A high-throughput sequencing library on-machine base balance system and method

Abstract

This invention relates to the field of high-throughput sequencing library preprocessing and sequencing technology, and discloses a high-throughput sequencing library base balancing system and method, including: a physicochemical parameter determination module to obtain library fragment length and concentration; a flow channel distribution control module to limit the proportion of unbalanced libraries to less than 30%; a concentration conversion module and a kinetic compensation module to calculate molar concentration and adjust the complexation coefficient, lowering the coefficient of small molecule ribonucleic acid libraries and increasing the coefficient of whole-genome bisulfite sequencing libraries; and a volume complexation module to mix and construct a base neutralization complexation system. This invention utilizes the synergistic complementarity of mass transfer resistance and affinity of nucleic acid fragments on the solid-phase surface to balance fluorescence throughput density, eliminate pixel overflow and algorithm errors, and maximize effective throughput while eliminating the physical space occupation of standards.

Description

Technical Field

[0001] This invention relates to a base balancing system and method for high-throughput sequencing libraries, belonging to the field of high-throughput sequencing library preprocessing and sequencing technology. Background Technology

[0002] Current high-throughput sequencing relies on multi-channel fluorescence detection systems to capture the radiation flux during single nucleotide monomer synthesis. Furthermore, multi-sample mixed sequencing requires mixing target libraries with different sequence characteristics to maintain base ratio balance within the sequencing cycle. Nucleic acid molecules bound to the surface of the solid-phase carrier follow specific diffusion laws and hybridization thermodynamics, resulting in a negative correlation between fragment mass transfer rate and molecular weight. The double-strand melting temperature depends on the symmetry of base distribution within the sequence. When processing heterogeneous combinations of small nucleotide libraries and whole-genome methylation sequencing libraries, the inherent differences in fragment length and complexity translate into non-uniform competition for surface primers during the solid-phase bridge amplification stage. Short fragments rapidly occupy space due to their high spatial diffusion rate, leading to local signal saturation. Libraries experiencing asymmetric base loss due to bisulfite conversion suffer hybridization efficiency degradation due to lower melting temperatures. This kinetic bias causes pixel overflow or signal loss in multi-channel fluorescence detection cameras and poses potential risks of algorithmic errors and data loss during low-complexity base recognition.

[0003] Conventional improvement approaches typically attempt to neutralize base biases by crudely adjusting the molar concentration of each component or incorporating a high proportion of standard libraries. However, simply changing the concentration cannot reverse the inherent thermodynamic differences of molecules, and library components that do not produce effective sequences will inevitably physically crowd out spatial channels. This leads to an inherent constraint between maintaining signal balance and the effective throughput of the chip. In addition to the limitations of solid-phase occupancy and spatial channel crowding in the sequencing chip's flow channels, pre-loading library standardization and pre-processing control methods for mixing samples also suffer from kinetic imbalances. For example, Chinese invention patent application CN109680042A discloses a sequencing library standardization method using magnetic beads for DNA mixing and processing. The kit uses a fixed amount of magnetic beads to specifically bind to the sticky sequences at the ends of the library, obtaining a fixed amount of DNA molecules during the elution stage to eliminate the need for quantitative detection and achieve equimolar mixing of diverse libraries. However, the establishment of this static homogenization control scheme implicitly relies on the ideal premise of highly consistent sample molecular weight and thermodynamic stability. In the face of real-world conditions with high heterogeneity including small RNAs and whole-genome methylation sequencing, equimolar capture ignores the objective differences in fragment mass transfer resistance and melting temperature. Relying solely on magnetic bead saturation capture for equimolar distribution cannot offset the competitive advantage of short fragment mass transfer and the deterioration of hybridization efficiency of methylated libraries. Instead, it may induce space occupancy imbalance and fluorescence flux overflow on the surface of the solid-phase flow channel.

[0004] Therefore, the technical problem to be solved by this invention is how to balance the multi-channel fluorescence radiation flux density by the synergistic complementarity of the mass transfer resistance and binding affinity of heterogeneous nucleic acid fragments on the solid surface without exceeding the rigid boundary of a specific equivalent ratio, thereby eliminating pixel overflow and algorithm error of optical sensors. Summary of the Invention

[0005] To address the problems in the background art, the technical solution of the present invention is as follows: A high-throughput sequencing library base balancing system, comprising: The physicochemical parameter determination module is used to obtain the library type, average fragment length, and mass concentration of the nucleic acid library of the sample to be tested. The library types include small molecule ribonucleic acid libraries and whole genome bisulfite sequencing libraries. The flow channel distribution control module is used to distribute the nucleic acid library of the test sample and limit the initial equivalent proportion of the nucleic acid library of the test sample as an unbalanced library in the same solid phase flow channel to be less than 30%. The nominal molar concentration conversion module is used to calculate the nominal molar concentration of each nucleic acid library to be tested based on the mass concentration and average fragment length. The hybridization kinetics compensation module is used to adjust the mating coefficient according to the library type. The initial equivalent ratio is multiplied by the mating coefficient to determine the final input amount. The mating coefficient of small molecule ribonucleic acid libraries is down-adjusted, while the mating coefficient of whole genome bisulfite sequencing libraries is up-adjusted. The physical volume mixing module is used to determine the physical pipetting volume of each nucleic acid library to be tested, and to physically mix the libraries corresponding to each physical pipetting volume to construct a base neutralization mixing system, thereby realizing the hybridization and amplification of nucleic acid fragments with solid-phase primers.

