Methodologically, the sequencing of DNA is straightforward. Most technologies are based on an enzymatic sequencing by synthesis. Here, modified DNA polymerases from thermophilic bacteria synthesize DNA in vitro using the genomic DNA of the target organism as template. The methods then differ, in part substantially, in the way of how they detect the incorporated nucleotide, and by doing so, determine the sequence of the DNA. A recent poster summarizing the ‘evolution of sequencing technology ’ provides a concise entry into the field. Common to all contemporary sequencing methods is, however, that the length of genomic DNA that can be consecutively sequenced, often referred to as the read length, is tiny to small compared to the genome size. Typical read lengths range from 75 base pairs (bp) up to a few thousand bp, depending on the technology. Figure 1 provides an overview of the existing technologies together with their average read lengths and output per run.

Over the years, the 3rd generation sequencing technologies have improved dramatically both with respect to read length and data quality. See for example the publication Opportunities and challenges in long-read sequencing data analysis by Amarasinghe et al. (2020). A major improvement were the development of the PacBio HiFi protocol that decreased the sequencing error of PacBio reads from around 15% down to less than 1%. This resembles almost the sequencing error of Illumina reads, however with the advantage of read lengths up to 20,000 - 30,000 bp.