With subsequent chemical steps, the chain termination moiety and fluorescent labels are removed and washed away, allowing for a next sequencing cycle to be performed.
After incorporation, excess reagents are washed away, the clusters are optically interrogated, and fluorescence is recorded. The dye-labeled nucleotides are incorporated according to sequence complementarity in each strand in a clonal cluster. Sequencing of the forward strands is initiated by hybridizing a primer complementary to adapter sequences followed by addition of polymerase and a mixture of four uniquely labeled, fluorescent “reversible” chain terminating nucleotides. For sequencing, clusters are denatured, and then a chemical cleavage reaction and wash are conducted, leaving only forward strands for single end sequencing. Multiple amplification cycles result in conversion of single-molecule DNA templates to clonally amplified arching clusters with each cluster containing approximately 1,000 clonal DNA molecules. In contrast to ePCR, DNA templates are amplified by an isothermal “bridge” amplification method mediated by formamide denaturation that relies upon captured DNA strands “arching” over and hybridizing to an adjacent anchor oligonucleotide. 59.2, the fragment library is denatured and, under limiting dilution conditions, is added to the flow cell and immobilized by hybridization to the oligonucleotide anchors. As with the 454 method, genomic template DNA first is converted into a randomly overlapping fragment library using either Covaris fragmentation followed by enzymatic end repair and Illumina adapter ligation, or via Nextera transposition (described below). The Genome Analyzer utilized a flow cell comprised of an optically transparent slide with eight individual microfluidic lanes on which oligonucleotide anchors are bound to the slide surface. One of the recognized strengths of the 454 technology is the progressive increase in sequencing read lengths that have been achieved, beginning with 100 base length reads on the GS-20 that have now been extended up to 1,000 bases. The collected data is algorithmically translated into a linear sequence output. With each dNTP reagent flow, wells are imaged and analyzed for signal-to-noise parameters.
A nucleotide-incorporation event in a well containing a clonally amplified template results in pyrophosphate release, and the luminescence generated in the well is transmitted through the fiber-optic picotiter plate and recorded by a charge-coupled device (CCD) camera. When loaded into the GS FLX instrument, the picotiter plate functions as a flow cell wherein iterative pyrosequencing is performed by successive microfluidic addition of polymerase and dNTPS (i.e., A followed by C, then G, then T, etc.). Beads containing clonal amplicons are deposited into individual picotiter wells that contain enzymatic components for pyrosequencing with luminescence signal generation. Specifically, the 454 flow cell is referred to as a “picotiter” plate and is made from a fused fiber-optic bundle into which millions of individual wells are etched into the surface.
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