DNA Extraction for WGS, Long-Read, and Targeted Sequencing

Introduction: Why DNA Extraction Strategy Matters for Sequencing

No matter how powerful your sequencing technology is, your results will only be as good as your DNA sample. DNA extraction is the foundation of every sequencing project, and the method you choose can make or break the outcome.

Different sequencing applications—such as whole genome sequencing (WGS), long-read sequencing, or targeted sequencing—have unique technical needs. These differences influence the ideal DNA extraction strategy. For instance:

• WGS demands consistent DNA quality across the whole genome.
• Long-read sequencing requires ultra-long, intact DNA fragments that are free from shearing.
• Targeted sequencing depends on DNA purity and sufficient concentration for enrichment protocols.

Each platform (like Illumina, PacBio, or Oxford Nanopore) has its own expectations for DNA integrity, fragment length, and purity metrics. Using the wrong extraction method may lead to poor coverage, sequencing failure, or biased results.

In short, choosing the right DNA extraction method is not optional—it’s essential. By understanding how each application differs, you can avoid delays, improve data quality, and reduce costs in the long run.

This guide will walk you through the core requirements, compare popular extraction methods, and help you select the most suitable approach for your downstream goals—whether you’re planning high-throughput WGS, resolving structural variants with long reads, or performing gene panel sequencing.

Core Requirements of DNA Extraction for Major Sequencing Platforms

To select the right DNA extraction method, it’s essential to understand what each sequencing platform requires in terms of DNA quality, quantity, and fragment length.

Short-Read NGS (Illumina)

• Input quantity: Typically requires 100–500 ng for human genomes; as low as 1 ng for microbial samples.
• Fragment size: Libraries are sheared to 300–600 bp, since longer fragments tend to increase error rates, especially in the second read.
• Purity: A 260/280 ratio around 1.8 and 260/230 around 2.0 are ideal; contaminants such as phenol or EDTA may inhibit tagmentation.

Long-Read Sequencing (PacBio HiFi & Oxford Nanopore)

• Input quantity:
  • PacBio HiFi: Standard ≥3 µg per Gb, ~15 µg for human; low-input workflows support 300 ng–3 µg for small genomes, ultra-low down to ~5 ng.
  • PacBio QC: Samples should have ≥50–200 ng/µL, especially for large insert sizes.
• Fragment size: High molecular weight (HMW) DNA (>10–20 kb) is crucial; at least 70% of fragments >10 kb for good library prep.
• Purity & integrity: Use fluorometric quantitation (e.g., Qubit) and spectrophotometry; purity metrics like 260/280 ≈1.8 and 260/230 ≥2.0 are essential to avoid contaminants.

Targeted Sequencing (Enrichment Panels, Exome)

• Input quantity: Illumina enrichment requires 10–1000 ng of high-quality DNA, or 50–1000 ng for challenging samples like FFPE.
• Fragment size: Enrichment works best with sheared DNA around 150–200 bp, matching probe designs. Quality controls screen out overly long fragments .
• Purity: Similar purity standards apply; low levels of contaminants ensure efficient capture and amplification.

Why It Matters

• Quantity ensures sufficient material for library prep and redundancy.
• Fragment length determines whether you preserve ultra-long DNA for structural genomics or optimize short fragments for read quality and uniformity.
• Purity prevents reaction failures and data biases.

Understanding these requirements lets you choose or customize extraction protocols that align with your sequencing goals—whether you’re preparing short-read libraries, ultra-long reads, or focused targeted panels.

Method Comparison Table: DNA Extraction for WGS vs Long-Read vs Targeted Sequencing

Below is a side-by-side comparison of common DNA extraction protocols, highlighting sample compatibility, yield, fragment length, processing time, automation potential, and best-suited sequencing applications.