[0006] Preferably, it also includes a boundary constraint module; the boundary constraint module configures an initial equivalent ratio for each library type according to the overall base fluorescence balance requirement of the solid phase channel in the sequencing cycle.

[0007] Preferably, the hybridization kinetics compensation module includes a ribonucleic acid library regulation submodule; the ribonucleic acid library regulation submodule identifies the test sample whose library type is a small molecule ribonucleic acid library, and lowers and corrects the matching coefficient corresponding to the small molecule ribonucleic acid library.

[0008] Preferably, the hybridization kinetics compensation module includes a methylation sequencing library regulation submodule; the methylation sequencing library regulation submodule identifies the test sample whose library type is a whole genome bisulfite sequencing library, and up-regulates and amplifies the matching coefficient corresponding to the whole genome bisulfite sequencing library to compensate.

[0009] Preferably, the physical volume mixing module is connected to the automatic pipetting unit and outputs the calculated physical pipetting volumes to the automatic pipetting unit.

[0010] Preferably, it also includes a fluorescence signal monitoring module; the fluorescence signal monitoring module receives the base fluorescence intensity signal released by the sequencer in the sequencing cycle, and adjusts the allocation ratio of the flow channel allocation control module when the base fluorescence intensity signal exceeds the set saturation threshold to avoid pixel overflow of the sequencing image.

[0011] Preferably, it also includes a sequencing cycle attenuation analysis module; the sequencing cycle attenuation analysis module records the cumulative number of sequencing cycles performed by the sequencer, and calculates the cycle signal attenuation coefficient, which characterizes the attenuation trend of solid primer hybridization efficiency, based on the cumulative number of cycles; when the cycle signal attenuation coefficient exceeds the set attenuation threshold, the hybridization kinetics compensation module adds a basic gain value to the matching coefficient of each nucleic acid library to be tested.

[0012] Preferably, it also includes a library quality assessment module; before the physicochemical parameter determination module obtains the mass concentration of each nucleic acid library to be tested, the library quality assessment module receives the input initial mass concentration sequence, and outputs a quality abnormality warning when it detects that the initial mass concentration sequence of any nucleic acid library is lower than the set lower limit threshold, and at the same time terminates the physical pipetting volume output of the physical volume reconstitution module.

[0013] Preferably, it also includes a base balance verification module; the base balance verification module receives the base distribution composition of each nucleic acid library to be tested in the sequencing cycle, and calculates the fluorescence complementarity of the four bases with reversible termination ends on the surface of the sequencing chip. When the fluorescence complementarity deviates from the set balance range, the matching coefficient is corrected by the hybridization kinetics compensation module to make the fluorescence complementarity stable within the set balance range.

[0014] A method for base balancing in high-throughput sequencing libraries, implemented using a high-throughput sequencing library base balancing system, includes the following steps: Step S1: Obtain the library type, average fragment length, and mass concentration of the nucleic acid library of the sample to be tested through the physicochemical parameter determination module. The library types include small molecule ribonucleic acid libraries and whole genome bisulfite sequencing libraries. Step S2: Distribute the nucleic acid library of the test sample through the flow channel distribution control module, and limit the total initial equivalent proportion of the nucleic acid library of the test sample as an unbalanced library in the same solid phase flow channel to be less than 30%; Step S3: Calculate the nominal molar concentration of each test sample nucleic acid library based on the mass concentration and average fragment length using the nominal molar concentration conversion module. Step S4: The hybridization kinetics compensation module adjusts the matching coefficient according to the library type, and the initial equivalent ratio is multiplied by the matching coefficient to determine the final input amount. The matching coefficient of small molecule ribonucleic acid libraries is lowered, and the matching coefficient of whole genome bisulfite sequencing libraries is raised. Step S5: The physical pipetting volume of each nucleic acid library to be tested is determined by the physical volume mixing module, and the libraries corresponding to each physical pipetting volume are physically mixed to construct a base neutralization mixing system, thereby realizing the hybridization and amplification of nucleic acid fragments with solid primers.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. In the base equilibration of high-throughput sequencing libraries, the nominal molar concentration is established by simultaneously collecting the average fragment length and mass concentration of different nucleic acid libraries. Based on this, the matching coefficient is dynamically adjusted according to the sequence structure characteristics of each nucleic acid library and the differences in hybridization amplification kinetics on the solid-phase surface of the flow channel. This reduces the matching coefficient of small molecule nucleic acid libraries with high spatial diffusion rates due to their shorter fragments, thereby suppressing excessive competition of their clonal populations and space overload. At the same time, it increases the matching coefficient of whole-genome methylated sequencing libraries with lower melting temperatures due to base asymmetry loss after bisulfite conversion, thereby offsetting the deterioration of their hybridization efficiency on solid-phase primers. Through the synergistic complementarity of nucleic acid fragments with different physicochemical properties in terms of mass transfer resistance and binding affinity on the solid-phase flow channel surface, the heterogeneous nucleic acid mixed system forms a spatially uniform and highly interleaved clonal microarray during the solid-phase bridge amplification stage.

