Devin ONT Protocol: Enhancing Microbial Read Capture on Nanopore

Metagenomic next-generation sequencing (mNGS) on Oxford Nanopore Technologies (ONT) platforms promises rapid, real-time pathogen detection from clinical specimens. In practice, however, human host DNA overwhelms microbial signal, and host-depleted samples often yield DNA concentrations too low for standard nanopore library preparation. An August 2024 protocol document from Micronbrane Medical addresses both problems in a single integrated workflow: the Devin Host Depletion Filter removes the majority of human cells before extraction, and the ONT Rapid PCR Barcoding Kit 24 V14 amplifies the remaining microbial DNA into a sequencing-ready library. The result is a sample-to-sequencer pipeline that fits within one working day using the low-cost Flongle flow cell.

Download the full protocol: ONT Protocol (PDF)

Key Takeaways

• Host-depleted samples processed through the Devin filter are compatible with ONT sequencing and increase the proportion of microbial reads in the final output
• The Rapid PCR Barcoding Kit 24 V14 recovers sequenceable library even from samples with DNA concentrations below the Qubit detection limit — do not discard low-yield extractions
• A ZymoBIOMICS spike-in control added during depletion provides an internal benchmark for tracking microbial recovery across runs
• The three-stage workflow — host depletion, PCR-based library prep, and Flongle sequencing — can be completed within a single working day
• Libraries with an average fragment size of approximately 4 kb and roughly 20 ng total mass deliver the highest output on the Flongle flow cell

Stage 1: Host Depletion From Whole Blood

The protocol begins with human whole blood processed through the Devin Microbial DNA Enrichment Kit (MEK-01). A syringe is attached to the Devin Host Depletion Filter, and the blood sample is passed through the filter at a controlled rate of 1 mL per 10 seconds. This step physically removes the majority of intact human cells while allowing smaller microbial cells to pass through.

Before filtration, ZymoBIOMICS Spike-in Control I (High Microbial Load) is added at a concentration of 1×10^4 per mL of blood. This spike-in serves as a quantitative internal control for tracking depletion efficiency and microbial recovery in downstream analysis.

After filtration, the eluate undergoes two sequential centrifugation steps. The first spin at 400 x g for 15 minutes separates plasma from residual cellular material. The plasma layer is transferred to a fresh tube and centrifuged again at 16,000 x g for 15 minutes to pellet microbial cells and cell-free microbial DNA. The supernatant is carefully removed — leaving approximately 20 uL if no visible pellet is present — and the pellet is carried forward into genomic DNA extraction following the MEK-01 kit protocol. The result is a concentrated microbial DNA fraction with substantially reduced human background, ready for library construction.

Stage 2: Library Preparation With Rapid PCR Barcoding

A critical insight from this protocol is that DNA with undetectable Qubit concentration can still produce a usable library through the PCR amplification step. Laboratories should not abandon samples that appear to have failed extraction. However, if DNA is measurable, no more than 5 ng of total input should be used.

Library construction begins with tagmentation: 3 uL of template DNA is combined with 1 uL of Fragmentation Mix (FRM) and incubated at 30 degrees C for 2 minutes, followed by enzyme inactivation at 80 degrees C for 2 minutes. Optionally, the input DNA should be at least 4 kb in average fragment length to ensure correct tagmentation and PCR amplification for long reads.

The tagmented DNA then enters a PCR reaction with barcoding primers. Each 50 uL reaction contains 20 uL nuclease-free water, 4 uL tagmented DNA, 1 uL of the appropriate Rapid Ligation Barcode (RLB) at 10 uM, and 25 uL LongAmp Taq 2X master mix. Thermal cycling proceeds through initial denaturation at 95 degrees C for 3 minutes, then 20 cycles of 95 degrees C for 15 seconds, 56 degrees C for 15 seconds, and 65 degrees C for 6 minutes, followed by a final extension at 65 degrees C for 6 minutes.

After PCR, 4 uL of EDTA is added and incubated for 5 minutes at room temperature before quantification. Barcoded samples are pooled in equimolar ratios targeting 200-400 fmol (approximately 400-800 ng), then cleaned with AMPure XP beads at a 0.6x ratio and eluted in 15 uL of Elution Buffer. This bead ratio selects for fragments above approximately 500 bp, removing primer dimers and short amplification artifacts. The protocol supports multiplexing up to 24 samples per sequencing run through the barcoding scheme.

Stage 3: Flongle Sequencing

The final stage loads the prepared library onto a Flongle flow cell for sequencing. Between 5 and 25 fmol of the eluted library is transferred to a DNA LoBind tube and brought to 5 uL total volume with Elution Buffer. The Rapid Adapter (RA) is diluted by combining 0.6 uL RA with 1.4 uL Adapter Buffer, and 0.5 uL of this diluted adapter is added to the library and incubated for 5 minutes at room temperature.

The Flongle flow cell is primed with a mixture of 117 uL flow cell flush and 3 uL flow cell tether. The sequencing mix is then prepared by combining 15 uL Sequencing Buffer, 5 uL of the adapter-ligated library, and 10 uL Library Beads, and loaded through the Flongle sample port.

For optimal output, the protocol notes that approximately 20 ng of library with a 4 kb average fragment size delivers the highest read yield on the Flongle. Because the Flongle is substantially more affordable than standard MinION flow cells, this workflow makes nanopore-based mNGS accessible for routine use and smaller laboratories. The MinION or GridION instrument serves as the sequencing platform, with the Flongle adapter providing the low-cost per-run economics. Laboratories already running the optimized ONT library prep protocol will find this workflow directly compatible, as both protocols share the same Rapid PCR Barcoding chemistry and can address the challenge of overcoming library prep failure in low-biomass samples.

Related Reading

• Devin Host Depletion Filter
• Devin Microbial DNA Enrichment Kits
• Optimized ONT Library Prep Protocol (Webinar)
• Long-Read mNGS on Flongle: Host Depletion + PCR
• Benchmarking Host Depletion: 5 Methods Compared
• Unison Ultralow Library Prep Kit
• Overcoming Library Prep Failure in Low-Biomass Samples
• NGS Library Prep Kits for Metagenomic Sequencing