Analysis of Read-Length Limiting Factors in Pyrosequencing Chemistry

摘要

Pyrosequencing is a bioluminometric DNA sequencing technique that measures the release of pyrophosphate during DNA synthesis. The amount of pyrophosphate is proportionally converted into visible light by a cascade of enzymatic reactions. Pyrosequencing has thus far been used for generating short sequence reads (1-100 nucleotides), as certain factors limit the system’s ability to accurately perform longer reads. In this study, we have characterized the main read-length limiting factors in both three-enzyme and four-enzyme Pyrosequencing systems. A new simulation model was developed to simulate the read-length of both systems, based on the inhibitory factors in the chemical equations governing each enzymatic cascade. Our results indicate that non-synchronized extension limits the obtained read-length; however, to a different extent for each system. In four-enzyme system, non-synchronized extension due mainly to a decrease in apyrase’s efficiency in degrading excess nucleotides proves to be the main limiting factor of read-length. Replacing apyrase with a washing step for removal of excess nucleotide proves essential in improving the read-length of Pyrosequencing. The main limiting factor of the three-enzyme system is shown to be loss of DNA fragments during the washing step. If this loss is minimized to 0.1% per washing cycle, the read-length of Pyrosequencing would be well beyond 300 bases.