Methods and compositions for base calling nucleic acids

• US 8,612,161 B2
• Filed: 03/17/2009
• Issued: 12/17/2013
• Est. Priority Date: 03/19/2008
• Status: Active Grant

• Current Assignee: QIAGEN Sciences, LLC (Qiagen NV)
• Original Assignee: Intelligent Bio-Systems Incorporated. (Qiagen NV)
• Inventors: Gordon, Steven, Veatch, Phillip A.
• Primary Examiner(s): Lin, Jerry
• Application Number:
  US12/405,694
• Publication Number:
  US 20100063743A1
• Time in Patent Office: 1,736 Days
• Field of Search: 702/20
• US Class Current: 702/20
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2537/165 Mathematical modelling, e.g. logarithm, ratio
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression

Abstract

The invention provides methods and compositions, including, without limitation, algorithms, computer readable media, computer programs, apparatus, and systems for determining the identity of nucleic acids in nucleotide sequences using, for example, data obtained from sequencing by synthesis methods. The methods of the invention include correcting one or more phenomena that are encountered during nucleotide sequencing, such as using sequencing by synthesis methods. These phenomena include, without limitation, sequence lead, sequence lag, spectral crosstalk, and noise resulting from variations in illumination and/or filter responses.

Claims

1. A method for determining an identity of a nucleic acid at an interrogation position in a nucleotide sequence from data acquired from one or more channels in parallel, comprising:

a) sequencing a nucleic acid by DNA sequencing by synthesis utilizing nucleotides with fluorescent dyes which are incorporated into a complementary strand,

b) obtaining a data set for one or more dye intensities at one or more nucleic acid positions in said complementary sequence, wherein each dye corresponds to a nucleotide,

c) determining the ratio contribution to dye intensity at said interrogation position from dye intensities at the interrogation position and at one or both of

i) at least one subsequent nucleic acid positions in said complementary sequence, and

ii) at least one preceding nucleic acid positions in said complementary sequence, and

d) applying said ratio contribution to said data set to arrive at an identity for a nucleotide at said interrogation position in said nucleotide sequence.

2. The method of claim 1, wherein said determining the ratio contribution to dye intensity comprises measuring the rate at which a lag occurs at one or more nucleotide position in said nucleotide sequence.

3. The method of claim 2, wherein said determining the ratio contribution to dye intensity comprises measuring the rate at which a lag occurs at each nucleotide position in said nucleotide sequence.

4. The method of claim 1, wherein said determining the ratio contribution to dye intensity comprises measuring the rate at which a lead occurs at one or more nucleotide positions in said nucleotide sequence.

5. The method of claim 1, further comprising calling a nucleic acid base at said interrogation position in said nucleotide sequence.

6. The method of claim 1, further comprising repeating steps c) and d) to arrive at an identity for a nucleic acid at more than one interrogation position in said nucleotide sequence.

7. The method of claim 1, further comprising

a) applying a sequence lead-lag compensation equation to determine the ratio contribution to intensity from a dye at

i) said interrogation position,

ii) each position preceding said interrogation position, and

iii) each position subsequent to said interrogation position, and

b) summing up said ratio contribution to dye intensity.

8. The method of claim 1, further comprising applying a sequence lead-lag compensation equation to said ratio contribution to dye intensity at a plurality of positions in said sequence.

9. The method of claim 8, wherein said sequence lead-lag compensation equation is determined by applying equation

10. The method of claim 8, wherein said sequence lead-lag compensation equation is determined by applying equation

11. The method of claim 10, wherein said sequence lead-lag compensation equation is determined by applying equation

12. The method of claim 1, wherein the nucleic acid comprises a base selected from the group consisting of adenine (A), guanine (G), cytosine (C), and thymine (T).

13. The method of claim 1, further comprising field flattening of background data for said data set.

14. The method of claim 13, wherein said field flattening comprises

a) obtaining a first data set for a plurality of pixel intensities of a first raw image of a dye at a first concentration on a solid support, wherein said first raw image is produced using a first spectral filter for detecting a first dye,

b) obtaining a second data set for a plurality of pixel intensities of a second smoothed image of said dye on said solid support, wherein said second smoothed image is produced using a low pass filter,

c) determining a field flattening intensity value for a plurality of pixels of said first raw image, and

d) generating a third field flattened image of said dye on said solid support using said field flattening intensity of said plurality of pixels, wherein the correlation of intensity of a plurality of pixels to their spatial location on said third field flattened image is reduced compared to the intensity of a plurality of pixels at a corresponding location on said first raw image.

