EP-4735870-A1 - AUTOMATIC SPECTRAL DYE MATRIX ADJUSTMENT TO REDUCE SPECTRAL CROSSTALK IN MULTIPLEXED QPCR ASSAYS

EP4735870A1EP 4735870 A1EP4735870 A1EP 4735870A1EP-4735870-A1

Abstract

A method for reducing spectral crosstalk in a multiplexed assay is provided. The method includes receiving filter signal data from a multiplexed fluorescence assay and an initial dye matrix including calibrated spectral dye data. The method further includes generating an updated dye matrix based on the initial dye matrix and estimated crosstalk proxies, and then adjusting the updated dye matrix to meet a calculated optimization value. The calculated optimization value is calculated based on at least the estimated crosstalk proxies and the filter signal data. The calculated optimization value may be further calculated based on each adjustment of the updated dye matrix, and a cross-correlation between dyes used in the assay. The method further includes generating an improved dye matrix based on the adjusted updated dye matrix, and then generating spectral adjusted data based on the improved dye matrix.

Inventors

GEORGE, WALLACE
MARKS, JEFFREY
COX, Conor
YAN, Xinyi
KLOPFER, Connor
VARGAS-VORACEK, RENE
WESSEL, THOMAS
CHU, YONG
LIU, Sitong

Assignees

Life Technologies Corporation

Dates

Publication Date: 20260506
Application Date: 20240617

Claims (1)

Docket No. TP386609WO1 CLAIMS What is claimed is: 1. A method for reducing spectral crosstalk in a multiplexed assay, the method comprising: receiving filter signal data from a multiplexed fluorescence assay; receiving an initial dye matrix, wherein the initial dye matrix includes calibrated spectral dye data; generating an updated dye matrix based on the initial dye matrix and estimated crosstalk proxies; adjusting the updated dye matrix to meet a calculated optimization value, wherein a calculated optimization value is calculated based on the estimated crosstalk proxies and the filter signal data; generating an improved dye matrix based on the adjusted updated dye matrix; and generating spectral adjusted data based on the improved dye matrix. 2. The method of claim 1, wherein adjusting the updated dye matrix further includes meeting a plurality of calculated optimization values, wherein each calculated optimization value is calculated based on each fluorescent dye used in the multiplexed fluorescence assay. 3. The method of any one of the claims 1 to 2, wherein the calculated optimization value is further calculated based on an intermediate updated dye matrix, and a cross-correlation between dyes used in the multiplexed fluorescence assay. 4. The method of any one of the claims 1 to 3, further comprising: deconvolving the filter signal data to determine individual dye data; adjusting the baseline of the discontinuity fixed data to reduce measurement noise; calculating a cross-correlation matrix for each fluorescent dye signal used in the multiplexed fluorescent assay; and Docket No. TP386609WO1 generating the calculated optimization value using the estimated crosstalk proxies for weighting data in the cross-correlation matrix. 5. The method of any one of the claims 1 to 4, wherein the improved dye matrix (DMimproved) approximates the true dye matrix, DM0, and is: DM improved ≈DM - DM e , where DM improved is the improved dye matrix, DM is the initial dye matrix, and DM e is the error in the initial dye matrix. 6. The method of any one of the claims 1 to 5, wherein adjusting the updated dye matrix is an iterative process. 7. The method of any one of the claims 1 to 6, further comprising: generating a quantification result using the spectral adjusted data. 8. The method of any one of the claims 1 to 7, further comprising: detecting discontinuities in the filter signal data, wherein a discontinuity is a significant increase or decrease between two temporal points of the filter signal data; and generating adjusted filter signal data based on the detected discontinuities, wherein the calculated optimization value is further based on the adjusted filter signal data. 9. The method of claim 8, wherein generating the adjusted filter signal data based on the detected discontinuities includes using a polynomial fit curve. 10. A system for reducing spectral crosstalk in a multiplexed assay, the system comprising: a detector configured to receive filter signal data from a multiplexed fluorescence assay; Docket No. TP386609WO1 a memory configured to store an initial dye matrix, wherein the initial dye matrix includes calibrated spectral dye data; a processor configured to: generate an updated dye matrix based on the initial dye matrix and estimated crosstalk proxies; adjust the updated dye matrix to meet a calculated optimization value, wherein a calculated optimization value is calculated based on the estimated crosstalk proxies and the filter signal data; generate an improved dye matrix based on the adjusted updated dye matrix; and generate spectral adjusted data based on the improved dye matrix. 11. The system of claim 10, wherein the processor is further configured to: adjust the updated dye matrix to meet a plurality of calculated optimization values, wherein each calculated optimization value is calculated based on each fluorescent dye used in the multiplexed fluorescence assay. 12. The system of any one of the claims 10 to 11, wherein the calculated optimization value is further calculated based on an intermediate updated dye matrix, and a cross-correlation between dyes used in the multiplexed fluorescence assay. 13. The system of any one of the claims 10 to 12, wherein the processor is further configured to: deconvolve the filter signal data to determine individual dye data; adjust the baseline of the discontinuity fixed data to reduce measurement noise; calculate a cross-correlation matrix for each fluorescent dye signal used in the multiplexed fluorescent assay; and generate the calculated optimization value using the estimated crosstalk proxies for weighting data in the cross-correlation matrix. 14. The system of any one of the claims 10 to 13, Docket No. TP386609WO1 wherein the improved dye matrix (DM improved ) approximates the true dye matrix, DM 0 , and is: DMimproved ≈ DM - DMe , where DMimproved is the improved dye matrix, DM is the initial dye matrix, and DMe is the error in the initial dye matrix. 15. The system of any one of the claims 10 to 14, wherein the processor is configured to adjust the updated dye matrix in an iterative process. 16. The system of any one of the claims 10 to 15, wherein the processor is further configured to: generate a quantification result using the spectral adjusted data. 17. The system of any one of the claims 10 to 16, wherein the processor is further configured to: detect discontinuities in the filter signal data, wherein a discontinuity is a significant increase or decrease between two temporal points of the filter signal data; and generate adjusted filter signal data based on the detected discontinuities, wherein the calculated optimization value is further based on the adjusted filter signal data. 18. The system of claim 17, wherein the processor generates the adjusted filter signal data based on the detected discontinuities includes using a polynomial fit curve. 19. A computer-readable medium encoded with computer-readable instructions, which when executed by a processor of a computer, causes the computer to carry out the method of any one of the claims 1 to 9. Docket No. TP386609WO1 20. A system comprising a processor, and a storage medium storing instruction, which when executed by a processor, causes the system to carry out the method of any one of claims 1 to 9.

