A method, apparatus and medium for determining an inter-channel crosstalk correction strength

Abstract

The application discloses a method, device and medium for determining the strength of inter-channel crosstalk correction, comprising: based on the multi-channel microscopic imaging system, detecting multiple rounds of reactions for edge synthesis and sequencing, so that in each round of reaction, a first channel signal and a second channel signal are generated, the first nucleotide and the second nucleotide are nucleotides containing different types of bases, and a part of the first channel signal is second channel crosstalk related noise; detecting the first channel signal and the second channel signal of each round of reaction, the first channel signal and the second channel signal both have strength; using a predetermined coefficient and the second channel signal strength to correct the first channel signal strength for inter-channel crosstalk, to determine the first channel signal strength after crosstalk correction, wherein the predetermined coefficient is a fixed coefficient related to the multi-channel microscopic imaging. The application can correct the inter-channel crosstalk, and improve the accuracy and efficiency of the signal correction and / or base recognition method.

Description

Technical Field

[0001] This application relates to the field of data processing technology, and more specifically to a method, apparatus and medium for determining the crosstalk correction strength between channels. Background Technology

[0002] Base calling is commonly used in nucleic acid sequencing. Specifically, on platforms that utilize optical imaging systems to acquire images of base extension signals multiple times and then process these images for nucleic acid sequencing, optical effects, spatial effects, or chemical reactions such as color difference, crosstalk, and / or phasing can significantly impact image acquisition, localization, or signal intensity, making accurate base calling difficult based on these images or image signals. The accuracy and efficiency of existing signal correction and / or base calling methods need improvement. Summary of the Invention

[0003] In view of the above, this application provides the following technical solution:

[0004] A method for determining the crosstalk correction strength between channels includes:

[0005] The multi-channel microscopic imaging system detects multiple rounds of sequencing-by-synthesis reactions, resulting in the generation of a first channel signal and a second channel signal in each round of the reaction. The first channel signal indicates the incorporation of a first nucleotide into multiple identical polynucleotide molecules in that round of the reaction, and the second channel signal indicates the incorporation of a second nucleotide into the multiple identical polynucleotide molecules in that round of the reaction. The first nucleotide and the second nucleotide are nucleotides containing different types of bases, and a portion of the first channel signal is noise related to crosstalk in the second channel.

[0006] The first channel signal and the second channel signal are detected in each round of reaction, and both the first channel signal and the second channel signal have intensity.

[0007] The crosstalk correction of the first channel signal intensity is performed using a predetermined coefficient and the second channel signal intensity to determine the crosstalk-corrected first channel signal intensity, wherein the predetermined coefficient is a fixed coefficient associated with the multi-channel microscopic imaging system.

[0008] An apparatus for determining the strength of inter-channel crosstalk correction, comprising:

[0009] The signal acquisition unit is used to detect multiple rounds of sequencing-by-synthesis reactions based on a multi-channel microscopic imaging system, so that a first channel signal and a second channel signal are generated in each round of reaction. The first channel signal indicates the signal of a first nucleotide being incorporated into multiple identical polynucleotide molecules in the round of reaction, and the second channel signal indicates the signal of a second nucleotide being incorporated into the multiple identical polynucleotide molecules in the round of reaction. The first nucleotide and the second nucleotide are nucleotides containing different types of bases, and a portion of the first channel signal is noise related to crosstalk in the second channel.

[0010] A signal detection unit is used to detect the first channel signal and the second channel signal in each round of reaction, both of which have intensity.

[0011] A correction unit is used to perform inter-channel crosstalk correction on the signal strength of the first channel using a predetermined coefficient and the signal strength of the second channel, so as to determine the crosstalk-corrected signal strength of the first channel, wherein the predetermined coefficient is a fixed coefficient associated with the multi-channel microscopic imaging system.

[0012] A computer-readable storage medium storing a program that can be executed by a processor to implement a method for determining the strength of inter-channel crosstalk correction as described in any one of the preceding descriptions.

[0013] As can be seen from the above technical solution, this application discloses a method and apparatus for determining the intensity of inter-channel crosstalk correction, comprising: detecting multiple rounds of sequencing-by-synthesis reactions based on a multi-channel microscopic imaging system, such that a first channel signal and a second channel signal are generated in each round of reaction, the first channel signal indicating the signal of a first nucleotide incorporated into multiple identical polynucleotide molecules in that round of reaction, and the second channel signal indicating the signal of a second nucleotide incorporated into multiple nucleotide molecules in that round of reaction, wherein the first nucleotide and the second nucleotide are nucleotides containing different types of bases, and a portion of the first channel signal is noise related to second channel crosstalk; detecting the first channel signal and the second channel signal in each round of reaction, both of which have intensity; and performing inter-channel crosstalk correction on the intensity of the first channel signal using predetermined coefficients and the intensity of the second channel signal to determine the crosstalk-corrected intensity of the first channel signal, wherein the predetermined coefficients are fixed coefficients associated with multi-channel microscopic imaging. This application can correct inter-channel crosstalk, improving the accuracy and efficiency of signal correction and / or base identification methods. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only embodiments of this application. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 A schematic flowchart of a base recognition method provided in an embodiment of this application;

[0016] Figure 2 A crosstalk diagram before correction is provided in an embodiment of this application;

[0017] Figure 3 A corrected crosstalk diagram provided for an embodiment of this application;