Summary of Key Findings

• Organic extraction yields ultra-high molecular weight DNA (HMW) ideal for long-read WGS, but it is time-consuming, toxic, and hard to automate.
• CTAB/salting-out offers inexpensive HMW DNA, particularly useful in plant and microbial genomics, but requires more manual hands-on time.
• Silica spin-columns deliver reliable DNA for Illumina WGS/targeted sequencing, with fast processing and high potential for automation.
• Magnetic-bead kits are highly scalable, support gentle handling (for HMW) and are well-suited to long-read and targeted library prep.
• Enzymatic lysis plus beads—as shown in DNA HMW MagBead kit—performs well on long-read metagenomic samples, providing good yield and fragment length with moderate hands-on time.
• Robotic platforms offer reproducibility at scale, suitable for clinical and high-throughput projects, including plasmid and long-read prep.

Implications for Sequencing Applications

• Want ultra-long DNA for HiFi or Nanopore? Consider organic or CTAB methods when automation isn’t critical.
• Doing standard Illumina WGS or targeted libraries? Silica spin-columns balance quality, speed, and automation.
• Aiming for long-read libraries with throughput? Magnetic-bead or enzymatic-bead kits offer the best mix of scale, fragment size, and yield.
• Need high throughput and consistency? Robotic systems produce reproducible DNA for both WGS and long-read projects.

DNA Extraction Best Practices for Whole Genome Sequencing (WGS)

To ensure successful WGS, it’s crucial to follow best practices that guarantee high yield, purity, and consistency. Below are the key steps supported by current literature:

1. Start with a High-Quality Sample

• Use fresh or flash-frozen tissue, ideally stored at –80 °C, to minimize DNA degradation.
• Choose sample types known for stability, like blood, muscle, or lymphoid tissues, which release fewer DNases than, say, liver tissue.

2. Optimize Lysis & Extraction

• Use detergents (e.g., SDS) and enzymes (e.g., proteinase K) to break open cells efficiently.
• For hard-to-lyse samples, include bead-beating early in the workflow—this boosts yield and purity.
• Particularly, early bead-beating in high-SDS lysis buffer achieved superior purity with no extra cleanup in mycobacterial DNA.
• Avoid excessive mechanical force; DNA fragmentation impairs library prep.

3. Choose an Appropriate Method

• Silica spin-columns are fast (~1 hour) and well-suited to WGS, delivering clean DNA with good throughput.
• Organic (phenol–chloroform) or CTAB-based methods are best when you need high molecular weight DNA (>10 kb), though they are less automation-friendly.
• Magnetic-bead kits offer a balance of high quality and scalability; handle gently to preserve longer fragments.

4. Quantify and Check Purity

• Use fluorometric assays (e.g., Qubit) for accurate double-stranded DNA concentration—more reliable than Nanodrop, which can overestimate.
• Ideal A260/280 ≈ 1.8 and A260/230 ≈ 2.0–2.2; these ratios indicate minimal protein and salt contamination.

5. Assess DNA Integrity

• Run 1% agarose gels to inspect size distribution and degradation for short-read WGS.
• For ultra-high integrity needs (long-read or hybrid prep), use PFGE, FIGE, or Femto-Pulse to confirm presence of large fragments.

6. Optional Cleanup

• If purity ratios fall short, re-clean using ethanol or isopropanol precipitation or silica columns to eliminate solvents or salts.
• Avoid excessive cleanups, which can reduce DNA recovery and fragment length.

7. Shearing & Library Prep Considerations

• For Illumina, fragment DNA to 350–650 bp before library prep; mechanical shearing is preferred to reduce bias and maintain uniform coverage.
• When aiming for PCR-free libraries, which minimize amplification bias:
  • Start with ≥ 500 ng of DNA
  • Avoid extra cleaning steps post-extraction.

8. Quality Control Checklist

✅ DNA concentration (Qubit) meets or exceeds kit requirements (typically 500 ng+).

✅ A260/280 and A260/230 ratios are within acceptable range.

✅ Gel electrophoresis / PFGE data shows intact, high-molecular-weight DNA.

✅ Confirm library size distribution and purity after shearing.

 Summary of WGS Prep Workflow

• Collect & freeze sample quickly
• Lyse with detergent + enzyme, adding bead-beating if needed
• Extract DNA using column, bead, or organic method
• Measure purity (Nanodrop) and quantify (Qubit) the sample
• Check integrity via gel or PFGE
• Clean if necessary, but avoid over-processing
• Shear clean DNA to desired fragment size
• Proceed with library prep, ideally using PCR-free protocols

Following this workflow improves consistency, reduces bias, and helps ensure that your WGS data is reliable and high quality.

