How to optimize your DNA/RNA extraction to improve long-read sequencing results in WGS and beyond.

Clinical genomics is reaching an inflection point: the whole genome sequencing (WGS) market is projected to grow from 3 billion to over 9 billion by 2030, while long-read sequencing is solving 15-40 % of previously undiagnosable cases by detecting structural variants that short-read WGS misses¹.

Third-generation sequencing technologies like Oxford Nanopore and PacBio HiFi sequencing have revolutionized our ability to resolve complex genomic regions. The improved accuracy (95 - 99.9 %) and declining costs of these technologies have increased their adoption by healthcare systems in the areas of rare diseases, oncology, and carrier screening. Numerous population-scale studies have also incorporated long-read methods to provide more reliable, actionable genomic data to improve precision medicine and health outcomes across diverse populations.

However, realizing the full potential of these platforms critically depends on high-quality sample preparation. Fragmented DNA limits read lengths, defeating the very purpose of these technologies. High molecular weight (HMW) DNA, defined as intact DNA fragments exceeding 50 kb, is essential for providing accurate reads of > 10 kb performed in long-read sequencing (LRS). This blog post describes how you can achieve optimal long-read sequencing results in a rapid, automated, and cost-efficient manner.

The challenge: extraction and preservation 

DNA is surprisingly fragile. Mechanical shearing from pipetting, physical agitation, heating, enzymatic degradation from nucleases, and chemical damage from improper storage can quickly fragment HMW DNA into unusable pieces. 

Best practices for sample collection: 

• Use sample collection tubes that inhibit nucleases (e.g. K2EDTA, K3EDTA)
• Avoid heparin tubes which can inhibit downstream enzymatic reactions
• During transport, avoid excessive physical agitation or long-term storage at warm temperatures
• Process samples within 24-48 h of collection or freeze until analysis, avoiding freeze-thaw cycles
• Avoid vortexing or excessive pipetting and perform mixing by gentle inversion of tubes
• Gently handle samples with wide-bore pipette tips 
• Ensure sufficient sample input to obtain enough yields for downstream assay (1 ml of blood typically yields around 20 – 50 µg of DNA)

Extraction tips:

• Avoid the use of columns – these would shear DNA/RNA to < 10 kb
• Avoid heated lysis or elution steps
• Choose extraction methods that gently handle samples, excessive physical agitation should be avoided
• Perform extraction in large batches, ideally with automation, to enhance consistency and reproducibility
• To enrich for long fragments, perform an optional size selection step to remove < 10 kb fragments
• Shearing isolated HMW DNA to ~20 kb fragments can help increase long-read sequencing efficiency (increases flow cell longevity for ONT and increases efficiency of SMRTbell adapter ligation for PacBio)

Revvity’s chemagic™ extraction technology automates the extraction of HMW DNA at fast run-times with standard chemagic extraction kits on chemagic instruments. The proprietary M-PVA Magnetic Beads with gentle chemagic separation technology has proven superior to other automated platforms in isolating higher yields of HMW DNA with fewer hands-on steps.² This not only avoids laborious manual sample processing steps but has helped biobanks and genetic testing labs achieve a consistent, high quality of nucleic acids at scale, suited for present and future day challenging assays.

Revvity’s chemagic HMW DNA extraction was also found to work with archived blood samples, with samples collected in 2014 working for PacBio HiFi long-read sequencing performed in 2024.³ Figure 1. chemagic separation technology gently but efficiently resuspends magnetic particles, avoiding DNA fragmentation. Movement of beads instead of liquids avoids pipetting and lowers sample cross-contamination risk.