The cluster fragments are denatured, annealed with a sequencing primer and subjected to DNA synthesis with four differentially reversible labeled fluorescent nucleotides that have their 3′-end chemical termination to ensure that only a single base is extended. Selleckchem Nutlin 3a After a single base is incorporated into the DNA strand, the terminator nucleotide is detected via its labeled fluorescent dye by the CCD camera. Then, the labeled fluorescent dye and 3′-end chemical terminations are removed and the next DNA synthesis cycle

is repeated. The Genome Analyzer IIx can obtain 30–100 nucleotide read lengths and data output per paired-end run from 1–3 Gb.[16-18] Moreover, Illumina released a HiSeq sequencer series which enabled higher throughput and a desktop MiSeq sequencer type which could sequence more rapidly in 2013. THE ABI SOLID sequencer was introduced in October 2007. SOLiD is an abbreviation for “sequencing by oligo ligation CP-868596 and detection”. It uses a unique sequencing method catalyzed by DNA ligase. The universal P1 adaptor-linked DNA fragments are attached to magnetic beads. Emulsion PCR is conducted in microreactors containing the reagents of the PCR reaction. The magnetic beads are covalently attached to the surface of a specially treated glass slide that is placed into a fluidic cassette within the sequencer. The universal sequence primers

hybridize to the P1 adapter within the library template.

The set of four fluorescent-labeled di-base 8-mer probes are annealed to the sequencing primer and library template. Identification of the nucleotide sequence by the 8-mer probe is achieved by interrogating every first and second base in each ligation reaction. When there is a matching of the 8-mer probe to the library template adjacent to the universal primer of the 3′-end, DNA ligase seals the phosphate backbone. After the ligation, the probe is enzymatically removed together with the last three bases attaching the linkage between base 5 and 6. Then, the same probe hybridizing process is conducted and the sequence data of each library template can be obtained at five nucleotide click here intervals. Following a series of ligation cycles, the library template is reset with five rounds of universal primers complementary to the n to n-4 position for a multistep round of ligation cycles. Through the primer reset process, each base is interrogated in two independent ligation reactions by two different primers and the nucleotide sequence is defined by this repetition. The ABI SOLiD 2.0 platform, produced in 2008, can obtain data output from 3–10 Gb per run.[16-18] HOWEVER, MUCH IMPROVED the NGS systems have already become, the competition in technology development is intensifying. The demand for low cost, high speed and highly accurate systems has spurred development beyond third-generation NGS systems (Table 1).