Template construction method for gene sequencing, product, gene sequencer and medium

Abstract

The application provides a template construction method, product, gene sequencer and medium for gene sequencing, the method comprising: acquiring multiple channel images corresponding to the response of the sequencing signal of each base type respectively, which are collected in the current cycle of gene sequencing; constructing a channel template corresponding to each base type channel according to the channel images; acquiring the number of base signal acquisition units included in each channel template; determining whether each channel template has been successfully constructed according to whether the maximum and minimum values in the number of base signal acquisition units of each channel template satisfy a preset condition and the maximum number of rounds; and if successfully constructed, merging each channel template to obtain a sequencing template.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene sequencing, in particular to a template construction method for gene sequencing, a product, a gene sequencer and a medium. BACKGROUND

[0002] At present, gene sequencing technology can be mainly divided into three generations. The first generation sequencing technology Sanger method is a sequencing technology based on DNA synthesis reaction, also known as the terminal termination method, the core of which is to realize sequencing through ddNTP (dideoxynucleotide triphosphate, dideoxynucleotide triphosphate) mediated chain extension termination. Sanger proposed it in 1975, and published the first complete organism genome sequence in 1977. The second generation sequencing technology is represented by the Illumina platform, which realizes high-throughput sequencing, makes large-scale parallel sequencing a reality, and greatly promotes the development of genomics in the field of life sciences. The third generation sequencing technology is Nanopore nanopore sequencing technology, which is a new generation of single molecule real-time sequencing technology, which mainly realizes real-time sequencing through the change of electric signal caused by ssDNA or RNA template molecule passing through the nanopore.

[0003] The second generation sequencing technology uses different fluorescent molecules with different fluorescence emission wavelengths. When these fluorescent molecules are irradiated by laser, they will emit fluorescent signals of corresponding wavelengths. In DNA sequencing, the commonly used fluorescent labels are four kinds. These four fluorescent labels are added into a cycle at the same time, and the image of the fluorescent signal is captured by using a camera. Since each fluorescent label corresponds to a specific wavelength, we can separate the fluorescent signals corresponding to different fluorescent labels in the image, and then obtain the corresponding fluorescent image. Among them, the determination of the base cluster position through image processing and base cluster positioning technology is an important reference for the accurate positioning of all base cluster positions on the chip subsequently, therefore, the image processing and base cluster positioning technology is one of the core algorithms of the second generation gene sequencing technology, which will have a direct relationship with the accuracy of the subsequent base sequence data obtained.

[0004] Therefore, how to construct a sequencing template more accurately based on the currently known image processing and base cluster positioning technology will greatly affect the accuracy of subsequent DNA sequencing. SUMMARY

[0005] To solve the existing technical problems, the present application provides a template construction method for gene sequencing with higher accuracy, a product, a gene sequencer and a medium.

[0006] In a first aspect, the embodiments of the present application provide a template construction method for gene sequencing, comprising:

[0007] acquire multiple channel images corresponding to the sequencing signal responses of each base type in the current cycle of gene sequencing, and construct a channel template corresponding to each base type according to the channel images;

[0008] acquire the number of base signal acquisition units included in each channel template;

[0009] determine whether each channel template has been successfully constructed according to whether the maximum and minimum values of the number of base signal acquisition units in each channel template satisfy a preset condition and a maximum number of cycles;

[0010] if successfully constructed, combine each channel template to obtain a sequencing template.

[0011] In a second aspect, an embodiment of the present application provides a gene sequencer, including a memory and a processor;

[0012] the processor stores a computer program;

[0013] the processor is configured to execute the computer program to implement the template construction method for gene sequencing according to any embodiment of the present application.

[0014] In a third aspect, an embodiment of the present application provides a computer program product, including a computer program, which, when executed by a processor, implements the template construction method for gene sequencing according to any embodiment of the present application.

[0015] In a fourth aspect, an embodiment of the present application provides a computer-readable storage medium, which stores a computer program, and the computer program, when executed by a processor, implements the template construction method for gene sequencing according to any embodiment of the present application.

[0016] In the above embodiments, the template construction method is to acquire multiple channel images corresponding to the sequencing signal responses of each base type in the current cycle of gene sequencing, and construct a channel template corresponding to each base type according to the channel images, acquire the number of base signal acquisition units included in each channel template, and determine whether each channel template has been successfully constructed according to whether the maximum and minimum values of the number of base signal acquisition units in each channel template satisfy a preset condition and a maximum number of cycles. In this way, the channel templates corresponding to each base type are constructed in real time according to the channel images obtained in the current cycle, and the successful construction criteria are set by using the difference in the number of base signal acquisition units in different channel templates and the number of cycles, so that the template construction scheme with a dynamic strategy is realized. For the unbalanced sample or balanced sample that may occur in the actual application of gene sequencing, the accuracy and efficiency of the sequencing template construction can be better balanced.