Description

Docket No. TP386609WO1 AUTOMATIC SPECTRAL DYE MATRIX ADJUSTMENT TO REDUCE SPECTRAL CROSSTALK IN MULTIPLEXED QPCR ASSAYS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/511,569, filed on June 30, 2023, which is incorporated herein in its entirety by reference. BACKGROUND [0002] The present disclosure is directed to biological analysis such as, for example, a polymerase chain reaction (PCR), and biological analysis devices such as, for example, instruments for PCR, especially to reduce spectral crosstalk in multiplexed assays and computer systems and computer software relating to methods for reducing spectral crosstalk in multiplexed assays. [0003] There is an increasing diagnostic need for quick and large volume testing of different viral strains, co-infections with different organisms, or surveying large panels of microbes. As such, there is a demand for qPCR testing instruments with much higher throughput and reduced per-sample costs. This requires much greater qPCR dye multiplexing. However, with multiplexing, the dye spectra get increasingly overlapped resulting in an increased tendency for spectral crosstalk. Accordingly, it is important to reduce or eliminate the resulting tendency to have spectral crosstalk. [0004] As such, there is an increasing need to have an algorithmic tool to help achieve lower limits of detection in multiplexed assays. SUMMARY [0005] In one exemplary embodiment, a method for reducing spectral crosstalk in a multiplexed assay is provided. The method includes receiving filter signal data from a multiplexed fluorescence assay and an initial dye matrix including calibrated spectral dye data. The method further includes generating an updated dye matrix based on the initial dye matrix Docket No. TP386609WO1 and estimated crosstalk proxies, and then adjusting the updated dye matrix to meet a calculated optimization value. The calculated optimization value is calculated based on at least the estimated crosstalk proxies and the filter signal data. The calculated optimization value may be further calculated based on each adjustment of the updated dye matrix, and a cross-correlation between dyes used in the assay. The method further includes generating an improved dye matrix based on the adjusted updated dye matrix, and then generating spectral adjusted data based on the improved dye matrix. [0006] In another exemplary embodiment, a system for reducing spectral crosstalk in a multiplexed assay is provided. The system includes a detector configured to receive filter signal data from a multiplexed fluorescence assay, and a memory configured to store an initial dye matrix including calibrated spectral dye data. The system further includes a processor configured to generate an updated dye matrix based on the initial dye matrix and estimated crosstalk proxies and adjust the updated dye matrix to meet a calculated optimization value. The calculated optimization value is calculated based on the estimated crosstalk proxies and the filter signal data. The calculated optimization value may be further calculated based on each adjustment of the updated dye matrix, and a cross-correlation between dyes used in the assay. The processor is further configured to generate an improved dye matrix based on the adjusted updated dye matrix, and then generate spectral adjusted data based on the improved dye matrix. [0007] In yet another exemplary embodiment, a computer-readable medium encoded with computer-readable instructions is provided. The computer-readable instructions, which when executed by a processor of a computer, causes the computer to carry out a method for reducing spectral crosstalk in a multiplexed assay. The method includes receiving filter signal data from a multiplexed fluorescence assay and an initial dye matrix including calibrated spectral dye data. The method further includes generating an updated dye matrix based on the initial dye matrix and estimated crosstalk proxies, and then adjusting the updated dye matrix to meet a calculated optimization value. The calculated optimization value is calculated based on at least the estimated crosstalk proxies and the filter signal data. The calculated optimization value may be further calculated based on each adjustment of the updated dye matrix, and a cross-correlation between dyes used in the assay. The method further includes generating an improved dye matrix Docket No. TP386609WO1 based on the adjusted updated dye matrix, and then generating spectral adjusted data based on the improved dye matrix. DESCRIPTION OF THE FIGURES [0008] FIG. 1 illustrates a flowchart showing a method of reducing spectral crosstalk in a multiplexed assay according to various embodiments described herein. [0009] FIG. 2 is a block diagram that illustrates a PCR instrument upon which embodiments of the present teachings may be implemented. [0010] FIG. 3 depicts an exemplary op