Single light source, two optical channel sequencing

Abstract

A system for determining the nucleotide sequence of a polynucleotide is provided. [Solution] In one aspect, the system of the present invention includes a single light source configured to stimulate the generation of fluorescence; at least one detector configured to detect four different fluorescent emissions from different fluorophores attached to the nucleotide; a processor configured to execute instructions to perform a method including generating light on the nucleotide from the light source; and identifying the nucleotide as a first, second, third, and fourth type when first, second, third, and fourth fluorescent emissions are detected by the at least one detector, respectively, wherein the detection of the first fluorescent emission, the second fluorescent emission, the third fluorescent emission, and the fourth fluorescent emission is determined based on four different wavelengths.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 468,242, filed March 7, 2017, the contents of which are incorporated herein by reference in their entirety.

[0002] background Field The present disclosure relates generally to the field of DNA sequencing, and more particularly to systems and methods for DNA sequencing utilizing a single light source and at least two dyes (e.g., two fluorescent labels). [Background technology]

[0003] Related Field Description Existing DNA sequencing systems and methods utilize two or more light sources to excite deoxyribonucleic acid analogs conjugated with fluorescent labels. However, during operation, the power consumption of the light sources is high and they can generate a significant amount of heat that needs to be dissipated. Fluorescent labels that can be efficiently excited by one light source can be susceptible to crosstalk, whereby each label emits light at wavelengths that overlap with other labels. If not corrected, this crosstalk can make it difficult for DNA sequencing systems to properly call the correct nucleotide base during a sequencing run. Summary of the Invention [Means for solving the problem]

[0004] Abstract Disclosed herein is a system and method for determining the nucleotide sequence of polynucleotide.In one example, the system includes a single light source, such as a laser or a light-emitting diode, configured to generate light, such as light of a predetermined wavelength; at least one detector configured to detect the fluorescent emission from the fluorophore attached to nucleotide, and the at least one detector configured to detect the fluorescent emission at a first wavelength and a second wavelength; a processor, configured to execute instructions to carry out a method comprising the steps of: generating light from the light source on nucleotide; when the fluorescent emission is not detected by the at least one detector, identifying the nucleotide as a first type; when the fluorescent emission at the first wavelength of light is detected by the at least one detector, identifying the nucleotide as a second type; when the fluorescent emission at the second wavelength of light is detected by the at least one detector, identifying the nucleotide as a third type; and when the fluorescent emission at the first wavelength and the second wavelength of light is detected by the at least one detector, identifying the nucleotide as a fourth type.

[0005] Another example is a computer-implemented method comprising the steps of generating light on a fluorophore attached to a nucleotide using a light source; detecting fluorescent emissions from the fluorophore attached to the nucleotide at a first wavelength and a second wavelength using at least one detector; and identifying the nucleotide, the steps including identifying the nucleotide as a first type if no fluorescent emission is detected by the at least one detector; identifying the nucleotide as a second type if fluorescent emission at the first wavelength of light is detected by the at least one detector; identifying the nucleotide as a third type if fluorescent emission at the second wavelength of light is detected by the at least one detector; and identifying the nucleotide as a fourth type if fluorescent emission at the first wavelength and the second wavelength of light are detected by the at least one detector.

[0006] In another example, a system includes a single light source configured to generate light; at least one detector configured to detect four substantially different fluorescent emissions from different fluorophores attached to nucleotides; and a processor configured to execute instructions to perform a method comprising the following steps: generating light from the light source onto the nucleotide; identifying the nucleotide as a first type if a first fluorescent emission is detected by the at least one detector; identifying the nucleotide as a second type if a second fluorescent emission is detected by the at least one detector; identifying the nucleotide as a third type if a third fluorescent emission is detected by the at least one detector; and identifying the nucleotide as a fourth type if a fourth fluorescent emission is detected by the at least one detector, wherein the first fluorescent emission, the second fluorescent emission, the third fluorescent emission, and the fourth fluorescent emission have substantially different wavelengths. In particular embodiments, for example, the following items are provided: (Item 1) 1. A system for determining a nucleotide sequence of a polynucleotide, the system comprising: a single light source configured to stimulate the emission of fluorescence; at least one detector configured to detect fluorescent emissions from fluorophores attached to nucleotides, the at least one detector configured to detect the fluorescent emissions at a first wavelength and a second wavelength; A processor comprising the steps of: generating light from said light source onto the nucleotide; identifying the nucleotide as a first type if no fluorescent emission is detected by the at least one detector; identifying the nucleotide as a second type when a fluorescent emission at the first wavelength of light is detected by the at least one detector; identifying the nucleotide as a third type when a fluorescent emission at the second wavelength of light is detected by the at least one detector; and identifying the nucleotide as a fourth type when fluorescent emissions at the first wavelength and the second wavelength of light are detected by the at least one detector; a processor configured to execute instructions to perform a method comprising: A system including: (Item 2) 2. The system of claim 1, wherein the processor is further configured to determine an intensity of one or more of the fluorescent emissions. (Item 3) 3. The system of claim 2, wherein the processor is further configured to determine the intensity of one or more of the fluorescent emissions by color correcting the intensities. (Item 4) 4. The system of claim 3, wherein the step of color correcting the intensities comprises estimating a color matrix. (Item 5) The step of estimating a color matrix comprises: generating an area-weighted angular histogram from a scatter plot of the intensities observed in the two channels; and estimating the angles of two outer maxima θ1 and θ2 in the area-weighted angle histogram, where the color matrix is

number

[0007] [Figure 1] Figure 1 is a schematic diagram illustrating an exemplary single light source-two optical channel sequencer.

[0008] [Diagram 2] Figure 2 shows a functional block diagram of an exemplary computer system for performing single light source-two optical channel sequencing.

[0009] [Diagram 3] Figure 3 is a flow chart of an exemplary method for sequencing-while-synthesizing utilizing a single light source and two optical channel sequencing.

[0010] [Figure 4] Figure 4 is a flow chart of an exemplary method for performing single light source-two optical channel sequencing base calling.

[0011] [Diagram 5] FIG. 5 is a flow chart of an exemplary method for performing single light source-two optical channel sequencing.

[0012] [Figure 6] Figure 6 shows an overview of nucleic acid clusters and their sequencing using a single light source-two optical channel sequencing.

[0013] [Figure 7] Figure 7A-D are schematic plots showing chromatic and phase correction for single light source-two optical channel sequencing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify similar components unless the context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It is readily understood that aspects of the present disclosure, as generally described herein and illustrated in the drawings, may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein.