[0017] In the above embodiments, the gene sequencer, the computer program product and the computer readable storage medium belong to the same concept as the corresponding template construction method for gene sequencing, thus having the same technical effects as the corresponding template construction method for gene sequencing, which will not be described here. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 An application scenario of gene sequencing in an embodiment.

[0019] Figure 2 A flowchart of a template construction method for gene sequencing in an embodiment.

[0020] Figure 3 A schematic diagram of constructing a sequencing template based on four channel templates in an embodiment.

[0021] Figure 4 A flowchart of constructing a sequencing template based on four channel templates in an embodiment.

[0022] Figure 5 A flowchart of constructing a single channel template in an embodiment.

[0023] Figure 6 A schematic diagram of channel images of the same channel in two consecutive cycle rounds in an embodiment.

[0024] Figure 7 A flowchart of correction in constructing a channel template in an embodiment.

[0025] Figure 8 A schematic diagram of a near neighbor signal unit group in an embodiment.

[0026] Figure 9 A schematic diagram of a gene sequencer in an embodiment. DETAILED DESCRIPTION

[0027] The technical solutions of the present application will be further described in detail below in combination with the accompanying drawings and specific embodiments.

[0028] In order to make the purposes, technical solutions and advantages of the present application clearer, the present application will be further described in detail below in combination with the accompanying drawings, and the described embodiments should not be regarded as limiting the present application. All other embodiments obtained by those skilled in the art without making creative efforts fall within the scope of protection of the present application.

[0029] In the following description, the expression “some embodiments” describes a subset of all possible embodiments, and it should be noted that “some embodiments” can be the same subset or different subsets of all possible embodiments, and can be combined with each other without conflict.

[0030] In the following description, the terms "first", "second", "third", etc. are merely used to distinguish similar objects, and do not represent a specific order or sequence of the objects. It is understood that the "first", "second", "third", etc. can be interchanged in a specific order or sequence as permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0031] The second-generation sequencing technology, also known as Next-generation Sequencing (NGS), can sequence hundreds of thousands to millions of DNA molecules at a time. Known second-generation sequencers generally record base information by optical signal, and convert the optical signal into base sequence. The base cluster position generated by image processing and fluorescence positioning technology is a reference for the template point position in the subsequent chip, so the image processing and base signal acquisition unit positioning technology are directly related to the accuracy of the base sequence data. The template construction method for gene sequencing provided in the embodiments of the present application, in one optional example, refers to taking the channel fluorescence images corresponding to different base types respectively collected by the sequencing chip in the fluorescence-labeled dNTP-based gene sequencing as input data, and is mainly applied to the second-generation gene sequencing technology.

[0032] Among them, fluorescence labeling is a measurement technology using optical signals, which is commonly used in the fields of DNA sequencing, cell labeling, drug research, etc. in industry. The gene sequencing optical signal method used by the second-generation sequencer is to use different waveband fluorescence labels to mark different bases, filter through an optical filter, and connect a specific base to excite a specific wavelength of light, and finally identify the DNA base sequence to be measured. This technology of generating images by collecting optical signals and then converting them into base sequences is the main principle of the second-generation gene sequencing technology.

[0033] The second-generation sequencer, taking the Illumina sequencer as an example, has a sequencing process mainly including four stages of sample preparation, cluster generation, sequencing, and data analysis.

[0034] Sample preparation, also known as library construction, refers to breaking the basic group DNA to be measured into a large number of DNA fragments, adding adapters to both ends of each DNA fragment, and the adapters respectively contain a sequencing binding site, indices (information identifying the source of the DNA segment), and a specific sequence complementary to the oligonucleotide on the sequencing chip (Flowcell).

[0035] Cluster generation, also known as library seeding on the Flowcell, uses bridge DNA amplification to form a base cluster from a DNA fragment.

[0036] Sequencing refers to sequencing reads for each base cluster on the flowcell. Sequencing adds a sequencing primer with a fluorescently labeled dNTP. The dNTP chemical formula has an azido group attached to one end, which can prevent polymerization during chain extension during sequencing, ensuring that only one base is extended per cycle, corresponding to the generation of a sequencing read, i.e. sequencing by synthesis. In one cycle, each base cluster is identified by a fluorescently labeled dNTP, and the corresponding base type is identified by a specific color of fluorescent signal response. The color of the emitted fluorescence can be used to determine which base corresponds to each base cluster in the current cycle by laser scanning. In one cycle, millions of base clusters are sequenced simultaneously on the flowcell. A fluorescent point represents the fluorescence emitted by a base cluster, and a base cluster corresponds to a read in fastq. In the sequencing stage, the fluorescent image of the flowcell surface is captured by an infrared camera. The base cluster is detected by image processing and base signal acquisition unit position positioning of the fluorescent image. The template is constructed according to the base cluster detection results of the multiple channel images corresponding to the sequencing signal response of different base types. The positions of all base cluster template points on the flowcell are constructed. In the subsequent gene sequencing process, the fluorescence intensity of the filtered image is extracted according to the template, and then the fluorescence intensity is corrected. Finally, the score is calculated according to the maximum intensity of each base cluster template point position to identify the base, and the fastq base sequence file is output.

