EP-4735635-A1 - SYSTEMS AND METHODS OF SEQUENCING POLYNUCLEOTIDES WITH ALTERNATIVE SCATTERPLOTS

Abstract

The application relates to DNA sequencing systems and methods. Systems and methods for determining the nucleotide sequence of a polynucleotide may include introducing a fourth labelled nucleotide in a two-channel sequencing by synthesis system to create alternative X-Y scatterplot shapes where the x-axis represents the intensity of fluorescence at one wavelength (channel 1), and the y-axis represents intensity of fluorescence at another wavelength (channel 2 The alternative scatterplot shapes may allow for additional encoding space for empty well detection.

Inventors

• CALLINGHAM, MICHAEL JAMES
• HONG, Stanley Seokjong
• ANNETTE, Callum Mark

Assignees

• Illumina, Inc.

Dates

Publication Date

20260506

Application Date

20240625

Claims (20)

1. WHAT IS CLAIMED IS: 1. A method of sequencing polynucleotides bound to a flowcell, comprising: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength and a second intensity, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths at third and fourth intensities; detecting fluorescent emissions from a fourth labeled nucleotide at the first and second wavelengths at fifth and sixth intensities; and determining the sequence of the polynucleotides based on the detected fluorescent emissions and intensities.
2. 2. The method of claim 1, wherein the first labeled nucleotide and the second labeled nucleotide are labeled with fluorescent dyes having the same excitation wavelength.
3. 3. The method of claim 1 or 2, wherein the first labeled nucleotide and the second labeled nucleotide are labeled with fluorescent dyes having different stokes shifts.
4. 4. The method of claim 1, wherein at least two of the first through sixth intensities are greater than zero.
5. 5. The method of claim 1, wherein at least three of the first through sixth intensities are greater than zero.
6. 6. The method of claim 1, wherein at least four of the first through sixth intensities are greater than zero.
7. 7. The method of claim 1, wherein at least five of the first through sixth intensities are greater than zero.
8. 8. The method of claim 1, wherein all of the first through sixth intensities are greater than zero.
9. 9. The method of claim 8, wherein the ratio of the third intensity to the fourth intensity is approximately equal to the ratio of the sixth intensity to the fifth intensity.
10. 10. The method of claim 9, wherein the first intensity is greater than the third intensity and the fifth intensity; further wherein the second intensity is greater than the fourth intensity and the sixth intensity.
11. 11. The method of claim 9, wherein the first intensity is less than the third intensity; further wherein the second intensity is less than the sixth intensity; and wherein four labeled nucleotides form a diamond configuration.
12. 12. The method of claim 1, further comprising determining the presence of an empty well based on a substantial absence of the detected fluorescent emissions and intensities.
13. 13. The method of claim 1, further comprising detecting fluorescent emissions from a fifth labeled nucleotide at the first and second wavelengths at seventh and eighth intensities.
14. 14. A kit for determining the sequence of a polynucleotide, comprising: a first mixture of a first nucleotide-first fluorescent dye conjugate detectable in a first wavelength channel and a first nucleotide-second fluorescent dye conjugate detectable in a second wavelength channel; a second mixture of a second nucleotide-first fluorescent dye conjugate detectable in the first wavelength channel and a second nucleotide-second fluorescent dye conjugate detectable in the second wavelength channel; a third mixture of a third nucleotide-first fluorescent dye conjugate detectable in the first wavelength channel and a third nucleotide-second fluorescent dye conjugate detectable in the second wavelength channel; and a fourth mixture of a fourth nucleotide-first fluorescent dye conjugate detectable in the first wavelength channel and a fourth nucleotide-second fluorescent dye conjugate detectable in the second wavelength channel.
15. 15. The kit of claim 14, wherein the ratio of intensity of emissions in the first channel versus the second channel for the third mixture is approximately equal to the ratio of intensity of emissions in the second channel versus the first channel for the fourth mixture.
16. 16. A system for determining the sequence of a polynucleotide, comprising: a machine-readable memory; and a processor configured to execute machine-readable instructions, which, when executed by the processor, cause the system to perform steps including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength and a second intensity, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths at third and fourth intensities; detecting fluorescent emissions from a fourth labeled nucleotide at the first and second wavelengths at fifth and sixth intensities; and determining the sequence of the polynucleotides based on the detected fluorescent emissions and intensities.
17. 17. The system of claim 16, further comprising: a first mixture of a first nucleotide-first fluorescent dye conjugate detectable in a first wavelength channel and a first nucleotide-second fluorescent dye conjugate detectable in a second wavelength channel; a second mixture of a second nucleotide-first fluorescent dye conjugate detectable in the first wavelength channel and a second nucleotide-second fluorescent dye conjugate detectable in the second wavelength channel; and a third mixture of a third nucleotide-first fluorescent dye conjugate detectable in the first wavelength channel and a third nucleotide-second fluorescent dye conjugate detectable in the second wavelength channel.
18. 18. The system of claim 16, further comprising, a fourth mixture of a fourth nucleotide- first fluorescent dye conjugate detectable in the first wavelength channel and a fourth nucleotide- second fluorescent dye conjugate detectable in the second wavelength channel.
19. 19. The system of claim 18, further comprising, a fifth mixture of a fifth nucleotide-first fluorescent dye conjugate detectable in the first wavelength channel and a fifth nucleotide-second fluorescent dye conjugate detectable in the second wavelength channel.
20. 20. The system of claim 19, wherein all of the mixtures have different ratios of first fluorescent dye conjugate to second fluorescent dye conjugate.