[0018] Figure 4 A schematic diagram of the excitation and emission spectra of different fluorescent groups provided in the embodiments of this application;

[0019] Figure 5 A schematic diagram of the spectrum of emitted light from different fluorescent groups entering a microscopic imaging system, provided as an embodiment of this application;

[0020] Figure 6 This is a schematic diagram of the structure of a base recognition device provided in an embodiment of this application;

[0021] Figure 7 Another crosstalk diagram before correction is provided for an embodiment of this application;

[0022] Figure 8 This is another corrected crosstalk diagram provided for an embodiment of this application. Detailed Implementation

[0023] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0024] In the embodiments of this application, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features; features defined with "first" or "second" may explicitly or implicitly include one or more of the stated features. Unless otherwise stated, "a group" or "a plurality" refers to two or more. Unless otherwise stated, the singular forms "a," "an," "the," etc. (a / an / the) include both singular and plural (more than one).

[0025] In this document, unless otherwise stated, the terms “comprising” or “including” are open-ended expressions and do not exclude the inclusion of other content or situations that fall under the description but are not listed or exemplified herein.

[0026] In this document, unless otherwise stated, nucleotide refers to the four natural nucleotides (such as dATP, dCTP, dGTP, and dTTP, or ATP, CTP, GTP, and UTP) or their derivatives, and is sometimes directly referred to by the bases they contain (A, T / U, C, G). Those skilled in the art will understand the reference to nucleotides or bases in the specific embodiments based on the context.

[0027] In this article, unless otherwise stated, polynucleotide molecules include single-stranded nucleic acid molecules, double-stranded nucleic acid molecules, or single-stranded-double-stranded complexes.

[0028] In this article, "sequencing" refers to sequence determination, the same as "nucleotide sequencing," which refers to the determination of the base order in a nucleotide sequence; including sequencing by synthesis (sequencing-while-synthesizing, SBS); including DNA sequencing and / or RNA sequencing; including long fragment sequencing and / or short fragment sequencing. The terms "long fragment" and "short fragment" are relative. For example, nucleotide molecules longer than 1Kb, 2Kb, 5Kb, or 10Kb can be called long fragments, while those shorter than 1Kb or 800bp can be called short fragments; including paired-end sequencing, single-end sequencing, and / or paired-end sequencing, etc. Paired-end sequencing or paired-end sequencing can refer to the readout of any two segments or parts of the same nucleotide molecule that do not completely overlap.

[0029] In this application, sequencing can be performed using a sequencing platform. According to the embodiments of this application, the sequencing platform that can be used to perform the above sequencing methods can be selected, but is not limited to, Illumina's HiSeq / Miseq / Nextseq / Novaseq sequencing platform, Thermo Fisher / Life Technologies' Ion Torrent platform, BGI GISEQ and MGISEQ / DNBSEQ platforms of BGI Genomics, and single-molecule sequencing platforms; the sequencing method can be single-end sequencing or paired-end sequencing.

[0030] In sequencing based on sequencing-by-synthesis (SBS), sequencing involves the process of linking, binding, or incorporating nucleotides (including nucleotide analogs) into a template under the catalysis of polymerases and collecting the corresponding reaction signals. Specifically, in platforms that implement SBS based on microscopic imaging systems, such as ILLUMINA's Miseq, Nextseq, and Novaseq platforms, and BGI Genomics' MGISEQ or DNBSEQ platforms, modified nucleotides are used to controllably achieve polymerase chain reactions to perform single-base extensions and collect the corresponding reaction signals. This process is repeated multiple times to determine the base sequence of the nucleic acid molecule to be tested (also known as the template). The repeated single-base extensions and corresponding signal collection mentioned here are generally referred to as a sequencing round or a sequencing cycle.

[0031] In this application, a sequencing round or a sequencing run refers to the process of extending one base and determining the type of the extended base. That is, in a sequencing round or a sequencing run, the determination of the base type at a given position on the template nucleotide molecule is completed. This method is also known as sequencing-by-synthesis (SBS). Generally, multiple sequencing runs are used to determine the order of multiple nucleotides / bases on the template.

[0032] A sequencing round, or one round of sequencing, can be defined as a single base extension of four nucleotides / bases. In other words, it's the process of determining the type of base at any specified position on the template. For SBS-based sequencing platforms, a sequencing round or one round of sequencing involves the binding of four nucleotides to the template and the acquisition of the corresponding reaction signals. The reaction system includes sequencing primers, reaction substrates (nucleotides), polymerase, and a template. Each sequencing round includes the following steps: binding a pre-defined sequence (sequencing primer) to the template; based on base pairing principles and polymerization, the added reaction substrate (nucleotide), catalyzed by the polymerase, controllably ligating to the 3' end of the sequencing primer, achieving base pairing with the corresponding position on the template. Typically, the added reaction substrate nucleotides carry specific fluorescent groups, which are detected using a microscopic imaging system to determine the extended base type.

[0033] In this application, a sequencing round or a single sequencing run may include one or more base extension reactions (repeat). For example, in one extension reaction, a mixture of all four nucleotides can be added to the reaction system, and the extension reaction and reaction signal can be collected, thereby determining the extended bases in a single extension reaction. Alternatively, the four nucleotides can be combined arbitrarily (e.g., in pairs) to determine the type of extended bases through at least one (e.g., two) extension reactions and reaction signal collection.