[0037] Data analysis involves analyzing millions of reads representing all DNA fragments. For each sample, the base sequence from the same library can be clustered by the unique index introduced in the adapter during library construction. Reads are paired to generate continuous sequences, which are aligned to the reference genome for mutation identification.

[0038] It should be noted that the above is to illustrate the sequencing process by taking Illumina sequencing technology as an example of a massively parallel sequencing technology (MPS). The DNA molecules to be tested are amplified by a specific amplification technique, and a base cluster is formed for each DNA fragment (single-stranded library molecule). The base cluster detection result is used to construct the template point of the base cluster on the sequencing chip, so that subsequent base recognition operations can be performed based on the template point of the base cluster, thereby improving the base recognition efficiency and accuracy. It can be understood that the template construction method for gene sequencing provided by the embodiments of the present application is based on the positioning detection and base type recognition of the base cluster after amplification of the single-stranded library molecule on the sequencing chip. Here, each base cluster refers to a base signal acquisition unit, so it is not limited to which amplification technique is used for the single-stranded library molecule. That is, the template construction method for gene sequencing provided by the embodiments of the present application can also be applied to the positioning detection and base type recognition of the base signal acquisition unit of the sequencing chip in other massively parallel sequencing technologies. For example, the base signal acquisition unit corresponding to each fluorescent point in the channel fluorescence image can refer to the base cluster obtained by bridge amplification technology in Illumina sequencing technology, and also includes nanoballs obtained by rolling circle amplification (RCA, Rolling Circle Amplification), etc. The present application does not limit this.

[0039] In view of the problem of how to improve the accuracy of template construction for gene sequencing based on the currently known image processing and base cluster positioning technology in the current gene sequencing process, the present inventors have made the following research:

[0040] Since the distribution of base clusters on the sequencing chip is random, and there is a color difference between the channel images of the four channels corresponding to the four base types, there are two important goals for template construction: 1) confirming the coordinates of each cluster; 2) confirming the color difference between the channels.

[0041] DNA sequencing is to measure one base per cycle, so it needs to add bases for many cycles, and different cycles need to take pictures of FOV (Field of View) positions on the sequencing chip. According to whether there is fluorescence in the picture position, it is judged whether the base reaction is connected, and the final sequence is obtained. Each cycle reaction takes a picture of the same position FOV, but the platform movement of the stage (a platform that controls the movement of the sequencing chip) has a precision limit, so the picture of the same position taken by each cycle may not be completely aligned, and registration needs to be performed according to the image and the bright spot. Only in this way can the light-emitting situation of the same single-stranded DNA in different cycles be connected in series to obtain a read. At the same time, four images of the same position are taken in each cycle, and four channel images corresponding to four base types are taken.

[0042] As shown in Figure 1 The sequencing chip 11 (slide / flowcell) to be scanned and photographed is grabbed by a mechanical arm to the stage 12, the camera 13 is fixed, and the sequencing chip 11 below is moved. One camera 13 contains two spectrometers 14, which can obtain two different channel images at the same time. After one shot, another spectrometer 14 is changed for re-scanning and photographing. Because the movement of the stage 12 in each round causes the movement of the Fov position, image registration is needed. At the same time, because there is a deviation in the camera lens, there is a chromatic aberration between the channel images of the two channels, and chromatic aberration correction is also needed. Therefore, the channel images of the four fluorescence channels have chromatic aberration, the deformation and distortion parameters of the channel images of different channels are different, the four channel images cannot be superimposed, and the offset needs to be corrected, the chromatic aberration needs to be corrected, and the four channel images need to be superimposed to extract the fluorescence brightness of the four channels at the same position, which lays the foundation for subsequent base recognition.

[0043] According to the fluorescent bright spots in the registered channel images, all fluorescent clusters on the FOV surface of the sequencing chip need to be found, that is, the DNA sequence set obtained by gene sequencing.

[0044] In the same cycle, in theory, the four channels have no intersection of base signal acquisition units, and the union of the base signal acquisition units of the four channels is the full set of base signal acquisition units in the sequencing template. In principle, through the four channel images of one cycle, that is, the union of the base signal acquisition units of the four base types, all DNA sequences can be found, but in actual application, due to the fact that some DNA may be too dark, or due to noise, or image blur, etc., part of the base signal acquisition unit may not be detected, so it is necessary to construct a sequencing template through multiple cycle channel images.