Description

ILLINC.766WO / IP-2551-PCT PATENT SYSTEMS AND METHODS OF SEQUENCING POLYNUCLEOTIDES WITH ALTERNATIVE SCATTERPLOTS INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No.63/511,382 filed June 30, 2023, the content of which is incorporated by reference in its entirety. BACKGROUND Field [0002] The present disclosure relates to DNA sequencing systems and methods. In particular, this disclosure relates to improved detection methods using alternative scatterplot shapes in two-channel detection systems. Background [0003] Current sequencing technologies involve determining DNA or RNA sequences by deciphering four natural bases in the genome, A, T (U), G, and C. One approach to polynucleotide sequencing involves using two optical channels to detect fluorescent emissions from dyes attached to different types of nucleotides. Illumina sequencing systems use onboard real-time analysis (RTA) to turn raw image data into basecalls (ACGT). This process can be massively parallelized and occurs in real time on the instrument. [0004] In a standard two-channel detection method, scatterplots may be used for analysis and determination of a polynucleotide sequence where the x-axis represents the intensity of fluorescence at one wavelength (channel 1), and the y-axis represents intensity of fluorescence at another wavelength (channel 2). Each data point on the scatterplot represents a single nucleotide that has been detected by the sequencing instrument, and the position of the dot on the scatterplot indicates the signal intensity for each channel. By analyzing the pattern of dots on the scatterplot, researchers can determine the sequence of nucleotides in the sample. The use of two channels for polynucleotide sequencing allows for the simultaneous detection of multiple nucleotides, which can increase the speed and accuracy of the sequencing process. [0005] For two-channel base-calling systems, the four DNA bases are encoded using two bits of information from each color channel, where the G base is encoded using two channels being in the off state. In a typical sequencing system, the seeding and amplification of reads is not expected to be completely efficient, which results in some empty wells. In 2-channel SBS on patterned flowcells, wells not occupied by a DNA cluster may be incorrectly called G because the G nucleotide is not labeled. This also occurs to a lesser extent with random flowcells due to spurious spots that are assigned as clusters. An “empty-detection” algorithm is usually required to distinguish “G” nucleotides and empty wells. SUMMARY [0006] An aspect of the disclosure is directed to a method of sequencing polynucleotides bound to a flowcell, including: detecting fluorescent emissions from a first labeled nucleotide at a first wavelength and a first intensity; detecting fluorescent emissions from a second labeled nucleotide at a second wavelength and a second intensity, wherein the first wavelength is different from the second wavelength; detecting fluorescent emissions from a third labeled nucleotide at the first and second wavelengths at third and fourth intensities; detecting fluorescent emissions from a fourth labeled nucleotide at the first and second wavelengths at fifth and sixth intensities; and determining the sequence of the polynucleotides based on the detected fluorescent emissions and intensities. I [0007] In some embodiments, the flowcell may comprise wells configured to bind polynucleotides. In some embodiments, systems and methods may be configured for one or both of one excitation, two channel chemistry or two excitation, two channel chemistry. In some embodiments, the first labeled nucleotide and the second labeled nucleotide may be labeled with fluorescent dyes having the same excitation wavelength. In some embodiments, the first labeled nucleotide and the second labeled nucleotide may be labeled with fluorescent dyes having different stokes shifts. [0008] In some embodiments, at least two of the first through sixth intensities are greater than zero. In some embodiments, at least three of the first through sixth intensities are greater than zero. In some embodiments, at least four of the first through sixth intensities are greater than zero. In some embodiments, at least five of the first through sixth intensities are greater than zero. In some embodiments, all of the first through sixth intensities are greater than zero. In some embodiments, the first intensity is less than the third intensity; further wherein the second intensity is less than the sixth intensity; and wherein four labeled nucleotides form a diamond configuration. In some embodiments, the ratio of the third intensity to the fourth intensity is approximately equal to the ratio of the sixth intensity to the fifth intensity. [0009] In some embodiments, methods may comprise the step of detecting fluorescent emissions from a fifth labeled nucleotide at t