Third‑generation sequencing is primarily represented by single‑molecule nanopore sequencing (ONT), a real‑time single‑molecule technology that differs fundamentally from first‑generation Sanger sequencing and second‑generation NGS. Leveraging the electrical‑signal‑sensing properties of nanopores, this approach eliminates the need for PCR amplification and fluorescent labeling, enabling real‑time direct sequencing of native full‑length single‑stranded DNA and RNA molecules. Characteristic current fluctuations as nucleic acids traverse the nanopore allow accurate identification of base identities as well as epigenetic modifications. It has become the mainstream technology for long‑read sequencing.
Suitable for animal‑plant, microbial and human genome research, this technology leverages ultra‑long read lengths to overcome second‑generation sequencing limitations. It captures repetitive sequences, centromeres, telomeres and high‑GC‑rich complex regions that short‑read sequencing fails to resolve. It supports near‑complete, gap‑free genome assembly and enables accurate detection of large‑scale structural variations: deletions, duplications, inversions and translocations.
Free from fragmentation bias, this approach directly captures intact full-length mRNA and lncRNA sequences. It enables accurate identification of full-length gene transcripts, alternative splicing isoforms, fusion genes and novel transcripts. Compared with second-generation transcriptome sequencing, it effectively eliminates the issues of erroneous fragment assembly and isoform misclassification.
This technique requires no bisulfite or other chemical conversions. It enables direct identification of native 5mC and 6mA DNA methylation modifications during sequencing. Genetic sequence data and epigenetic modification profiles are acquired simultaneously, supporting integrated analysis of gene sequences and epigenetic regulation. It is widely applicable to studies on epigenetic mechanisms, environmental stress responses, and microbial epigenetic regulation.
For scenarios including ultra-long target fragments, complex mutation regions, gene fusion sites, HLA typing, and microbial full-length typing, precise sequencing verification is performed to compensate for the shortcomings of short-read Sanger sequencing and complex structural variation limitations of NGS. It serves as the core technology for identifying complex genetic loci.
This approach obtains accurate full-length microbial gene sequences, enables precise identification of closely related species and strain typing, and supports high-quality microbial genome assembly. It efficiently characterizes the species composition, functional gene profiles and structural variations of bacterial communities from intestinal, soil, and aquatic environments.
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