[0015] An embodiment of the present invention relates to a next-generation nucleotide sequencing system that can distinguish all four nucleotide bases using a single light source and only two different optical channels. The sequencing system can utilize a sequencing-while-synthesis process. During each sequencing cycle, four types of nucleotide analogs can be incorporated onto a growing primer hybridized to the polynucleotide being sequenced. In some embodiments, the four types of nucleotide analogs can include a deoxyguanosine triphosphate triphosphate (dGTP) analog not conjugated with any fluorescent dye, a deoxythymidine triphosphate (dTTP) analog conjugated with a first fluorescent dye, a deoxycytidine triphosphate (dCTP) analog conjugated with a second fluorescent dye, and a deoxyadenosine triphosphate (dATP) analog conjugated with both fluorescent dyes (or a mixture of two dATP analogs, one dATP with its first fluorescent dye and the other dATP analog with its second fluorescent dye). The fluorescent dyes conjugated to the four types of nucleotide analogs are intended to be illustrative and not limiting. For example, the dTTP analog may not be conjugated to any fluorescent dye, the dCTP analog may be conjugated to a first fluorescent dye, the dATP analog may be conjugated to a second fluorescent dye, and the dGTP analog may be conjugated to both of the fluorescent dyes (or a mixture of two dGTP analogs, one dGTP analog with a first fluorescent dye and the other dGTP analog with a second fluorescent dye). As another example, the dCTP analog may not be conjugated to any fluorescent dye, the dATP analog may be conjugated to a first fluorescent dye, the dTTP analog may be conjugated to a second fluorescent dye, and the dGTP analog may be conjugated to both of the fluorescent dyes (or a mixture of two dGTP analogs, one dGTP analog with a first fluorescent dye and the other dGTP analog with a second fluorescent dye).As yet another example, the nucleotide analog not conjugated with any fluorescent dye can be dGTP, dTTP, dCTP, or dATP. The nucleotide analog conjugated with the first fluorescent dye or the second fluorescent dye can be dGTP, dTTP, dCTP, or dATP. The nucleotide analog conjugated with two fluorescent dyes can be dGTP, dTTP, dCTP, or dATP. The dGTP, dTTP, dCTP, or dATP analog can include a mixture of two analogs, one with a first fluorescent dye and the other with a second fluorescent dye.

[0016] The light source (e.g., laser or light-emitting diode) can excite the two fluorescent dyes. The first fluorescent dye can fluoresce at a first wavelength and be captured in a first fluorescent image. The second fluorescent dye can fluoresce at a second wavelength and be captured in a second fluorescent image. The captured fluorescent emission intensity is extracted from the two fluorescent images. In some embodiments, the two fluorescent dyes may be subject to crosstalk, and the fluorescent emission of the dTTP analog and the dCTP analog may be captured in both of the fluorescent images. Thus, the extracted intensity needs to be corrected, for example, by color correction. In some embodiments, the two fluorescent dyes may have a large Stokes shift, and the fluorescent emission may have a minimal Stokes shift or no crosstalk.

[0017] In some embodiments, one of the two fluorescent dyes can be a normal Stokes shift dye and the other of the fluorescent dyes can be a long Stokes shift dye. Non-limiting examples of normal Stokes shift dyes include Alexa 488 or its dye analogs (e.g., 3,6-bis(ethylamine)-2,7-dimethyl-[2-carboxylato-5-(3-carboxypropyloxy)phenyl]quinanthylium betaine (dye I-3) and 3,6-bis(ethylamine)-2,7-dimethyl-[2-carboxylato-4-(3-carboxypropyloxy)phenyl]quinanthylium betaine (dye I-4) disclosed in U.S. Pat. No. 8,754,244, the contents of which are incorporated herein by reference in their entirety. The normal Stokes shift dye is excited with a laser or light-emitting diode (LED) light source having a wavelength of 488 nm. and may have an emission peak at 520 nm. The long Stokes shift dye may be the dye NR520LS in PCT Patent Application No. PCT / GB2016 / 051474, the contents of which are incorporated by reference in their entirety. The long Stokes shift dye may have an emission peak at 590 nm. In some embodiments, the two fluorescent dyes may be Cy3 (with an emission peak at around 575 nm) and the fluorescence resonance energy transfer (FRET) pair dye Cy3-Cy5 (with an emission peak at 670 nm).

[0018] Color correction may utilize a color matrix to adjust the extracted intensities by taking advantage of characteristics of the underlying intensity distribution in each fluorescent image. The color matrix may apply the extracted intensities from the first fluorescent image to the positions (x i , y i ) against the extracted intensity from the corresponding position in the second fluorescent image. i and y i denote the intensity extracted from position i of the growing primer-polynucleotide in the second and first fluorescent images, respectively. i , y iThe plotted intensity in polar coordinates (r i , θ i ) and the angle θ i A region-weighted histogram of is calculated. Two maxima in the region-weighted histogram, θ1 and θ2, may be used to infer the color matrix. The color matrix may be:

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[0019] The inverse of that color matrix is i , y i ) the base of the incorporated nucleotide can be determined. For example, if no fluorescence emission is detected, the incorporated nucleotide can be a dGTP analog. If fluorescence emission is detected in the second fluorescence image and not in the first fluorescence image, the incorporated nucleotide can be a dTTP analog. If fluorescence emission is detected in the first fluorescence image and not in the second fluorescence image, the incorporated nucleotide can be a dCTP analog. If fluorescence emission is detected in both fluorescence images, the incorporated nucleotide can be a dATP analog.

[0020] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd Edition, J. Wiley & Sons (New York, NY 1994);Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989).For the purposes of this disclosure, the following terms are defined below.

[0021] Single Light Source - Two Optical Channel Sequencing Machine Disclosed herein are systems and methods for determining the nucleotide sequence of a polynucleotide using a single light source (e.g., a laser or LED). In one embodiment, at least two dyes are used to sequence the polynucleotide. FIG. 1 is a schematic diagram illustrating an exemplary single light source-two optical channel sequencing system 100. The single light source-two optical channel sequencing system 100 may be configured to utilize a sequencing method based on two dyes, e.g., a first fluorescent label and a second fluorescent label. Non-limiting examples of the sequencing methods utilized may include sequential synthesis sequencing and Heliscope single molecule sequencing. The single light source-two optical channel sequencing system 100 may include an optical system 102 configured to generate raw sequencing data using sequencing reagents provided by a fluidic system 104 that is part of the single light source-two optical channel sequencing system 100. The raw sequencing data may include a fluorescent image captured by the optical system 102. A computer system 106 that is part of the single light source, two optical channel sequencing system 100 can be configured to control the optical system 102 and the fluidic system 104 via communication channels 108A and 108B. For example, the computer interface 110 of the optical system 102 can be configured to communicate with the computer system 106 through communication channel 108A.

[0022] During the sequencing reaction, the fluidic system 104 may direct the flow of reagents through one or more reagent tubes 112 to and from a flow cell 114 disposed on a mounting stage 116. The reagents may be, for example, fluorescently labeled nucleotides, buffers, enzymes, and cleavage reagents. The flow cell 114 may include at least one fluidic channel. The flow cell 114 may be a patterned array flow cell or a random array flow cell. The flow cell 114 may include multiple clusters of single-stranded polynucleotides to be sequenced in at least one fluidic channel. The length of the polynucleotides may vary, for example, ranging from 200 bases to 1000 bases. The polynucleotides may be attached to one or more fluidic channels of the flow cell 114. In some embodiments, the flow cell 114 may include multiple beads, where each bead may include multiple copies of the polynucleotide to be sequenced. The mounting stage 116 can be configured to allow for proper alignment and movement of the flow cell 114 in relation to other components of the optical system 102. In one embodiment, the mounting stage 116 can be used to align the flow cell 114 with the lens 118.

[0023] The optical system 102 may include a single light source 120, such as a single laser or a single LED source configured to generate light at a predetermined wavelength (e.g., 532 nm). The light generated by the light source 120 may pass through a fiber optic cable 122 to excite fluorescent labels in the flow cell 114. A lens 118 (mounted on a focuser 124) may move along the z-axis. The focused fluorescent emission may be detected by a detector 126, such as a charge-coupled device (CCD) sensor or a complementary metal-oxide semiconductor (CMOS) sensor.

[0024] The filter assembly 128 of the optical system 102 may be configured to filter the fluorescent emission of the fluorescent label in the flow cell 114. The filter assembly 128 may include a first filter and a second filter. Each filter may be a long pass filter, a short pass filter, or a band pass filter depending on the type of fluorescent molecule being used in the system. The first filter may be configured to detect the fluorescent emission of the first fluorescent label by the detector 126. The second filter may be configured to detect the fluorescent emission of the second fluorescent label by the detector 126. With two filters in the filter assembly 128, the detector 126 may detect two different wavelengths of light. The two wavelengths of light may be from the same fluorescent label or from different fluorescent labels. The two wavelengths of light may be separated by, for example, at least 20 nm.

