One of the most groundbreaking developments in recent years has been the emergence of long-read sequencing technology. While traditional short-read sequencing methods have been instrumental in decoding the human genome since the completion of the Human Genome project in 2003, they do have limitations. Long-read sequencing is changing the game, providing scientists with a more comprehensive view of the genome and offering new insights into complex genetic structures that were previously inaccessible.
The Rise of Long-Read Sequencing
Short-read sequencing, the predominant method for decades, involves sequencing short fragments of DNA, typically around 150-300 base pairs in length. While this method is incredibly useful in many applications, and NGS is just about gaining popularity due to decreasing sequencing costs, it still has limitations when it comes to resolving repetitive regions, structural variants, and complex genomic rearrangements. This is where long-read sequencing comes in.
Long-read sequencing technologies, such as those developed by Pacific Biosciences (PacBio) and Oxford Nanopore Technologies, produce much longer sequence reads, ranging from thousands to tens of thousands of base pairs. This extended read length enables scientists to directly sequence through repetitive regions and structural variations that were previously difficult or impossible to resolve with short-read sequencing.
PacBio: Single molecule real-time (SMRT) sequencing
In this method, a lengthy DNA chain is synthesized utilizing the DNA slated for sequencing as a template. Fluorescence signals are detected upon the integration of nucleotides into the elongating DNA strand. The process is outlined briefly as follows:
The target DNA is circularized.
The circular DNA is applied to a surface known as a "SMRT Cell," featuring numerous tiny wells called "zero mode waveguides." Each well accommodates a single DNA polymerase enzyme operating on an individual circular DNA molecule.
Fluorescently labeled nucleotides are utilized to initiate the synthesis of a new DNA strand from the circular DNA. As each nucleotide is added by the polymerase to the growing strand, its fluorescence is recorded. This occurs within each of the numerous wells.
Since there is no termination point for sequencing, each circular DNA molecule undergoes multiple sequencing cycles.
Oxford Nanopore: In this technique, DNA bases are identified as they traverse a minuscule aperture, termed a nanopore, within a membrane. The process is described below:
Linear patient DNA is coupled with an enzyme that unwinds the double helix, enabling a single strand to be guided into the nanopore.
Nanopores are embedded in a thin membrane immersed in a salt solution, and an electrical potential is applied. This action prompts salt ions to traverse the pore, thereby establishing a current.
As the single-stranded DNA traverses the nanopore, each base (A, C, G, or T) obstructs the current flow in a distinct manner. The sequence of these current disruptions is detected and converted into the sequence of bases within the DNA strand.
The Advantages of Long-Read Sequencing
Resolving Complex Genomic Regions: In the human genome, repetitive sequences and structural variations are abundant and play crucial roles in genetic diversity and disease. The capability of Long-Read Sequencing to identify these regions has led to the discovery of novel genetic elements and provided insights into the role of repetitive regions in the genome.
Detecting Structural Variants: Structural variants (SVs), such as insertions, deletions, duplications, and inversions, are another area where long-read sequencing excels. Short-read sequencing often fails to fully characterize SVs, especially those in highly repetitive regions. Long-read sequencing can directly span these variants, providing a more complete picture of the genome. This is particularly important in the study of genetic diseases, as SVs are known to play a significant role in many disorders, including cancer and neurological conditions.
Genome Assembly: Genome assembly, the process of reconstructing the complete genome sequence from short DNA fragments, is another area where long-read sequencing shines. While short-read sequencing can generate high coverage, the fragmented nature of the data makes it challenging to accurately assemble complex genomes. Long-read sequencing produces longer reads, simplifying the assembly process and enabling the reconstruction of large, contiguous stretches of DNA. This has profound implications for species with large, complex genomes, as well as for metagenomics and microbiome research.
Characterizing RNA Transcripts: Long-read sequencing is also revolutionizing the field of transcriptomics, the study of RNA transcripts produced by the genome. Traditional short-read RNA sequencing methods struggle with isoform-level transcriptome analysis due to the difficulty in accurately reconstructing full-length transcripts. Long-read RNA sequencing overcomes this limitation by providing full-length transcript sequences, enabling the precise characterization of alternative splicing events and isoform diversity. This has the potential to enhance our understanding of gene regulation and gene expression dynamics.
Other possible clinical uses encompass:
Analyzing genes containing pseudogenes.
Identifying modified DNA bases, like methylation.
Establishing the distribution of variants on each chromosome copy (haplotype phasing).
Sequencing RNA transcripts for alternative splicing detection.
Utilizing portable devices for advantageous applications in Nanopore sequencing, like the MinION.
Challenges and Future Directions
While long-read sequencing holds tremendous promise, several challenges still need to be addressed.
The high error rates and relatively high cost of long-read sequencing are significant barriers to widespread adoption. Improvements in sequencing accuracy, reductions in cost and better sample throughput will be essential to realizing the full potential of long-read sequencing.
Computational methods for analyzing long-read sequencing data are still in development. As read lengths continue to increase, new algorithms and software tools will be needed to process and interpret the data effectively.
Long-read sequencing faces challenges in clinical labs due to the need for training and new workflows. Additionally, obtaining input DNA in long fragments, such as high molecular weight DNA, can be difficult using standard automated laboratory DNA isolation techniques.
Despite these challenges, the future of long-read sequencing is bright. As technology continues to improve and costs decline, long-read sequencing is expected to become the standard for genomic analysis. Its ability to provide a more comprehensive view of the genome will revolutionize our understanding of genetics and have far-reaching implications for human health, evolutionary biology, and beyond.
References:
Amarasinghe, S.L. et al. (2020) ‘Opportunities and challenges in long-read sequencing data analysis’, Genome Biology, 21(1). doi:10.1186/s13059-020-1935-5.
Marx, V. Method of the year: long-read sequencing. Nat Methods 20, 6–11 (2023). https://doi.org/10.1038/s41592-022-01730-w
-Written by Sohni Tagore
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