by entering the sequences as custom primers; V4-V5 needs 2×300 bp reads.

Can you compare results from different regions?

Not directly. The same organism produces a different ASV in each region, so ASV tables from different regions cannot be merged. Genus-level comparisons are possible but still carry each primer pair's own biases. For any study you plan to analyze together, sequence every sample with the same primers on the same platform.

What about full-length 16S sequencing?

Long-read platforms (PacBio HiFi and Oxford Nanopore) can sequence the entire 16S gene and resolve more taxa to species. They cost more per sample and need different analysis settings. Short-read regions remain the standard for most studies because they are cheap, well-characterized and comparable with published data.

Summary

Pick the region your read length can merge, and that matches the literature in your field. If in doubt and you have 2×300 bp reads, V3-V4 is a safe, widely comparable default. With 2×150 bp reads, V4 is the only option, and only if your reads start after the primer. The full list of supported presets and their primer sequences is on the 16S analysis page.

References

1. Apprill A, McNally S, Parsons R, Weber L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquatic Microbial Ecology. 2015;75:129–137.
2. Klindworth A, Pruesse E, Schweer T, et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Research. 2013;41:e1.
3. Meisel JS, Hannigan GD, Tyldsley AS, et al. Skin microbiome surveys are strongly influenced by experimental design. Journal of Investigative Dermatology. 2016;136:947–956.
4. Parada AE, Needham DM, Fuhrman JA. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environmental Microbiology. 2016;18:1403–1414.
5. Walters W, Hyde ER, Berg-Lyons D, et al. Improved bacterial 16S rRNA gene (V4 and V4-5) and fungal internal transcribed spacer marker gene primers for microbial community surveys. mSystems. 2016;1:e00009-15.