[0025] In some embodiments, the optical system 102 may include a dichroic element configured to split the fluorescent emission. The optical system 102 may include two detectors, a first detector coupled with a first filter for detecting the fluorescent emission at a first wavelength, and a second detector coupled with a second filter for detecting the fluorescent emission at a second wavelength. After splitting the fluorescent emission with the dichroic element, the optical system 102 may detect the fluorescent emission at two wavelengths simultaneously (or closely spaced in time) using the two detectors coupled with different filters. The configuration may accelerate the imaging process. Thus, multiple flow cells may be processed simultaneously, with one flow cell undergoing imaging while nucleotide analogs are incorporated into polynucleotide clusters in one or more other flow cells.

[0026] In use, a sample having polynucleotides to be sequenced is loaded into the flow cell 114 and placed on the mounting stage 116. The computer system 106 then operates the fluidics system 104 to initiate a sequencing cycle. During the sequencing reaction, the computer system 106 commands the fluidics system 104 through the communication interface 108B to deliver reagents (e.g., nucleotide analogs) to the flow cell 114. Through the communication interface 108A and the computer interface 110, the computer system 106 controls the light source 120 of the optical system 102 to generate light at a predetermined wavelength and configured to shine on the nucleotide analogs incorporated into the growing primer that is hybridized to the polynucleotide being sequenced. The computer system 106 controls the detector 126 of the optical system 102 to capture the emission spectrum of the nucleotide analogs in a fluorescent image. The computer system 106 receives the fluorescent image from the detector 126 and processes the received fluorescent image to determine the nucleotide sequence of the polynucleotide being sequenced.

[0027] Light Sources and Filters The single light source, two optical channel sequencing system 100 may utilize a light source (e.g., a laser or LED) capable of exciting two fluorescent labels with sufficiently non-overlapping emission spectra. The wavelength of the light generated by the light source 120 may range, for example, from 400 nm to 800 nm. In some embodiments, the wavelength of the light generated by the light source 120 can be or may be about 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a value or range between any two of these values. In some embodiments, the wavelength of the light generated by the light source 120 can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm.

[0028] Detector 126, along with filter assembly 128, may be configured to detect light at or about two different wavelengths (e.g., a first wavelength and a second wavelength). The first wavelength and the second wavelength may be separated from each other, for example, ranging from 10 nm to 100 nm. In some embodiments, the first wavelength and the second wavelength may be separated by or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 nm, or a value or range between any two of these values. In some embodiments, the first wavelength and the second wavelength may be at least, or at most, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nm apart.

[0029] The number of filters in the filter assembly 128 can range from 1 to 10. In some embodiments, the number of filters in the filter assembly 128 can be or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a range between any two of these values. In some embodiments, the number of filters in the filter assembly 128 can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0030] The filter may be a bandpass filter and may have a peak transmittance ranging from 400 nm to 800 nm, hi some embodiments, the peak transmittance may be at or about 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a value or range between any two of these values. In some embodiments, the peak transmittance can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm. The width of the filter can vary, for example, over a range of 1 nm to 50 nm. In some embodiments, the width of the filter may be or may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nm, or a value or range between any two of these values. In some embodiments, the width of the filter may be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nm.

[0031] Fluorescent labeling Fluorescent labels utilized by the systems and methods disclosed herein may have a variety of peak absorption wavelengths, ranging from, for example, 400 nm to 800 nm. In some embodiments, the peak absorption wavelength of the fluorescent label may be at or about 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a value or range between any two of these values. In some embodiments, the peak absorption wavelength of a fluorescent label can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm.

[0032] The fluorescent labels may have various peak emission wavelengths, ranging from, for example, 400 nm to 800 nm, In some embodiments, the peak emission wavelength of the fluorescent label may be at or about 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or a value or range between any two of these values. In some embodiments, the peak emission wavelength of the fluorescent label can be at least, or at most, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm.

[0033] The fluorescent labels may have various Stokes shifts, ranging from, for example, 10 nm to 200 nm. In some embodiments, the Stokes shift may be or may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm, or a value or range between any two of these values. In some embodiments, the Stokes shift may be at least, or at most, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm.

[0034] The systems and methods disclosed herein may utilize two fluorescent labels, e.g., a first fluorescent label and a second fluorescent label may have overlapping emission spectra and may be susceptible to crosstalk. In some embodiments, the peak emission wavelengths of the two fluorescent labels may vary, e.g., ranging from 10 nm to 200 nm. In some embodiments, the peak emission wavelengths of the two fluorescent labels may be at or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm, or a value or range between any two of these values. In some embodiments, the peak emission wavelengths of the two fluorescent labels may be at least, or at most, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. The detector 126 may detect the fluorescent emission of the first fluorescent label with one of the filters in the filter assembly 128. The detector 126 may detect the fluorescent emission of the second fluorescent label with another filter in the filter assembly 128.

[0035] In some embodiments, one of the two fluorescent dyes can be a normal Stokes shift dye and the other of the fluorescent dyes can be a long Stokes shift dye. Non-limiting examples of normal Stokes shift dyes include Alexa 488 or its dye analogs in U.S. Pat. No. 8,754,244, such as 3,6-bis(ethylamine)-2,7-dimethyl-[2-carboxylato-5-(3-carboxypropyloxy)phenyl]quinanthylium betaine (dye I-3) and 3,6-bis(ethylamine)-2,7-dimethyl-[2-carboxylato-4-(3-carboxypropyloxy)phenyl]quinanthylium betaine (dye I-4). The normal Stokes shift dye can be used with a laser or light emitting dye having a wavelength of 488 nm. The long Stokes shift dye may be excited with an LED light source and have an emission peak at 520 nm. The long Stokes shift dye may be the dye NR520LS in PCT Patent Application No. PCT / GB2016 / 051474. The long Stokes shift dye may have an emission peak at 590 nm. In some embodiments, the two fluorescent dyes may be Cy3 (emission peak at around 575 nm) and the fluorescence resonance energy transfer (FRET) pair dye Cy3-Cy5 (emission peak at 670 nm). [ka]

[0036] Computer Systems The computer system 106 of the single light source, two optical channel sequencing system 100 may be configured to control the optical system 102 and the fluidic system 104, as discussed above. While many configurations are possible for the computer system 106, one embodiment is illustrated in Figure 2. As shown in Figure 2, the computer system 106 may include a processor 202 in electronic communication with a memory 204, a storage device 206, and a communication interface 208.

[0037] The processor 202 may be configured to execute instructions that cause the fluidics system 104 to deliver reagents to the flow cell 114 during a sequencing reaction. The processor 202 may execute instructions to control the light source 120 of the optical system 102 to generate light at a predetermined wavelength. The processor 202 may execute instructions to control the detector 126 of the optical system 102 and receive data from the detector 126. The processor 202 may execute instructions to process data received from the detector 126 (e.g., a fluorescent image) and determine a nucleotide sequence of a polynucleotide based on the data received from the detector 126.

[0038] The memory 204 may be configured to store instructions for configuring the processor 202 to perform the functions of the computer system 106 when the single light source-two optical channel sequencing system 100 is powered on. The storage device 206 may store instructions for configuring the processor 202 to perform the functions of the computer system 106 when the single light source-two optical channel sequencing system 100 is powered off. The communication interface 208 may be configured to facilitate communication between the computer system 106 and the optical system 102 and the fluidic system 104.