[0045] Therefore, in the whole process of constructing the sequencing template, it is necessary to ensure that the four channels corresponding to the channel templates can be successfully constructed, and there are enough common points among the four channel templates to calculate the chromatic aberration matrix. In actual application, there may be a type of base imbalance sample in the sample of the gene sequencer. In each cycle, only one or two or three types of bases (for base balanced samples, four types of bases corresponding to four channels exist in each cycle), and in the known template construction method, the number of cycles on which the gene sequencer depends for template construction in the gene sequencing process is fixed, such as constructing four channel templates according to the channel images collected in six cycles, but in actual application, the sequencing sample is flexible and variable, and it cannot be ensured that four channel templates can be collected in six cycles. For example, for a type of base imbalance sample, there may be no base type A in six cycles.

[0046] In order to solve the problem of the influence of the construction of the channel template on the accuracy of the final sequencing template, the cycle number for template construction is increased, for example, the channel images obtained by 10 cycles are used for channel template construction, which can effectively improve the adaptability of the gene sequencer to the sequencing sample. However, it is difficult to determine how many cycles are required to solve the problem of inaccurate construction of channel templates caused by base imbalance samples. Moreover, the more the number of cycles for constructing templates, the more the accumulated computing amount, which obviously increases the downtime of the gene sequencer. For base balanced samples, the computing amount and the downtime of the gene sequencer are also increased unnecessarily.

[0047] In addition, the base balanced sample is also added to the sequencing sample to change the base imbalance sample into a base balanced sample, but the addition of more than 20% balanced samples is generally required to solve the problem of sample balance, which will cause the loss of the throughput of the gene sequencer.

[0048] Based on the above research, the inventors of the present application propose a design idea of channel template construction based on a dynamic strategy, which constructs the channel templates corresponding to each channel in real time according to the channel images in each cycle, and sets the judgment rule for ending the construction of the channel template by using the difference between the number of base signal acquisition units corresponding to different types of bases and the upper limit value of the preset cycle number, so that the accuracy and efficiency of the sequencing template construction can be better considered for the imbalance sample or balanced sample that may occur in the actual application of the gene sequencing. The following will be further described in conjunction with the embodiments.

[0049] Please refer to Figure 2The template construction method for gene sequencing provided by an embodiment of the present application comprises the following steps:

[0050] S101, acquiring multiple channel images corresponding to the sequencing signal responses of different base types collected in a current cycle of gene sequencing, and constructing channel templates corresponding to channels of different base types according to the channel images.

[0051] The base type generally refers to A, C, G and T four base types. Since different base types correspond to the fluorescence signals of different fluorescently labeled dNTPs, there is no intersection between the base clusters of different fluorescently labeled dNTPs. The channel image corresponding to the sequencing signal response of each base type includes the base clusters of the same base type contained in the corresponding part of the sequencing chip. Multiple channel images corresponding to the sequencing signal responses of different base types of the target part of the sequencing chip are acquired, each channel image includes the position information of the base clusters of one base type, and the position information of the complete multiple base clusters contained in the target part of the sequencing chip is obtained according to the position information of the base clusters respectively included in the multiple channel images.

[0052] The channel image, taking the fluorescently labeled dNTP gene sequencing as an example, refers to the original fluorescence grayscale image of the sequencing chip surface shot in the sequencing stage in the sequencing process. In the embodiment, the A, C, G and T bases correspond to the fluorescence signals of four different fluorescently labeled dNTPs. In theory, there is no intersection between the base clusters of the four different fluorescently labeled dNTPs in the same cycle. Acquiring multiple channel images corresponding to the sequencing signal responses of different base types of the target part of the sequencing chip refers to shooting four fluorescence grayscale images corresponding to the fluorescence signals of four different fluorescently labeled dNTPs for the same target part of the sequencing chip, collecting the corresponding fluorescence grayscale images (four original fluorescence grayscale images) of the A, C, G and T four bases excited by the fluorescence signals (four environments) of the four different fluorescently labeled dNTPs in the same field of view (the same target part of the sequencing chip) according to the different brightness of the A, C, G and T four base types under the irradiation of light of different wave bands, as the multiple channel images corresponding to the sequencing signal responses of different base types.

[0053] It should be noted that in the description of the embodiments of the present application, the base signal acquisition unit in the channel image is mainly referred to as a fluorescence spot, taking the fluorescently labeled dNTP gene sequencing as an example. In other optional embodiments, the channel image can also refer to an image including base signal acquisition units formed based on non-fluorescently labeled sequencing response signals, which is not limited in the present application.

[0054] S103, acquiring the number of base signal acquisition units included in each channel template.

[0055] For the current cycle, according to the channel images of the corresponding channels corresponding to each base type collected in the current cycle, a channel template of the current cycle is constructed in real time, and an image recognition algorithm can be used to recognize the base signal acquisition units contained in the channel template, and the number of base signal acquisition units included in each channel template is counted.