DNA Extraction for Long-Read Sequencing: Meeting High Molecular Weight Demands

Long-read sequencing platforms like PacBio HiFi and Oxford Nanopore require DNA that is not only high quality but also long and intact, often tens of kilobases in length. Here’s how to meet those demands:

1. Aim for Ultra-High Molecular Weight DNA

• Target fragments consistently >10–20 kb, with PacBio HiFi preferring >40–50 kb average lengths required for optimal reads (Circular Consensus Sequencing).
• This preserves long-range information essential for structural variant detection and complete genome assemblies.

2. Use Gentle Lysis and Nuclei Isolation

Avoid harsh physical disruption; instead, isolate intact nuclei using a buffer-based protocol, followed by gentle CTAB extraction, as shown with diverse plant taxa producing DNA five times longer than kit-based methods.

For recalcitrant plants (e.g., Streptocarpus), a CTAB + buffer method without phenol improved fragment length and sequencing performance on PacBio HiFi.

3. Selective Enzyme Buffers & No Harsh Chemicals

• Remove chaotropic agents like guanidine, phenol, and chloroform, as they can inhibit Polymerase Binding on SMRTbell adapters.
• Use CTAB lysis at moderate temperature (e.g., 58 °C for 4 hours) to release high-quality DNA without fragmentation.

4. Validate DNA Quality & Fragment Size

• Use Femto Pulse or PFGE analysis to confirm that ≥70% of fragments exceed 10–20 kb.
• Ensure high purity: A260/280 ≈ 1.8, A260/230 ≥ 2.0 to avoid impurities that affect adapter ligation and sequencing performance.

5. Optimize for Low Input Where Needed

• For limited starting material (<0.1 g), protocols adapted for Nanopore (e.g., PromethION) can still yield high-quality DNA in ~2.5 hours with successful output.

6. Outcomes

• Plant genomics: Nuclei + CTAB extraction delivered DNA five times longer than commercial kits, enriching long-read yield in plant samples.
• Tropical plants: A CTAB-based protocol without phenol/guanidine yielded 14–17 kb N50 reads on Streptocarpus, enabling draft assemblies with contig N50s of ~49 Mb.
• Microbial metagenomics: Magnetic-bead protocols applied to oral microbiome samples outperformed several commercial kits, delivering longer human tongue DNA suitable for long-read assembly .

Best Practices Summary

By adopting nuclei isolation, CTAB lysis, and gentle purification, labs consistently obtain long, pure, high-molecular-weight DNA—perfectly suited for PacBio HiFi and Oxford Nanopore runs.

Schematic illustration of the steps involved in the PacBio HiFi DNA extraction protocol. (Kanae Nishii et al,.2023)

DNA Extraction for Targeted and Exome Sequencing

Targeted and exome sequencing workflows rely on high DNA purity, consistent fragment sizes, and sufficient input material to ensure specific and efficient capture of genomic regions like exons or gene panels.

1. Know Your Input Requirements

• Most hybrid-capture kits require 50–500 ng of high-quality DNA; some low-input kits function with as little as 10 ng (especially for FFPE or degraded samples).
• For deep whole-exome sequencing (e.g., 400× coverage), starting with 50 ng of high-molecular-weight gDNA is a reliable baseline.

2. Focus on Purity and Fragment Size

• Aim for A₂₆₀/₂₈₀ ≈ 1.8 and A₂₆₀/₂₃₀ ≈ 2.0–2.2 to remove proteins, salts, or phenol that may inhibit probe hybridization.
• Shear DNA to ~150–200 bp, compatible with probe design and PCR amplification steps—crucial to avoid capture bias.

3. Choose the Right Extraction Strategy

• Silica-based spin-column kits are preferred for exome workflows due to their clean yields, speed, and ease of automation.
• For low-input, degraded, or FFPE-derived DNA, enzymatic repair kits and optimized lysis protocols effectively restore DNA integrity—one enriched capture method (OS-Seq) performed well with as little as 10 ng and without extensive PCR bias.