[0039] The computer system 106 may include a user interface 210 configured to communicate with a display device (not shown) for displaying sequencing results of the single light source-two optical channel sequencing system 100. The user interface 210 may be configured to receive input from a user of the single light source-two optical channel sequencing system 100. The optical system interface 212 and the fluidic system interface 214 of the computer system 106 may be configured to control the optical system 102 and the fluidic system 104 through communication links 108A and 108B illustrated in FIG. 1. For example, the optical system interface 212 may communicate with the computer interface 110 of the optical system 102 through communication link 108A.

[0040] The computer system 106 may include a nucleobase determiner 216 configured to determine a nucleotide sequence of a polynucleotide using data received from the detector 126. The nucleobase determiner 216 may include one or more of the following: a template generator 218, a position recorder 220, an intensity extractor 222, an intensity corrector 224, a base caller 226, and a quality score determiner 228. The template generator 218 may be configured to generate a template of the location of the polynucleotide cluster in the flow cell 114 using a fluorescent image captured by the detector 126. The position recorder 220 may be configured to record the location of the polynucleotide cluster in the flow cell 114 in the fluorescent image captured by the detector 126 based on the position template generated by the template generator 218. The intensity extractor 222 may be configured to extract the intensity of the fluorescent emission from the fluorescent image to generate extracted intensities. The intensity corrector 224 may be configured to reduce or eliminate crosstalk between the fluorescent labels, for example, by color correcting the extracted intensities to generate corrected intensities. In some embodiments, the intensity corrector 224 may phase correct or pre-phase correct the extracted intensities. The base caller 226 may be configured to determine a base of the polynucleotide from the corrected intensities. The base of the polynucleotide determined by the base caller 226 may be associated with a quality score determined by the quality score determiner 228.

[0041] Methods for performing sequential synthetic sequencing FIG. 3 is a flow chart of an exemplary method 300 for performing sequential synthetic sequencing utilizing the sequencing system 100. After the method 300 starts at block 305, a flow cell 114 containing fragmented polynucleotide fragments (e.g., fragmented single-stranded or double-stranded polynucleotide fragments) is received at block 310. The fragmented double-stranded polynucleotide fragments may be generated from a deoxyribonucleic acid (DNA) sample. The DNA sample may be derived from a variety of sources, such as a biological sample, a cell sample, an environmental sample, or any combination thereof. The DNA sample may include one or more of biological fluids, tissues, and cells derived from a patient. For example, the DNA sample may be taken from or may include blood, urine, cerebrospinal fluid, pleural fluid, amniotic fluid, semen, saliva, bone marrow, a biopsy sample, or any combination thereof.

[0042] The DNA sample may include DNA from a cell of interest. The cell of interest may vary and, in some embodiments, may express a malignant phenotype. In some embodiments, the cell of interest may include tumor cells, bone marrow cells, cancer cells, stem cells, endothelial cells, virally infected cells, pathogenic, parasitic cells, or any combination thereof.

[0043] The length of the fragmented polynucleotide fragments can range from 200 bases to 1000 bases. Once the flow cell 114 containing the fragmented polynucleotide fragments is received at block 310, the process 300 moves to block 320 where the polynucleotide fragments are bridge amplified into clusters of polynucleotide fragments attached to the inner surface of one or more channels of the flow cell (e.g., flow cell 114). The inner surface of one or more channels of the flow cell can include two types of primers (e.g., a first primer type (P1) and a second primer type (P2)) and the DNA fragments can be amplified by well known methods.

[0044] After generating the clusters in the flow cell 114, the process 300 may begin a process of sequential synthetic sequencing, which may include determining the nucleotide sequence of the cluster of single-stranded polynucleotide fragments. To determine the sequence of the cluster of single-stranded polynucleotide fragments having the sequence 5'-P1-F-A2R-3', a primer having the sequence A2F (which is complementary to the sequence A2R) may be added in block 325 with 0, 1, or 2 labeled nucleotide analogs and extended by a DNA polymerase to form a growing primer-polynucleotide.

[0045] During each sequencing cycle, four types of nucleotide analogs can be added and incorporated into the growing primer-polynucleotide. The four types of nucleotide analogs can have different modifications. For example, the first type of nucleotide can be an analog of deoxyguanosine triphosphate (dGTP) that is not conjugated to any fluorescent label. The second type of nucleotide can be an analog of deoxythymidine triphosphate (dTTP) that is conjugated to a first type of fluorescent label via a linker. The third type of nucleotide can be an analog of deoxycytidine triphosphate (dCTP) that is conjugated to a second type of fluorescent label via a linker. The fourth type of nucleotide can be an analog of deoxyadenosine triphosphate (dATP) that is conjugated to both the first type of fluorescent label and the second type of fluorescent label via one or more linkers. The linker can include one or more cleavage groups. Prior to a subsequent sequencing cycle, the fluorescent label can be removed from the nucleotide analog. For example, the linker attaching the fluorescent label to the nucleotide analog may contain, for example, an azide and / or alkoxy group on a carbon such that the linker can be cleaved after each cycle of incorporation by a phosphine reagent, thereby releasing the fluorescent label from subsequent sequencing cycles.

[0046] The nucleotide triphosphates can be reversibly blocked at the 3' position so that sequencing can be controlled and only one nucleotide analog can be added onto each extending primer-polynucleotide in each cycle. For example, the 3' ribose position of the nucleotide analog can contain both alkoxy and azido functional groups, which can be removable by cleavage with a phosphine reagent, thereby creating a nucleotide that can be further extended. After incorporation of the nucleotide analog, the fluidic system 104 can wash one or more channels of the flow cell 114 to remove any unincorporated nucleoside analogs and enzymes. Before a subsequent sequencing cycle, the reversible 3' block can be removed so that another nucleotide analog can be added onto each extending primer-polynucleotide.

[0047] In block 330, a single light source (e.g., laser 120 or LED source) may excite two fluorescent labels at a predetermined wavelength. The laser or LED source may be non-adjustable. In block 335, the signal from the fluorescent labels may be detected. Detecting the fluorescent labels may include, for example, capturing the fluorescent emission in two fluorescent images at a first wavelength and a second wavelength by detector 126 using two filters. The fluorescent emission of the first fluorescent label may be at or near the first wavelength, and the fluorescent emission of the second fluorescent label may be at or near the second wavelength. The fluorescent images may be stored for later offline processing. In some embodiments, the fluorescent images may be processed to determine the sequence of the growing primer-polynucleotide in each cluster in real time.

[0048] In decision block 340, a decision can be made whether to detect more nucleotides, for example, based on the quality of the signal or after a predefined number of bases. If more nucleotides are to be detected, the nucleotide decision of the next sequencing cycle can be made starting again in block 325, and nucleotide analogs with 0, 1, or 2 labels are added to extend the primer-polynucleotide. Before the next sequencing cycle, the fluorescent label can be removed from the incorporated nucleotide analog, and the reversible 3' block can be removed so that another nucleotide analog can be added onto the primer-polynucleotide during each extension.

[0049] In offline fluorescent imaging processing, if there are no further nucleotides to be detected in decision block 340, the fluorescent image containing the detected fluorescent signal may be processed in block 345 to determine the base of the incorporated nucleotide. For each determined nucleotide base, a quality score may be determined in block 350. After all of the fluorescent images have been processed, the process 300 may terminate in block 355.