[0016] 2. This method constructs a sequencing library pool with its own fluorescence complementarity by configuring a composite system containing balanced and unbalanced libraries within the sequencing channel and setting the total equivalent proportion of unbalanced libraries below a certain limit as a rigid boundary constraint. Through the spatial interleaving and mutual neutralization of the balanced and kinetically calibrated unbalanced libraries in terms of molecular base distribution logic, a sequencing library pool with its own fluorescence complementarity is constructed. This eliminates the need for the artificial introduction of commercial standards that do not contribute effective target sequence data for optical dilution, and eliminates the physical occupation of the solid-phase carrier channel resources of the sequencing chip by hybridization fragments with no business value. This ensures that every sequence read generated on the sequencing matrix belongs to the target sample to be tested, thereby maximizing the output of the effective throughput of the solid-phase channel while ensuring base fluorescence balance.

[0017] 3. This method calculates the physical input volume based on the nominal molar concentration and the final input amount after affinity bias compensation. Heterogeneous nucleic acid libraries are physically mixed according to the physical input volume to construct a complex system. By precisely controlling the clonal cluster density and base spatial crossover on the physical surface of the solid flow channel, the fluorescence signal radiation flux density released by the single nucleotide monomers with reversible fluorescent termination ends at each spatial coordinate node is maintained within the equilibrium range in each cycle step of the sequencing instrument during synthesis-while-sequencing. This avoids pixel overflow of the optical sensor camera caused by local fluorescence signal oversaturation, eliminates the risk of deadlock and misjudgment of the base recognition algorithm due to the lack of fluorescence signal in specific channels, ensures the recognition accuracy of the optical signal of the multi-channel fluorescence detection system, and improves the data output quality in the process of full-read sequence determination. Attached Figure Description

[0018] Figure 1 This is a flowchart of the high-throughput sequencing library base balancing and hybridization amplification method of the present invention; Figure 2 This diagram illustrates the influencing factors of fluorescence signal imbalance and pixel overflow in high-throughput sequencing according to the present invention.

[0019] The objectives, features, and advantages of this invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0020] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0021] A high-throughput sequencing library base balancing system includes: The physicochemical parameter determination module is used to obtain the library type, average fragment length, and mass concentration of the nucleic acid library of the sample to be tested. The library types include small molecule ribonucleic acid libraries and whole genome bisulfite sequencing libraries. The flow channel distribution control module is used to distribute the nucleic acid library of the test sample and limit the initial equivalent proportion of the nucleic acid library of the test sample as an unbalanced library in the same solid phase flow channel to be less than 30%. The nominal molar concentration conversion module is used to calculate the nominal molar concentration of each nucleic acid library to be tested based on the mass concentration and average fragment length. The hybridization kinetics compensation module is used to adjust the mating coefficient according to the library type. The initial equivalent ratio is multiplied by the mating coefficient to determine the final input amount. The mating coefficient of small molecule ribonucleic acid libraries is down-adjusted, while the mating coefficient of whole genome bisulfite sequencing libraries is up-adjusted. The physical volume mixing module is used to determine the physical pipetting volume of each nucleic acid library to be tested, and to physically mix the libraries corresponding to each physical pipetting volume to construct a base neutralization mixing system, thereby realizing the hybridization and amplification of nucleic acid fragments with solid-phase primers.

[0022] Preferably, it also includes a boundary constraint module; the boundary constraint module configures an initial equivalent ratio for each library type according to the overall base fluorescence balance requirement of the solid phase channel in the sequencing cycle.

[0023] Preferably, the hybridization kinetics compensation module includes a ribonucleic acid library regulation submodule; the ribonucleic acid library regulation submodule identifies the test sample whose library type is a small molecule ribonucleic acid library, and lowers and corrects the matching coefficient corresponding to the small molecule ribonucleic acid library.

[0024] Preferably, the hybridization kinetics compensation module includes a methylation sequencing library regulation submodule; the methylation sequencing library regulation submodule identifies the test sample whose library type is a whole genome bisulfite sequencing library, and up-regulates and amplifies the matching coefficient corresponding to the whole genome bisulfite sequencing library to compensate.