15. The method of claim 14, wherein said field flattening intensity value of a pixel is determined by equation

F_x,y=R_x,yM_x0,y0/M_x,y

where

F_x,y is a field flattening intensity value,

R_x,y is the intensity of a pixel of the plurality of pixels on the first raw image,

M_x,y is the intensity of a pixel of the plurality of pixels on the second smoothed image at a corresponding spatial location to the pixel on the first raw image, and

M_x0,y0 is the intensity of a reference pixel on said second smoothed image or is an arbitrary scale factor.

16. The method of claim 14, further comprising repeating steps a) to d), using a second spectral filter for detecting a second dye.

17. The method of claim 14, further comprising repeating steps a) to d), using said dye at a second concentration on said solid support.

18. The method of claim 13, wherein said solid support comprises a microscope slide.

19. The method of claim 13, wherein said solid support comprises a silicon chip.

20. The method of claim 1, further comprising reducing spectral crosstalk in said one or more channels.

21. The method of claim 20, wherein said reducing spectral crosstalk comprises

a) determining spectral crosstalk factors for each of said one or more dyes in its corresponding channel from one or more adjacent channels,

b) applying said spectral crosstalk factors to determine a spectral crosstalk matrix, and

c) applying said spectral crosstalk matrix to said data set for said one or more dye intensities.

22. The method of claim 20, wherein said reducing spectral crosstalk comprises

a) determining intensities for one or more dyes from one or more channels, wherein each channel corresponds to a dye;

b) determining the ratios of said intensities in said one or more channels to arrive at a signature ratio for said dye intensity in its corresponding channel,

c) applying said signature ratios in a matrix equation, and

d) inverting said matrix equation to arrive at an inverted matrix.

23. The method of claim 22, further comprising e) applying said inverted matrix to data from said one or more channels.

24. The method of claim 21, wherein said determining spectral crosstalk matrix comprises using equation

25. The method of claim 24, wherein said equation is solved to determine spectral crosstalk matrix K⁻

1 using equation

26. A method for processing data for nucleic acids in a nucleotide sequence, wherein said data is acquired from one or more channels in parallel, the method comprising:

a) sequencing a nucleic acid by DNA sequencing by synthesis utilizing nucleotides with fluorescent dyes which are incorporated into a complementary strand,

b) determining the ratio contribution to intensity in said one or more channels for one or more interrogation positions, from dye intensities at the interrogation position and at one or both of

i) at least one subsequent nucleic acid positions in said complementary sequence, and

ii) at least one preceding nucleic acid positions in said complementary sequence,

c) processing data from said one or more channels to correct for sequence lead and sequence lag, and

d) reconstructing said data in said one or more channels.

27. The method of claim 26, wherein said processing data comprises applying said ratio contribution to intensity to determine, for said dye at said one or more interrogation positions, a sequence lead-lag compensation equation.

28. The method of claim 27, wherein said sequence lead-lag compensation equation is determined by applying equation

29. The method of claim 26, wherein said sequence lead-lag compensation equation is determined by applying equation

30. The method of claim 29, wherein said sequence lead-lag compensation equation is determined by applying equation

31. The method of claim 26, further comprising field flattening of background data.

32. The method of claim 26, further comprising reducing spectral crosstalk between said data comprised in a plurality of channels.

33. An apparatus that processes data for nucleic acids in a nucleotide sequence to determine an identity of a nucleic acid at an interrogation position in said nucleotide sequence in parallel DNA sequencing by synthesis utilizing nucleotides with fluorescent dyes, said apparatus comprising

a) means for inputting data from one or more channels for one or more intensities, wherein each channel corresponds to a dye, and each dye corresponds to a nucleic acid base,

b) means for applying to the input data a sequence lead-lag compensation equation to correct for sequence lead and sequence lag,

c) means for comparing intensities in said one or more channels that have been corrected for sequence lead and sequence lag,

d) means for determining the highest intensity of the compared dye intensities, and

e) means for identifying a nucleic acid base at said interrogation position according to said highest intensity.

34. The apparatus of claim 33, further comprising f) means for applying field flattening of background data.

35. The apparatus of claim 33, further comprising f) means for reducing spectral crosstalk between data comprised in said one or more channels.

36. A system for processing data to determine an identity of a nucleic acid at an interrogation position in a nucleotide sequence in parallel DNA sequencing by synthesis utilizing fluorescent nucleotides, said system comprising

a) a processor, and

b) a computer readable medium readable by said processor, said computer readable medium storing a computer program that comprisescode that receives as input a plurality of nucleic acid base calls at an interrogation position in a nucleotide sequence,

code that applies to the input data a sequence lead-lag compensation equation to correct for sequence lead and sequence lag, andcode that identifies a nucleic acid at said interrogation position according to the corrected data.

37. The system of claim 36, wherein said computer readable medium further comprises 4) code that applies field flattening of background data.

38. The system of claim 36, said computer readable medium further comprises 4) code that reduces spectral crosstalk between data comprised in said one or more channels.