[0056] S105, whether the maximum and minimum values of the number of base signal acquisition units of each channel template meet the preset condition and the maximum number of rounds, determine whether each channel template has been successfully constructed.

[0057] According to whether the maximum and minimum values of the number of base signal acquisition units in each channel template meet the preset condition, the number of cycles of the current cycle or the cumulative value of the number of cycles that meet the preset condition, a dynamic judgment strategy for judging whether the channel template has been successfully constructed is set, and for each channel image collected after the current cycle round, the corresponding channel template is constructed. After that, the dynamic judgment strategy is used for real-time judgment. In this way, if the current sequencing sample is a balanced sample, there is a greater probability that the channel template construction success condition can be reached after the first two cycles or three cycles. If the current sequencing sample is an unbalanced sample, the channel template construction can be completed through more cycle rounds of channel images that do not exceed the maximum number of rounds, so as to greatly ensure that the unbalanced sample can be normally sequenced.

[0058] S107, if successfully constructed, merging each channel template to obtain a sequencing template.

[0059] If the channel templates corresponding to the channels of different base types constructed based on the current cycle have been successfully constructed, the union of the base signal acquisition units in the multiple channel templates is the base signal acquisition unit set, so that merging each channel template can obtain a sequencing template.

[0060] Please refer to Figure 3 , for the schematic diagram of merging four channel templates corresponding to four base types to obtain a sequencing template. In the sequencing template, the union of the base signal acquisition units in the multiple channel templates is included, and according to the union of the base signal acquisition units, all DNA sequences on the sequencing chip can be found.

[0061] In the above embodiments, the template construction method is to obtain multiple channel images corresponding to the sequencing signal response of each base type in the current cycle of gene sequencing, construct channel templates corresponding to each base type, obtain the number of base signal acquisition units included in each channel template, determine whether the maximum and minimum values of the number of base signal acquisition units in the multiple channel templates satisfy a preset condition, and determine whether the cycle number of the current cycle reaches a preset cycle number value, to determine whether each channel template has been successfully constructed. In this way, the channel templates corresponding to each base type are constructed in real time based on the channel images obtained in the current cycle, and the difference in the number of base signal acquisition units in different channel templates and the cycle number value are used to set the successful construction judgment criteria, so that the dynamic strategy template construction scheme is realized. For the unbalanced sample or balanced sample that may occur in the actual application of gene sequencing, the accuracy and efficiency of the sequencing template construction can be better balanced.

[0062] In some embodiments, referring to Figure 4 , before the step S107, the merging of each channel template to obtain the sequencing template, includes:

[0063] S1071, determining a reference channel, and constructing a channel template of the reference channel based on the channel image of the reference channel;

[0064] S1072, for each other channel, respectively constructing a corresponding channel template, determining a correction parameter between the channel template of the other channel and the channel template of the reference channel, and correcting the channel template of the other channel through the correction parameter;

[0065] S1073, merging each channel template to obtain a sequencing template based on the channel templates of the reference channel and the other channels after correction.

[0066] The reference channel can be any one of the multiple channels corresponding to multiple base types. After selecting one of the multiple channels corresponding to multiple base types as the reference channel, the unselected channels are the other channels. For the same channel, the fluorescence signals of the base clusters of different base types are excited in different cycle numbers, and the fluorescence signals of the same base cluster position are excited and brightened, forming common base signal acquisition units between the channel images collected in different cycle numbers of the same channel. The construction of the channel template for each channel is performed by using the channel images corresponding to the same channel collected in the current cycle, as shown in Figure 5 .

[0067] In this embodiment, the key idea for constructing the sequencing template is to locate the positions of all base clusters. After the sequencing template is completely constructed, the base types can be identified by detecting the brightness values ​​of the corresponding base cluster positions using the located base cluster positions in the sequencing template. At this time, the base types can be identified by comparing the brightness values ​​of the base signal acquisition units corresponding to the base cluster positions in different channels. Thus, in this embodiment, during the sequencing template construction process, when constructing the channel template in real time for the channel image acquired in the current cycle, it fully utilizes the fact that different cycles within the same channel contain a sufficient number of common base signal acquisition units, such as... Figure 6 As shown, this is a schematic diagram of the channel image of the same channel, such as channel A, in two consecutive cycle rounds. There is usually only a major offset between different cycle rounds of the same channel, thus constructing channel templates corresponding to each channel. For the channel templates of different channels, the common base signal acquisition unit is used to calculate the correction parameters, and the correction parameters are used to correct the channel templates of different channels to improve the accuracy of channel template construction.

