Illumina is the clear leader in next-generation sequencing (NGS) technology. They purchased their core “massively parallel” sequencing-by-synthesis (SBS) technology from Solexa in the 2000s and have optimizing it for use in their MiniSeq, MiSeq, NextSeq, HiSeq, and NovaSeq platforms ever since. If Illumina represents the next generation of sequencing technology to be developed after the traditional “Sanger” sequencing approach (that was ubiquitous from the 1970s-1990s), there is now a “third-generation” of sequencing technologies that strive to get information from single molecules using reads that are much longer than what is currently offered on any Illumina platform.
With each new “generation” of DNA sequencing technologies, the quality of each individual base that is sequenced (known as a “basecall”) goes down. However, in each case the lower per base quality is a trade off for clear benefits over the previous generation’s technology. In the case of Illumina’s short-read NGS technology, the massive increase in total output compared to Sanger sequencing outweighed the drop in per base quality (Sanger sequencing remains the gold standard for highest quality basecalls across sequencing technologies). For the new third-generation technologies, it is the long read lengths that are seen as the benefit that outweighs the relatively lower per base quality when compared to Illumina sequencing. The use of these longer reads in “scaffolding” for de novo assembly is essential when studying highly repetitive genomes.
Chief among current third-generation sequencing companies is Pacific Biosciences (or “PacBio”). The PacBio RSII and Sequel sequencing platforms offer read lengths of many 1,000s of bases in length using PacBio’s proprietary “Single Molecule, Real Time” (SMRT) chemistry. The RSII platform can get reads up to 40-60kb in length, and the Sequel offers a higher output and better pricing with read lengths that currently fall below 20kb.
In addition to PacBio’s long read technology, there are also “linked-reads” technologies that exist somewhere in between current Illumina NGS technology and the third-generation platforms. For linked-reads, high-molecular weight (HMW) DNA is used for library construction. The long range information about the HMW DNA is maintained during a library construction process that ultimately yields a library capable of being sequenced on an Illumina platform. Advanced microfluidic technology and complex library indexing strategies make this possible. The most common linked-reads technology we use is the Chromium platform made by 10x Genomics.