[0025] Preferably, the physical volume mixing module is connected to the automatic pipetting unit and outputs the calculated physical pipetting volumes to the automatic pipetting unit.

[0026] Preferably, it also includes a fluorescence signal monitoring module; the fluorescence signal monitoring module receives the base fluorescence intensity signal released by the sequencer in the sequencing cycle, and adjusts the allocation ratio of the flow channel allocation control module when the base fluorescence intensity signal exceeds the set saturation threshold to avoid pixel overflow of the sequencing image.

[0027] Preferably, it also includes a sequencing cycle attenuation analysis module; the sequencing cycle attenuation analysis module records the cumulative number of sequencing cycles performed by the sequencer, and calculates the cycle signal attenuation coefficient, which characterizes the attenuation trend of solid primer hybridization efficiency, based on the cumulative number of cycles; when the cycle signal attenuation coefficient exceeds the set attenuation threshold, the hybridization kinetics compensation module adds a basic gain value to the matching coefficient of each nucleic acid library to be tested.

[0028] Preferably, it also includes a library quality assessment module; before the physicochemical parameter determination module obtains the mass concentration of each nucleic acid library to be tested, the library quality assessment module receives the input initial mass concentration sequence, and outputs a quality abnormality warning when it detects that the initial mass concentration sequence of any nucleic acid library is lower than the set lower limit threshold, and at the same time terminates the physical pipetting volume output of the physical volume reconstitution module.

[0029] Preferably, it also includes a base balance verification module; the base balance verification module receives the base distribution composition of each nucleic acid library to be tested in the sequencing cycle, and calculates the fluorescence complementarity of the four bases with reversible termination ends on the surface of the sequencing chip. When the fluorescence complementarity deviates from the set balance range, the matching coefficient is corrected by the hybridization kinetics compensation module to make the fluorescence complementarity stable within the set balance range.

[0030] A method for base balancing in high-throughput sequencing libraries, implemented using a high-throughput sequencing library base balancing system, includes the following steps: Step S1: Obtain the library type, average fragment length, and mass concentration of the nucleic acid library of the sample to be tested through the physicochemical parameter determination module. The library types include small molecule ribonucleic acid libraries and whole genome bisulfite sequencing libraries. Step S2: Distribute the nucleic acid library of the test sample through the flow channel distribution control module, and limit the total initial equivalent proportion of the nucleic acid library of the test sample as an unbalanced library in the same solid phase flow channel to be less than 30%; Step S3: Calculate the nominal molar concentration of each test sample nucleic acid library based on the mass concentration and average fragment length using the nominal molar concentration conversion module. Step S4: The hybridization kinetics compensation module adjusts the matching coefficient according to the library type, and the initial equivalent ratio is multiplied by the matching coefficient to determine the final input amount. The matching coefficient of small molecule ribonucleic acid libraries is lowered, and the matching coefficient of whole genome bisulfite sequencing libraries is raised. Step S5: The physical pipetting volume of each nucleic acid library to be tested is determined by the physical volume mixing module, and the libraries corresponding to each physical pipetting volume are physically mixed to construct a base neutralization mixing system, thereby realizing the hybridization and amplification of nucleic acid fragments with solid primers.

[0031] Example 1: The system of this invention is applied to the library construction and sequencing process of high-throughput sequencing. When performing heterogeneous library pooled sequencing, the average fragment length and mass concentration of the balanced and unbalanced libraries are obtained through the physicochemical parameter measurement module, and these physical quantities are used as the input basis for solid-phase bridge amplification of nucleic acid sequences. In the flow channel allocation control module, the system pre-configures each sample to be tested according to the rated data output of 250G / lane per flow channel of the sequencing chip. When the system receives a mixed sample sequence containing a small molecule ribonucleic acid library and a whole genome bisulfite sequencing library, the preset control logic triggers the monitoring of the proportion of unbalanced libraries. The nominal molar concentration of each component is calculated by the nominal molar concentration conversion module based on the mass concentration and average fragment length of each library. This concentration represents the number of nucleic acid molecules per unit volume.

