In order to determine factors limiting read-length in Pyrosequencing, we investigated the effect of product accumulation for each enzymatic reaction by varying reagent concentrations and studying sequence signal peak responses. Product accumulation decreases the catalytic efficiency of the enzymatic reactions, thereby limiting Pyrosequencing read-length. The cascade of reactions is initiated by DNA polymerase, when a nucleotide complementary to the target strand is incorporated. No major factors inhibiting the DNA polymerase reaction have been observed when using natural nucleotides or the efficiently incorporated isomers of α-thio dATP, which is used in Pyrosequencing.
ATP sulfurylase inhibition by byproduct accumulation was investigated by adding different amounts of the substrates PPi and APS to the Pyrosequencing reaction mixtures. The sequence signals from these experiments were compared with standard Pyrosequencing. Five hundred, 1000, and 2000 pmol quantities of PPi and APS were added to three different reaction wells. As shown in , signal intensities decrease by 20, 30, and 52 percent, respectively.
Effect of product inhibition on ATP sulfurylase
In similar experiments as described above, amounts of 500, 1000, and 2000 pmol of AMP were added to Pyrosequencing reaction mixtures, and the light signals observed were compared to a standard control. demonstrates the effects of adding these quantities of AMP to the reaction mixtures. As shown, addition of AMP over this concentration range has a minimal effect on sequence signals. The higher signal intensities shown in over the standard control were found to be due to instrument error, rather than the effect of AMP inhibition. This experiment was performed five times (data not shown), and the peak heights were not observed to have any inhibition correlation with AMP. Therefore, the results suggest that AMP is not a limiting factor in Pyrosequencing read length.
Effect of AMP inhibition on luciferase
To investigate the effects of possible oxyluciferin inhibition on luciferase, the same experiments as above were carried out but instead 500, 1000, and 2000 pmol of ATP were added to different reaction mixtures. The results of this experiment are presented in . Compared to the control signal (), signal peaks were decreased by 5, 10, and 22%, respectively, when 500, 1000, and 2000 pmol of ATP were added.
Effect of oxyluciferin inhibition on luciferase
To study the effect of byproduct accumulation on apyrase inhibition, signal quality was observed following the addition of varying nucleotide concentrations. The Pyrosequencer PSQ™96MA dispenses 0.2 μl, or approximately 100 pmoles, of a given dNTP per cycle. In this experiment, five standard Pyrosequencing reactions were performed in parallel. The first sequence reaction contained DNA template as control, while the other four reaction mixtures did not contain any DNA template. Iterative nucleotide dispensations of 10, 20, 40, and 80 cycles were performed on wells two, three, four, and five, respectively. Subsequently, DNA template was added to each well. The effect of accumulated byproducts on apyrase activity was studied by observing the baseline broadness of signal peaks (the wider the baseline, the more inhibition) (). illustrates signals from the standard Pyrosequencing reaction. Arrows highlight positions where nucleotide inhibition of apyrase can be observed. As shown, an increase in the number of cycles of nucleotide dispensations causes increasing byproduct inhibition of apyrase catalytic activity ().
Effect of product inhibition on apyrase
Another potential factor limiting read-length is nucleotide misincorporation by DNA polymerase. To test the misincorporation rate of Klenow DNA polymerase, two reactions containing all standard Pyrosequencing reagents and enzymes except apyrase were examined. In one reaction well (), a mismatched nucleotide was dispensed. After 20 minutes of observation, the correct nucleotide was dispensed to both solutions. The misincorporation rate for dGTP was calculated to be 0.17/1200 seconds () by comparing the height of the signal for correct incorporation and misincorporation within the same pyrogram.
Effect of misincorporation by DNA polymerase on Pyrosequencing signals
To measure the effects of dilution and evaporation, 100 dispensations of 0.2 μl of water were made in 10 different wells each of which initially contained 50 μl of water. Afterwards, the average volume in the wells was measured to be 54.0 μl ± 0.5 μl. The volume of water in each well after 100 dispensations should have been 70 μl. This indicates that 22% (16 μl) of the reaction mixture evaporates over a period of 100 minutes. In other words, on average, the sequencing reaction mixture is being diluted 0.07% (or 0.04 μl) after each nucleotide dispensation, and this dilution phenomenon will gradually affect the concentration balance of enzymes and reagents in the Pyrosequencing reaction.
Based on the obtained results from the above-mentioned experiments, the following reactions were added to the previously described Pyrosequencing model (25
) to represent the inhibition effects of byproduct accumulation in Pyrosequencing reactions.
- polymerase.DNA + dNMP ↔ polymerase.DNA.dNMP
- apyrase + dNMP ↔ apyrase.dNMP
Furthermore, the effect of dilution on each enzyme after each cycle was incorporated into the proposed simulation model.
demonstrates the result of simulating the final four-enzyme model for 150 nucleotide dispensations. After approximately 80 dispensations, the signal-to-noise ratio decreases until it becomes more difficult to distinguish signals and noise as well as single, double, and triple base signal peaks. If only the sequencing data from the first 80 nucleotide dispensations are considered, an accurate base-calling of about 60 bases can be obtained. This result is in alignment with previously reported experimental results (22
). However, more promising results were obtained from simulating the three-enzyme Pyrosequencing reaction (see below).
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.
Next, we examined the effects of manual washing for nucleotide removal, as opposed to enzymatic degradation. To study the washing efficiency in the three-enzyme system, 100% and 90% washing efficiencies were performed in 200 nucleotide dispensations. demonstrate that even 90% washing efficiency is able to generate sequencing reads of more than 400 nucleotides. presents the simulation results of the 90% washing efficiency system for 300 nucleotide dispensation. Noise in the lower panel of remains insignificant even after 250 nucleotide dispensations. Three-enzyme Pyrosequencing with 99% and 99.9% washing efficiencies produced very similar data to 100% washing efficiency (data not shown). It is worth noting that the decrease in signal intensity (evident in the top portion of ) results from the assumption that 0.1% of DNA fragments are lost during each washing cycle. These simulation results point to the ability of the three-enzyme system to generate much longer read lengths.
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 (more ...)
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 (more ...)