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Using TruSeq sequencing by synthesis (SBS) chemistry, hundreds of millions of these DNA fragment clusters are sequenced in parallel using a proprietary reversible-terminator method that detects single nucleotide bases as they are incorporated into growing DNA strands. During each sequencing cycle, a single fluorescently labeled deoxynucleoside triphosphate (dNTP) is added to the nucleic acid chain. The nucleotide label serves as a terminator for polymerization, so after each dNTP incorporation, the fluorescent dye is imaged to identify the base and then enzymatically cleaved to allow incorporation of the next nucleotide.
Since all four reversible terminator-bound dNTPs (A, G, T, C) are present as single, separate molecules, natural competition minimizes incorporation bias. Base calls are made directly from signal intensity measurements during each sequencing cycle, which greatly reduces raw mismatch rates compared to other technologies. The end result is highly accurate base-by-base sequencing that greatly reduces sequence-context specific errors, enabling robust base calling across the genome, including repetitive sequence regions and within homopolymers.
(A) A thymine base is incorporated into the growing DNA strand. At this point, the reversible terminator (red dot) blocks further polymerization. (B) Once the labeled thymine has been imaged, the reversible terminator is cleaved, allowing incorporation of the next nucleotide.