[0032] The hybridization kinetics compensation module dynamically adjusts the mating coefficient: for small molecule ribonucleic acid libraries, the system adjusts the mating coefficient to 0.8 based on the space overload phenomenon caused by the high diffusion rate on the solid phase surface of the flow channel; for whole-genome bisulfite sequencing libraries, the system adjusts the mating coefficient to 1.2 based on the deterioration of solid phase primer hybridization efficiency caused by the low melting temperature after bisulfite conversion. The physical volume mating module calculates the final input amount of each library based on the nominal molar concentration, equivalent ratio, and adjusted mating coefficient, thereby determining the corresponding... Specifically, regarding the pipetting volume, the physical volume mixing module determines the pipetting input volume by multiplying the initial equivalent proportion of each nucleic acid library in the test sample by its corresponding mixing coefficient to obtain the final target molar proportion of the library. Next, combining this with the preset total molar load for a single flow channel, the required final target absolute molar volume of the library is calculated. This final target absolute molar volume is then divided by the nominal molar concentration of the library output by the nominal molar concentration conversion module to derive the corresponding absolute value of the physical pipetting input volume. Finally, if the total volume exceeds or falls short of the flow channel's rated loading volume, a proportional adjustment is made. The scaling factor normalizes the volume of all components to ensure the total volume is precisely equal to the rated loading volume of 15 μL. An automated pipetting unit mixes the libraries according to volume ratios to construct a base-neutralized complex system, which is then injected into the sequencer. During sequencing-while-synthesizing, the sequencer's optical sensors monitor the fluorescence signals at each spatial coordinate node. When the intensity signal of a specific channel in a given cycle deviates from the preset four-color balance threshold range, a feedback loop drives the multidimensional complexing pooling unit to fine-tune the complexing coefficients in subsequent batches in real time, preventing pixel overflow in local fluorescence channels. To ensure stable sequencing data output quality throughout the cycle, the multidimensional compounding pooling unit is structurally composed of a multi-channel microfluidic pipetting matrix and an array of electromagnetic microvalve groups. Its control logic integrates a library matrix mapping algorithm. After receiving the compounding coefficient fine-tuning command from the feedback loop, the unit uses its internal microprocessor to convert the one-dimensional coefficient variable into a control voltage signal for the multi-channel liquid flow, dynamically changing the opening time step of the microvalve. This allows for direct adjustment of the mixing throughput of different library components at the microfluidic level, achieving real-time online conversion from data computation to physical pipetting actions.

[0033] Example 2: This invention was systematically validated in a sequencing production environment for a multiplex sequencing scenario containing heterogeneous libraries. The experiment selected a composite sample containing mRNA, exon sequencing, small RNA, and whole-genome bisulfite sequencing libraries as the research object. Before the experiment, the physicochemical parameter measurement module determined the initial mass concentration and average fragment length of each library. The average fragment length of the small RNA library was 25.4 bp, and the mass concentration was 2.52 ng / μl; the average fragment length of the whole-genome bisulfite sequencing library was 302.3 bp, and the mass concentration was 15.18 ng / μl. The flow channel allocation control module set rigid boundaries within the flow channels to ensure that the total initial equivalent proportion of the imbalanced libraries was controlled within a threshold of 28.4%, thus avoiding the nonlinear physical crowding effect of high-proportion imbalanced fragments on the solid surface of the flow channels. The initial equivalent ratio of 30% is a rigid boundary determined experimentally based on the spatial steric hindrance limit of the spatial arrangement of clonal clusters on the solid-phase flow channel surface and the linear response range of the optical imaging system. Experimental tests show that when the total proportion of the unbalanced library is controlled below 30%, the competitive occupancy of primers on the solid-phase surface of the flow channel exhibits a locally controllable linear state, and the base bias can be completely neutralized by subsequent adjustment of the kinetic coefficients. However, once the proportion reaches or exceeds 30%, the rapid diffusion of short fragments and the dense aggregation of low-complexity fragments will cause a nonlinear surge in the spatial congestion effect, resulting in the spatial overlap of clonal clusters on the flow channel surface exceeding the dissociation limit of the image segmentation algorithm, which increases the pixel overflow frequency of the sequencer camera by more than 3 times. Even with the use of dynamic coefficient adjustment, it is impossible to maintain the full-cycle base interpretation accuracy above 95%. Therefore, 30% is established as the absolute rigid boundary upper limit to ensure the efficiency of flow channel data output.

[0034] During the experiment, two sets of control verification experiments were designed: one set was the control sample group, which did not apply the compensation logic of this invention and was simply mixed in equal mass proportions; the other set was the experimental sample group, which applied the kinetic compensation logic of this invention. In the hybridization kinetic compensation stage, for small molecule ribonucleic acid libraries, based on their high diffusion rate characteristics on the solid-phase surface, the matching coefficient was set to 0.81 through control logic to reduce their spatial occupancy weight at the amplification node; for whole-genome bisulfite sequencing libraries, based on the hybridization rate loss caused by the low melting temperature after transformation, their matching coefficient was set to 1.18 to enhance their binding competitiveness on solid-phase primers. The experimental monitoring indicators focused on the optical sensor pixel saturation and base interpretation accuracy within the sequencing cycle range of 1 to 150 cycles.