[0068] Please see Figure 7 The following is a flowchart of channel image correction in an optional example. Taking the A, C, G, and T channels corresponding to the 4-base type as an example, channel A is selected as the reference channel, while channels C, G, and T are other channels. For channel A, a channel template A is constructed based on the corresponding acquired channel image. For channel C, a channel template C is constructed based on the channel image. Based on the characteristic data of the common base signal acquisition unit between the channel template C and the channel template A, the correction parameters corresponding to channel C are calculated. The channel template C is then corrected using the correction parameters to obtain the corrected channel template C. Similarly, for channel A... For channel G, a channel template is constructed based on the channel image of channel G. Based on the characteristic data of the common base signal acquisition unit between the channel template of channel G and the channel template of channel A, the correction parameters corresponding to channel G are calculated. The channel template of channel G is then corrected using the correction parameters between A and G to obtain the corrected channel template G. For channel T, a channel template is constructed based on the channel image of channel T. Based on the characteristic data of the common base signal acquisition unit between the channel template of channel T and the channel template of channel A, the correction parameters corresponding to channel T are calculated. The channel template of channel T is then corrected using the correction parameters between A and T to obtain the corrected channel template T.

[0069] In the above embodiment, in the construction process of the sequencing template, the channel template corresponding to each current cycle of channel image collected can be constructed in the first few cycles of the sequencing process, and the channel images obtained by different cycles of the same channel often contain enough common base signal acquisition units, and the main difference is the offset. By constructing the corresponding channel template for each channel first, and then correcting the images between different channels, the accuracy of channel template construction is improved.

[0070] In some embodiments, in step S1072, for each other channel, a corresponding channel template is constructed, and correction parameters between the channel template of the other channel and the channel template of the reference channel are determined, including:

[0071] In the channel template of the reference channel, m neighbor signal unit groups are determined; each neighbor signal unit group includes n base signal acquisition units.

[0072] Each other channel is taken as a target channel to be corrected, and each neighbor signal unit group is taken as a matching object. It is determined whether the channel template of the target channel to be corrected contains qualified base signal acquisition units corresponding to the current neighbor signal unit group. According to the qualified base signal acquisition units, a set of candidate offset vectors corresponding to the current neighbor signal unit group is determined. According to the set of candidate offset vectors of the m neighbor signal unit groups, offset correction parameters corresponding to the channel template of the target channel to be corrected are determined.

[0073] The number of neighbor signal unit groups can be determined by a base cluster positioning algorithm on the channel template of the reference channel to identify the positions of all base signal acquisition units in the channel template, and then by a clustering algorithm.

[0074] In an optional example, there are 1000 neighbor signal unit groups, and each neighbor signal unit group contains 5 base signal acquisition units. As shown in Figure 8 , it is an optional schematic diagram of a neighbor signal unit group.

[0075] Taking the reference channel as the A channel and the other channels as the current target channel to be corrected, for example, the 1000 groups of near neighbor signal unit groups in the channel template A are traversed, the matching of each near neighbor signal unit group with the channel template C is determined respectively, and the corresponding qualified base signal acquisition unit of each near neighbor signal unit group is determined according to the matching, and the corresponding candidate offset vector set of each near neighbor signal unit group is determined accordingly. After the traversal of the near neighbor signal unit groups is completed, the offset correction parameter corresponding to the channel template C is determined according to the candidate offset vector set corresponding to all the near neighbor signal unit groups.

[0076] In the above embodiment, by introducing the near neighbor signal unit group, taking the near neighbor signal unit group as the matching object, the matching between the channel templates corresponding to the two channels is performed to determine the offset correction parameter, which can improve the correction accuracy between the channel templates of different channels, thereby improving the accuracy of the constructed channel template.

[0077] In some embodiments, taking each near neighbor signal unit group as the matching object, whether the qualified base signal acquisition unit corresponding to the current near neighbor signal unit group is contained in the channel template of the target channel to be corrected is determined, including:

[0078] Taking each near neighbor signal unit group as the matching object respectively, taking each base signal acquisition unit in the near neighbor signal unit group as the base point and its n-1 base signal acquisition units as the control points in sequence, whether the matching points meeting the requirements exist in the channel template of the target channel to be corrected after the distance value between the base point and any matching base signal acquisition unit in the target channel to be corrected is offset by the distance value is judged.

[0079] If the control points associated with the base point all have matching points meeting the requirements, the current matching base signal acquisition unit is confirmed as the qualified base signal acquisition unit corresponding to the current near neighbor signal unit group.

[0080] Still taking the reference channel as the A channel and the other channels as the current target channel to be corrected, for example, the matching of each near neighbor signal unit group with the channel template C is determined respectively, for example, the near neighbor signal unit group is peakSet1, the base signal acquisition units in the near neighbor signal unit group peakSet1 are composed of coordinates (x1, y1), (x2, y2), (x3, y3), (x4, y4), and (x5, y5), and the matching of the near neighbor signal unit group peakSet1 with the channel template of the C channel is determined. The channel template of the C channel is composed of (Xp1, Yp1), (Xp2, Yp2),..., and (Xpn, Ypn).

[0081] With (x1, y1) as the base point, (x2, y2), (x3, y3), (x4, y4), and (x5, y5) are all corresponding control points.

