Best Practices for High-Molecular-Weight DNA Extraction for Long-Read Sequencing

Why High-Molecular-Weight DNA Matters for Long-Read Sequencing

Long-read sequencing technologies, such as PacBio and Oxford Nanopore Technologies (ONT), rely heavily on the integrity and length of input DNA to deliver high-quality reads. Unlike short-read platforms, which process DNA fragments of 100–300 base pairs, long-read systems can handle fragments that span tens to hundreds of kilobases (kb)—provided the input DNA is sufficiently intact.

High-molecular-weight (HMW) DNA, typically defined as fragments greater than ~50 kb (and potentially exceeding 100 kb), offers several crucial advantages:

• Enhanced genome assembly contiguity.

In their landmark study, Jain et al. (2018) (DOI: https://doi.org/10.1038/nbt.4060) used Nanopore sequencing with ultra-long reads (N50 > 100 kb, up to 882 kb) to assemble a human reference genome (GM12878) with significantly improved contiguity. The addition of these long reads boosted the NG50 metric from ~3 Mb to ~6.4 Mb, enabling more complete coverage and gap resolution.

Nanopore sequencing and assembly of a human genome with ultra-long reads(Jain et al. 2018).

• Improved structural variant detection.

Longer reads bridge complex or repetitive regions, allowing detection of large insertions, deletions, and rearrangements—often missed by fragmented short reads.

• High-confidence phasing and epigenetic insights.

Long contiguous fragments support phasing of alleles across kilobases and preserve native DNA modifications (e.g., methylation), essential for advanced genomic studies.

In essence, acquiring HMW DNA is not optional—it's the foundation for generating ultra-long reads that unlock the full capabilities of long-read sequencing. Techniques that compromise DNA length or integrity intrinsically limit read length, assembly quality, and genomic insight.

Challenges in Isolating HMW DNA

Extracting HMW DNA suitable for long-read sequencing is technically demanding. Several critical challenges can compromise DNA integrity and affect downstream data quality:

Mechanical Shearing & DNA Fragmentation

High shear forces from aggressive pipetting, vortexing, or rapid centrifugation can significantly fragment long DNA strands. A comprehensive protocol for Chlamydomonas reinhardtii demonstrated that minimizing physical handling—especially eliminating vortexing and using wide-bore pipette tips—helps preserve fragment sizes over 50 kb, verified via pulsed-field gel electrophoresis (PFGE)(Frédéric Chaux et al,. 2024).

Freeze–Thaw Cycles

Repeated cycles of freezing and thawing weaken DNA molecules through ice crystal formation, causing breakage. Preservation guidelines recommend limiting freeze–thawing and favor storage at 4 °C for short-term use or constant −80 °C for long-term storage .

Contaminants from Lysis and Sample Matrix

Substances like proteins, polysaccharides, phenolics, or residual lysis reagents can inhibit sequencing or lead to fragmented DNA. Plant and fungal samples are especially prone: protocols using CTAB + β-mercaptoethanol have shown improved purity and high integrity in samples with high polysaccharide content(https://doi.org/10.3389/fpls.2022.883897).

Inadequate Lysis and Chemical Damage

Incomplete lysis leads to low yields, but harsh buffers or prolonged procedures can degrade DNA. For example, β-mercaptoethanol helps reduce oxidative damage and inhibits DNases—but must be balanced to avoid harming DNA with excessive reagents.

Standard Kits Not Optimized for HMW DNA

Many spin column or bead-based commercial kits are efficient for small fragments but result in sheared DNA far below the desired 50–100 kb range. Oxford Nanopore's and PacBio's technical notes recommend HMW-specific protocols (e.g., Nanobind) to preserve ultra-long fragments.

Summary of Failure Points & Risks

Together, these factors underscore that HMW DNA extraction is not routine—it requires optimized protocols, sample-specific adjustments, and deliberate handling to maintain DNA integrity for long-read sequencing.

Explore our general DNA extraction methods →

Sample Preparation: Choosing the Right Starting Material

Careful selection and handling of the starting biological material are essential for successful HMW DNA extraction. Below are key factors to consider:

Sample Type and Collection

Different sample types—such as fresh tissue, frozen samples, cell pellets, blood, and microbial or plant material—vary in their ease of HMW DNA extraction.

Vertebrate tissues (e.g., muscle, spleen): PacBio recommends either fresh tissue processed within 24 hours at 4 °C or flash-frozen samples stored at –80 °C to preserve DNA integrity.

Blood samples: Full blood with anticoagulants like K2-EDTA should be processed or frozen quickly and used within a few days at 4 °C, or stored at –80 °C (pacb.com).

