16S rRNA V1-V3 amplicon analysis
Upload V1-V3 amplicon reads amplified with 27F/534R and get ASVs, SILVA v138.2 taxonomy and diversity statistics in a publication-ready report. The V1-V3 insert is about 470 bp, so trimmed read pairs need about 490 bp combined to merge.
V1-V3 primer sequences
| Region | Forward primer | Reverse primer | Insert length | Reads needed to merge |
|---|---|---|---|---|
| V1-V3 | 27FAGAGTTTGATCMTGGCTCAG | 534RATTACCGCGGCTGCTGG | 470 bp | R1 + R2 ≥ 490 bp |
These are the sequences the V1-V3 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 470 bp, the truncated reads must together reach about 490 bp.
2×300 bp (MiSeq v3) required. Shorter reads cannot reach the 490 bp needed to merge.
The pipeline starts from default truncation of 280 bp (R1) and 220 bp (R2), then adapts them to the quality profile of your run and re-checks that the pairs can still merge.
When to use V1-V3
Skin microbiome studies, where it separates Staphylococcus and Cutibacterium better than V4.
- The longest of the 16S presets, so it carries the most taxonomic information per read pair.
- It also has the least margin for merging, so low read-end quality affects it first.
Comparing regions? Read V3-V4 vs V4 vs V1-V3: choosing a 16S region.
What the V1-V3 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
V1-V3 analysis FAQ
What are the V1-V3 16S primer sequences?
The V1-V3 preset uses 27F (AGAGTTTGATCMTGGCTCAG) as the forward primer and 534R (ATTACCGCGGCTGCTGG) 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 V1-V3?
The V1-V3 insert between the primers is about 470 bp. DADA2 needs the trimmed forward and reverse reads to overlap by at least 20 bp, so together they must cover about 490 bp. 2×300 bp (MiSeq v3) required. Shorter reads cannot reach the 490 bp needed to merge.
What is V1-V3 best used for?
Skin microbiome studies, where it separates Staphylococcus and Cutibacterium better than V4. The longest of the 16S presets, so it carries the most taxonomic information per read pair.
What happens if my V1-V3 reads are too short to merge?
The pipeline checks merge feasibility before denoising. If truncated reads cannot reach 490 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 V1-V3 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.