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Microfluidic Sanger sequencing is a lab-on-a-chip application for DNA sequencing, in which the Sanger sequencing steps (thermal cycling, sample purification, and capillary electrophoresis) are integrated on a wafer-scale chip using nanoliter-scale sample volumes. This technology generates long and accurate sequence reads, while obviating many ...
English: The Sanger (chain-termination) method for DNA sequencing. (1) A primer is annealed to a sequence, (2) Reagents are added to the primer and template, including: DNA polymerase, dNTPs, and a small amount of all four dideoxynucleotides (ddNTPs) labeled with fluorophores.
Traditional Sanger sequencing and next-generation sequencing are used to sequence viruses in basic and clinical research, as well as for the diagnosis of emerging viral infections, molecular epidemiology of viral pathogens, and drug-resistance testing.
The classical shotgun sequencing was based on the Sanger sequencing method: this was the most advanced technique for sequencing genomes from about 1995–2005. The shotgun strategy is still applied today, however using other sequencing technologies, such as short-read sequencing and long-read sequencing.
A typical human cell consists of about 2 x 3.3 billion base pairs of DNA and 600 million mRNA bases. Usually, a mix of millions of cells is used in sequencing the DNA or RNA using traditional methods like Sanger sequencing or next generation sequencing.
Instead, the cDNA is randomly fragmented and the 3'ends are sequenced from the 5' end of the cDNA molecule that carries the poly-A tail. The sequencing length of the tag can be freely chosen. Because of this, the tags can be assembled into contigs and the annotation of the tags can be drastically improved.
SOLiD applies sequencing by ligation and dual base encoding. The first SOLiD system was launched in 2007, generating reading lengths of 35bp and 3G data per run. After five upgrades, the 5500xl sequencing system was released in 2010, considerably increasing read length to 85bp, improving accuracy up to 99.99% and producing 30G per 7-day run. [10]
These techniques for sequencing DNA generate shorter fragments than Sanger sequencing; Ion Torrent PGM System and 454 pyrosequencing typically produces ~400 bp reads, Illumina MiSeq produces 400-700bp reads (depending on whether paired end options are used), and SOLiD produce 25–75 bp reads. [23]