[0034] It should be noted that those skilled in the art will understand that after each extension reaction, images can be taken using a microscopic imaging system, and different channels can be used to detect different colors of fluorescence. Therefore, for each sequencing round or each sequencing cycle, at least one base image, or even multiple base images, needs to be collected to determine the type of extended bases. These base images can then be used for subsequent sequencing analysis.

[0035] Crosstalk, also known as laser-crosstalk, spectral crosstalk, or spectral crossover, refers to the phenomenon in sequencing where the signal corresponding to one base diffuses into the signal of another base. For example, in sequencing platforms that use different fluorescently labeled groups to identify different bases, if the emission spectra of two or more selected fluorescent groups overlap, it is possible to detect the diffusion of the signal of one fluorescent molecule from one channel to another in a sequencing run.

[0036] Phase discontinuity (phasing / prephasing), also known as phase imbalance, phase discontinuity, or phase difference, refers to the phenomenon in a chemical reaction where multiple identical polynucleotide molecules react asynchronously, including phasing (lagging) and prephasing (leading).

[0037] See Figure 1 This is a flowchart illustrating a method for determining the crosstalk correction strength between channels, provided in an embodiment of this application. The method may include the following steps:

[0038] S101. Based on the detection of a multi-channel microscopic imaging system, a multi-round reaction of synthesis and sequencing is performed, so that the first channel signal and the second channel signal are generated in each round of reaction.

[0039] A multi-channel microscopy system was used to detect sequencing information from multiple rounds of sequencing-by-synthesis reactions. The first channel signal indicates the incorporation of the first nucleotide into multiple identical polynucleotide molecules in that round, while the second channel signal indicates the incorporation of the second nucleotide into multiple identical polynucleotide molecules in that round. The first and second nucleotides are nucleotides containing different types of bases, and a portion of the first channel signal is noise related to crosstalk in the second channel. For example, in sequencing platforms that use different fluorescent molecules to identify different bases, if the emission spectra of two or more fluorescent groups overlap, it may be possible to detect the diffusion of fluorescence signals from one channel to another during a sequencing round.

[0040] S102, Detect the first channel signal and the second channel signal in each round of reaction.

[0041] Both the first channel signal and the second channel signal have intensity. Intensity can be a characterization of light intensity. Light intensity data can include pixel-related information of the first channel signal and the second channel signal on the base image, such as pixel grayscale values.

[0042] S103. Perform inter-channel crosstalk correction on the first channel signal strength using a predetermined coefficient and the second channel signal strength to determine the crosstalk-corrected first channel signal strength.

[0043] The predetermined coefficients are fixed parameters associated with the multi-channel microscopy system, that is, fixed parameters determined based on the configuration information of the multi-channel microscopy system.

[0044] In this embodiment of the application, crosstalk correction is performed on the first channel signal strength using a predetermined coefficient and the second channel signal strength to determine the crosstalk-corrected first channel signal strength, including:

[0045] The crosstalk correction signal strength of the first channel is determined by I1' = I1 - K * I2;

[0046] Wherein, I1' is the crosstalk-corrected first channel signal strength, I1 is the detected first channel signal strength, I2 is the detected second channel signal strength, and K is a predetermined coefficient.

[0047] When both the first channel signal and the second channel signal are fluorescence signal channels, determining the predetermined coefficients includes:

[0048] The possible range of values ​​for the predetermined coefficients is determined based on the spectral overlap region of the first and second channels.

[0049] Within the range of possible values, the values ​​that can achieve the preset correction requirements are determined as predetermined coefficients.

[0050] In this embodiment, the possible range of values ​​for the predetermined coefficients is determined based on the spectral maps corresponding to the first channel signal and the second channel signal, and then the final predetermined coefficients are determined by parameter traversal.

[0051] Furthermore, the process of determining the predetermined coefficient within the range of possible values ​​that can achieve the preset correction requirements may include:

[0052] Based on the configuration information of the multi-channel microscopic imaging system, the preset calibration requirements are determined;

[0053] The traversal parameters iterate through the corresponding values ​​within the possible range to determine the values ​​that meet the preset correction requirements, thus establishing a predetermined coefficient. The traversal parameters include at least a step size parameter, which represents the interval between two values ​​during the traversal process.

[0054] When determining preset calibration requirements based on the configuration information of a multi-channel microscopy imaging system, it is mainly related to the imaging configuration information of the multi-channel microscopy imaging system, such as the hardware configuration information of the multi-channel microscopy imaging system, or the optically detectable markers used during imaging. Correspondingly, the preset calibration requirements include alignment rate and / or error rate. Alignment rate represents the ratio of reads aligned to the reference genome to the total number of reads, i.e., alignment rate = (number of reads aligned to the reference genome / total number of reads) × 100%; "number of reads aligned to the reference genome" refers to the number of reads generated by sequencing that match the reference genome, and "total number of reads" refers to the total number of reads generated by sequencing; error rate represents the percentage of bases in the sequenced reads that do not match the reference genome.

[0055] In determining the predetermined coefficients, target sample data with known base identification results can be used. The base image obtained through a multi-channel microscopic imaging system can contain multiple signal points. The optical detection marker can be a fluorescent group; that is, the fluorescence signal emitted by the base carrying the fluorescent group under the illumination of a mixed-wavelength laser beam can be collected. The collected fluorescence signal is then imaged using the multi-channel microscopic imaging system to obtain the base image. Multiple signal points, along with their position and light intensity, can be obtained from the base image. The position can be a coordinate parameter, and the light intensity data can include pixel-related information of the signal point on the base image, such as pixel grayscale values.

