The use of high-throughput sequencing in plant health research and diagnostics

High throughput sequencing (HTS), also referred to as Next Generation Sequencing (NGS) has revolutionized biology and medicine during the past decade. These techniques, on the market since 2005, allow the sequencing of millions of DNA molecules in parallel at a low cost per base pair. The resulting sequences are referred to as “reads”. Two main technologies are currently dominating the market: 1) a sequencing-by-synthesis method commercialized by Illumina (San Diego, CA, USA), called Illumina sequencing and 2) single molecule sequencing commercialized by Oxford Nanopore Technologies (ONT) (Oxford, UK), referred to as nanopore sequencing (Figure 1). Illumina sequencing needs an amplification of the DNA molecules to so-called clusters that are attached to a sequencing flowcell. These clusters are then used as template to synthesize complementary DNA, and each time a fluorescently labelled nucleotide is built in, it is detected by a laser. In order to obtain enough fluorescent signal, the DNA fragments need to be amplified (copied) in situ (on the sequencing flow cell). The amplification step, also called cluster generation, is needed to obtain enough fluorescent signal and is typical for second generation sequencing techniques. In third generation sequencing techniques, such as nanopore sequencing, there is no amplification step involved, and DNA fragments are sequenced directly. In nanopore sequencing, the DNA fragments are pulled through a pore-forming protein (nanopore) which creates a tiny hole in a membrane with an electric potential applied to it. Each time one of the bases passes through the pore, a small disruption occurs in the electric current, and this small disruption can be measured with a sensor. The disruption in the current is different for the different DNA bases when they pass, hence from these signals the corresponding bases can be deduced