rRNA Depletion and Your Sequencing Budget

Team Dynamic Matrices | 12 July 2026

Ribosomal RNA is 80 to 90 percent of the RNA in a cell. Removing it before sequencing is not optional; it is the difference between a library full of signal and one full of reads you pay to generate and then discard. What most labs do not do is put a number on how much residual rRNA is costing them. We did, and it changed how we think about the depletion step.

Where the residual comes from

No depletion method removes 100 percent. Published and vendor data for common approaches typically show:

• Probe hybridisation with bead pulldown (Ribo-Zero style): 1 to 5 percent residual on intact human RNA, rising to 10 to 30 percent on degraded, FFPE or non-model samples where probes miss fragments or sequence variants (Adiconis et al., Nature Methods, 2013; Zhao et al., BMC Genomics, 2018)
• RNase H digestion (NEBNext, RiboErase style): 2 to 10 percent on intact RNA, higher on fragmented input where short rRNA fragments escape
• Poly(A) selection: effectively removes rRNA but discards non-polyadenylated transcripts and fails on fragmented RNA, so it is a different trade rather than a solution

The reads that survive depletion map to rRNA, get filtered in the first step of analysis, and never appear in your results. They are pure cost.

A worked cost model

Assume a 200-sample study, 30 million reads per sample, on a platform costing about €5 per million reads at current list prices for a shared high-output run. Total sequencing spend: €30,000.

At 20 percent residual, which is ordinary for FFPE or degraded samples with probe-based depletion, you are paying €6,000 for reads that go straight to the bin, or, put the other way, you would need to sequence 25 percent deeper to reach the usable depth you planned for. Across a core facility running thousands of samples a year, this is the largest hidden line item in the RNA-seq budget.

Why efficiency drops on hard samples

Probe panels are designed against reference rRNA. Fragments that do not span the probe site, and variants in non-model organisms, escape. RNase H requires a formed hybrid and does not cut efficiently on very short fragments. Both effects are worst exactly where sample material is most precious.

What riboLASSO changes

riboLASSO captures rRNA with a library of sequence-specific DNA catcher strands attached to a soluble polymer. Hybridisation triggers a phase transition that condenses the polymer network and the bound rRNA into a pellet. No enzyme, no bead, no dependence on a fixed probe spanning an intact site; short fragments are captured as long as they carry any catcher-complementary sequence, and the catcher library (195 strands for human, derived from the Adiconis et al. 2013 tiling) covers cytoplasmic and mitochondrial rRNA. In our benchmarks residual rRNA is under 2 percent on intact RNA and stays low on degraded input, and the reagent cost per sample is about one euro versus tens of euros for commercial kits (Dynamic Matrices, Angewandte Chemie, 2025).

We would rather you measured this than took our word for it. A qPCR before and after depletion on your own samples gives a residual percentage in an hour.

Recommendations

1. Measure residual rRNA on your current pipeline for your actual sample types. Use the sequencing report's rRNA-mapping fraction, or qPCR before sequencing.
2. Put that percentage into the table above with your read cost. That is your annual waste.
3. If it is above 5 percent for intact RNA or above 10 percent for degraded RNA, test an alternative chemistry side by side.
4. Once residual is low, consider whether you can reduce depth. Usable reads, not total reads, set statistical power.