Microbial, plant, or insect samples: Each requires protocol adjustments—PacBio highlights gentle homogenization methods such as TissueRuptor for insects and liquid nitrogen grinding for tough plant tissues (pacb.com).

Best Practice: Flash freezing or immediate cold storage minimizes enzyme activity and DNA degradation across all sample types.

Input Quantity and Quality

The ideal input amount varies alongside sample type and extraction method:

Blood, cultured cells, bacterial cells: PacBio's Nanobind PanDNA kit works well with 1–5 million cells or 200–400 µL of whole blood, yielding 3–30 µg of HMW DNA with mode fragment sizes over 100 kb.

Plant tissues: Sample prep with nuclei isolation yields the best results. Input volumes of 0.25–5 g of plant nuclei typically yield large DNA fragments (>100 kb) .

Key Insight: Adequate input mass ensures both yield and DNA integrity, supporting downstream long-read sequencing.

Sample-Specific Guidance

Protocol Selection: Proven Methods for HMW DNA Extraction

Selecting the right extraction protocol is critical for preserving high-molecular-weight (HMW) DNA suitable for long-read sequencing. Here are proven, research-backed methods optimized for PacBio and Oxford Nanopore platforms:

1. Nanobind Magnetic Disk Kits (PacBio)

PacBio recommends Nanobind CBB and PanDNA kits with magnetic disk technology to gently isolate HMW DNA across a variety of sample types—blood, cells, tissue, plant nuclei, and insects—all within ~1–2.5 hours and yielding fragments of 50–300+ kb.

Key advantages: Minimal mechanical stress due to disk-based binding, inclusion of a Short Read Eliminator (SRE) to remove smaller fragments (<10 kb), and compatibility with both PacBio HiFi and Nanopore sequencing workflows.

2. Gentle Lysis with Nuclei Isolation and CTAB/Pipetting Control

For plant and insect samples, initial nuclei isolation (e.g., via CTAB buffer and beta-mercaptoethanol) followed by Nanobind-based extraction can preserve ultra-long DNA. A case study on Fraxinus excelsior and Taxus baccata used this protocol to efficiently recover plant nuclei and successfully obtain ~100 kb fragments for both ONT and PacBio HiFi sequencing.

Use wide-bore tips or pipette mixing; vortex only during early resuspension—no harsh agitation thereafter .

3. Phenol-Chloroform Extraction with Agarose Plug Encasement

Historically used for microbial genomes and challenging metagenomic samples, this method involves embedding samples in agarose plugs, performing gentle lysis, phenol-chloroform extraction, and agarase digestion to release DNA.

• Pros: Capable of generating extremely long DNA fragments (>200 kb).
• Cons: Labor-intensive, risk of UV damage, phenol oxidation, and incomplete removal of contaminants.

4. Commercial Spin-Column Kits: Not Recommended for HMW DNA

Conventional spin kits (e.g., silica columns or magnetic bead kits) often introduce mechanical shearing, yielding fragmented DNA unsuitable for long-read sequencing.

5. Best-Practice Handling Techniques

Across all methods, adhere to these essential practices:

Use wide-bore pipette tips and avoid vortexing after initial lysis to reduce shearing.

Use low-binding tubes (e.g., Eppendorf Protein LoBind) to reduce DNA loss.

Minimize freeze–thaw cycles, and resuspend DNA gently at room temperature over several hours for improved solubility.

Monitor extraction buffer use before and after lysis to maintain optimal yielding and integrity.

Overview Table: Protocols Compared

These optimized methods ensure you obtain HMW DNA of sufficient length and purity, enabling high-quality long-read sequencing results.

Learn more about our Nanobind extraction services and sequencing packages →

Explore FFPE DNA extraction and limitations →

Quality Control: Measuring Size and Integrity of Extracted DNA

Ensuring high-molecular-weight (HMW) DNA is intact and pure before sequencing is essential for reliable long-read data. Here are the key QC steps and measurements used in research laboratories and core facilities:

1. Pulsed-Field Gel Electrophoresis (PFGE)

PFGE remains the gold standard for separating large DNA fragments—ranging from tens to hundreds of kilobases. It resolves fragments up to ~2 megabases, providing visual confirmation of HMW DNA integrity. PFGE is especially useful early in the prep to detect shearing, guiding adjustments to extraction methods.

2. Fragment Analyzer (Capillary Electrophoresis)

Systems like Agilent TapeStation and Fragment Analyzer automate DNA sizing and purity assessment.

The Fragment Analyzer provides a Genomic Quality Number (GQN)—the percentage of DNA above a set threshold—offering quantitative QC for HMW extraction.

These tools are widely used due to speed (15–30 minutes per run), multiplexing capabilities, and minimal sample requirements (~1–5 µL) .