[0056] When determining the base category corresponding to a signal point, the signal point in the base image for each type of base is obtained by laser excitation of a marker (such as a fluorophore) on the base to generate a fluorescence signal, which is then acquired and imaged by a microscopic imaging system. Each type of base emits fluorescence of a specific wavelength upon excitation. The microscopic imaging system acquires and images signals from each channel. If the fluorescence spectra emitted by two or more fluorophores overlap, crosstalk between channels will occur, causing other base signals to appear on the base image of a certain base. That is, a portion of the first channel signal described in this embodiment is noise related to crosstalk in the second channel, thus preventing correct base identification. Therefore, crosstalk correction processing is required for the light intensity data of each channel signal. After obtaining a predetermined coefficient, crosstalk correction can be performed on the first channel signal based on a common crosstalk correction method to determine the crosstalk-corrected first channel signal intensity.

[0057] When determining the predetermined coefficients for crosstalk correction based on target sample data, the target sample data consists of the light intensity data of the first and second channel signals generated on the target sample. This light intensity data can then be used to construct a crosstalk diagram. Each point in the crosstalk diagram represents a signal generated on the target sample. The horizontal axis of the crosstalk diagram represents the light intensity of the first channel signal, and the vertical axis represents the light intensity of the second channel signal. In the diagram, this is represented by two arms, or double arms, composed of multiple points, with an angle between them. Repeated verification revealed that when crosstalk correction meets the preset correction requirements—that is, when the preset comparison rate and / or error rate is met—the angle between the double arms is the target angle value corresponding to 90 degrees, with 90 degrees being the optimal value. To achieve this preset correction requirement, the corresponding values ​​within the possible range are iterated according to the traversal parameters to determine the values ​​that meet the preset correction requirements, which are then used as predetermined coefficients.

[0058] Specifically, the most accurate predetermined coefficient can be obtained when the angle between the two arms is 90 degrees. However, in practical applications, it has been found that when the predetermined coefficient obtained by the angle between the two arms fluctuates within a small range above and below 90 degrees, a relatively accurate crosstalk correction result can also be obtained. Therefore, in this embodiment, the preset correction requirement includes the angle between the two arms being a target angle value corresponding to 90 degrees. This target angle value includes 90 degrees, or it can include a value within a small range above and below 90 degrees. This range can be determined according to the actual application requirements. For example, it can be within the range of 90 degrees ± 1 degree or 0.5 degrees. Specifically, the first target angle value can be within the range of [89°, 91°]. During the traversal process, verification can be performed after each value is taken. However, if the range is large and there are many points that need to be verified, in order to improve the traversal efficiency, a preset step size parameter can be used to select the next value to be traversed. This step size parameter represents the interval parameter between two values ​​during the traversal process. Specifically, based on the crosstalk diagram, the strength values ​​of the first channel signal and the second channel signal corresponding to each point on the crosstalk diagram are determined. Based on the strength values ​​of the first channel signal and the second channel signal at each point, the range of possible values ​​is determined, and then the predetermined parameters are further determined by traversing within this range.

[0059] In this embodiment, the first channel can correspond to the signal channel of the first base, and the second channel can correspond to the signal channel of the second base. On the crosstalk graph, each point may include signal intensity information of two bases, such as A and C bases, or A and G bases, etc. The intensity value of the base corresponding to each point in the crosstalk graph can be the pixel grayscale value of that point in the base image. Based on the engineering approach of debugging and finding the optimal value of a parameter, the processing idea of ​​an evaluation function is usually adopted. That is, finding an index y that can evaluate the influence of the parameter, traversing or sampling the parameter x to obtain the distribution curve or partial distribution curve of the function y = f(x), thereby finding the global optimum or local optimum value as the setting value of parameter x, thus ensuring that the evaluation index y is in a relatively optimal range. Corresponding to the two methods for evaluating crosstalk correction provided in this application embodiment (i.e., two y indices), both can be used to determine the predetermined coefficients for crosstalk correction.

[0060] Method 1 is as described above. Crosstalk originates from the crosstalk of signal energy between channels. There are six possible combinations between the four bases of ACGT. See [link to relevant documentation]. Figure 2 , Figure 2The crosstalk diagram is shown. For example, there is significant energy crosstalk between AT and CG, meaning the angle between the two arms is acute; however, there is no crosstalk between AC, meaning the angle between the corresponding two arms is 90 degrees. It is evident that after crosstalk correction, it should be ensured that there is no crosstalk between any base combinations, achieving the effect of a 90-degree angle between signal arms (the arms are parallel to the corresponding coordinate axes), such as... Figure 2 The effect of the crosstalk diagram corresponding to AC bases. For details, please refer to... Figure 3 The crosstalk diagram shown shows that the angle between the two arms reaches 90 degrees after crosstalk correction. Although some corrections do not reach 90 degrees, they are still close to 90 degrees. In summary, whether the angle between any two arms reaches 90 degrees and is parallel to the coordinate axis can be used as an evaluation index for the effectiveness of crosstalk correction.