[0050] Base calling Base calling is described in U.S. Patent No. 8,965,076, the contents of which are incorporated herein by reference in their entirety. Briefly summarized, base calling can refer to the process of determining the base of a nucleotide incorporated into a growing primer-polynucleotide cluster that is being sequenced to be guanine (G), thymine (T), cytosine (C), or adenine (A). FIG. 4 is a flow chart of an exemplary method 400 for making a base call utilizing a sequencing system 100. Processing the detected signal in block 345 illustrated in FIG. 3 can include making the base call of method 400. After starting in block 405, light of a predetermined wavelength can be generated using a light source and can shine on the nucleotide analog in block 410. For example, the computer system 106 can cause the light source 120 to generate light at a predetermined wavelength through its optical system interface 212 and communication channel 108A.

[0051] The light generated by the light source can shine on the nucleotide analogs incorporated into the growing primer-polynucleotide attached on the inner surface of one or more channels of the flow cell (e.g., flow cell 114). The primer-polynucleotide can include a cluster of single-stranded polynucleotide fragments hybridized to a sequencing primer. The nucleotide analogs can each include zero, one, or two fluorescent labels. The two fluorescent labels can be a first fluorescent label and a second fluorescent label. The fluorescent labels can emit fluorescent emissions after being excited by the light generated by the light source. For example, the first fluorescent label can produce fluorescent emissions at a first wavelength that can be captured, for example, in a first fluorescent image. The second fluorescent label can produce fluorescent emissions at a second wavelength that can be captured, for example, in a second fluorescent image.

[0052] The nucleotide analogues may include a first type of nucleotide, a second type of nucleotide, a third type of nucleotide, and a fourth type of nucleotide. The first type of nucleotide, for example, an analogue of deoxyguanosine triphosphate (dGTP), is not conjugated to the first or second fluorescent label. The second type of nucleotide, for example, an analogue of deoxythymidine triphosphate (dTTP), may be conjugated to the first type of fluorescent label, but not to the second type of fluorescent label. The third type of nucleotide, for example, an analogue of deoxycytidine triphosphate (dCTP), may be conjugated to the second type of fluorescent label, but not to the first type of fluorescent label. The fourth type of nucleotide, for example, an analogue of deoxyadenosine triphosphate (dATP), may be conjugated to both the first and second type of fluorescent labels.

[0053] In block 415, the fluorescent emission of the nucleotide analog at the first wavelength and the second wavelength can be detected using at least one detector. For example, the detector 126 can capture two fluorescent images (a first fluorescent image at the first wavelength and a second fluorescent image at the second wavelength). After receiving the two fluorescent images from the optical system 102, the nucleobase determiner 216 can determine the presence or absence of fluorescent emission in the two fluorescent images.

[0054] Because the first type of nucleotide is not conjugated to either the first or second fluorescent label, the first type of nucleotide may generate no or minimal fluorescence emission at the first or second wavelength. If no fluorescence emission is detected at decision block 420, the nucleotide may be determined to be a first type of nucleotide (e.g., dGTP). If any or more than minimal fluorescence emission is detected, method 400 may proceed to decision block 425.

[0055] Since the second type of nucleotide is conjugated with the first type of fluorescent label and not conjugated with the second type of fluorescent label, the second type of nucleotide may generate fluorescent emission at the first wavelength and may generate no or minimal fluorescent emission at the second wavelength. At decision block 425, if fluorescent emission at the second wavelength is not detected in the second fluorescent image, and from decision block 420, fluorescent emission at the first wavelength is detected in the first fluorescent image, the nucleotide may be determined to be a second type of nucleotide (e.g., dTTP). If fluorescent emission is detected at the second wavelength, method 400 may proceed to decision block 430.

[0056] Because the third type nucleotide is conjugated to a second type fluorescent label and not to a first type fluorescent label, the third type nucleotide may generate fluorescent emission at a second wavelength and no or minimal fluorescent emission at the first wavelength. If, at decision block 430, no fluorescent emission at the first wavelength is detected in the first fluorescent image, and from decision block 425, fluorescent emission at the second wavelength is detected in the second fluorescent image, the nucleotide may be determined to be a third type nucleotide (e.g., dCTP).

[0057] Because the fourth type nucleotide is conjugated with both the first type fluorescent label and the second type fluorescent label, the fourth type nucleotide may generate fluorescent emission at the first wavelength or the second wavelength. At decision block 430, if fluorescent emission can be detected at the first wavelength in the first fluorescent image, and from decision block 425, fluorescent emission can be detected at the second wavelength in the second fluorescent image, the nucleotide may be determined to be a fourth type nucleotide (e.g., dATP).

[0058] The flow cell 114 may contain a growing cluster of primer-polynucleotides to be sequenced. If, at decision block 435, there is at least one more cluster with fluorescent emissions to be processed for a given sequencing cycle, the method 400 may continue at block 415. If no more clusters of single-stranded polynucleotides are to be processed, the method 400 may end at block 440.

[0059] Single Light Source-Two Optical Channel Sequencing Workflow Cycle 1: Template generation, position recording, and intensity extraction FIG. 5 is a flow chart of an exemplary method 500 for performing single light source-two optical channel sequencing. The single light source-two optical channel sequencing system 100 may perform the method 500. After beginning at block 505, the light source may generate light of a predetermined wavelength onto the nucleotide at block 510. At block 515, the fluorescent emission from the first fluorescent label at the first wavelength and from the second fluorescent label at the second wavelength may be detected, for example, using at least one detector, to generate a first fluorescent image and a second fluorescent image. Detecting the fluorescent emission may include determining an intensity of the fluorescent emission. After receiving the two fluorescent images, a position template may be generated at block 520, for example, by the template generator 218.

[0060] Generating position templates may be necessary during the first sequencing cycle to determine the location of clusters of single-stranded polynucleotides. FIG. 6 shows an overview of nucleic acid clusters and their sequencing using single light source-two optical channel sequencing. During the first sequencing cycle, the location of the clusters is unknown. A flow cell may contain four clusters, clusters 1-4. During the first sequencing cycle, the template generator 218 may determine the presence of clusters 1, 2, and 4 in the flow cell.

[0061] During the first sequencing cycle, a first fluorescent image 602 and a second fluorescent image 604 of the flow cell in a first state 606 (corresponding to a first sequencing cycle) may be generated. The nucleotide analog incorporated into the growing primer-polynucleotide cluster may vary. For example, the nucleotide incorporated into cluster 1 may be an analog of deoxyadenosine triphosphate (dATP) conjugated with both a first type of fluorescent label and a second type of fluorescent label. The first fluorescent image 602 may capture the fluorescent emission of the first type of fluorescent label on the dATP analog. The second fluorescent image 604 may capture the fluorescent emission of the second type of fluorescent label on the dATP analog. The template generator 218 may determine the presence of the cluster 1 at a particular cluster 1 location from the first fluorescent image 602 or the second fluorescent image 604.

[0062] The nucleotide incorporated into cluster 2 may be an analog of deoxycytidine triphosphate (dCTP) conjugated to a second type of fluorescent label and not conjugated to the first type of fluorescent label. The second fluorescent image may capture the fluorescent emission of the second type of fluorescent label on the dCTP analog. If the first fluorescent label and the second fluorescent label are subject to crosstalk, the cluster 2 may have some fluorescent emission on the first fluorescent image. The template generator 218 may determine the presence of cluster 2 at a particular cluster 2 location from the second fluorescent image 604.

[0063] The nucleotide incorporated into cluster 3 may be an analog of deoxyguanosine triphosphate (dGTP) that is not conjugated to the first or second fluorescent label. The first fluorescent image 602 and the second fluorescent image 604 therefore have no or minimal fluorescent emission from cluster 3. The template generator 218 may not be able to determine the presence of cluster 3 at a particular cluster 3 location from the first fluorescent image 602 and the second fluorescent image 604.