3. Spectrophotometry and Fluorometry (Nanodrop & Qubit)

Nanodrop provides purity ratios: OD260/280 (~1.8) and OD260/230 (~2.0–2.2). These indicate protein or organic contaminants, which can inhibit library prep or reduce read length.

Qubit assays measure double-stranded DNA concentration precisely, unlike Nanodrop, which can be influenced by RNA or free nucleotides .

Recommended QC Metrics

Tip for Long-Read Prep

Combine qualitative (PFGE) and quantitative (DIN/GQN, Nanodrop/Qubit) assessments to fully verify DNA integrity. Early detection of shearing allows remediation—such as re-extraction or size selection—to preserve long-read yield.

Storage and Handling Tips to Preserve DNA Integrity

Proper storage and handling are pivotal to maintaining high-molecular-weight (HMW) DNA quality for long-read sequencing. Below are evidence-based best practices for preserving DNA fragment length and purity:

1. Minimize Freeze–Thaw Cycles

Freezing and thawing DNA samples repeatedly can fragment high-molecular-weight strands due to ice-crystal formation and mechanical stress. Research on long-read DNA extraction emphasizes avoiding such cycles by aliquoting samples, storing them consistently at –20 or –80 °C, and working with just one aliquot at a time.

2. Optimal Temperature Storage

• Short-term (weeks to a few months): Store HMW DNA at 4 °C in a buffered solution like 10 mM TrisHCl (pH 8–9) to maintain stability without freeze–thaw damage.
• Long-term (several months and beyond): Use –20 °C or –80 °C storage, ensuring aliquoting to prevent repeated thaw cycles.

3. Gentle Handling Practices

• Always use wide-bore pipette tips and pipette slowly and minimally to reduce mechanical damage to DNA fragments.
• Avoid vortexing; mix samples by gentle inversion or flicking.
• Use low-bind or Protein LoBind tubes to prevent DNA sticking to tube walls, especially during prolonged storage.

4. Use of Buffer and Shipping Considerations

• Store DNA in a neutral, buffered solution (e.g., Tris-HCl buffer) rather than water to prevent degradation from pH fluctuations.
• For shipping or longer-term storage, dry ice is recommended, and samples should remain frozen to reduce agitation and mechanical stress.

Storage Strategy Summary

Practical Tip for Lab Workflows

Establish a sample management protocol that includes labelling aliquots with date/volume, tracking freeze–thaw history, and using pre-chilled consumables to maintain consistent temperatures during transfer or preparation.

These best practices ensure your HMW DNA maintains the integrity required for ultra-long reads, maximizing sequencing accuracy and downstream assembly quality.

Application Spotlight: PacBio vs. Nanopore Platform Requirements

When designing a long-read sequencing project, understanding each platform's DNA input needs and quality requirements is key. Below is a concise comparison of PacBio and Oxford Nanopore, highlighting how high-molecular-weight (HMW) DNA supports each system's optimal performance.

PacBio (SMRTbell Library Requirements)

PacBio's HiFi sequencing workflow demands high-quality, HMW DNA:

• DNA Integrity: Most fragments should exceed 30–50 kb, as measured by Femto Pulse or PFGE.
• Minimum Input Mass:
• Sequel I: ~150 ng gDNA (>30 kb)
• Sequel II/IIe: ~400 ng gDNA (>30 kb) per singleplex library.
• Library Yield: A typical single-cell or multiplex library with ~400–1,000 ng input can generate sufficient template for one SMRT Cell 8M, delivering >10 Gb data.
• Whole Genome Amplicons: For 10 kb amplicon libraries (e.g., Revio platform), input of ≥100 ng amplified DNA is adequate for ≥2 SMRT Cells with SPRQ chemistry.

PacBio's protocol emphasizes QC of fragment length (>30 kb) and accurate input quantification to ensure optimal yield and high-fidelity sequencing.

Oxford Nanopore (Ligation vs. Rapid Kits)

Oxford Nanopore offers flexible kits tailored for long-read applications:

• Ligation Sequencing Kit:
• Input: ~1 µg HMW gDNA; can work with 100–500 ng.
• Library Prep Time: ~65 minutes; fragmentation is optional if HMW DNA is used.
• Loading Strategy: For >10 kb libraries, loading 800–1,500 ng improves pore occupancy and data yield (~8–10 Gb) on high-throughput platforms.
• Rapid Sequencing Kits:
• Input: ~100–400 ng HMW gDNA (>30 kb) for long and ultra-long fragments.
• Workflow: Transposase-based, <10-minute prep; fewer pipetting steps preserve fragment integrity.

Nanopore's mean and median read lengths (20–30 kb, sometimes >50 kb) and library yield depend heavily on initial DNA quality and minimization of handling for ultra-long fragments.