[0061] Furthermore, to reduce the number of traversals and the consumption of computational resources, a specific range can be determined based on the intensity values ​​corresponding to the bases represented by the horizontal and vertical axes in the current crosstalk graph. Then, a specific step size parameter for traversal can be determined based on this specific range. For example, if the specific range is large, the corresponding specific parameter step size is also large; if the specific range is small, the corresponding specific parameter step size should be small to improve accuracy. For example, all correction values ​​between 0.01 and 1 can be traversed with a step size of 0.01. See also the section on crosstalk correction for AT crosstalk graphs. Figure 3 The optimal AT correction value is 0.27, which means that the arm corresponding to the A base on the AT graph is parallel to the horizontal axis.

[0062] In this embodiment of the application, the value that can meet the preset correction requirements is a predetermined coefficient. The preset correction requirements include alignment rate and / or error rate. The alignment rate represents the ratio of the number of reads aligned to the reference genome to the total number of reads. The error rate represents the proportion of bases that do not match the reference genome when the reads generated by sequencing are aligned with the reference genome.

[0063] Since the purpose of crosstalk correction is to obtain more accurate base identification results, it is part of the grayscale value correction in base images. Therefore, the quality of crosstalk correction can be judged by the key indicators of base identification: alignment rate and error rate. Specifically, it involves identifying DNA inserts of known species (such as E. coli) or specific known tag sequences (such as barocde / index sequences), and then determining the accuracy of the DNA insert or tag sequence through alignment to obtain the corresponding alignment rate and / or error rate indicators, thereby judging the quality of the predetermined coefficients. Without changing other algorithm modules, the closer the predetermined coefficients are to the real situation, the more reliable the identification results and the better the alignment indicators.

[0064] Furthermore, in order to quickly obtain reliable predetermined coefficients for crosstalk correction, the crosstalk ratio between channels can be calculated and the predetermined coefficients can be determined by utilizing known spectral information and optical path design information of the multi-channel microscopic imaging system.

[0065] Specifically, such as Figure 4 and Figure 5 As shown. Figure 4 Excitation and emission spectra of different fluorescent groups are provided. Figure 5 A schematic diagram of the emission light from different fluorescent groups entering a microscopic imaging system is provided. Figure 4 In the schematic diagram of the spectrum, the dashed lines represent the excitation spectrum, emitted by the exciter; the solid lines represent the emission spectrum, emitted by different fluorescent groups (e.g., CY3, ROX, CY5, IF700). Figure 5 In the spectral diagram, the four bases ATCG are labeled with different fluorescent groups, for example, CY3 labels the A base, ROX labels the T base, CY5 labels the G base, and IF700 labels the C base. From Figure 5 It can be seen that the emission spectra of different fluorophores overlap. For example, there is partial overlap between the emission spectra of CY3 and ROX. When a microscopic imaging system detects the first and second channel signals generated by CY3 and ROX respectively, the first channel signal may crosstalk into the second channel, and / or the second channel signal may crosstalk into the first channel, i.e., inter-channel crosstalk occurs. Although the microscopic imaging system is equipped with dichroic mirrors and filters to disperse and filter the fluorescence signals generated by these two fluorophores (for example, filter 2 is used to transmit the fluorescence signal generated by ROX and filter the fluorescence signal generated by CY3; filter 1 is used to transmit the fluorescence signal generated by CY3 and filter the fluorescence signal generated by ROX), inter-channel crosstalk still occurs. This may be related to the optical path design of the microscopic imaging system. Therefore, the relevant inter-channel energy crosstalk ratio can be theoretically calculated using known spectral information and the optical path design information of the multi-channel microscopic imaging system. Figure 5 The gray and black shaded areas shown provide a theoretical reference value for the predetermined coefficients, which can be fine-tuned near the theoretical value during practical applications. The fine-tuning results are based on the highest comparison rate and the lowest error rate. Alternatively, only one of the indicators can be used as the correction target.

[0066] This application employs multiple methods to determine predetermined coefficients, and uses these predetermined coefficients to correct inter-channel crosstalk, thereby improving the accuracy and efficiency of signal correction and / or base identification methods.

[0067] It should be noted that, in order to apply the predetermined coefficients for crosstalk correction to more scenarios requiring crosstalk correction, this application can also use special data to verify the initially obtained predetermined coefficients. If the verification passes, they are confirmed as predetermined coefficients; otherwise, the corresponding initially obtained predetermined coefficients can be fine-tuned to obtain the final predetermined coefficients. In higher sequencing epochs, the phase correction value will decrease significantly, while in lower sequencing epochs, the phase correction value remains essentially unchanged (because the interference between these two types of crosstalk is not yet significant). To be applicable to different numbers of sequencing epochs and corresponding scenarios, this application's embodiments utilize verification sample data with specific characteristics to verify the determined fixed predetermined coefficients. These specific characteristics include at least the presence of missing arms in the crosstalk graph. For example... Figure 7 and Figure 8 As shown, this embodiment of the application uses validation sample data with missing arms in the crosstalk graph to validate the determined fixed predetermined coefficients. Successful validation, conversely, demonstrates that the predetermined coefficients used for crosstalk correction are applicable to crosstalk correction scenarios with missing arms. Furthermore, the validation sample data with specific characteristics used for validation may, for example, include sequencing epochs with specific characteristics. These sequencing epochs may be, for example, a specific number of sequencing epochs, which can represent higher or lower sequencing epochs; for example, it could be the 100th sequencing epoch or the 10th sequencing epoch.

