Decoding Cancer's Genetic Map: Next-Generation Long-Read Sequencing Technologies

Why Do We Need New Sequencing Technologies?

Cancer arises from a complex combination of genetic mutations and progresses according to the principles of cellular evolution. Most genetic analyses used to guide treatment for millions of patients with cancer to date have relied on short-read sequencing technologies. However, this technology has significant limitations, particularly in detecting complex genetic rearrangements and large-scale structural alterations. This is where third-generation sequencing (TGS), also known as long-read sequencing (LRS), comes into play.

Thanks to new technologies, particularly platforms such as PacBio and Oxford Nanopore (ONT), complex genomic regions, epigenetic changes, and the full transcriptome structure can now be examined more accurately and comprehensively. This article explores why long-read sequencing is a revolutionary development in cancer research and where it offers particular advantages.

NOTE: Epigenetic changesare chemical modifications, such as DNA methylation, that affect how genes function without changing the DNA sequence. The full transcriptome structure is the complete set of RNA molecules actively expressed in a cell, particularly mRNA isoforms. Analyzing these features helps us understand how gene expression changes in diseases such as cancer and how cells respond to environmental influences.
It also plays a critical role in developing targeted treatments and discovering biomarkers.

The Limits of Short-Read Sequencing: Where Does NGS Fall Short?

Developed after the turn of the millennium, short-read sequencing technologies (Next-Generation Sequencing – NGS)reveal genetic information by sequencing short DNA fragments billions of times . However, read length is usually limited to just a few hundred base pairs. This makes them inadequate for:

• Detecting complex structural variants (SVs),

• Genome assembly,

• Transcriptome analyses such as RNA splicing variants,

• And directly detecting epigenetic modifications.

For example, errors introduced by PCR amplification and low read coverage in GC-rich regions can compromise the completeness of RNA sequencing results obtained with NGS.

Table 1.Summary of the advantages and disadvantages of long- and short-read technologies.

What Is Long-Read Sequencing, and Why Does It Matter?

TGS/LRS technologies are platforms that can read DNA or RNA molecules in much longer fragments. Read lengths generally exceed 10000 bp and can even reach 2 million bases in some reads. This enables the analysis of:

• Complex genomic rearrangements,

• Full-length mRNA transcripts,

• Epigenetic changes, such as DNA methylation,

• Phasing at the single-cell level and many other details previously inaccessible with NGS.

With reads generated by this technology, it is now possible to construct complete, individual-specific genome assemblies rather than aligning reads to a reference genome.

What Does Each Technology Offer?

The limitations of conventional short-read sequencing technologies (NGS) have led scientists toward new solutions capable of more comprehensive and accurate analyses. Third-generation sequencing technologies (TGS) provide tools powerful enough to revolutionize both genome and transcriptome analyses. The schematic below summarizes how the three most widely used platforms work and how they differ:

A) PacBio HiFi Sequencing Technology: The PacBio platform joins DNA fragments using special “hairpin-like” adapters to form a circular structure called SMRTbell . This structure is loaded into an SMRT cell, and the same DNA molecule is read repeatedly in succession. Errors in these reads are corrected using circular consensus algorithms to produce a highly accurate final read.

(B) Oxford Nanopore (ONT) Sequencing Technology: In the ONT platform, DNA is tagged with adapters containing motor proteins at its ends. A single DNA molecule is passed through a nanopore, and each base causes a distinctive change in electrical current. These changes are measured to decode the genetic sequence. The platform stands out for its real-time sequencing and ultralong-read capabilities.

(C) Illumina (Short-Read) Sequencing Technology: In the Illumina system, DNA is broken into small fragments and tagged with adapters. After library preparation, each DNA molecule is sequenced, generating many short reads. Although this technology offers high accuracy and throughput, it is limited in terms of length and complexity.

Where Does Long-Read Sequencing Make a Difference?

1. Detecting Point Mutations and Their Phase in Cancer

• As illustrated by EGFR mutations, knowing which allele carries the L858R and T790M mutations is critical for treatment response.

• PacBio and ONT can phase these mutations at the level of individual alleles.

• Phasing makes it possible to predict which patients will benefit from which targeted treatments.

2. Detailed Mapping of Structural Variants (SVs)

• SVs have been shown to account for 55% of driver mutations in cancer.

• Studies using PacBio have discovered more than 20000 SVs undetectable with short reads .

• Events such as deletion of tumor suppressor genes or amplification of oncogenes are now better understood.

3. Identifying New Gene Fusions

• In addition to known fusions such as RET, ALK, and ROS1, new fusions can also be identified.

• ONT has enabled the detection of gene fusions in less than 24 hours in samples enriched using CRISPR–Cas9.

4. Full-Length Transcriptome Analysis

• Studies in gastric and colorectal cancers have revealed more than 60% previously unknown isoforms.

• These isoforms show promise for identifying new biomarkers and therapeutic targets.

5. DNA Methylation and Epigenetic Analyses

• TGS can directly read methylation marks such as 5-mC and 6-mA without requiring chemical treatment.

• In brain tumors, methylation profiling of intraoperative samples within 2 hours has enabled real-time diagnosis.

6. Liquid Biopsy and cfDNA Analysis

• Unlike NGS, an approach producing results in 24 hours can provide real-time results with low coverage.

• TP53 mutations can be detected even at mutation frequencies as low as 0.02%.

• Long cfDNA fragments have also been shown to carry tumor-specific information.

Table 2.Summary of data complexity in TGS and NGS.

Bioinformatics Challenges and Future Prospects

• Because long-read data are larger and noisier, specialized analysis software is required.

• New algorithms, such as Minimap2 and Mapquik, have been optimized for these data.

• The large volume of data may place cost pressures on research centers in terms of storage and analytical infrastructure.

What Can We Expect in the Future?

• PacBio HiFi sequencing is ideal for SNVs and phasing, while ONT offers advantages in read length and speed.

• As prices fall and accuracy improves, integration into clinical practice will accelerate.

• Complete, individual-specific genome maps will become more common in place of a reference genome.

• Genomic information will be available together with epigenomic and transcriptomic information.

Conclusion: Long-Read Technologies Are Ushering in a New Era in Cancer Treatment

It is now possible not only to detect genetic variants but also to interpret them in context. Long-read sequencing technologies enable a more comprehensive understanding of cancer biology and more precise development of personalized treatment approaches. This revolutionary development appears likely to move beyond research laboratories and into clinical practices in the near future.