Figure 3

Principles for sequencing and imaging. (a) Illumina sequencing of three template molecules. All four nucleotides, carrying terminating moieties and unique fluorescent labels, and DNA polymerase are added, and one complementary nucleotide becomes incorporated at each template molecule (1). After washing, fluorescence is registered at four wavelengths (2). Fluorescent dyes and terminating groups are cleaved off. A new set of nucleotides is added (3), and imaged (4). Sequence reads of equal length are obtained (5). (b) 454 sequencing of three template molecules. One type of natural non-terminating deoxynucleotides and DNA polymerase are added and a pyrophosphate molecule is released at each nucleotide incorporation (1). Pyrophosphate is converted into light using sulfurylase and luciferase, and the light intensity is measured in each well (2). Free deoxynucleotides are destroyed with apyrase before adding the next type of deoxynucleotide (3) and imaging (4). Light signals are converted to flowgrams with higher signal intensity bars in homopolymer regions (5). Sequence reads that may differ in length are obtained (6). (c) SOLiD sequencing of one template molecule. A sequencing primer, DNA ligase and 1,024 unique probes, which are fluorescently labeled according to their first two bases, are added, and the complementary probe is ligated to the template (1). After washing, fluorescence is registered at four wavelengths. The three universal bases and the fluorophor are cleaved off (2). Addition of a new probe set is repeated for the desired number of cycles (3,4). The newly built strand is melted off. A new sequencing primer is added, which anneals one base off from the first primer and therefore interrogates different positions (5). Sequencing is repeated for the desired number of cycles (6). Additional primers are added, until each base is sequenced twice. The colors from all sequencing rounds are merged and can be converted to nucleotides (7).