4. Apply Stringent Quality Control

• Measure concentration with Qubit, which only detects double-stranded DNA and avoids overestimation from contaminants.
• Assess purity via spectrophotometer and inspect fragment size visually on a gel or digitally with tools like Bioanalyzer.
• If using degraded or low-input samples, look for kits that include DNA repair enzymes and single-stranded library prep, which help reduce capture bias.

5. Common Pitfalls & Troubleshooting

• Low A₂₆₀/₂₃₀ suggests salt carryover from column or ethanol; re-clean sample using a spin-column or ethanol precipitation.
• Over-shearing reduces fragment size below probe length, leading to inefficient capture. Aim for 200 bp ± 20 bp in library prep.
• Low-complexity libraries (e.g., high duplication rates) often result from too little input DNA—sticking to recommended input ranges can prevent this.

🚀Summary: Targeted & Exome Prep Essentials

By optimizing DNA input, purity, and library prep, labs can obtain high-quality targeted and exome data—even on challenging sample types. Quality at this stage directly influences capture success, sequencing uniformity, and variant detection.

How to Choose the Right DNA Extraction Method

To simplify method selection for your specific sequencing goals, here’s a clear decision tree—ask yourself the following questions step-by-step:

1. What type of sequencing are you planning?

• Ultra-long reads (PacBio/Nanopore) → Go to Step 2
• Short-read WGS or targeted panels (Illumina) → Go to Step 4

2. For long-read platforms: Is preserving high molecular weight (HMW) DNA essential?

• Yes → Choose gentle protocols with minimal shearing
  • Option A: Nuclei isolation + CTAB extraction
  • Option B: Organic extraction (phenol-chloroform) (ideal if automation is not a priority)
  • Option C: Gentle bead-beating + magnetic-bead cleanup (for scalable HMW extraction)
• No (fragment length less critical) → Consider magnetic-bead kits or automated platforms to balance convenience and output

3. Do you need scalability or automation?

• High throughput → Prefer magnetic-bead kits (e.g., Zymo Quick-DNA HMW) or robotic platforms
• Lower throughput with maximum size/purity → Use manual CTAB or organic methods

4. For Illumina WGS or targeted sequencing: Do you have enough intact DNA?

• Yes (>100–500 ng of good-quality DNA) → Silica spin-column kits are fast, clean, and automatable
• No (e.g., degraded or FFPE samples) → Use enzymatic repair + kits designed for low-input (e.g., OS-Seq, ultra-low-input kits)

5. Final Quality Assurance

• Check purity (A₆₂₀/₂₈₀ ≈ 1.8; A₂₆₀/₂₃₀ ≥ 2.0)
• Confirm fragment size (via gel, Bioanalyzer, Femto Pulse)
• Quantify accurately (Qubit preferred over NanoDrop)

Why This Matters

• Aligns protocol with sequencing goals—avoids wasted effort
• Balances purity, fragment size, yield, and throughput effectively
• Ensures reproducibility—especially crucial for CROs and pharma pipelines

This decision framework is adapted from industry protocols such as PacBio’s “Sequencing 101” guide and validated by extraction method comparisons across sample types.

Common Pitfalls in DNA Extraction for Sequencing and How to Avoid Them

Even with the best protocols, DNA extraction can be fraught with challenges that compromise sequencing outcomes. Below are common pitfalls and practical solutions to ensure high-quality DNA for your sequencing projects.

1. Pitfall: DNA Shearing During Extraction

• Problem:
  Mechanical processes like bead-beating or vortexing can break DNA into smaller fragments, especially in long-read sequencing, where high molecular weight (HMW) DNA is crucial.
• Solution:
  • Use gentle methods: Opt for manual or semi-automated protocols that minimize mechanical stress.
  • Choose appropriate kits: Select extraction kits designed for long-read sequencing, such as those utilizing Nanobind disks, which protect DNA integrity during extraction.

