June 13, 2024

Global Long Read Sequencing: Demystifying DNA through Third Generation Sequencing Technologies

Long read sequencing, also known as third generation sequencing, refers to a set of DNA sequencing technologies that can determine nucleotide sequences in a single read of up to hundreds of kilobases in length. This represents a significant improvement over traditional short read second generation technologies that are limited to reads only a few hundred base pairs long. The ability to generate long reads allows for more complete characterization of complex genomic regions that were difficult to assemble using short reads alone.

History and Development of Long Read Technologies

While next generation short read sequencing revolutionized genomics starting in the mid-2000s, these technologies still faced limitations in sequencing repetitive regions and structurally complex DNA. Long Read Sequencing The development of single-molecule real-time (SMRT) sequencing by Pacific Biosciences in 2011 marked the first commercially available long read platform. SMRT sequencing works by detecting fluorescently labeled nucleotides as they are incorporated into DNA polymerase-synthesized complementary strands in real-time. Read lengths averaging 10kb initially enabled more contiguous reference genome assemblies.

Oxford Nanopore Technologies launched the MinION portable sequencer in 2014 utilizing a novel nanopore-based approach. Individual DNA or RNA molecules are electrophoretically threaded through biological nanopores and changes in electrical current are measured as each unique nucleotide base passes through. Nanopore reads have improved to routinely exceed 50kb with some reads over 1Mb in length. Third generation technologies have continued advancing to make long reads accessible for a wide range of genomic applications.

Advantages of Long Range Information

A major limitation of short reads is their inability to resolve complex repeats and segmental duplications in genomes. Long reads can reliably span these repetitive regions to correctly place them in reference assemblies. Finished reference genomes with no gaps are important resources for evolutionary and biomedical studies.

Long reads are also valuable for studying structural variants like inversions, copy number variations, and translocations that are difficult to detect using short read data alone. They enable phasing of genes and variants to determine alleles inherited together, providing insights into haplotypes and disease risk. Additionally, long range information from individual DNA molecules benefits metagenomic analysis of microbial communities and isoform detection in transcriptomics.

Clinical Applications of Third Generation Sequencing

The capacity of long reads sequencing to meticulously characterize complex genomic regions has made them indispensable for clinical analysis. They are improving diagnostics for difficult genetic disorders and cancers by enabling detection of pathogenic structural variants. Long range haplotyping with Nanopore or SMRT sequencing is proving valuable for diseases linked to defective repeat expansions like Huntington’s disease.

Applications in microbial genomics using portable Nanopore devices are revolutionizing infectious disease diagnostics. Rapid real-time sequencing aboard has potential for outbreak surveillance and antimicrobial resistance monitoring in resource-limited settings. These technologies are deepening understanding of human microbiomes and their role in health. Long reads may also advance noninvasive prenatal testing and fetal genome analysis by resolving placental-specific structural mutations.

Challenges and Future Outlook

While third generation technologies have vastly extended read lengths, throughput remains more limited than second generation platforms. Ongoing work aims to boost yields and commercial platforms now integrate short and long reads. Error rates are also higher than short read sequencing currently. However, continuous technological innovations are steadily improving accuracy profiles for most applications.

Looking ahead, long read sequencing capabilities are expected to be broadly incorporated into clinical genetic testing in the coming years. Widespread adoption may be accelerated as costs decrease further. Nanopore’s rapid real-time sequencing especially holds immense promise for surveillance of infectious outbreaks and diagnostic applications in point-of-care settings. Long read resolution of structural variation will revolutionize cancer genomics and population prehistories. Continued expansion of reference genomes is also changing our perspective of genetic diversity among individuals and species. Overall, third generation sequencing is empowering unprecedented biological insights by elucidating once illegible regions of genomes and transcriptomes.

1. Source: Coherent Market Insights, Public sources, Desk research
2. We have leveraged AI tools to mine information and compile it