Data analysis and display system for ligation-based DNA sequencing

• US 20030224419A1
• Filed: 04/02/2003
• Published: 12/04/2003
• Est. Priority Date: 05/23/1997
• Status: Abandoned Application

• Current Assignee: Solexa Incorporated (Illumina Incorporated)
• Original Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Inventors: Corcoran, Kevin C., Eletr, Sam
• Application Number:
  US10/407,089
• Publication Number:
  US 20030224419A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/107 Maxam and Gilbert method, i.e. sequential release and detection of nucleotides
• CPC Class Codes: G16B 45/00 ICT specially adapted for bioinformatics-related data visualisation, e.g. displaying of maps or networks

Abstract

A data analysis system for ligation-based sequencing is provided. The system includes a base calling algorithm that may be implemented with a program of instructions (e.g., software) for determining a signature of a nucleotide sequence. A graphical user interface (GUI) enables a user to interact with and control various aspects of the base calling algorithm. The base calling algorithm and associated software may be used in connection with a technique that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 μm diameter microbeads.

Claims

1. A method for determining a signature of a nucleotide sequence, comprising:

(a) obtaining optical measurements having values ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4 indicative of each nucleotide in each of a j^th group of nucleotide positions i, for i equal 1 through k and for j equal 1 through m;

(b) for every group of nucleotide positions from j equal 2 through m, and every position from i equal 1 through k, adjusting the values ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4 by repeatedly subtracting from each a first predetermined fraction of <sup>j−

1</sup>v_i1, <sup>j−

1</sup>v_i2, <sup>j−

1</sup>v_i3, and <sup>j−

1</sup>v_i4, respectively, until the ratio of the highest value in the set of ^jv_i1 through ^jv_i4, to the next highest value in the same set is greater than or equal to a predetermined factor, or until the repeatedly subtracted fractions have a sum equal to a second predetermined fraction; and

(c) generating a base call for position i in the j^th group based on results of the adjusting in (b).

2. The method of claim 1, wherein said base call generating (c) comprises

assigning a base code corresponding to the highest value to position i in the j^th group whenever the highest value is greater than or equal to a predetermined minimum value and the ratio of the highest value in the set of ^jv_i1 through ^jv_i4, to the next highest value in the same set is greater than or equal to the predetermined factor, and

assigning a two-base ambiguity code corresponding to the highest value and the next highest value whenever the ratio is less than the predetermined factor and the highest value and the next highest value are each greater than or equal to the predetermined minimum value.

3. The method of claim 2, further comprising rejecting the signature whenever the number of ambiguity codes assigned is greater than one.

4. The method of claim 2, wherein said obtaining (a) comprises adjusting values ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4, for i equal 1 through k and for j equal 1 through m, for background noise.

5. The method of claim 4, wherein the background noise is computed as the average of the lowest three of ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4, and wherein the computed background noise is subtracted from each of ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4.

6. The method of claim 2, wherein the groups of positions, j=1 through m, are contiguous.

7. The method of claim 2, wherein m=3, 4 or 5.

8. The method of claim 7, wherein m=4.

9. The method of claim 2, wherein k=1, 2, 3, 4 or 5.

10. The method of claim 9, wherein k=2, 3 or 4.

11. The method of claim 10, wherein k=4.

12. The method of claim 2, wherein the predetermined factor is between about 2 and about 5.

13. The method of claim 4, wherein the predetermined minimum value is greater than 125% of the background noise.

14. The method of claim 2, wherein the first predetermined fraction is {fraction (1/50)}.

15. The method of claim 4, wherein the second predetermined fraction is set such that the highest value does not fall below 125% of the background noise.

16. An apparatus for determining a signature of a nucleotide sequence, comprising:

(a) a storage medium that stores a plurality of sets of digital signal values ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4 indicative of each nucleotide in each of a j^th group of nucleotide positions i, for i=1 through k and for j equal 1 through m; and

(b) a processor in communication with the storage medium to;

(i) adjust the values ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4, for every nucleotide position from i equal 1 through k in every group of nucleotide positions from j equal 2 through m, by repeatedly subtracting from each a first predetermined fraction of <sup>j−

1</sup>v_i1, <sup>j−

1</sup>v_i2, <sup>j−

1</sup>v_i3, and <sup>j−

1</sup>v_i4, respectively, until the ratio of the highest value in the set of ^jv_i1 through ^jv_i4, to the next highest value in the same set is greater than or equal to a predetermined factor, or until the repeatedly subtracted fractions have a sum equal to a second predetermined fraction, and

(ii) generate a base call for position i in the j^th group based on results of the adjusting in (i).