[0068] After signal crosstalk correction by a predetermined coefficient, phase crosstalk between different sequencing epochs still exists. Therefore, in this embodiment, phase correction can be performed after crosstalk correction by a predetermined coefficient. That is, fixed crosstalk correction is performed first, and then phase correction is performed.

[0069] In this embodiment of the application, after performing inter-channel crosstalk correction on the first channel signal strength using a predetermined coefficient and the second channel signal strength, the method further includes:

[0070] Phase correction is performed on the first channel signal strength after inter-channel crosstalk correction using the detected first channel signal strength of the previous round of response and / or the next round of response, so as to obtain the phase-corrected first channel signal strength.

[0071] After crosstalk correction between channels, the first nucleotide incorporated into multiple identical nucleotide molecules in this round of reaction can be identified based on the signal intensity of the first channel after crosstalk correction. This enables base identification using signal data after phase correction.

[0072] Further, identifying the first nucleotide incorporated into multiple identical nucleotide molecules in this round of reaction based on the phase-corrected first channel signal intensity includes: determining a normalization parameter based on the signal intensity of each channel; normalizing the phase-corrected first channel signal intensity based on the normalization parameter to obtain a normalized first channel signal intensity; and identifying the first nucleotide incorporated into multiple identical nucleotide molecules in this round of reaction based on the normalized first channel signal intensity.

[0073] Because crosstalk correction requires fixed parameters, it has been found that differences in excitation power between different instruments and variations in the devices or samples used to support the test sample can lead to differences in signal intensity. After fixing the crosstalk correction, the signal intensity of a certain channel may exhibit exceptionally high values, which is detrimental to calculating the final corrected channel signal intensity. Therefore, it is necessary to normalize the intensity of each channel signal after phase correction. The specific process is shown in the following formula:

[0074] newInts=K'*(rawInts–pMvalue(AllTempBase)) / (pNvalue(AllTempBase)-pMvalue(AllTem pBase));

[0075] Where rawInts refers to the signal strength before normalization, newInts is the signal strength after normalization, K' is a constant, pMvalue(AllTempBase) represents the channel signal strength at the M quantile, pNvalue(AllTempBase) represents the channel signal strength at the N quantile, and N>M>0.

[0076] The above normalization will unify the final corrected channel signal strength to a preset range for comparison.

[0077] It should be noted that, in order to improve the processing effect in this embodiment, a normalization process can be performed before crosstalk correction. This normalization process can employ a zero-stress method to ensure that the background values ​​of each channel are the same. In one implementation, the normalization can be based on a template point at the image center, for example, 800*800 pixels, and a zero-stress translation can be performed. Each channel performs the following operations independently:

[0078] newlnts1=rawInts1-plvalue(midTemp);

[0079] Here, "this process" refers to the normalization process before crosstalk correction, "newlnts1" represents the normalized channel signal strength in this process, "rawInts1" refers to the channel signal strength before normalization in this process, and "p1value(midTemp)" represents the channel signal strength at the 1st quantile.

[0080] This application performs phase correction and normalization operations after crosstalk correction to complete base identification, further improving the accuracy of base identification.

[0081] In another embodiment of this application, a device for determining the crosstalk correction strength between channels is also provided, see [link to relevant documentation]. Figure 6 The device may include:

[0082] The signal acquisition unit 601 is used to detect multiple rounds of sequencing-by-synthesis reactions based on a multi-channel microscopic imaging system, so that a first channel signal and a second channel signal are generated in each round of reaction. The first channel signal indicates the signal of a first nucleotide being incorporated into multiple identical polynucleotide molecules in the round of reaction, and the second channel signal indicates the signal of a second nucleotide being incorporated into the multiple identical polynucleotide molecules in the round of reaction. The first nucleotide and the second nucleotide are nucleotides containing different types of bases, and a portion of the first channel signal is noise related to crosstalk in the second channel.

[0083] The signal detection unit 602 is used to detect the first channel signal and the second channel signal in each round of reaction. Both the first channel signal and the second channel signal have intensity.

[0084] The correction unit 603 is used to perform inter-channel crosstalk correction on the signal strength of the first channel using a predetermined coefficient and the signal strength of the second channel, so as to determine the crosstalk-corrected signal strength of the first channel, wherein the predetermined coefficient is a fixed coefficient associated with the multi-channel microscopic imaging system.

[0085] Optionally, the correction unit includes:

[0086] The first determining subunit is used to determine the crosstalk-corrected first channel signal strength by I1' = I1 - K * I2;

[0087] Where I1' is the signal strength of the first channel after crosstalk correction, I1 is the detected signal strength of the first channel, I2 is the detected signal strength of the second channel, and K is a predetermined coefficient.

[0088] Optionally, it also includes:

[0089] The phase correction subunit is used to perform phase correction on the first channel signal strength after inter-channel crosstalk correction using the first channel signal strength detected in the previous round of response and / or the next round of response, so as to obtain the phase-corrected first channel signal strength.

[0090] Optionally, the device further includes:

[0091] The recognition unit is used to identify the first nucleotide incorporated into multiple identical nucleotide molecules in this round of reaction based on the intensity of the first channel signal after phase correction.

