16S Strikes Out

We’ve explained why qPCR offers a compelling alternative to sequencing and how we validate Branchpoint assays to ensure target specificity, but how do our assays compare with other microbiome profiling approaches?

We benchmarked more than 100 of our species-specific assays against both 16S and deep metagenomic sequencing using stool samples from 3 healthy human donors. In our next two posts we’ll dive into head-to-head comparisons against alternative profiling methods so you can judge for yourself. 

First up, let’s compare Branchpoint qPCR to 16S amplicon sequencing.

A Species Mess with 16S

Even after applying the most advanced techniques available for 16S analysis, out of the 923 amplicon sequence variants (ASVs) only 292 had assignments to the species-level (31.6%). But a species assignment is only useful if it means something – after filtering out non-specific species identifiers or placeholder names (e.g., Alistipes sp. or uncultured bacterium, respectively) 74 ASVs (8.0%) could be assigned to species in our communities. With all this effort, 16S provided relative abundance information for only 7 of the more than 100 species we quantified using qPCR.

Even worse, 16S butchered our positive control spike-ins. We amended our samples with three positive controls – at 1%, 0.1%, and 0.001% of the total DNA – Porphyromonas somerae, P. uenonis, and P. asaccharolytica, respectively.

• P. asaccharolytica was below the detection limit in all three samples
• P. uenonis (amended at 0.1% total DNA) was undetected in one of the three samples
• P. somerae was simultaneously over- and underestimated across the samples (A: 1.53%, B: 1.39%, C: 0.76%)

Community composition with taxonomic precision

Meanwhile, using our 100+ qPCR assays we were able to quantify 81 species across the three donors. What’s more, we accomplished this by delivering absolute counts rather than relative sequence abundance, including measuring our 0.001% spike-in at 7 genome equivalents per reaction. Together, our assays illuminated the overall donor community composition while also providing accurate species-level resolution.

Primed for Success

When species matter, you need more than 16S. Branchpoint assays deliver accurate species-level profiles, but how do we compare with shotgun metagenomic sequencing? Stay tuned for a head-to-head comparison in our next blog.

Summary of 16S Methodology.

We used DADA2 to convert the 16S sequencing data to amplicon sequencing variants (ASVs) – which offer more accuracy than operational taxonomic units  (OTUs) and have become the gold-standard for 16S analysis. ASVs were then assigned taxonomy using the pre-trained Naive Bayes classifier built using the Silva R138 99% OTUs full-length dataset – another top line, publicly available database.