16S rRNA V4 amplicon analysis
Upload V4 amplicon reads amplified with 515F (Parada)/806R (Apprill) and get ASVs, SILVA v138.2 taxonomy and diversity statistics in a publication-ready report. The V4 insert is about 253 bp, so trimmed read pairs need about 273 bp combined to merge.
V4 primer sequences
| Region | Forward primer | Reverse primer | Insert length | Reads needed to merge |
|---|---|---|---|---|
| V4 | 515F (Parada)GTGYCAGCMGCCGCGGTAA | 806R (Apprill)GGACTACNVGGGTWTCTAAT | 253 bp | R1 + R2 ≥ 273 bp |
These are the sequences the 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 253 bp, the truncated reads must together reach about 273 bp.
2×250 bp recommended. Reads must still contain the primers; once they are removed, 2×150 bp reads fall just short of the 273 bp needed to merge.
The pipeline starts from default truncation of 240 bp (R1) and 160 bp (R2), then adapts them to the quality profile of your run and re-checks that the pairs can still merge.
When to use V4
Large studies, environmental samples and anything following the Earth Microbiome Project (EMP) protocol.
- The short amplicon gives a long read overlap, so merging is robust even when read ends lose quality.
- The Parada/Apprill primer updates fixed the original 515F/806R bias against SAR11 and Thaumarchaeota.
Comparing regions? Read V3-V4 vs V4 vs V1-V3: choosing a 16S region.
What the 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
V4 analysis FAQ
What are the V4 16S primer sequences?
The V4 preset uses 515F (Parada) (GTGYCAGCMGCCGCGGTAA) as the forward primer and 806R (Apprill) (GGACTACNVGGGTWTCTAAT) 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 V4?
The V4 insert between the primers is about 253 bp. DADA2 needs the trimmed forward and reverse reads to overlap by at least 20 bp, so together they must cover about 273 bp. 2×250 bp recommended. Reads must still contain the primers; once they are removed, 2×150 bp reads fall just short of the 273 bp needed to merge.
What is V4 best used for?
Large studies, environmental samples and anything following the Earth Microbiome Project (EMP) protocol. The short amplicon gives a long read overlap, so merging is robust even when read ends lose quality.
What happens if my V4 reads are too short to merge?
The pipeline checks merge feasibility before denoising. If truncated reads cannot reach 273 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 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.