[0035] Experimental data showed that during cycles 30 to 60, the optical sensor pixels in the control group frequently overflowed, causing the base interpretation accuracy to drop from 99.2% at the beginning to 94.5%. Data analysis revealed that the root cause of this accuracy decline was excessive competition during the cloning and amplification process of the small molecule ribonucleic acid library, leading to local oversaturation of the fluorescence signal. This was accompanied by the loss of key signals due to insufficient hybridization efficiency in the whole-genome bisulfite sequencing library. In the same cycle interval, the experimental group maintained the optical signal-to-noise ratio at a preset stable level by utilizing the differential compensation logic of the matching coefficient. Within a defined range, under this compensation mechanism, the base interpretation accuracy throughout the entire cycle is maintained between 99.1% and 99.3%, and the effective data output ratio of each type of sample deviates from the target value by no more than 1.8%. This data fluctuation range meets the tolerance requirements of industrial production, confirming that without introducing additional physical doping standards, this invention effectively optimizes the data output efficiency of the sequencer channel by physically adjusting the mass transfer resistance and binding affinity of nucleic acid fragments, thus solving the problem of fluorescence signal imbalance during the sequencing process of heterogeneous library mixing.

[0036] Example 3: This example combines Figures 1 to 2 This document describes a base balancing system and method for high-throughput sequencing libraries, such as... Figure 1 As shown, steps S1 to S5 are as follows: Step S1 involves obtaining the library type, average fragment length, and mass concentration of the nucleic acid library of the test sample through the physicochemical parameter determination module. The library type includes small molecule ribonucleic acid libraries and whole-genome bisulfite sequencing libraries. Then, step S2 is executed, in which the nucleic acid library of the test sample is allocated through the flow channel allocation control module, and the initial equivalent proportion of the nucleic acid library of the test sample as an unbalanced library in the same solid phase flow channel is limited to less than 30%. Then, step S3 is executed, in which the nominal molar concentration conversion module calculates the mass concentration and average fragment length of each test sample. The nominal molar concentration of the sample nucleic acid library is determined, and then step S4 is executed. The hybridization kinetics compensation module adjusts the mixing coefficient according to the library type. The initial equivalent ratio is multiplied by the mixing coefficient to determine the final input amount. The mixing coefficient of small molecule ribonucleic acid libraries is lowered, and the mixing coefficient of whole genome bisulfite sequencing libraries is raised. Finally, step S5 is completed. The physical volume mixing module determines the physical pipetting volume of each sample nucleic acid library to be tested, and the libraries corresponding to each physical pipetting volume are physically mixed to construct a base neutralization mixing system to achieve hybridization amplification of nucleic acid fragments with solid-phase primers.

[0037] like Figure 2As shown, the factors leading to fluorescence signal imbalance and pixel overflow in high-throughput sequencing involve mass transfer resistance and diffusion characteristics, channel allocation and boundary constraints, hybridization thermodynamics and efficiency degradation, and cycle monitoring and dynamic verification. Among them, mass transfer resistance and diffusion characteristics include short fragments of small molecule ribonucleic acid libraries, high spatial diffusion rates and rapid site occupancy, and excessive competition for signal saturation by clonal populations. Channel allocation and boundary constraints include the initial equivalent ratio of unbalanced libraries, a single solid-phase channel being less than 30%, and avoiding physical crowding by commercial standards. Hybridization thermodynamics and efficiency degradation include the degradation of solid-phase primer binding hybridization efficiency, low melting temperature of asymmetric base loss, and whole-genome bisulfite libraries. Cycle monitoring and dynamic verification include the cumulative cycle number signal attenuation coefficient, deviation of fluorescence chip surface complementarity, and base fluorescence intensity signal oversaturation.

[0038] Example 4: In a high-throughput sequencing library construction scenario, for heterogeneous nucleic acid mixture samples containing short-sequence fragment small-molecule ribonucleic acid libraries and whole-genome bisulfite sequencing libraries, the system is configured with a physicochemical parameter measurement module to measure and record the average fragment length of the small-molecule ribonucleic acid library as 25.4 bp and the mass concentration as 2.52 ng / μl, and the average fragment length of the whole-genome bisulfite sequencing library as 302.3 bp and the mass concentration as 15.18 ng / μl. It should be noted that here, the small-molecule ribonucleic acid... The average fragment length of the acid library is 25.4 bp, specifically referring to the net length of the target small RNA insert fragment before adapter ligation. In actual sequencing processes, both ends of the library molecule are pre-ligated with standardized sequencing adapter sequences totaling 120 bp, making the actual total physical length of the library 145.4 bp, thus fully meeting the spatial geometry and sequence complementarity requirements for solid-phase primer hybridization and bridge amplification. In kinetic calculations, the system independently extracts this 25.4 bp insert fragment length as the basis for evaluating the diffusion rate. The parameter measurement module transmits the acquired fragment length data and mass concentration data to the nominal molar concentration conversion module. This module derives the nominal molar concentration of small molecule ribonucleic acid libraries and whole-genome bisulfite sequencing libraries based on the average molecular weight of double-stranded nucleic acid molecules, providing a quantitative material basis for subsequent base balancing. The hybridization kinetics compensation module dynamically adjusts the matching coefficients of each library type based on a preset solid-phase hybridization mass transfer resistance database. For small molecule ribonucleic acid libraries with short fragment lengths and extremely high diffusion rates, which have a spatial competitive advantage on the solid-phase carrier surface, the hybridization kinetics compensation module sets their matching coefficient to 0.8 to suppress the local clonal population density of this component during the solid-phase amplification stage, avoiding the risk of signal overflow from the photosensitive device. For whole-genome bisulfite sequencing libraries with reduced sequence complexity and lower melting temperatures due to bisulfite treatment, the hybridization kinetics compensation module sets their matching coefficient to 1.2 to enhance the hybridization capture and amplification driving force at the solid-phase primers, thereby compensating for the attenuation of fluorescence signal intensity caused by insufficient hybridization efficiency.

