Third-Generation Sequencing: The Spearhead towards the Radical Transformation of Modern Genomics

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.

The Notable Achievements and the Prospects of Bacterial Pathogen Genomics

“…These can achieve read length orders of magnitude higher than those produced by the short-read platforms. However, their initial error-rates were very high (10–15%) [ 20 ]. The latest PacBio Sequel IIe system generates reads of average length 10–15 kb, producing 500 Gb of data within 30 h of running [ 20 ].…”

“…However, their initial error-rates were very high (10–15%) [ 20 ]. The latest PacBio Sequel IIe system generates reads of average length 10–15 kb, producing 500 Gb of data within 30 h of running [ 20 ]. The PacBio circular consensus sequencing (CCS) can now generate high fidelity (HiFi) long-reads with an error-rate of less than 1% [ 21 ].…”

“…The PacBio circular consensus sequencing (CCS) can now generate high fidelity (HiFi) long-reads with an error-rate of less than 1% [ 21 ]. As a comparison, the Oxford Nanopore PromethION 48 generates reads with a maximum length of more than 4 Mb, producing 14 Tb of data within 72 h of running [ 20 ]. The new ONT R.10 chemistry allows for a mean consensus single molecule error-rate of less than 1%.…”

Using nanopore sequencing to obtain complete genomes from saliva samples

“…The short-reads from within these regions all map non-specifically, therefore one cannot tell how many repeats exist within the repeat structure, or what part of the genome connects on the other side of the repeat (Figure 1). Consequently, these regions cannot be elucidated with confidence during assembly, causing the production of separate fragments, known as contigs, rather than a complete chromosome in the resulting draft genome (Athanasopoulou, Boti, Adamopoulos, Skourou, & Scorilas, 2021). When dealing with metagenomes, rather than isolate genomes, several additional problems are introduced (Figure 1).…”

“…In ONT sequencing, native nucleic acids are ratcheted through a nanopore embedded in a synthetic membrane, and the changes in electrical current are monitored (Jain et al, 2015). Sequences of bases or windows of bases cause specific and predictable perturbations in the electrical potential, therefore the sequence of bases passing through the nanopore can be reconstructed by analyzing the current (Amarasinghe et al, 2020; Athanasopoulou et al, 2021). Although nanopore sequencing technology is not new, its applicability was limited for many years by a high error rate.…”