Abstract
Although next-generation sequencing (NGS) technology revolutionized sequencing, offering a tremendous sequencing capacity with groundbreaking depth and accuracy, it continues to demonstrate serious limitations. In the early 2010s, the introduction of a novel set of sequencing methodologies, presented by two platforms, Pacific Biosciences (PacBio) and Oxford Nanopore Sequencing (ONT), gave birth to third-generation sequencing (TGS). The innovative long-read technologies turn genome sequencing into an ease-of-handle procedure by greatly reducing the average time of library construction workflows and simplifying the process of de novo genome assembly due to the generation of long reads. Long sequencing reads produced by both TGS methodologies have already facilitated the decipherment of transcriptional profiling since they enable the identification of full-length transcripts without the need for assembly or the use of sophisticated bioinformatics tools. Long-read technologies have also provided new insights into the field of epitranscriptomics, by allowing the direct detection of RNA modifications on native RNA molecules. This review highlights the advantageous features of the newly introduced TGS technologies, discusses their limitations and provides an in-depth comparison regarding their scientific background and available protocols as well as their potential utility in research and clinical applications.
Highlights
Looking back in the late 1970s, when Frederic Sanger and his colleagues developed the first established method for DNA sequencing in the history of molecular biology [1,2], no one could imagine what would follow (Figure 1)
(~1.5 kb) that were characterized by high error rates (~13%) [12], the technology was improved over the years, leading to the release of a new sequencer, the Sequel System, which was quickly established as the basic sequencing machine of Pacific Biosciences (PacBio) for genome analysis
All PacBio platforms are based on single-molecule, real-time sequencing technology that detects alterations in light emission when a nucleotide is incorporated by the DNA
Summary
Looking back in the late 1970s, when Frederic Sanger and his colleagues developed the first established method for DNA sequencing in the history of molecular biology [1,2], no one could imagine what would follow (Figure 1). The identification of the DNA sequence is based on the emission of the light that is detected via imaging after the incorporation of a fluorescent nucleotide [10] This method simplified the library preparation process, due to the avoidance of PCR amplification steps, it demonstrated serious limitations regarding the time-consuming sequencing, its high cost, the high error rates and, last but not least, the production of short reads (~32 bp). The initial sequencer led to the generation of relatively short average read lengths (~1.5 kb) that were characterized by high error rates (~13%) [12], the technology was improved over the years, leading to the release of a new sequencer, the Sequel System, which was quickly established as the basic sequencing machine of PacBio for genome analysis. Through the rapid evolution of ONT chemistries, a significant increase in throughput has been achieved, resulting in the establishment of Nanopore sequencing in multiple research fields
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