[0064] The nucleotide incorporated into cluster 4 may be an analog of deoxythymidine triphosphate (dTTP) conjugated to a first type of fluorescent label and not conjugated to a second type of fluorescent label. The first fluorescent image 602 may capture the fluorescent emission of the first type of fluorescent label on the dTTP analog. If the first fluorescent label and the second fluorescent label are subject to crosstalk, the cluster 4 may have some fluorescent emission on the second fluorescent image 604. The template generator 218 may determine the presence of cluster 4 at a particular cluster 4 location from the first fluorescent image.

[0065] The template generator 218 may generate location templates for the clusters 1, 2, and 4 based on the first fluorescent image 602 and the second fluorescent image 604 in the first sequencing cycle. In some embodiments, generating the location templates may include detecting crosstalk between the first fluorescent label and the second fluorescent label. The crosstalk may advantageously make image recording more robust, especially in low diversity situations, because the emission of the fluorescent label may be captured in both the first fluorescent image 602 and the second fluorescent image 604.

[0066] Cycle 2: Template generation and position recording Generating a position template may be necessary during the second sequencing cycle to determine the position of a cluster of single-stranded polynucleotides when a random flow cell is used. After the first sequencing cycle, the position of cluster 3 may be unknown. The nucleotide incorporated into cluster 3 during the second sequencing cycle may be an analog of deoxycytidine triphosphate (dTTP) conjugated with the first type of fluorescent label and not conjugated with the second type of fluorescent label. A first fluorescent image 612 may capture the fluorescent emission of the first type of fluorescent label on the dTTP analog. During the second sequencing cycle, the template generator 218 may determine the presence of cluster 3 at a particular cluster 3 position from the first fluorescent image 612. Template generation when a patterned flow cell is used is described in U.S. Patent Application Serial No. 14 / 530,299, the contents of which are incorporated herein by reference in their entirety.

[0067] Location recording and intensity extraction 5, in block 525, the positions of the clusters in the position template may be recorded for the captured fluorescent images for the first and subsequent sequencing cycles. Fluorescence intensities of the growing primer-polynucleotide clusters at the recorded positions, e.g., positions 1, 2, and 4, may be extracted in block 530. The extracted intensities may be corrected in 535 to generate corrected intensities. For example, extracting the extracted intensities by the intensity corrector 224 may include one or more of spatial normalization in block 540, color correction in block 545, or phase synchronization correction in block 550.

[0068] Spatial normalization, color correction, and phase synchronization correction Spatial normalization can include normalizing the intensity of the fluorescent emission in different fluorescent images of a sequencing cycle to generate spatially normalized intensity. For example, in each sequencing cycle, 5% and 95% of the intensity of its first fluorescent image and its second fluorescent image can be normalized to 0 and 1. If the sequencing cycle is within an indexed read, the 95th percentile from the last cycle of the non-indexed read can be used for normalization. Spatial normalization can reduce cycle-dependent intensity variation.

[0069] Figures 7A-D are schematic plots showing chromatic and phase correction for single light source-two optical channel sequencing. Figure 7A shows the chromatic and phase correction of the extracted or spatially normalized intensity from the first fluorescent image as a function of position (x,y) when there is no crosstalk between the first fluorescent label and the second fluorescent label. i , y i ) is a scatter plot of the extracted intensities from the corresponding positions in the second fluorescent image at x i y denotes the spatially normalized intensity of the growing primer-polynucleotide cluster i in the second fluorescence image. idenotes the spatially normalized intensity of cluster i of growing primer-polynucleotide in its first fluorescent image. A dGTP analog does not contain either the first or second fluorescent label and therefore has no fluorescent emission in its first or second fluorescent image. Thus, the population of dGTP analogs is at position (0, 0) of its scatter plot. A dTTP analog contains the first fluorescent label and therefore has fluorescent emission in its first fluorescent image and does not have it in its second fluorescent image. Thus, the population of dTTP analogs is at position (0, 1) of its scatter plot. A dCTP analog contains the second fluorescent label and therefore has fluorescent emission in its second fluorescent image and does not have it in its first fluorescent image. Thus, the population of dCTP analogs is at position (1, 0) of its scatter plot. The dATP analog contains the first fluorescent label and the second fluorescent label and therefore has fluorescence emission in the first fluorescent image and the second fluorescent image. The population of dATP analogs is at position (1,1) in the scatter plot because there is no crosstalk between the first fluorescent label and the second fluorescent label.

[0070] FIG. 7B shows a schematic diagram of a scatter plot in which the two fluorescent labels have overlapping emission spectra and are susceptible to crosstalk. Since the first fluorescent label and the second fluorescent label are susceptible to crosstalk, the dTTP analog has a stronger emission in the first fluorescent image and a weaker emission in the second fluorescent image. Thus, the cloud of points corresponding to the fluorescent emission from the dTTP analog population is at a position around (0, 1), e.g., (0.2, 0.8). The dCTP analog has a stronger emission in the second fluorescent image and a weaker emission in the first fluorescent image. Thus, the cloud of points corresponding to the fluorescent emission from the dCTP analog population is at a position around (1, 0), e.g., (0.8, 0.2). The cloud of points corresponding to the fluorescent emission from the dATP analog population is at a position around (1, 1), e.g., (0.9, 0.9).

[0071] To reduce or eliminate crosstalk between the first and second fluorescent labels, the extracted intensities or the spatially normalized intensities may be color corrected at 545. Color correction may utilize a color matrix to adjust the extracted intensities taking advantage of characteristics of the underlying intensity distribution within each fluorescent image.

[0072] The two-channel color matrix can be a 2×2 matrix used to correct for crosstalk between two-channel capture, e.g., a first channel and a second channel. The first channel can capture the first and second fluorescent images in a sequencing cycle. For example, if a cluster becomes bright in the first channel corresponding to the first fluorescent image, some of the emission is also collected in the second channel corresponding to the second fluorescent image. Color correction can include using the two-channel color matrix to generate a matrix-corrected intensity that can reduce or eliminate the crosstalk. The color matrix can also balance any differences in overall intensity between color channels. The color matrix M,

number

[0073] The color matrix can be inferred based on the cluster intensities collected for an early sequencing cycle, e.g., a configurable set of sequencing cycles 1 through 10. This color matrix can be used for the remainder of the sequencing cycles, along with a normalization of the relative intensities that is cycle-dependent.

[0074] The color matrix can be used to estimate crosstalk between the pair of channels because they have overlapping emission spectra. In some embodiments, estimating the color matrix is ​​done by using the i,チャネル2 , a i,チャネル1 ) into polar coordinates (where i denotes the cluster number and a i, チャネル1 denotes the intensity of the i-th cluster in the first channel, and a i, チャネル2 indicates the intensity of the i-th cluster in the second channel). The estimation of the color matrix is ​​done by estimating the position (a i, チャネル2 , a i, チャネル1 ) from the plotted intensity in the range [0, 90] i In the second fluorescent image, the area weighted histogram of a i, チャネル2 and the intensity of a in the first fluorescence image i, チャネル1 For cluster i with strength of size r i is the intensity a i, チャネル1 and a i, チャネル2 (e.g., (a i, チャネル1 2 + a i, チャネル2 2 ) 1 / 2 ). Angle θ i tan -1 (a i, チャネル1 / a i, チャネル2 7C shows a schematic diagram of a zone-weighted histogram when the two fluorescent labels have overlapping emission spectra and are susceptible to crosstalk. i, チャネル1 , a i, チャネル2) can be converted to the area-weighted angle histogram in Figure 7C. For single source, two channel sequencing, the area-weighted angle histogram contains three peaks in Figure 7B, which correspond to the clusters of dTTP, dATP, and dCTP analog dots, respectively. The central peak, which corresponds to the cluster of dATP analog dots, is at an angle of approximately 45°.

