SFD-HT: An Automation-Compatible Approach to DNA Size Redistribution

A post-isolation step for long-read workflows

Obtaining HMW DNA is essential for long-read sequencing, but isolation alone may not produce the fragment-size distribution a project needs. Shorter molecules can remain after isolation or arise during handling, storage, and transportation. A post-isolation step can help reshape that distribution before library preparation.

SFD-HT (Short Fragment Depletor-High Throughput) is MagBio Genomics’ patent-pending, magnetic bead-based DNA purification and size-redistribution reagent. It is designed to selectively reduce short DNA fragments while enriching longer molecules in previously isolated HMW DNA or genomic DNA.

 

What SFD-HT is; and what it is not

SFD-HT is not an HMW DNA isolation kit. It does not extract DNA directly from a biological specimen. The recommended sequence is to isolate HMW DNA or genomic DNA, apply SFD-HT for short-fragment depletion, and then proceed to long-read library preparation and sequencing.

Keeping this distinction clear helps laboratories place the reagent at the correct point in the workflow and evaluate it against the appropriate performance measures.

 

A configurable molecular filter

SFD-HT uses paramagnetic bead chemistry to create a configurable high-pass effect. Depending on the selected workflow condition, it is designed to deplete fragments below 10 kb, 25 kb, or 40 kb while enriching the longer molecules retained for downstream use.

A tunable threshold matters because long-read projects do not all have the same objective. A whole-genome sequencing workflow, a metagenomic study, and an ultra-long-read project may require different balances among fragment length, DNA recovery, input mass, and library yield.

 

Performance across configurable thresholds

Current internal data support the intended size-redistribution effect at all three settings. At the <10 kb condition, treatment of human-blood gDNA increased the fraction measured above the threshold from 91.8% to 97.8% and increased average fragment size from approximately 60.6 kb to 90.1 kb. ONT MinION read N50 increased from 12.6 kb to 27.8 kb in that study.

At the <25 kb condition, 88.5% of measured DNA fell in the 25–300 kb target window after SFD-HT treatment, compared with 76.1% in the untreated input. At the <40 kb condition, 76.1% fell in the 40–400 kb window after treatment, compared with 60.5% in the untreated HMW DNA input. DQN increased from 7.6 to 8.9 in the <25 kb experiment and from 6.1 to 7.6 in the <40 kb experiment.

Because these figures come from separate internal experiments with different inputs and conditions, they should not be used to rank the thresholds against one another. Each setting should be evaluated with representative samples and the intended downstream workflow.

 

SFD-HT - Three Configurable Depletion Thresholds

Threshold-specific results across three separate internal SFD-HT experiments. Values summarize input-to-treatment changes; cross-threshold ranking is not appropriate.

 

Designed for automation

The magnetic bead workflow follows a familiar bind-wash-elute structure and requires no centrifugation. This architecture supports integration with liquid-handling systems and plate-based laboratory operations. Reducing centrifugation and manual tube handling may also simplify scaling and standardization across larger sample batches.

Automation compatibility does not eliminate the need for method verification. Laboratories should still optimize aspiration behavior, mixing conditions, magnet timing, and liquid-handling parameters for their instruments and sample characteristics.

 

Compatible long-read applications

SFD-HT is optimized for Oxford Nanopore and PacBio workflows. Potential applications include long-read whole-genome sequencing, structural variant analysis, genome assembly, ultra-long-read sequencing, and long-read metagenomics.

The intended outcome is a DNA input with fewer unwanted short fragments and a greater relative representation of longer molecules. The magnitude of any downstream sequencing benefit will depend on the starting material, the depletion threshold, library preparation, sequencing platform, and study design.

 

Evaluate SFD-HT in the context of your samples

A useful evaluation should measure the input and output fragment-size distributions, DNA recovery, library yield, read-length distribution, read N50, and workflow repeatability. Comparing these measures across representative samples is more informative than relying on a single idealized input.

MagBio Genomics is inviting qualified laboratories and biotechnology companies to participate in the SFD-HT Early Adopter & Collaboration Program. Participants can work with MagBio R&D to evaluate SFD-HT in their own long-read workflows and provide feedback during development.

 

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