System and method for adaptive reagent control in nucleic acid sequencing

• US 20090053724A1
• Filed: 06/27/2008
• Published: 02/26/2009
• Est. Priority Date: 06/28/2007
• Status: Abandoned Application

• Current Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Nobile, John Richard, Chen, Zhoutao, Gomes, Xavier Victor, Nealis, James Matthew, Srinivasan, Maithreyan, Roth, George Thomas
• Application Number:
  US12/215,455
• Publication Number:
  US 20090053724A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)

Abstract

An embodiment of a method for adaptive reagent control is described that comprising a) introducing a first concentration of an enzyme reagent into a reaction environment with a reaction substrate, where the enzyme reagent and reaction substrate are constituent parts of a sequencing process; b) measuring a level of activity of the first concentration of the enzyme reagent in the reaction environment, where the level of activity comprises a measurable product of a reaction between the enzyme reagent and the reaction substrate; c) identifying an optimal concentration using the measured level of activity of the first concentration; and d) performing the sequencing process in the reaction environment using the optimal concentration of the enzyme reagent, where the sequencing process comprises an iterative series of sequencing reactions.

Claims

1. A method for adaptive reagent control, comprising:

a) introducing a first concentration of an enzyme reagent into a reaction environment with a reaction substrate, wherein the enzyme reagent and reaction substrate are constituent parts of a sequencing process;

b) measuring a level of activity of the first concentration of the enzyme reagent in the reaction environment, wherein the level of activity comprises a measurable product of a reaction between the enzyme reagent and the reaction substrate;

c) identifying an optimal concentration using the measured level of activity of the first concentration; and

d) performing the sequencing process in the reaction environment using the optimal concentration of the enzyme reagent, wherein the sequencing process comprises an iterative series of sequencing reactions.

2. The method of claim 1, further comprising:

before step d), repeating steps a) and b) using the optimal concentration as the first concentration; and

verifying the optimal concentration of the enzyme reagent using the measured level of activity.

3. The method of claim 1, further comprising:

introducing a second concentration and a third concentration of the enzyme reagent into the reaction environment with the reaction substrate;

measuring a level of activity of the second and third concentrations of the enzyme reagent in the reaction environment; and

identifying the optimal concentration using the measured level of activity of the first, second and third concentrations.

4. The method of claim 3, wherein:

measuring the level of activity for each of the first, second, and third concentrations two or more times, wherein the optimal concentration is identified using an average of the two or more measured levels of activity at each of the first, second and third concentrations.

5. The method of claim 1, further comprising:

e) repeating steps a)-c) one or more times during the sequencing process.

6. The method of claim 1, wherein:

the enzyme reagent comprises apyrase and the reaction substrate comprises ATP, wherein the ATP is introduced into the reaction environment with the apyrase.

7. The method of claim 6, wherein:

the iterative series of sequencing reactions are performed with a template nucleic acid, and the optimal concentration of the apyrase is introduced into the reaction environment prior to a subsequent iteration of sequencing reaction to reduce an introduced error in sequence composition of the template nucleic acid generated in the subsequent iteration.

8. The method of claim 7, wherein:

the introduced error comprises a carry forward error when a concentration of the apyrase in the reaction environment is lower than the optimal concentration.

9. The method of claim 7, wherein:

the introduced error comprises an incomplete extension error when a concentration of the apyrase in the reaction environment is higher than the optimal concentration.

10. The method of claim 7, wherein:

the introduced error comprises a low reaction product from the sequencing reaction when a concentration of the apyrase in the reaction environment is higher than the optimal concentration.

11. The method of claim 1, wherein:

the enzyme reagent comprises PPi-ase and the reaction substrate comprises PPi, wherein the PPi is introduced into the reaction environment with the PPi-ase.

12. The method of claim 1, wherein:

the enzyme reagent comprises apyrase and the reaction substrate comprises PPi, wherein the PPi is introduced into the reaction environment with the apyrase.

13. The method of claim 1, wherein:

the enzyme reagent is sensitive to an environmental condition, wherein the environmental condition changes the level of activity of the enzyme reagent.