Key Comparison Table

Choosing the Right Path

• For accuracy-centric projects (e.g., de novo assembly, variant calling), PacBio's HiFi offers unmatched precision—provided input DNA meets integrity requirements.
• For ultra-long reads (e.g., telomere resolution, structural variants), Nanopore's Rapid/Ultra-long kits paired with ultra-gentle prep deliver the longest reads available.
• Sample availability may dictate kit choice: Ligation workflows tolerate lower input, while HiFi and ultra-long methods require larger, intact DNA.

Explore our PacBio & Nanopore sequencing services and get a quote →

Troubleshooting: Low Yield or Fragmentation? Here's What to Do

Even with optimized protocols, challenges like low DNA yield or excessive fragmentation can arise during HMW DNA extraction. Here's a practical troubleshooting guide to address common issues and improve outcomes:

1. Low DNA Yield

Possible Causes & Fixes:

• Inefficient Lysis / Nuclease Degradation
• Ensure complete cell or tissue lysis by incubating sufficiently with Proteinase K or RNase A.
• Prolonged digestion can boost yield and reduce interference from nucleases.

Sample Loss During Cleanup

• When post-extraction DNA concentration (Qubit) is significantly lower than NanoDrop, residual RNA or single-stranded DNA may be inflating NanoDrop readings.
• Treat with RNase and Exonuclease VII, then purify with 0.45× AMPure PB beads to remove contaminants (per PacBio recommendations).

Low Input Quantity

For samples containing rare cells or tiny tissue samples, consider pooling or using low-input extraction protocols like the Nanobind PanDNA kit or specialist plant/insect workflows .

2. DNA Fragmentation or Shearing

Detection & Solutions:

• Detecting Fragmentation
• Run PFGE, TapeStation, or Fragment Analyzer to assess size distribution. A smear below target indicates shearing.
• Common Mechanical Stress Sources
• Avoid vortexing, rapid pipetting, or centrifugation at high speeds. Use wide-bore tips and gentle inversion .
• Enzymatic or Chemical Shearing
• Overdigestion or harsh reagents can nick DNA. Use buffers with antioxidants (e.g., beta-mercaptoethanol) and limit exposure time.

3. Contamination from RNA, Proteins, or Reagents

Identification & Cleanup:

• Purity Metrics
• A260/280 < 1.8 or A260/230 < 2.0 indicates contamination. Clean up with bead-based purification or phenol-chloroform extraction (with caution).
• Protein or Reagent Carryover
• Visible pellet during extraction suggests incomplete removal. Include extra ethanol-wash or use Nanobind's standardized washes.

4. Platform-Specific Yield Issues

PacBio SMRT Sequencing

Low polymerase-read length or total yield may be due to library contamination or overloading. Monitor P0, P1, P2 metrics in SMRT Link and optimize DNA quantity per SMRT Cell.

ONT Sequencing

Suboptimal pore occupancy and throughput could indicate too many short fragments. Use DNA repair kits and optional gentle fragmentation (e.g., Covaris g-Tubes) to improve read N50 suggestions by ONT.

5. Inhibitors or Environmental Contamination

Environmental DNA Contamination

• Low-input samples are at risk of background contamination; always include negative/control blanks during extraction.

Reagent Quality

• Use nuclease- and DNA-free certified reagents. Filter or autoclave buffers to prevent microbial or chemical contamination.

Troubleshooting Reference Table

Proper troubleshooting ensures your HMW DNA preparations consistently meet the quality standards needed for high-fidelity long-read sequencing.

Refer to FFPE DNA extraction troubleshooting →

References:

1. Jain, M., Koren, S., Miga, K. H., Quick, J., Rand, A. C., Sasani, T. A., Tyson, J. R., … Loose, M. (2018). Nanopore sequencing and assembly of a human genome with ultra-long reads. Nature Biotechnology, 36(4), 338–345.
2. Pacific Biosciences (2024). Guide & overview – Nanobind PanDNA kit. Retrieved from PacBio documentation.
3. Pacific Biosciences (2024). Guide & overview – Nanobind CBB kit. Retrieved from PacBio documentation.
4. Protocols.io (2019). Pulse-Field Gel Electrophoresis for Long-Read Sequencing [Protocol].
5. PacBio Blog (2025). DNA extraction tips and best practices for HiFi sequencing.
6. Bellott, D., Ting-Jan, C., Ting-Jan, C., Skaletsky, H., Hughes, J., & Page, D. (2022). SHIMS 3.0: Highly efficient single-haplotype iterative mapping and sequencing using ultra-long nanopore reads. PLoS One, 17(6), e0269692.
7. DNA extraction tips and best practices for HiFi sequencing - PacBio. https://www.pacb.com/blog/dna-extraction-tips-and-best-practices-for-hifi-sequencing/