Analysis of read length limiting factors in Pyrosequencing chemistry

Abstract

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 heretofore been used for generating short sequence reads (1-100 nucleotides) because certain factors limit the system's ability to perform longer reads accurately. 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 nonsynchronized extension limits the obtained read length, albeit to a different extent for each system. In the four-enzyme system, nonsynchronized 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 to be 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.

Figure 1

Enzymatic reactions of the four-enzyme Pyrosequencing system.

Figure 2. Effect of product inhibition on ATP sulfurylase

(a) signals from the control solution. Amounts of 500 pmol (b), 1000 pmol (c), and 2000 pmol (d) of PPi and APS were dispensed into three different solutions. The signal intensities decrease by 20%, 30%, and 52% in (b), (c), and (d) respectively as compared to signal intensities in (a).

Figure 3. Effect of AMP inhibition on luciferase

(a) control solution 500 pmol, 1000 pmol, and 2000 pmol of AMP were added to solution in (b), (c), and (d) respectively. The light signals’ peak heights and shapes do not change significantly in these solutions compared to the signal from the control solution.

Figure 4. Effect of oxyluciferin inhibition on luciferase

(a) is the control solution. 500 pmol, 1000 pmol, and 2000 pmol of ATP were added to (b), (c), and (d) respectively prior to Pyrosequencing. Signal peaks decreased by 5, 10, and 22% in (b), (c), and (d) respectively compared to the control signal.

Figure 5. Effect of product inhibition on apyrase

(a) is the control solution. DNA fragments were added to (b) after 10, to (c) after 20, to (d) after 40, and to (e) after 80 dNTP dispensations. More noise (vertical arrows) due to unsynchronized DNA extension is visible in solutions with more dNTP dispensations.

Figure 6. Effect of misincorporation by DNA polymerase on Pyrosequencing signals

(a) is the control solution; the solution in (b) was incubated with an unmatched nucleotide for 20 minutes (in the absence of apyrase) before the complementary nucleotide was dispensed. The light signal in (b) is 17% less than the control signal.

Figure 7

Simulation results of four-enzyme Pyrosequencing system on a 300-base long DNA fragment. Error-free base-calling is achieved for 60 bases in this simulation result.

Figure 8

Simulation results of three-enzyme system Pyrosequencing on a 300-base long DNA fragment with (a) 100% and (b) 90% washing efficiency. Noise remains minimal in both cases resulting in much longer error-free read-length compared to four-enzyme Pyrosequencing system.

Figure 9

Simulation results of the three-enzyme Pyrosequencing system on the same DNA fragment as above, but with 300 nucleotide dispensations. The signal intensity decreases slightly over 300 nucleotide dispensations (top); even during the later cycles and by 300th dispenstion, signal-to-noise ratio remains acceptable for an error free base-calling (bottom). This shows major improvement compared to simulation result obtained for the four-enzyme system.