[0075] Inferring the color matrix may involve identifying two outer maxima θ1 and θ2 in the area-weighted histogram. For a channel with no crosstalk, θ1 is 0° and θ2 is 90°. The crosstalk coefficients M 1,2 can be, for example, tan(θ1). The crosstalk coefficient M 2,1 may be, for example, tan(90-θ2). In some embodiments, if an insufficient number of clusters can be called with one of the four nucleotides, the color matrix guess may not be ideal and a discrimination matrix may be used instead. The diagonal elements of the matrix may be 1 and the color matrix may be

number

[0076] The color matrix may be normalized to have a determinant of 1. In some embodiments, the color matrix of an early sequencing cycle may be used for subsequent sequencing cycles. The corrected intensities may be calculated by multiplying the intensities plotted in FIG. 7B by the inverse of the color matrix to generate color-corrected intensities. FIG. 7D shows a schematic of the intensity scatter plot of FIG. 7B after color correction. With the corrected intensities, the individual clusters corresponding to dGTP, dTTP, dCTP, and dATP may be better separated. In some embodiments, the fluorescent image may be split into multiple tiles, and a color matrix may be estimated for each tile. In some embodiments, the color matrix may be estimated using the intensities of many sequencing cycles. The size and shape of the fluorescent emission dot clusters in FIG. 7B and 7D are for illustration only. For example, the dot clusters corresponding to the dot clusters of dATP analogs after color correction in FIG. 7D may be larger than the dot clusters corresponding to the dot clusters of dATP analogs before color correction in FIG. 7B.

[0077] Referring to FIG. 5, the color-corrected intensities can be phase-corrected in block 550. During the sequencing-while-synthesizing process, each primer or extended primer in a primer-polynucleotide cluster can be extended one base / cycle. A small percentage of the strands can be out of phase with the current sequencing cycle, either lagging bases (phase-locking) or leading bases (pre-phase-locking). For each cycle of sequencing, a phase-locking correction can be calculated, for example, by determining a phase-locking matrix and applying the phase-locking matrix to the extracted intensities to maximize data quality.

[0078] Base calling In block 555, the base of the nucleotide incorporated into the growing primer-nucleotide cluster may be determined, for example, by the base caller 226. A quality score may be determined for each called base. With reference to FIG. 6, in the first sequencing cycle, cluster 1 has a fluorescent emission in both the first and second fluorescent images, so its incorporated nucleotide is a dATP analog. Cluster 2 has a fluorescent emission only in the second fluorescent image, so its incorporated nucleotide is a dCTP analog. Cluster 4 has a fluorescent emission only in the first fluorescent image, so its incorporated nucleotide is a dTTP analog.

[0079] In the second sequencing cycle, the nucleotides incorporated into clusters 1-4 may be dGTP, dCTP, dTTP, and dATP, respectively. After determining the presence of cluster 3, the nucleotide incorporated into cluster 3 during the first sequencing cycle may be dGTP, which has no fluorescence emission in the first or second fluorescent images. After the third sequencing cycle, the clusters 1-4 may be determined to have the nucleotide sequences AGT, CCA, GTA, and TAG, respectively.

[0080] In some embodiments, the base call in block 555 may be based on the corrected intensities from block 535. The correspondence between the nucleotides and the populations on the scatter plot of Figure 7D may be defined as follows: if a population is off in its first channel and off in its second channel, the incorporated nucleotide is a dGTP analog; if a population is off in its second channel and on in its first channel, the incorporated nucleotide is a dTTP analog; if a population is on in its second channel and off in its first channel, the incorporated nucleotide is a dCTP analog; and if a population is on in its first channel and on in its second channel, the incorporated nucleotide base call is a dATP analog.

[0081] Base calling may include normalizing the corrected intensities to (0, 1) by the 5th and 95th percentiles. Four Gaussian distributions (one for each of dGTP, dTTP, dCTP, and dATP) may be fitted to the corrected and normalized intensity data via an expectation maximization algorithm. The expectation maximization algorithm may determine which means and distribution best fits the data. After calculating the Gaussian distributions, the likelihood of the population belonging to each Gaussian distribution may be calculated for each population. Base calling may be based on the maximum likelihood of the population belonging to a particular Gaussian distribution. For low diversity samples, the expectation maximization algorithm may be used to identify a covariance matrix to avoid overfitting data. Subsampling targets may be increased to sample a large amount of data for accuracy.

[0082] In some embodiments, the population can be filtered by a chastity metric. The chastity metric can be, for example, D1 / (D1+D2), where D1 can be the distance to the closest Gaussian mean and D2 can be the distance to the next closest distance. The distance can be measured, for example, using the Mahalanobis method, which can take into account the width of the distribution along the line defined by each Gaussian centriod and the point under consideration.

[0083] At block 560, one or more quality metrics can be determined before the method ends at block 565. Sequencing quality metrics can provide important information about the accuracy of each step in the process, including library preparation, base calling, read alignment, and variant calling. Base calling accuracy, as measured by Phred quality score (Q-score), can be used to assess the accuracy of a sequencing platform. It can indicate the probability that a given base is called incorrectly by the sequencer. The Q-score is a function of 10 log 10 P, where P is the base calling error probability.

[0084] Cluster Scaling In some embodiments, correcting the intensity may include cluster scaling. Clusters may have varying brightness. For example, some clusters may be bright and some clusters may be dim. The brightness of the clusters may be caused by the fragment length distribution of the sample. The varying brightness of the cluster population may have the effect of lengthening the "on" population in the base call scatter plot. It may be advantageous to normalize the intensity of each cluster by its average intensity in the first 10 cycles to reduce population intensity variation. For example, in the first 10 cycles, two zones may be calculated for every non-guanine (G) base call: the distance of the population intensity from the origin, and the distance of the corresponding Gaussian mean from the origin. Cluster scaling may include, for example, normalizing to the average of the ratio of these two zones averaged over the first 10 cycles. All cluster intensities may be normalized by this scaling factor before phase correction and base calling are performed. Cluster scaling may advantageously increase throughput and reduce error rates, for example, for samples with large fragment length distributions.

[0085] Single Light Source Multiple Optical Channel Sequencing Machine Disclosed herein are embodiments of a system or method for determining the nucleotide sequence of a polynucleotide. In one embodiment, the system may include or be in communication with a single light source (e.g., a laser or LED light source) configured to generate light (e.g., light at a predetermined wavelength). The system may include or be in communication with at least one detector configured to detect four substantially different fluorescent emissions from different fluorophores attached to the nucleotide. The system may cause the light source to generate light onto the nucleotide. The nucleotide may be identified as a first type if a first fluorescent emission is detected by the at least one detector. The nucleotide may be identified as a second type if a second fluorescent emission is detected by the at least one detector. The nucleotide may be identified as a third type if a third fluorescent emission is detected by the at least one detector. The nucleotide may be identified as a fourth type if a fourth fluorescent emission is detected by the at least one detector. At least two of the first fluorescent emission, the second fluorescent emission, the third fluorescent emission, and the fourth fluorescent emission may have substantially different wavelengths.

[0086] Different types of nucleotides may be attached to different fluorophores or may not be attached to fluorophores.For example, the first type of nucleotide may not be attached to a fluorophore that can be excited by a single light source, and the first fluorescent emission comprises no emission.In another example, the first type of nucleotide may be attached to two different fluorophores, and the first fluorescent emission comprises the emission from two different fluorophores.

