The Third-Generation Sequencing Challenge: Novel Insights for the Omic Sciences

Abstract

The understanding of the human genome has been greatly improved by the advent of next-generation sequencing technologies (NGS). Despite the undeniable advantages responsible for their widespread diffusion, these methods have some constraints, mainly related to short read length and the need for PCR amplification. As a consequence, long-read sequencers, called third-generation sequencing (TGS), have been developed, promising to overcome NGS. Starting from the first prototype, TGS has progressively ameliorated its chemistries by improving both read length and base-calling accuracy, as well as simultaneously reducing the costs/base. Based on these premises, TGS is showing its potential in many fields, including the analysis of difficult-to-sequence genomic regions, structural variations detection, RNA expression profiling, DNA methylation study, and metagenomic analyses. Protocol standardization and the development of easy-to-use pipelines for data analysis will enhance TGS use, also opening the way for their routine applications in diagnostic contexts.

Keywords: Oxford Nanopore Technologies; PacBio; RNA sequencing; epigenetics; genome sequencing; metagenomics; third-generation sequencing.

Figure 1

Third-generation sequencing timeline.

Figure 2

PacBio Sequencing method: The different instruments employ the same chemistry based on the use of a silicon chip, called an SMRT (Single-Molecule, Real-Time) cell, which hosts millions of wells for sequencing reactions (A). In each well, a single DNA molecule is immobilized and can be replicated following the injection of fluorescently labeled nucleotides (B). Fluorescent signals are registered and used for base-calling (C).

Figure 3

PacBio library preparation workflows: Using genomic DNA as starting material, the first step of the library preparation procedure is represented by DNA fragmentation. Then, DNA fragments are hairpin ligated to obtain an SMRTbell library suitable for polymerase binding and sequencing (A). Full-length mRNAs can also be used as an input sample. Indeed, the mRNAs are retro-transcribed, amplified, and hairpin ligated. The obtained SMRTbell library is ready for sequencing (B).

Figure 4

Oxford Nanopore Sequencing method: Several instruments featuring a different throughput are available, all based on the use of nanosensors capable of detecting changes induced by the DNA molecules in the electric current in real time (A). Indeed, the flowcell contains thousands of nanopores, each one able to measure the electric current flowing through; so, when a DNA molecule passes inside a pore, it modifies the current according to its sequence (B). This typical “squiggle” is used for subsequent base-calling (C).

Figure 5

ONT library preparation workflows: DNA libraries can be obtained by a rapid protocol that employs a transposase for both DNA cleavage and adapters ligation or by a high-throughput procedure requiring DNA fragmentation followed by adapters ligation (A). RNA libraries can be achieved by cDNA synthesis and adapter ligation or by direct adapter ligation to RNA molecules (B).

Figure 6

Third-generation sequencing applications and usefulness in different omic fields.