Next-generation sequencing (NGS) has revolutionized genomics, enabling the high-throughput sequencing of entire genomes at unprecedented speed and cost. Several NGS platforms have been developed, each with its unique characteristics, strengths, and limitations. This article explores the different types of next-generation sequencing instruments and how they differ from one another.
Perhaps the most widely used NGS platform, Illumina sequencing employs a sequencing-by-synthesis approach. DNA fragments are immobilized on a flow cell and amplified into clonal clusters. Fluorescently-labeled nucleotides are incorporated one by one into the growing DNA strand, with each incorporation event producing a fluorescent signal that is captured and translated into a base call. Illumina platforms offer high output, high accuracy, and relatively short read lengths (up to 600 bases). They are versatile and can be used for a wide range of applications, including whole-genome sequencing, exome sequencing, RNA-seq, and more.
Ion Torrent, developed by Thermo Fisher Scientific, uses a unique sequencing-by-synthesis approach that detects hydrogen ions released during the incorporation of a nucleotide into the growing DNA strand. This method does not require fluorescence or chemiluminescence, simplifying the sequencing process and reducing cost. Ion Torrent offers medium read lengths (up to 400 bases) and throughput, making it suitable for targeted sequencing, small genome sequencing, and RNA-seq.
Unlike Illumina and Ion Torrent, PacBio uses single-molecule real-time (SMRT) sequencing, which allows for the sequencing of individual DNA molecules in real-time. This results in significantly longer read lengths (up to 20,000 bases), enabling the resolution of complex genomic regions and structural variants that may be missed by short-read sequencing. However, PacBio sequencing has a higher error rate compared to Illumina and Ion Torrent.
ONT's MinION and PromethION devices use nanopore sequencing, a unique method that measures changes in electrical current as a single DNA molecule passes through a nanopore. This allows for extremely long read lengths (up to 2 million bases), making ONT sequencing particularly useful for de novo genome assembly and detection of structural variants. However, like PacBio, ONT sequencing tends to have a higher error rate compared to Illumina and Ion Torrent.
Choosing the right NGS platform depends on the specific needs of the project, such as the desired read length, throughput, accuracy, and cost. Each platform offers unique advantages and potential drawbacks. As technology continues to advance and new platforms are developed, researchers will have an increasingly diverse toolkit for exploring the genomic landscape.