[0039] The physical volume mixing module receives the aforementioned nominal molar concentration data and calibrated mixing coefficients. Driven by an automated liquid handling unit, it uses a pipetting pump to quantitatively mix the balanced library with the calibrated unbalanced library, constructing a base-neutralized mixing system that meets the optical signal balance requirements. The flow channel distribution control module monitors the equivalent percentage of each unbalanced library in real time and implements flow channel load limits to ensure that the total equivalent percentage of all unbalanced libraries in the same flow channel remains below 28%, thus ensuring that the spatial distribution of bases on the solid support surface meets the preset fluorescence radiation equilibrium state. The fluorescence signal monitoring module during sequencing acquires the four-color light intensity signals from the flow channel surface in real time and performs closed-loop regulation of the radiation flux density of each channel through a feedback calibration loop. During this process, the circulating signal... The attenuation coefficient is calculated by the system through the following steps: The arithmetic mean of the total fluorescence intensity of the four colors in the first five initial sequencing cycles is obtained as the initial baseline value. Then, the arithmetic mean of the total fluorescence intensity of the current five consecutive cycles is obtained as the real-time monitoring value. The difference between the initial baseline value and the real-time monitoring value is divided by the initial baseline value to obtain the cycle signal attenuation coefficient, which characterizes the degree of degradation in solid-phase primer hybridization efficiency. When this coefficient exceeds a set attenuation threshold of 0.15, a base gain value of 0.1 is automatically added. Simultaneously, fluorescence complementarity is calculated by uniformly evaluating the fluorescence intensity of four nucleotide monomers with reversible termination ends (adenine, thymine, cytosine, and guanine) excited on the chip surface: the system calculates the independent fluorescence intensity of each base. The absolute difference between the total fluorescence intensity of the four bases and the average fluorescence intensity of the four bases is summed, and then the sum is divided by the total fluorescence intensity of the four bases to obtain the fluorescence complementarity value. The closer this value is to 0, the higher the complementarity balance. The set balance range is 0.01 to 0.05. In each sequencing cycle, if the optical sensor of any channel produces an abnormal signal saturation, the system compares the fluorescence radiation flux density of the four color channels in the current cycle, calculates the signal contribution weight of each component on the solid surface, and dynamically drives the hybridization kinetics compensation module to correct the matching coefficient of subsequent batches. The signal contribution weight calculation here is achieved through the preset library feature signal dissociation matrix. Because small molecule ribonucleic acid libraries have fixed read length characteristics and Specific bases exhibit a high concentration preference in early cycles, while whole-genome bisulfite sequencing libraries suffer significant cytosine loss due to transformation, exhibiting extreme asymmetric signal loss in specific cycles. The system compares the fluorescence deviation vectors of the four channels monitored in three consecutive cycles with the standard cycle characteristic spectra of each library type in the database using least squares. This deconvolution yields the percentage contribution of each library component to the current local fluorescence oversaturation or loss, which is then used as the corresponding signal contribution weight. This eliminates the causal chain break where the overall mixed signal cannot be traced back to a specific surface library. In the sequencing cycle interval from 30 to 60, this compensation mechanism controls the radiation flux deviation of each channel to within 3.5%, and the base interpretation accuracy increases from 99% in the initial stage.The efficiency was maintained at 2% to 99.1%, and the effective data output ratio of the chip channel deviated from the target value by less than 1.5%, with the data throughput reaching 98.6% of the rated capacity. This demonstrates that the compound system and kinetic balance mechanism can effectively suppress optical imbalance in the sequencing channel.

[0040] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention.

[0041] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A base balancing system for high-throughput sequencing libraries, characterized in that, include: The physicochemical parameter determination module is used to obtain the library type, average fragment length, and mass concentration of the nucleic acid library of the sample to be tested. The library types include small molecule ribonucleic acid libraries and whole genome bisulfite sequencing libraries. The flow channel distribution control module is used to distribute the nucleic acid library of the test sample and limit the initial equivalent proportion of the nucleic acid library of the test sample as an unbalanced library in the same solid phase flow channel to be less than 30%. The nominal molar concentration conversion module is used to calculate the nominal molar concentration of each nucleic acid library to be tested based on the mass concentration and average fragment length. The hybridization kinetics compensation module is used to adjust the mating coefficient according to the library type. The initial equivalent ratio is multiplied by the mating coefficient to determine the final input amount. The mating coefficient of small molecule ribonucleic acid libraries is down-adjusted, while the mating coefficient of whole genome bisulfite sequencing libraries is up-adjusted. The physical volume mixing module is used to determine the physical pipetting volume of each nucleic acid library to be tested, and to physically mix the libraries corresponding to each physical pipetting volume to construct a base neutralization mixing system, thereby realizing the hybridization and amplification of nucleic acid fragments with solid-phase primers.