[0092] Optionally, the device further includes a coefficient determination unit, configured to determine predetermined coefficients when both the first channel signal and the second channel signal are fluorescence signal channels. The coefficient determination unit includes:

[0093] The second determining subunit is used to determine the possible range of values ​​for a predetermined coefficient based on the spectral overlap region of the first and second channels.

[0094] The third determining subunit is used to determine the value that can achieve the preset correction requirements within the range of possible values, which is a predetermined coefficient.

[0095] Optionally, the third determining subunit is specifically used for:

[0096] Based on the configuration information of the multi-channel microscopic imaging system, the preset calibration requirements are determined;

[0097] The corresponding values ​​within the range of possible values ​​are traversed according to the traversal parameters to determine the values ​​that can meet the preset correction requirements and the predetermined coefficients are determined. The traversal parameters include at least the step size parameter, which represents the interval between two values ​​during the traversal process.

[0098] Optionally, the preset correction requirements include alignment rate and / or error rate, wherein the alignment rate represents the ratio of the number of reads aligned to the reference genome to the total number of reads;

[0099] Error rate characterizes the percentage of bases that do not match the reference genome when the sequenced reads are compared with the reference genome.

[0100] Optionally, the identification unit includes:

[0101] The fourth determining subunit is used to determine the normalization parameters based on the signal strength of each channel;

[0102] The normalization subunit is used to normalize the corrected first channel signal strength based on the normalization parameters to obtain the normalized first channel signal strength.

[0103] The identification subunit is used to identify the first nucleotide incorporated into multiple identical nucleotide molecules in this round of reaction based on the normalized first channel signal intensity.

[0104] It should be noted that the specific implementation of each unit and subunit in this embodiment can be referred to the corresponding content above, and will not be described in detail here.

[0105] In another embodiment of this application, a computer-readable storage medium is also provided for storing a program executable by a computer, the program including a method for determining the inter-channel crosstalk correction strength as described in any of the preceding claims.

[0106] In another embodiment of this application, an electronic device is also provided, which may include:

[0107] Memory, used to store applications and the data generated by the running of the applications;

[0108] A processor for executing the application to implement the method for determining the inter-channel crosstalk correction strength as described in any one of the above descriptions.

[0109] It should be noted that the specific implementation of the processor in this embodiment can be referred to the corresponding content above, and will not be described in detail here.

[0110] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since it corresponds to the method disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0111] Those skilled in the art will further recognize that the units and algorithm steps of the various examples described in conjunction with the embodiments disclosed herein can be implemented in electronic hardware, computer software, or a combination of both. To clearly illustrate the interchangeability of hardware and software, the components and steps of the various examples have been generally described in terms of functionality in the foregoing description. Whether these functions are implemented in hardware or software depends on the specific application and design constraints of the technical solution. Those skilled in the art can use different methods to implement the described functions for each specific application, but such implementation should not be considered beyond the scope of this application.

[0112] The steps of the methods or algorithms described in conjunction with the embodiments disclosed herein can be implemented directly by hardware, a software module executed by a processor, or a combination of both. The software module can be located in random access memory (RAM), main memory, read-only memory (ROM), electrically programmable ROM, electrically erasable programmable ROM, registers, hard disk, removable disk, CD-ROM, or any other form of storage medium known in the art.

[0113] The above description of the disclosed embodiments enables those skilled in the art to make or use this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for determining the crosstalk correction strength between channels, characterized in that, include: The multi-channel microscopic imaging system detects multiple rounds of sequencing-by-synthesis reactions, resulting in the generation of a first channel signal and a second channel signal in each round of the reaction. The first channel signal indicates the incorporation of a first nucleotide into multiple identical polynucleotide molecules in that round of the reaction, and the second channel signal indicates the incorporation of a second nucleotide into the multiple identical polynucleotide molecules in that round of the reaction. The first nucleotide and the second nucleotide are nucleotides containing different types of bases, and a portion of the first channel signal is noise related to crosstalk in the second channel. The first channel signal and the second channel signal are detected in each round of reaction, and both the first channel signal and the second channel signal have intensity. The crosstalk correction of the first channel signal intensity is performed using predetermined coefficients and the second channel signal intensity to determine the crosstalk-corrected first channel signal intensity. The predetermined coefficients are fixed coefficients associated with the multi-channel microscopic imaging system, and the determination of the predetermined coefficients includes: When the first channel signal and the second channel signal are fluorescence signals, the possible range of the predetermined coefficient is determined based on the spectral overlap region of the first channel and the second channel, and the value that can achieve the preset correction requirement is determined within the possible range of the predetermined coefficient as the predetermined coefficient. The step of performing inter-channel crosstalk correction on the first channel signal strength using a predetermined coefficient and the second channel signal strength to determine the crosstalk-corrected first channel signal strength includes: via I1 ' =I1-K*I2 determines the signal strength of the first channel after crosstalk correction; Among them, I1 ' I1 is the detected first channel signal strength after crosstalk correction, I2 is the detected second channel signal strength, and K is the predetermined coefficient.

2. The method according to claim 1, characterized in that, After performing inter-channel crosstalk correction on the first channel signal strength using predetermined coefficients and the second channel signal strength, the following steps are also included: Phase correction is performed on the first channel signal strength after inter-channel crosstalk correction using the detected first channel signal strength of the previous round of response and / or the next round of response, so as to obtain the phase-corrected first channel signal strength.