[0082] In the channel template C, the distance value S1 between the to-be-matched base signal acquisition unit (Xp1, Yp1) and the base point is calculated. The distance value S1 can include the horizontal and vertical coordinate differences Xp1-x1=diffx and Yp1-y1=diffy.

[0083] According to the distance value S1, it is determined whether there is a matching point that meets the requirements in the channel template C after the distance value is offset for each control point (x2, y2), (x3, y3), (x4, y4), and (x5, y5).

[0084] If there is a matching point that meets the requirements in the channel template C for each control point (x2, y2), (x3, y3), (x4, y4), and (x5, y5), the to-be-matched base signal acquisition unit (Xp1, Yp1) in the channel template C is taken as a qualified base signal acquisition unit corresponding to the neighbor signal unit group peakSet1.

[0085] On the contrary, if there is no matching point that meets the requirements in the channel template C for any control point (x2, y2), (x3, y3), (x4, y4), and (x5, y5), the process is repeated, and it is determined again whether other to-be-matched base signal acquisition units (Xp2, Yp2)...(Xpn, Ypn) in the channel template C are qualified base signal acquisition units corresponding to the neighbor signal unit group peakSet1 until the current base point (x1, y1) is determined.

[0086] In the above embodiment, for each neighbor signal unit group, one base signal acquisition unit is taken as a base point, and other base signal acquisition units are taken as control points. For each base point and to-be-matched base signal acquisition unit in the channel template of the to-be-corrected target channel, other base signal acquisition units can find a matching point that meets the requirements in the channel template of the to-be-corrected target channel as a screening condition. In this way, each neighbor signal unit group is taken as a matching object, matching is performed between the channel templates corresponding to two channels, a qualified base signal acquisition unit corresponding to the neighbor signal unit group is determined, it is convenient to find the offset value between two channel templates more quickly and accurately, and the accuracy of channel template matching is improved.

[0087] In some embodiments, determining whether there is a matching point that meets the requirements in the channel template of the to-be-corrected target channel after the distance value is offset for each control point includes:

[0088] a control point is selected, and it is determined whether there is a matching point meeting the requirement in the channel template of the target channel to be corrected, with the distance value offset from the coordinates of the control point as the center;

[0089] If there is a matching point meeting the requirement, the next control point is selected, and the step of determining whether there is a matching point meeting the requirement in the channel template of the target channel to be corrected, with the distance value offset from the coordinates of the control point as the center, is performed again.

[0090] If there is no matching point meeting the requirement, the matching search of other control points associated with the current base point is ended.

[0091] Wherein, (x1, y1) is a base point, (x2, y2), (x3, y3), (x4, y4), and (x5, y5) are control points, the distance value between the base point (x1, y1) and the base signal acquisition unit (Xp1, Yp1) to be matched in the channel template C is S1, and the distance value S1 includes the horizontal and vertical coordinate differences Xp1-x1=diffx and Yp1-y1=diffy.

[0092] According to the distance value S1, it is determined whether there is a matching point meeting the requirement in the channel template C, with the distance value offset from each of the control points (x2, y2), (x3, y3), (x4, y4), and (x5, y5) as the center, which can include the following:

[0093] For the control point (x2, y2), the closest point to (x2+diffx, y2+diffy) in the channel template C is found, and the corresponding distance is S2; if the distance S2 is greater than 1.5 pixels, the search for (Xp1, Yp1) is ended, it is considered that (Xp1, Yp1) does not match peakSet1, and the matching search of other control points associated with the current base point (x1, y1) is ended; if the distance S2 is less than 1.5 pixels, the next control point, such as the control point (x3, y3), is selected.

[0094] For the control point (x3, y3), the closest point to (x3+diffx, y3+diffy) in the channel template C is found, and the corresponding distance is S3; if the distance S3 is greater than 1.5 pixels, the search for (Xp1, Yp1) is ended, it is considered that (Xp1, Yp1) does not match peakSet1, and the matching search of other control points associated with the current base point (x1, y1) is ended; if the distance S3 is less than 1.5 pixels, the next control point, such as the control point (x4, y4), is selected.

[0095] For the control point (x4, y4), find the nearest point of (x4+diffx, y4+diffy) in the channel template C, and the corresponding distance is S4; if the distance S4 is greater than 1.5 pixels, end the search of (Xp1, Yp1), and consider that (Xp1, Yp1) is not matched with peakSet1, that is, end the matching search of other control points associated with the current base point (x1, y1); if the distance S4 is less than 1.5 pixels, select the next control point, such as the control point (x5, y5).

[0096] For the control point (x5, y5), find the nearest point of (x5+diffx, y5+diffy) in the channel template C, and the corresponding distance is S5; if the distance S5 is greater than 1.5 pixels, end the search of (Xp1, Yp1), and consider that (Xp1, Yp1) is not matched with peakSet1, that is, end the matching search of other control points associated with the current base point (x1, y1); if the distance S5 is less than 1.5 pixels, it means that all control points associated with the base point have matching points that meet the requirements.