2. The high-throughput sequencing library base balancing system according to claim 1, characterized in that, It also includes a boundary constraint module; the boundary constraint module configures the initial equivalent ratio for each library type according to the overall base fluorescence balance requirement of the solid phase channel in the sequencing cycle.

3. The high-throughput sequencing library base balancing system according to claim 1, characterized in that, The hybridization kinetics compensation module includes a ribonucleic acid library regulation submodule; the ribonucleic acid library regulation submodule identifies the test sample whose library type is a small molecule ribonucleic acid library, and lowers and corrects the matching coefficient corresponding to the small molecule ribonucleic acid library.

4. The high-throughput sequencing library base balancing system according to claim 1, characterized in that, The hybridization kinetics compensation module includes a methylation sequencing library regulation submodule; the methylation sequencing library regulation submodule identifies the test sample whose library type is whole genome bisulfite sequencing library, and up-regulates and amplifies the matching coefficient corresponding to the whole genome bisulfite sequencing library to compensate.

5. The high-throughput sequencing library base balancing system according to claim 1, characterized in that, The physical volume mixing module is connected to the automatic pipetting unit and outputs the calculated physical pipetting volumes to the automatic pipetting unit.

6. The high-throughput sequencing library base balancing system according to claim 1, characterized in that, It also includes a fluorescence signal monitoring module; the fluorescence signal monitoring module receives the base fluorescence intensity signal released by the sequencer in the sequencing cycle, and adjusts the allocation ratio of the flow channel allocation control module when the base fluorescence intensity signal exceeds the set saturation threshold to avoid pixel overflow in the sequencing image.

7. The high-throughput sequencing library base balancing system according to claim 1, characterized in that, It also includes a sequencing cycle decay analysis module; the sequencing cycle decay analysis module records the cumulative number of sequencing cycles performed by the sequencer, and calculates the cycle signal decay coefficient, which characterizes the decay trend of solid primer hybridization efficiency, based on the cumulative number of cycles. When the cycle signal decay coefficient exceeds the set decay threshold, the hybridization kinetics compensation module adds a basic gain value to the matching coefficient of each nucleic acid library to be tested.

8. The high-throughput sequencing library base balancing system according to claim 1, characterized in that, It also includes a library quality assessment module; before the physicochemical parameter determination module obtains the mass concentration of each nucleic acid library to be tested, the library quality assessment module receives the input initial mass concentration sequence, and outputs a quality abnormality warning when it detects that the initial mass concentration sequence of any nucleic acid library is lower than the set lower limit threshold, and at the same time terminates the physical pipetting volume output of the physical volume reconstitution module.

9. The high-throughput sequencing library base balancing system according to claim 1, characterized in that, It also includes a base balance verification module; the base balance verification module receives the base distribution composition of each nucleic acid library to be tested in the sequencing cycle, and calculates the fluorescence complementarity of the four bases with reversible termination ends on the sequencing chip surface. When the fluorescence complementarity deviates from the set balance range, the hybridization kinetics compensation module corrects the matching coefficient to make the fluorescence complementarity stable within the set balance range.

10. A method for base balancing in high-throughput sequencing libraries, implemented by the high-throughput sequencing library base balancing system described in claim 1, characterized in that... Includes the following steps: Step S1: Obtain the library type, average fragment length, and mass concentration of the nucleic acid library of the sample to be tested through the physicochemical parameter determination module. The library types include small molecule ribonucleic acid libraries and whole genome bisulfite sequencing libraries. Step S2: Distribute the nucleic acid library of the test sample through the flow channel distribution control module, and limit the total initial equivalent proportion of the nucleic acid library of the test sample as an unbalanced library in the same solid phase flow channel to be less than 30%; Step S3: Calculate the nominal molar concentration of each test sample nucleic acid library based on the mass concentration and average fragment length using the nominal molar concentration conversion module. Step S4: The hybridization kinetics compensation module adjusts the matching coefficient according to the library type, and the initial equivalent ratio is multiplied by the matching coefficient to determine the final input amount. The matching coefficient of small molecule ribonucleic acid libraries is lowered, and the matching coefficient of whole genome bisulfite sequencing libraries is raised. Step S5: The physical pipetting volume of each nucleic acid library to be tested is determined by the physical volume mixing module, and the libraries corresponding to each physical pipetting volume are physically mixed to construct a base neutralization mixing system, thereby realizing the hybridization and amplification of nucleic acid fragments with solid primers.

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