Decoding the Power of Third-Generation Sequencing: Applications Beyond NGS

In recent years, second-generation sequencing (NGS) has rapidly shortened turnaround times and continues to dominate the market with its short-read technology. Yet since 2008, third-generation sequencing (TGS) has gained strong momentum. With its unique long-read capability and the ability to sequence single DNA molecules without PCR amplification, TGS opens new possibilities in genome assembly, pathogen research, and mutation detection.

Figure 1. Development of sequencing technology

The Principles of Third-Generation Sequencing

Third-generation sequencing, also known as single-molecule sequencing, leverages cutting-edge advances in optics, polymers, and nanotechnology to directly distinguish the signals of individual nucleotides. Unlike short-read platforms, TGS provides a direct readout of long DNA or RNA fragments.

Figure 2 . The Principles of TGS

Why Third-Generation Sequencing Matters

By providing ultra-long reads and direct single-molecule sequencing, TGS overcomes many NGS limitations. Its strengths include:

• Resolving repetitive or complex genomic regions.
• Accurate structural variation detection (insertions, deletions, inversions, CNVs).
• Full-length transcript and isoform sequencing.
• Real-time epigenetic (e.g., methylation) analysis without additional steps.
• Eliminating PCR bias for more native representation of nucleic acids.

Table 1. Comparison of Sequencing Technologies

Feature	First-Generation (Sanger)	Second-Generation (NGS)	Third-Generation (TGS)
Read Length	500–1000 bp	100–300 bp	10 kb – >100 kb
Throughput	Low	Very High	Moderate–High
Accuracy (per base)	~99.9%	~99% (Q30 typical)	Raw ~85–95% (consensus >99%)
Turnaround Time	Slow	Rapid	Moderate (improving)
PCR Requirement	Yes	Yes	No (single-molecule sequencing)
Cost per Base	High	Low	Decreasing, but higher than NGS
Best Applications	Gene cloning, small-scale validation	Whole-genome sequencing, RNA-seq, clinical panels	De novo genome assembly, structural variants, isoform discovery, epigenetics

Applications of Third-Generation Sequencing

TGS has already proven transformative in plant/animal genomics, microbiome studies, clinical diagnostics, and oncology research. Long-read TGS platforms are increasingly applied across multiple research and clinical domains, addressing limitations that short-read sequencing cannot easily overcome:

Table 2. Key Application Advantages of TGS

Application	NGS Limitation	TGS Advantage
De Novo Assembly	Fragmented assemblies	Near-complete genomes
Structural Variants	Misses large SVs	Accurate SV detection
Transcriptomics	Reconstruct isoforms computationally	Direct isoform sequencing
Epigenetics	Needs extra assays	Direct detection of modifications
Metagenomics	Strain-level resolution poor	High-resolution species profiling
Clinical Research	May miss complex events	Detects rare/complex variants

 

DNA/RNA QC for TGS Library Prep

The quality of extracted DNA/RNA is critical for long-read library preparation. Contaminants, degradation, or insufficient fragment length directly impact sequencing performance.

Key QC requirements for TGS library prep:

• Purity: High-purity DNA/RNA free of cross-contamination or protein. Specialized long-fragment extraction kits are recommended.
• Concentration: ≥30 ng/μL.
• Integrity: For DNA, pulsed-field gel analysis should show average fragment size >30 kb; for RNA, RIN ≥8.
• Input amount: DNA ≥1 μg (depending on desired sequencing depth).
• Storage: TE buffer is recommended for long-term storage of high molecular weight gDNA.

Data Quality Metrics in TGS

Unlike NGS, which uses base-calling accuracy metrics such as Q20/Q30, TGS has a different error profile. Single-base accuracy alone is not the best measure of quality. Instead, read length and distribution are more important indicators of library quality.

High-quality libraries → produce longer reads, better coverage, higher consensus accuracy.

Low-quality libraries → yield shorter reads, uneven coverage, and poor data output.

• PacBio QC metrics: Total yield (Gb), Polymerase read length, Insert size, Subread N50
• Oxford Nanopore QC metrics: Total yield (Gb), Average read length, Mean quality score (Q), Read length N50

Longest read length & corresponding Q-score

Yeasen Solutions for Third-Generation Sequencing: Hieff™ DNA Library Prep Kit for ONT(Cat#13301)

At Yeasen, we understand that high-quality long-read sequencing starts with robust library preparation. That’s why we’ve developed a suite of specialized library prep kits optimized for Oxford Nanopore platforms, designed to meet diverse research and clinical needs.

• Our solutions help scientists achieve:
• Reliable long-read library construction.
• Consistent high-yield data outputs.
• Flexible workflows for genomics, transcriptomics, and clinical diagnostics.

Figure 3. Rapid Workflow of Hieff™ DNA Library Prep Kit for ONT

Related Product

Cat. No.	Name	Notes
13301	Hieff™ DNA Library Prep Kit for ONT	Universal products
13309	Hieff™ DNA Fragmentation Reagent for ONT	DNA fragmentation module
12606	Hieff NGS™ FFPE DNA Repair Reagent	FFPE DNA treatment