[0087] In yet another example, a first fluorescent emission is from a first fluorophore attached to a first nucleotide of a first type, a second fluorescent emission is from a second fluorophore attached to a second nucleotide of a second type, a third fluorescent emission is from a third fluorophore attached to a third nucleotide of a third type, and a fourth fluorescent emission is from a fourth fluorophore attached to a fourth nucleotide of a fourth type. The four fluorophores can be excited using a light source. In one implementation, all four of the first fluorophore, the second fluorophore, the third fluorophore, and the fourth fluorophore are different. For example, the nucleotide sequence can be determined based on emission by four dyes at four different wavelengths. In another implementation, three of the first fluorophore, the second fluorophore, the third fluorophore, and the fourth fluorophore are different. For example, the nucleotide sequence can be determined based on emission by three dyes at three different wavelengths. In another implementation, two of the first fluorophore, the second fluorophore, the third fluorophore, and the fourth fluorophore are identical. For example, the nucleotide sequence can be determined based on the emission by two dyes at two different wavelengths.

[0088] Sequencing The methods described herein can be used with various nucleic acid sequencing techniques. Particularly applicable techniques are those in which the nucleic acids are attached to fixed positions in an array so that their relative positions do not change, and the array is imaged repeatedly. Particularly applicable are embodiments in which images are obtained in different color channels, e.g., with the simultaneous presence of different labels used to distinguish one nucleotide base type from another. In some embodiments, the process of determining the nucleotide sequence of the target nucleic acid can be an automated process. Preferred embodiments include sequential synthesis sequencing ("SBS") techniques.

[0089] "Sequential synthetic sequencing ("SBS") technology" generally involves the enzymatic extension of a nascent nucleic acid chain through the repeated addition of nucleotides to a template strand. In traditional methods of SBS, a single nucleotide monomer can be provided to a target nucleic acid in the presence of a polymerase in each delivery. However, in the methods described herein, more than one type of nucleotide monomer can be provided to a target nucleic acid in the presence of a polymerase in each delivery.

[0090] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such substitution is technically possible. It will be recognized by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0091] With respect to the use of virtually any plural and / or singular term herein, those of skill in the art may interpret the plural to the singular and / or the singular to the plural, as appropriate to the context and / or application. Various singular / plural permutations may be expressly set forth herein for clarity.

[0092] It will be understood by those skilled in the art that the terms used herein in general, and in particular in the appended claims (e.g., the body of the appended claims) are generally intended as "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "includes" should be interpreted as "includes but is not limited to", etc.). It will be further understood by those skilled in the art that where specific numerals of the introduced claim recitations are intended, such intention will be expressly set forth in the claims, and in the absence of such recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases "at least one" and "one or more" to introduce the claim recitations. However, the use of such phrases should not be construed as suggesting that introducing a claim recitation with the indefinite article "a" or "an" limits any particular claim that includes such an introduced claim recitation to embodiments that include only one such recitation, even if the same claim includes the introductory phrases "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"); the same applies with respect to the use of definite articles used to introduce claim recitations.Furthermore, even when a specific number of recitations of an introduced claim is explicitly recited, one of ordinary skill in the art will recognize that such recitation should be interpreted to mean at least the recitation number (e.g., the bare recitation "two recitations" means at least two recitations, or two or more recitations, without other modifiers). Furthermore, in those cases where a convention similar to "at least one of A, B, and C, etc." is used, such an interpretation is generally intended in the sense that one of ordinary skill in the art would understand that convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to "such as at least one of A, B, and C" is used, such an interpretation is generally intended in the sense that one of skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A only, B only, C only, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those of skill in the art that substantially any disjunct word and / or phrase expressing two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B," or "A and B."

[0093] Moreover, when features and aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described with respect to any individual members or subgroups of members of the Markush group.

[0094] As will be understood by those skilled in the art, for any and all purposes, for example, in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any recited range can be easily understood as being fully described and allowing that same range to be broken down into at least 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, etc. As a non-limiting example, each range discussed herein can be easily broken down into a lower 1 / 3, a middle 1 / 3, and an upper 1 / 3, etc. As will also be understood by those skilled in the art, all phrases such as "up to," "at least," "greater than," "less than," etc., refer to a range that includes the recited numbers and can be subsequently broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1-5 items refers to a group having 1, 2, 3, 4, or 5 items, etc.

[0095] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those of skill in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, the true scope and spirit of which is indicated by the following claims.

Claims

1. 1. A system for use in a method for determining the nucleotide sequence of a plurality of polynucleotides, the system comprising: a single light source configured to generate light of a single wavelength for stimulating fluorescent emissions from different fluorophores attached to nucleotides in the plurality of polynucleotides, the different fluorescent emissions of the different fluorophores being stimulated by the light of the single wavelength; at least one detector configured to detect four different fluorescent emissions from the different fluorophores attached to the nucleotides; and 1. A processor comprising the steps of: generating light of said single wavelength from said light source on a nucleotide in a polynucleotide in said plurality of polynucleotides; and identifying the four nucleotides corresponding to the four different fluorescent emissions when more than one of the four nucleotides is present in the plurality of polynucleotides, identifying the nucleotide as a first type if a first fluorescent emission is detected by the at least one detector; identifying the nucleotide as a second type if a second fluorescent emission is detected by the at least one detector; identifying the nucleotide as a third type if a third fluorescent emission is detected by the at least one detector; and identifying the nucleotide as a fourth type if a fourth fluorescent emission is detected by the at least one detector. a processor configured to execute instructions to perform the method, Including, the detection of the first fluorescent emission, the second fluorescent emission, the third fluorescent emission, and the fourth fluorescent emission is determined based on four different wavelengths. system.

2. The system of claim 1 , wherein the processor is further configured to determine an intensity of one or more of the fluorescent emissions.

3. The system of claim 2 , wherein the processor is further configured to color correct the intensities.

4. The system of claim 3 , wherein color correcting the intensities comprises inferring a color matrix.

5. 2. The system of claim 1, wherein the light source is a laser, and the single wavelength of light generated by the laser is between 400 nm and 800 nm.

6. 2. The system of claim 1, wherein the light source is a light emitting diode, and the single wavelength of light generated by the light emitting diode is between 400 nm and 800 nm.

7. 2. The system of claim 1, wherein the first type of nucleotides are attached to two different fluorophores and the first fluorescent emission comprises emission from the two different fluorophores.

8. 2. The system of claim 1, wherein the first fluorescent emission is from a first fluorophore attached to a first nucleotide of the first type, the second fluorescent emission is from a second fluorophore attached to a second nucleotide of the second type, the third fluorescent emission is from a third fluorophore attached to a third nucleotide of the third type, and the fourth fluorescent emission is from a fourth fluorophore attached to a fourth nucleotide of the fourth type.

9. 9. The system of claim 8, wherein all four of the first fluorophore, the second fluorophore, the third fluorophore, and the fourth fluorophore are different.

10. 2. The system of claim 1, wherein the method further comprises reducing crosstalk between two of the first fluorescent emission, the second fluorescent emission, the third fluorescent emission, and the fourth fluorescent emission.

11. 10. The system of claim 1, wherein the method further comprises receiving a flow cell having the polynucleotide attached at a fixed position.

12. The system of claim 1 , wherein the method further comprises repeatedly imaging the polynucleotide.

13. 2. The system of claim 1, wherein the detector is configured to capture a fluorescent image of the polynucleotide, and the method further comprises extracting an array of fluorescent emission intensities from the fluorescent image.

14. 2. The system of claim 1, wherein the processor is configured to identify the four nucleotides when multiple of the four nucleotides are present simultaneously.

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