The DNA is unzipped by a helicase to allow single-strand sequencing. a Nanopore sequencing: DNA is analyzed by threading it through a biological protein pore (e.g., Mycobacterium smegmatis porin A, MspA). Principle of nanopore and single-molecule real-time (SMRT) sequencing. The longest reads achieved with the current method comprise a length of more than 2 million bases of DNA in a row. The technology does not depend on a polymerase and allows sequencing of native DNA and RNA and the detection of various chemical modifications (e.g., methylation) of nucleic acids. When DNA or RNA passes through the pore via a helicase, this creates a characteristic change in the current, which provides information on the respective nucleotides in the nanopore (Fig. In nanopore sequencing, a tiny protein pore ( Mycobacterium smegmatis porin A, MspA, or Escherichia coli Curlin sigma S‑dependent growth subunit G, CsgG) is embedded in an electrically resistant polymer membrane and an ionic current is passed through this nanopore by setting a voltage across the membrane. The idea to sequence long fragments of DNA and RNA without PCR amplification and nucleotide labeling had its origins as early as the 1980s, but has only become feasible after a technology using nanopores recently reached market maturity (Oxford Nanopore Technologies®, ONT, Oxford, UK). ![]() Several of these shortcomings can be overcome by third-generation sequencing technologies (TGS), also referred to as long-read sequencing in the following. Moreover, PCR amplification of sequencing templates generates artefacts and precludes detection of native base modifications. The short-reads prevent analysis of complex genomic loci, repetitive elements, or variant phasing (haplotyping) and result in inefficient and incomplete genome assemblies. However, both Sanger sequencing and NGS technologies deliver only short-read DNA fragments within the range of 50–1000 bases. Different methods using semiconductors (Ion Torrent), pyrosequencing (Roche), sequencing by ligation (Applied Biosystems), and the widely used sequencing by synthesis with reversible terminators (Solexa, Illumina) enabled gene panel, whole-exome, and whole-genome sequencing within a few days at moderate costs. Within 10 years, NGS led to a dramatic increase in knowledge on genetic variation and allowed fast and accurate diagnostics of clinically relevant germline and somatic mutations. However, the advent of massively parallel sequencing (next-generation sequencing, NGS) turned out to be another game changer and revolutionized human genetics. Sanger sequencing culminated in the sequencing of the human genome and is still relevant for targeted resequencing. Subsequently, the widespread method using chain-terminating dideoxynucleotides by Frederick Sanger and colleagues has fostered sequencing since the mid-1970s. Back in 1968, Wu and Kaiser used primer extension methods to identify a short sequence of the bacteriophage lambda, whereas 5 years later, Maxam and Gilbert determined the sequence of the lactose-repressor binding site by chemical cleavage.
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