2. Pitfall: Contamination from Co-Extracted Materials

• Problem:
  Contaminants like phenolic compounds, polysaccharides, or proteins can co-purify with DNA, leading to inhibition in downstream applications.
• Solution:
  • Implement cleanup steps: Incorporate additional purification steps, such as magnetic bead-based cleanup or silica column purification, to remove contaminants.
  • Use appropriate buffers: Employ buffers that effectively remove contaminants without compromising DNA yield.

3. Pitfall: Inadequate Sample Storage Leading to DNA Degradation

• Problem:
  Improper storage conditions can lead to DNA degradation, affecting quality and yield.
• Solution:
  • Immediate processing: Process samples as soon as possible after collection.
  • Optimal storage conditions: Store samples at -80°C or in DNA stabilizing solutions to preserve DNA integrity.

4. Pitfall: Inconsistent DNA Yield Across Samples

• Problem:
  Variability in DNA yield can occur due to differences in sample type, extraction method, or handling procedures.
• Solution:
  • Standardize protocols: Follow consistent extraction protocols tailored to the specific sample type.
  • Quality control: Regularly assess DNA yield and quality using spectrophotometry or fluorometry.

5. Pitfall: Low DNA Purity Affecting Sequencing Performance

• Problem:
  Impurities in DNA can interfere with sequencing reactions, leading to poor data quality.
• Solution:
  • Assess purity: Measure DNA purity using A260/A280 and A260/A230 ratios.
  • Purification steps: Incorporate additional purification steps if purity ratios are outside the optimal range (A260/A280 ~1.8–2.0, A260/A230 >2.0).

6. Pitfall: Inadequate Fragment Size for Long-Read Sequencing

• Problem:
  DNA fragments that are too short can lead to poor performance in long-read sequencing platforms.
• Solution:
  • Optimize extraction methods: Use extraction methods that preserve DNA fragment length, such as those utilizing Nanobind disks.
  • Assess fragment size: Evaluate DNA fragment size using gel electrophoresis or capillary electrophoresis.

7. Pitfall: Lack of Automation Leading to Variability

• Problem:
  Manual extraction methods can introduce variability and are time-consuming.
• Solution:
  • Implement automation: Utilize automated extraction systems to increase throughput and consistency.
  • Standardize procedures: Develop and adhere to standard operating procedures (SOPs) to minimize variability.

Final Summary: Matching Your DNA Extraction Method to Your Sequencing Goals

Selecting the appropriate DNA extraction method is crucial for the success of your sequencing project. The method you choose directly impacts the quality and integrity of the DNA, which in turn affects the reliability and accuracy of your sequencing results. Here’s a recap of key considerations:

• Understand Your Sequencing Platform’s Requirements: Different sequencing technologies have specific DNA input requirements. For instance, long-read sequencing platforms like PacBio and Oxford Nanopore require high molecular weight (HMW) DNA to produce accurate and long reads.
• Choose the Right Extraction Method: Based on your sample type and the sequencing platform, select an extraction method that preserves DNA integrity and meets the required quality standards. For example, the Nanobind DNA extraction method is designed to isolate HMW DNA suitable for long-read sequencing.
• Implement Best Practices: Follow established protocols and best practices to minimize common pitfalls such as DNA shearing, contamination, and inadequate DNA yield. Regular quality control checks, like spectrophotometry and gel electrophoresis, can help assess DNA purity and integrity.
• Avoid Common Pitfalls: Be aware of potential issues like DNA shearing, contamination, and low yield. Implement strategies to mitigate these problems, such as using gentle lysis methods, proper sample storage, and appropriate cleanup steps.

By carefully selecting and optimizing your DNA extraction method, you can ensure high-quality DNA for your sequencing projects, leading to reliable and reproducible results.

References:

1. Nishii K, Möller M, Foster RG, Forrest LL, Kelso N, Barber S, Howard C, Hart ML. A high quality, high molecular weight DNA extraction method for PacBio HiFi genome sequencing of recalcitrant plants. Plant Methods. 2023 Apr 29;19(1):41. PMID: 37120601
2. Tan, G., Opitz, L., Schlapbach, R. et al. Long fragments achieve lower base quality in Illumina paired-end sequencing. Sci Rep 9, 2856 (2019). https://doi.org/10.1038/s41598-019-39076-7