3. The method according to claim 2, characterized in that, The method further includes: The first nucleotide incorporated into multiple identical nucleotide molecules in this round of reaction is identified based on the intensity of the first channel signal after phase correction.

4. The method according to claim 3, characterized in that, Both the first channel signal and the second channel signal are fluorescence signals. Determining the predetermined coefficient includes: The possible range of values ​​for the predetermined coefficient is determined based on the spectral overlap region of the first and second channels; Determining the predetermined coefficient as a value that can achieve the preset correction requirement within the range of possible values ​​includes: Based on the configuration information of the multi-channel microscopic imaging system, the preset correction requirements are determined; The corresponding values ​​within the range of possible values ​​are traversed according to the traversal parameters to determine the value that can meet the preset correction requirements, which is the predetermined coefficient; the traversal parameters include at least a step size parameter, which represents the interval parameter between two values ​​during the traversal process.

5. The method according to claim 1, wherein the preset correction requirement includes comparison rate and / or error rate, wherein, The alignment rate represents the ratio of the number of reads aligned to the reference genome to the total number of reads; The error rate represents the percentage of bases that do not match the reference genome when the reads generated by sequencing are compared with the reference genome.

6. The method according to claim 3, characterized in that, The step of identifying the first nucleotide incorporated into multiple identical nucleotide molecules in this round of reaction based on the phase-corrected first channel signal intensity includes: Determine the normalization parameters based on the signal strength of each channel; The normalized signal strength of the first channel after phase correction is normalized based on the normalization parameter to obtain the normalized signal strength of the first channel. The first nucleotide incorporated into multiple identical nucleotide molecules in this round of reaction is identified based on the normalized first channel signal intensity.

7. A device for determining the crosstalk correction strength between channels, characterized in that, include: The signal acquisition unit is used to detect multiple rounds of sequencing-by-synthesis reactions based on a multi-channel microscopic imaging system, so that a first channel signal and a second channel signal are generated in each round of reaction. The first channel signal indicates the signal of a first nucleotide being incorporated into multiple identical polynucleotide molecules in the round of reaction, and the second channel signal indicates the signal of a second nucleotide being incorporated into the multiple identical polynucleotide molecules in the round of reaction. The first nucleotide and the second nucleotide are nucleotides containing different types of bases, and a portion of the first channel signal is noise related to crosstalk in the second channel. A signal detection unit is used to detect the first channel signal and the second channel signal in each round of reaction, both of which have intensity. A correction unit is configured to perform inter-channel crosstalk correction on the first channel signal intensity using a predetermined coefficient and the second channel signal intensity, to determine the crosstalk-corrected first channel signal intensity, wherein the predetermined coefficient is a fixed coefficient associated with the multi-channel microscopic imaging system; the correction unit includes: The first defined subunit is used to pass I1 ' =I1-K*I2 determines the signal strength of the first channel after crosstalk correction; Among them, I1 ' I1 is the detected first channel signal strength after crosstalk correction, I2 is the detected second channel signal strength, and K is the predetermined coefficient.

8. The apparatus according to claim 7, further comprising: A phase correction subunit is used to perform phase correction on the first channel signal strength after inter-channel crosstalk correction using the detected first channel signal strength of the previous round of response and / or the next round of response, so as to obtain the phase-corrected first channel signal strength.

9. The apparatus according to claim 8, further comprising: The identification unit is used to identify the first nucleotide incorporated into multiple identical nucleotide molecules in this round of reaction based on the intensity of the first channel signal after phase correction.

10. The apparatus according to any one of claims 7-9, further comprising a coefficient determining unit, configured to determine the predetermined coefficient when both the first channel signal and the second channel signal are fluorescence signals, the coefficient determining unit comprising: The second determining subunit is used to determine the possible range of values ​​for the predetermined coefficient based on the spectral overlap region of the first channel and the second channel. The third determining subunit is used to determine the predetermined coefficient as a value that can achieve the preset correction requirements within the range of possible values.

11. The apparatus according to claim 10, wherein the third determining subunit is specifically used for: Based on the configuration information of the multi-channel microscopic imaging system, the preset correction requirements are determined; The corresponding values ​​within the range of possible values ​​are traversed according to the traversal parameters to determine the value that can meet the preset correction requirements, which is the predetermined coefficient; the traversal parameters include at least a step size parameter, which represents the interval parameter between two values ​​during the traversal process.

12. The apparatus according to claim 10, wherein the preset calibration requirement includes a comparison rate and / or an error rate, wherein, The alignment rate represents the ratio of the number of reads aligned to the reference genome to the total number of reads; The error rate represents the percentage of bases that do not match the reference genome when the reads generated by sequencing are compared with the reference genome.

13. The apparatus according to claim 9, wherein the identification unit comprises: The fourth determining subunit is used to determine the normalization parameters based on the signal strength of each channel; The normalization subunit is used to normalize the phase-corrected first channel signal strength based on the normalization parameters to obtain the normalized first channel signal strength. The identification subunit is used to identify the first nucleotide incorporated into multiple identical nucleotide molecules in this round of reaction based on the normalized first channel signal intensity.

14. A computer-readable storage medium, characterized in that, The medium stores a program that can be executed by a processor to implement the method for determining the inter-channel crosstalk correction strength according to any one of claims 1-6.

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