16S rRNA sequencing data analysis, online
Upload 16S rRNA amplicon FASTQ files from any sequencing provider and get amplicon sequence variants, SILVA taxonomy, diversity statistics and predicted function in a publication-ready report. It costs $25–45 CAD per sample, takes about an hour, and your data is processed and stored in Canada.
What is 16S rRNA analysis?
16S rRNA analysis identifies the bacteria and archaea in a sample by sequencing part of the 16S ribosomal RNA gene, which every bacterium carries and which varies between taxa. The bioinformatics step converts millions of raw reads into a table of exact sequence variants, names each one against a reference database, and measures how diverse the community is.
BioAnalysis.ca automates that step. It is built for microbiology and microbiome labs that have FASTQ files back from a sequencing core and need trustworthy results without a bioinformatician or a computing cluster.
What you get
- ASV table and taxonomy (SILVA v138.2), phylum to species where resolvable
- Read tracking through every DADA2 stage, so data loss is visible
- Alpha diversity metrics with rarefaction curves
- Quality score with pass, warn or fail per check
- Beta diversity, PERMANOVA and ANCOM-BC2 for projects
- PICRUSt2 pathway predictions
- PDF, interactive report, R figures and CSV/JSON data package
How the 16S analysis pipeline works
Each step uses an established, peer-reviewed tool. Every report records the exact versions and parameters used.
1. Input checks
Files are checked as they upload: FASTQ format, read pairing and file integrity. Before the run, the pipeline scans reads for your primers, detects R1/R2 files that were swapped and fixes them, and stops with a clear reason if the primers are missing.
2. Primer removal (Cutadapt v5.2)
Forward and reverse primers are removed, allowing for IUPAC ambiguity codes and a few leading spacer bases. Reads that do not carry the primer are discarded, so off-target reads do not become false ASVs.
3. Quality-adaptive truncation
Truncation lengths are chosen from the actual quality profile of your run, then checked so the forward and reverse reads still overlap by at least 20 bp across the amplicon. If they cannot, the run stops instead of silently losing most read pairs.
4. Denoising into ASVs (DADA2 v1.34.0)
DADA2 learns the error rates of your run, infers exact amplicon sequence variants, merges pairs and removes chimeras. The report shows how many reads survive each stage.
5. Taxonomy (SILVA v138.2)
Each ASV is classified with the DADA2 naive Bayes classifier against SILVA v138.2, from kingdom to genus, with exact-match species assignment where the sequence is unique.
6. Phylogeny (MAFFT v7.525 + FastTree v2.2.0)
ASVs are aligned and placed on a phylogenetic tree, which powers Faith's phylogenetic diversity and the interactive tree in the report.
7. Diversity
Alpha diversity (Shannon, Simpson, Chao1, observed ASVs, Faith's PD) with rarefaction curves. In projects: beta diversity (Bray-Curtis, Jaccard), PCoA and PERMANOVA with 999 permutations.
8. Differential abundance (ANCOM-BC2)
For multi-sample projects, ANCOM-BC2 tests which taxa differ between the groups defined in your metadata file, with bias correction for compositional data.
9. Functional prediction (PICRUSt2 v2.5.3)
PICRUSt2 predicts metabolic pathway abundances from the ASVs' phylogenetic placement. Predictions are labelled as inferred, not measured.
Supported 16S regions and primers
Choose a preset or enter your own primer sequences. Each region page explains read-length requirements and when to use it. How to choose a 16S region →
| Region | Forward primer | Reverse primer | Insert length | Reads needed to merge |
|---|---|---|---|---|
| V3-V4 | 341FCCTACGGGNGGCWGCAG | 805RGACTACHVGGGTATCTAATCC | 427 bp | R1 + R2 ≥ 447 bp |
| V4 | 515F (Parada)GTGYCAGCMGCCGCGGTAA | 806R (Apprill)GGACTACNVGGGTWTCTAAT | 253 bp | R1 + R2 ≥ 273 bp |
| V1-V2 | 27FAGAGTTTGATCMTGGCTCAG | 338RGCTGCCTCCCGTAGGAGT | 273 bp | R1 + R2 ≥ 293 bp |
| V1-V3 | 27FAGAGTTTGATCMTGGCTCAG | 534RATTACCGCGGCTGCTGG | 470 bp | R1 + R2 ≥ 490 bp |
16S analysis pricing
Per sample, in Canadian dollars, with no subscription.
Amplicon Basic
- Primer trimming (Cutadapt)
- DADA2 denoising and ASV table
- Taxonomy against SILVA v138.2 (16S) or UNITE v9.0 (ITS)
- Phylum, genus and species composition
- AI-assisted interpretation
- PDF and interactive web report
- Paired-end and single-end reads
Amplicon Complete
- Everything in Amplicon Basic
- Species-level assignment where the region resolves it
- Phylogenetic tree (MAFFT + FastTree)
- Alpha diversity: Shannon, Simpson, Chao1, Faith's PD
- Rarefaction curves
- Functional prediction (PICRUSt2)
- Multi-sample projects: beta diversity, PERMANOVA, ANCOM-BC2
- Research data package (ZIP)
16S rRNA analysis FAQ
What does a 16S rRNA analysis report include?
The report includes the DADA2 read-tracking summary, an ASV table, taxonomic composition at phylum, genus and species level, alpha diversity with rarefaction curves, a quality score, predicted functional pathways, AI-assisted interpretation and every pipeline parameter and tool version. Projects add beta diversity, PERMANOVA and ANCOM-BC2 results.
Is BioAnalysis.ca an alternative to QIIME 2?
It runs the same core methods QIIME 2 users typically choose, Cutadapt and DADA2 with a SILVA classifier, without installing software or writing commands. QIIME 2 offers more options for custom analyses; BioAnalysis.ca is faster for standard 16S studies and produces the report and figures automatically.
How many reads per sample do I need for 16S analysis?
Aim for at least 10,000 reads per sample after filtering. The quality score in each report flags samples below 5,000 reads as a warning and below 500 as a failure, and rarefaction curves show whether sequencing depth captured the community.
Can I analyze several samples together and compare groups?
Yes. Create a project, upload all samples and add a metadata CSV or TSV with your groups. The study pipeline denoises every sample with one shared error model, then runs beta diversity, PERMANOVA and ANCOM-BC2 differential abundance on those groups.
Is my 16S data kept in Canada?
Yes. Sequencing data is processed and stored on servers in Canada. The AI-written interpretation in each report is generated by Google's Gemini API from summary results (taxonomic composition, diversity and quality metrics); sequencing reads are never sent.
Upload your 16S FASTQ files
Your first 3 analyses are free, with no credit card. Upload FASTQ files and get a publication-ready report, processed and stored in Canada.