17. The apparatus of claim 16, wherein, to generate a base call for position i in the j^th group, the processor

assigns a base code corresponding to the highest value to position i in the j^th group whenever the highest value is greater than or equal to a predetermined minimum value and the ratio of the highest value in the set of ^jv_i1 through ^jv_i4, to the next highest value in the same set is greater than or equal to the predetermined factor, and

assigns a two-base ambiguity code corresponding to the highest value and the next highest value whenever the ratio is less than the predetermined factor and the highest value and the next highest value are each greater than or equal to the predetermined minimum value.

18. The apparatus of claim 17, further comprising a display in communication with the processor, wherein the processor renders a graphical representation of the digital signal values on the display upon user command.

19. The apparatus of claim 17, further comprising a display in communication with the processor, wherein the processor renders a graphical representation of a plurality of microbeads, each containing at least one copy of the nucleotide sequence, on the display upon user command.

20. A system for determining a signature of a nucleotide sequence, comprising:

(a) a processing and detection apparatus including an optical train operable to collect and convert a plurality of optical signals into corresponding digital signal values that comprise a plurality of sets digital signal values ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4 indicative of each nucleotide in each of a j^th group of nucleotide positions i, for i=1 through k and for j equal 1 through m;

(b) a storage medium that stores ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4; and

(c) a processor in communication with the storage medium and being operable to;

(i) adjust the values ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4, for every nucleotide position from i equal 1 through k in every group of nucleotide positions from j equal 2 through m, by repeatedly subtracting from each a first predetermined fraction of <sup>j−

1</sup>v_i1, <sup>j−

1</sup>v_i2, <sup>j−

1</sup>v_i3, and <sup>j−

1</sup>v_i4, respectively, until the ratio of the highest value in the set of ^jv_i1 through ^jv_i4, to the next highest value in the same set is greater than or equal to a predetermined factor, or until the repeatedly subtracted fractions have a sum equal to a second predetermined fraction, and

(ii) generate a base call for position i in the j^th group based on results of the adjusting in (i).

21. The system of claim 20, wherein, to generate a base call for position i in the j^th group, the processor

assigns a base code corresponding to the highest value to position i in the j^th group whenever the highest value is greater than or equal to a predetermined minimum value and the ratio of the highest value in the set of ^jv_i1 through ^jv_i4, to the next highest value in the same set is greater than or equal to the predetermined factor, and

assigns a two-base ambiguity code corresponding to the highest value and the next highest value whenever the ratio is less than the predetermined factor and the highest value and the next highest value are each greater than or equal to the predetermined minimum value.

22. The system of claim 21, further comprising a program of instructions for execution by the processor to carry out (i) and (ii).

23. The system of claim 22, wherein the program of instructions is embodied in software.

24. The system of claim 22, wherein the program of instructions is embodied in hardware formed integrally or in communication with the processor.

25. The system of claim 22, further comprising a display and a graphical user interface presented on the display for enabling a user to display and manipulate data and results.

26. The system of claim 21, further comprising a data base, in communication with the processor, for storing sequencing information.

27. The system of claim 26, further comprising a second processor in communication with the data base for performing quality control analysis on the sequence signature.

28. A processor-readable medium embodying a program of instructions for execution by a processor for performing a method of determining a signature of a nucleotide sequence, the program of instructions comprising instructions for:

(a) obtaining optical measurements having values ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4 indicative of each nucleotide in each of a j^th group of nucleotide positions i, for i equal 1 through k and for j equal 1 through m;

(b) for every group of nucleotide positions from j equal 2 through m, and every position from i equal 1 through k, adjusting the values ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4 by repeatedly subtracting from each a first predetermined fraction of <sup>j−

1</sup>v_i1, <sup>j−

1</sup>v_i2, <sup>j−

1</sup>v_i3, and <sup>j−

1</sup>v_i4, respectively, until the ratio of the highest value in the set of ^jv_i1 through ^jv_i4, to the next highest value in the same set is greater than or equal to a predetermined factor, or until the repeatedly subtracted fractions have a sum equal to a second predetermined fraction; and

(c) generating a base call for position i in the j^th group based on results of the adjusting in (b).

