16S rRNA V3-V4 amplicon analysis
Upload V3-V4 amplicon reads amplified with 341F/805R and get ASVs, SILVA v138.2 taxonomy and diversity statistics in a publication-ready report. The V3-V4 insert is about 427 bp, so trimmed read pairs need about 447 bp combined to merge.
V3-V4 primer sequences
| Region | Forward primer | Reverse primer | Insert length | Reads needed to merge |
|---|---|---|---|---|
| V3-V4 | 341FCCTACGGGNGGCWGCAG | 805RGACTACHVGGGTATCTAATCC | 427 bp | R1 + R2 ≥ 447 bp |
These are the sequences the V3-V4 preset removes with Cutadapt before denoising. If your facility used a variant of these primers, for example with extra degenerate bases, choose Custom primers on upload and paste the exact sequences from your sequencing report.
Read length and merging
DADA2 merges each forward and reverse read into one sequence, which needs at least 20 bp of overlap. With an insert of about 427 bp, the truncated reads must together reach about 447 bp.
2×300 bp (MiSeq v3) recommended. 2×250 bp works only while read-end quality holds. 2×150 bp cannot span this region.
The pipeline starts from default truncation of 280 bp (R1) and 200 bp (R2), then adapts them to the quality profile of your run and re-checks that the pairs can still merge.
When to use V3-V4
Human gut, oral and environmental bacterial communities; the Illumina 16S Metagenomic Sequencing Library Preparation protocol.
- The most widely used 16S target on Illumina MiSeq, so results compare easily with published studies.
- Genus-level assignment is reliable; species-level assignment is possible for some taxa but not for closely related species.
Comparing regions? Read V3-V4 vs V4 vs V1-V3: choosing a 16S region.
What the V3-V4 analysis includes
- Primer removal with Cutadapt and quality-adaptive truncation
- DADA2 denoising, read-pair merging and chimera removal
- Taxonomy against SILVA v138.2 with exact-match species assignment where possible
- Alpha diversity, rarefaction, phylogenetic tree and PICRUSt2 (Complete tier)
- Beta diversity, PERMANOVA and ANCOM-BC2 across groups in multi-sample projects
See pricing for what each tier includes.
Other 16S regions
V3-V4 analysis FAQ
What are the V3-V4 16S primer sequences?
The V3-V4 preset uses 341F (CCTACGGGNGGCWGCAG) as the forward primer and 805R (GACTACHVGGGTATCTAATCC) as the reverse primer. Letters such as N, W, Y and V are IUPAC ambiguity codes; the pipeline matches them against real bases when it looks for and removes the primers.
What read length do I need for V3-V4?
The V3-V4 insert between the primers is about 427 bp. DADA2 needs the trimmed forward and reverse reads to overlap by at least 20 bp, so together they must cover about 447 bp. 2×300 bp (MiSeq v3) recommended. 2×250 bp works only while read-end quality holds. 2×150 bp cannot span this region.
What is V3-V4 best used for?
Human gut, oral and environmental bacterial communities; the Illumina 16S Metagenomic Sequencing Library Preparation protocol. The most widely used 16S target on Illumina MiSeq, so results compare easily with published studies.
What happens if my V3-V4 reads are too short to merge?
The pipeline checks merge feasibility before denoising. If truncated reads cannot reach 447 bp combined, it first extends truncation within the safe quality range; if that is still not enough, the run stops with an explanation instead of silently discarding most read pairs.
Analyze your V3-V4 16S data
Your first 3 analyses are free, with no credit card. Upload FASTQ files and get a publication-ready report, processed and stored in Canada.