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-scoreYeasen Solutions for Third-Generation Sequencing
At Yeasen, we understand that high-quality long-read sequencing begins with robust and reliable library preparation. To support the growing demand for Oxford Nanopore Technologies (ONT) applications, we have developed a comprehensive portfolio of specialized library preparation kits designed to deliver flexibility, scalability, and performance across diverse research and clinical workflows.
Our solutions are engineered to help scientists achieve:
- Reliable long-read library preparation
- Consistent high-yield sequencing output
- Flexible workflows across genomics, transcriptomics, and clinical diagnostics
Genomic DNA (gDNA) Library Preparation
Cat#13301ES – Hieff™ DNA Library Prep Kit
Designed for whole-genome long-read sequencing, this solution enables high-quality library construction for:
Robust Long-Read Library Preparation Performance Across Diverse DNA Templates
|
Template |
Input DNA |
Library Recovery |
Data Yield |
Mean Read Length (bp) |
Max Read Length (bp) |
Read N50 (bp) |
|
Human gDNA (4–20 K) |
1 μg |
96% |
15 Gb |
9,136 |
113,518 |
12,633 |
|
1.7 K Fragment |
500 ng |
65% |
0.33 Gb |
1,791 |
23,493 |
1,771 |
|
3 K Fragment |
500 ng |
75% |
0.31 Gb |
2,557 |
26,165 |
3,005 |
|
1–16 K Fragment |
1 μg |
34.5% |
2.89 Gb |
4,189 |
41,457 |
5,893 |
|
Plasmid 1 |
300 ng |
>60% |
0.96 Gb |
2,413 |
23,637 |
2,658 |
|
Plasmid 2 |
300 ng |
>60% |
0.83 Gb |
2,129 |
35,855 |
2,808 |
QC Performance Comparison Case
|
Metric |
Yeasen 13301-1 |
Yeasen 13301-2 |
Supplier N*-1 |
Supplier N*-2 |
|
Total Reads |
300,000 |
300,000 |
300,000 |
300,000 |
|
Total Bases |
2,249,847,727 |
2,253,976,461 |
2,088,984,895 |
2,077,473,854 |
|
4 K < Bases < 12 K |
1,676,616,541 (74.52%) |
1,677,915,744 (74.44%) |
1,620,205,248 (77.56%) |
1,613,854,728 (77.68%) |
|
4 K < Bases < 20 K |
2,041,243,371 (90.73%) |
2,046,019,349 (90.77%) |
1,896,234,026 (90.77%) |
1,889,588,184 (90.96%) |
Comparable Sequencing QC Performance Between Yeasen 13301 and Supplier N*
Libraries prepared using Yeasen 13301 and Supplier N* under the same workflow generated highly consistent post-QC sequencing results, demonstrating comparable read distribution and base yield performance.
Full-Length Amplicon Library Preparation
Cat#13306ES + Cat#13304ES + Native Barcode Kits (13317–13320ES)
A complete workflow for amplicon-based long-read sequencing.
Microbial Amplicon library-barcoded library Case
Full-Length Plasmid Library Preparation
Cat#13305ES + Cat#13304ES + Native Barcode Kits (13317–13320ES)
Optimized for high-accuracy plasmid sequencing and structural verification.·
Full-Length Sequencing of AAV Plasmids Case
|
Kit |
Template |
Input DNA |
Library Recovery |
ONT Sequencing Yield(G) |
Reads Mean Length |
Reads N50 Length |
|
13305+13317/13318 |
Plasmid1 |
200 ng |
＞60% |
0.0531 |
3061 |
3109 |
|
Plasmid2 |
200 ng |
＞60% |
0.0834 |
2941 |
3082 |
|
|
Plasmid3 |
200 ng |
＞60% |
0.0419 |
3074 |
3095 |
|
|
Supplier N* |
Plasmid1 |
200 ng |
＞60% |
0.1949 |
3059 |
3107 |
|
Plasmid2 |
200 ng |
＞60% |
0.0805 |
3088 |
3108 |
|
|
Plasmid3 |
200 ng |
＞60% |
0.088 |
3067 |
3095 |
Full-Length RNA Transcriptome Library Preparation
Cat#12937ES + Cat#13302ES + Cat#13304ES
A streamlined workflow for full-length transcript sequencing.
Full-Length Transcriptome Sequencing Case
Figure . High-Quality Full-Length Transcriptome Sequencing Performance
Full-length transcriptome libraries prepared using the HieffTM Full-Length Transcriptome Library Prep Kit (Cat#12937ES) generated high-quality sequencing data with 88.02% full-length transcripts and a 98.01% mapping rate. Uniform gene body coverage without obvious 5′/3′ bias supports reliable full-length transcript analysis.
Related Products
|
Application |
Cat. No. |
Description |
|
Product Library Prep(Full-Length Amplicons) |
End Repair & Ligation Module |
|
|
Adapter Ligation Module |
||
|
Native barcode kits, 384 Barcode |
||
|
Full-Length Plasmid Library Prep |
Fragmentation & End Repair & Ligation Module |
|
|
Adapter Ligation Module |
||
|
Native barcode kits, 384 Barcode |
||
|
Full-Length RNA Library Prep |
Full-length cDNA Library Prep |
|
|
13302ES |
End Repair & Ligation Module |
|
|
Adapter Ligation Module |
||
|
rRNA Depletion |
12266ES |
rRNA Removing for Mammalian Samples(Magnetic method) |
|
12264ES |
rRNA Removing for Bacterial Samples(Magnetic method) |
|
|
12262ES |
rRNA Removing for Plant Samples(Magnetic method) |
|
|
Full-Length Microbial Sequencing |
DNA Library Prep |