29. The processor-readable medium of claim 28, wherein said base call generating instructions (c) comprises instructions for

assigning a base code corresponding to the highest value to position i in the j^th group whenever the highest value is greater than or equal to a predetermined minimum value and the ratio of the highest value in the set of ^jv_i1 through ^jv_i4, to the next highest value in the same set is greater than or equal to the predetermined factor, and

assigning a two-base ambiguity code corresponding to the highest value and the next highest value whenever the ratio is less than the predetermined factor and the highest value and the next highest value are each greater than or equal to the predetermined minimum value.

30. The processor-readable medium of claim 29, further comprising instructions for rejecting the signature whenever the number of ambiguity codes assigned is greater than one.

31. The processor-readable medium of claim 29, wherein said obtaining instructions (a) comprises instructions for adjusting values ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4, for i equal 1 through k and for j equal 1 through m, for background noise.

32. The processor-readable medium of claim 31, wherein the background noise is computed as the average of the lowest three of ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4, and wherein the computed background noise is subtracted from each of ^jv_i1, ^jv_i2, ^jv_i3, and ^jv_i4.

33. The processor-readable medium of claim 29, wherein the groups of positions, j=1 through m, are contiguous.

34. The processor-readable medium of claim 29, wherein m=3, 4 or 5.

35. The processor-readable medium of claim 34, wherein m=4.

36. The processor-readable medium of claim 29, wherein k=1, 2, 3, 4 or 5.

37. The processor-readable medium of claim 36, wherein k=2, 3 or 4.

38. The processor-readable medium of claim 37, wherein k=4.

39. The processor-readable medium of claim 29, wherein the predetermined factor is between about 2 and about 5.

40. The processor-readable medium of claim 31, wherein the predetermined minimum value is greater than 125% of the background noise.

41. The processor-readable medium of claim 29, wherein the first predetermined fraction is {fraction (1/50)}.

42. The processor-readable medium of claim 31, wherein the second predetermined fraction is set such that the highest value does not fall below 125% of the background noise.

43. A graphical user interface presented on a computer for facilitating interaction between a user and a computer-implemented method of determining a signature of a nucleotide sequence, the graphical user interface comprising:

(a) a data display area for displaying one or more displays of data; and

(b) a control area for displaying one or more selectable functions including

(i) a first function which when selected causes a graphical representation of the plurality of digital signal values to be displayed in the data display area, and

(ii) a second function which when selected causes a graphical representation of a plurality of sequence-containing microbeads to be displayed in the data display area.

44. The graphical user interface of claim 43, wherein the one or more selectable functions are represented by graphical push buttons displayed in the control area of the graphical user interface.

45. A graphical user interface presented on a computer for facilitating interaction between a user and a computer-implemented method of determining a signature of a nucleotide sequence, the graphical user interface comprising:

(a) an animation mode including a first main window having (i) a display area for displaying an animated image of a sequence-containing bead array, and a first control panel for displaying one or more selectable functions associated with the animation mode;

(b) an alignment mode including a second main window for aligning shifted images to show bead movement based on a comparison with a reference image, and a second control panel for displaying one or more selectable functions associated with the alignment mode; and

(c) a bead mode including a third main window for displaying a sequence-containing bead array, and one or more selectable functions for performing one or more base calling functions.

46. A method of determining a nucleotide sequence of a polynucleotide from a series of optical measurements comprising a plurality of groups, each group containing one or more sets of four optical measurements wherein each optical measurement of a set corresponds to a different one of deoxyadenosine, deoxyguanosine, deoxycytidine, or deoxythymidine, the groups of optical measurements being produced by successively ligating to and cleaving from the end of a polynucleotide signal-generating adaptor having protruding strands, and each optical measurement having a value, and each set of optical measurements corresponding to a separate nucleotide position of the protruding strand of a signal-generating adaptor, the method comprising the steps of:

adjusting the value of the optical measurement of each set within a group by repeatedly subtracting therefrom a predetermined fraction of the value of the corresponding optical measurement of the corresponding set obtained in the previous ligation until the ratio of the highest value to the next highest value in the same set is greater than or equal to a first predetermined fraction, or until the sum of the repeatedly subtracted fractions is less than or equal to a predetermined factor; and

assigning a base code to each set based on the results of the adjusting.