14. The method of claim 13, wherein:

the environmental condition comprises PH or temperature.

15. The method of claim 1, wherein:

the measurable product comprises light emitted from the reaction.

16. The method of claim 1, wherein:

the sequencing reactions produce a plurality of reaction products that comprise the reaction substrate.

17. The method of claim 1, wherein:

the enzyme reagent degrades the reaction substrate.

18. The method of claim 1, wherein:

the reaction environment comprises a flow cell.

19. The method of claim 1, wherein:

the reaction environment comprises a well of a plate.

20. The method of claim 19, wherein:

the plate comprises a fiber optic faceplate comprising an array of the wells.

21. A nucleic acid sequencing system, comprising:

a flow cell that comprises a reaction environment for performing a sequencing process comprising an iterative series of sequencing reactions;

a valve that introduces a first concentration of an enzyme reagent into a reaction environment with a reaction substrate, wherein the enzyme reagent and reaction substrate are constituent parts of the sequencing process;

a detector that measures a level of activity of the first concentration of the enzyme reagent in the reaction environment, wherein the level of activity comprises a measurable product of a reaction between the enzyme reagent and the reaction substrate;

wherein in response to the measured level of activity the valve provides an optimal concentration of the enzyme reagent into the reaction environment.

22. The system of claim 21, further comprising:

a computer having executable code stored thereon, wherein the executable code performs the steps of;

providing instructional control for the valve to introduce the first concentration of the enzyme reagent and the reaction substrate into the reaction environment;

receiving the measured level of activity of the first concentration from the detector;

identifying an optimal concentration using the measured level of activity of the first concentration; and

providing instructional control for the valve to provide the optimal concentration.

23. The system of claim 22, wherein:

the instructional control is provided to a microcontroller that controls timing functions of the valve.

24. The system of claim 23, wherein:

the timing functions of the valve include control of a pulse width.

25. The system of claim 21, wherein:

the valve is a multiport valve.

26. The system of claim 21, further comprising:

a first fluid reservoir comprising a stock solution of the enzyme reagent and a second fluid reservoir comprising a stock solution of the reaction substrate, wherein the valve introduces the first concentration of the enzyme reagent and the reaction substrate into the reaction environment via the first and second fluid reservoirs.

27. The system of claim 26, wherein:

the valve introduces the optimal concentration via the first fluid reservoir.

28. The system of claim 21, wherein:

the detector is a CCD detector.

29. The system of claim 21, wherein:

the enzyme reagent comprises apyrase and the reaction substrate comprises ATP, wherein the ATP is introduced into the reaction environment with the apyrase.

30. The system of claim 29, wherein:

the valve provides the optimal concentration of the apyrase in each subsequent iteration of sequencing reaction after a first iteration to reduce an introduced error in a sequence composition generated in the subsequent iterations

31. The system of claim 30, wherein:

the introduced error comprises a carry forward error when a concentration of the apyrase in the reaction environment is lower than the optimal concentration.

32. The system of claim 30, wherein:

the introduced error comprises an incomplete extension error when a concentration of the apyrase in the reaction environment is higher than the optimal concentration.

33. The system of claim 30, wherein:

the introduced error comprises a low reaction product from the sequencing reaction when a concentration of the apyrase in the reaction environment is higher than the optimal concentration.

34. The system of claim 21, wherein:

the enzyme reagent is sensitive to an environmental condition, wherein the environmental condition changes the level of activity of the enzyme reagent.

35. The system of claim 34, wherein:

the environmental condition comprises PH or temperature.

36. The system of claim 21, wherein:

the measurable product comprises light emitted from the reaction.

37. The system of claim 21, wherein:

the sequencing reactions produce a plurality of reaction products that comprise the reaction substrate.

38. The system of claim 21, wherein:

the enzyme reagent degrades the reaction substrate.

39. The system of claim 21, wherein:

the reaction environment comprises a well of a plate.

40. The system of claim 39, wherein:

the plate comprises a fiber optic faceplate comprising an array of the wells.