[0097] All control points (x2, y2), (x3, y3), (x4, y4), (x5, y5) associated with the base point (x1, y1) have matching points that meet the requirements, so that the current to-be-matched base signal acquisition unit (Xp1, Yp1) corresponding to the base point (x1, y1) is confirmed as a qualified base signal acquisition unit corresponding to the current neighbor signal unit group peakSet1.

[0098] In another optional example, when all control points (x2, y2), (x3, y3), (x4, y4), (x5, y5) associated with the base point (x1, y1) have matching points that meet the requirements, further calculate the sum S2+S3+S4+S5 of the distances between each control point and the nearest point thereof, and if the result of S2+S3+S4+S5 is less than or equal to 5, the distance value (diffx, diffy) between the base point (x1, y1) and the corresponding to-be-matched base signal acquisition unit (Xp1, Yp1) is included in the candidate offset vector set (peakSet1_offset1) of the current neighbor signal unit group peakSet1.

[0099] As described above, the matching conditions between all the to-be-matched base signal acquisition units (Xp2, Yp2)...(Xpn, Ypn) and the base point (x1, y1) in the channel template C are sequentially calculated and judged to determine whether the to-be-matched base signal acquisition units (Xp2, Yp2)...(Xpn, Ypn) are qualified base signal acquisition units corresponding to the current near neighbor signal unit group peakSet1, and one or more distance values that can be included in the candidate offset vector set (peakSet1_offset1) are obtained according to the determination result.

[0100] As described above, the candidate offset vector sets (peakSet1_offset2)...(peakSet1_offsetn) corresponding to the other near neighbor fluorescent point groups peakSet2...peakSetn are sequentially calculated.

[0101] In the above embodiment, the optional specific implementation manner of taking the near neighbor fluorescent point group as the matching object to determine whether the channel template of the to-be-corrected target channel contains the qualified base signal acquisition unit corresponding to the current near neighbor signal unit group is provided, which can accurately and effectively and more quickly screen out the qualified base signal acquisition unit, and is beneficial to improving the accuracy and efficiency of the correction between the channel templates.

[0102] In some embodiments, the offset correction parameter corresponding to the channel template of the to-be-corrected target channel is determined according to the candidate offset vector sets of the m near neighbor signal unit groups, including:

[0103] The channel template of the to-be-corrected target channel is divided into a plurality of image regions;

[0104] The offset value corresponding to each image region is determined according to the candidate offset vector set of the near neighbor signal unit group in each image region.

[0105] The offset correction parameter corresponding to the channel template of the to-be-corrected target channel is determined according to the offset values of the image regions.

[0106] The size of the image region can be adjusted as needed, such as being divided into a size of 250 pixels*250 pixels. According to the number of near neighbor signal unit groups contained in each image region, the candidate offset vector set in each image region is integrated, for example, the mode is taken with an accuracy of 2 pixels, so as to obtain the offset value of the corresponding image region. The offset values of the image regions in the whole image can fit the distortion field of the whole image.

[0107] In the above embodiment, further using the image region division manner, the image correction of the channel template is optimized according to the image region, the local features formed by the random part of the base signal acquisition unit can be fully utilized, the image distortion correction can be accurately completed, and the correction accuracy between the channel templates is improved.

[0108] In some embodiments, the step S105 comprises:

[0109] According to whether the difference between the product of the minimum value and the maximum value of the base signal acquisition unit quantity of each channel template and the preset ratio meets the preset condition, and according to whether the cycle number of the current cycle or the cycle number of the current accumulation that meets the preset condition reaches the maximum cycle number value, it is judged whether each channel template has been successfully constructed.

[0110] The preset ratio is not less than 0.25.

[0111] The difference between the product of the minimum value and the maximum value of the base signal acquisition unit quantity and the preset ratio meeting the preset condition means that the minimum value of the base signal acquisition unit quantity in the channel template is greater than the product of the maximum value and the preset ratio. By counting the number of different types of base signal acquisition units in the channel template, and using the relationship between the maximum value and the minimum value, a dynamic judgment standard for judging whether each channel template has been successfully constructed is set. In the construction process of the sequencing template, the channel template constructed under the balanced sample condition can be effectively identified as early as possible in the first few cycles of the sequencing process, and the construction of the channel template can be completed with fewer cycle numbers, thereby avoiding unnecessary increase of the sequencing time.

[0112] In some embodiments, the step S105 comprises:

[0113] If the maximum value and the minimum value of the base signal acquisition unit quantity of each channel template meet the preset condition, it is considered that each channel template is successfully constructed; and / or,

[0114] If the maximum value and the minimum value of the base signal acquisition unit quantity of each channel template meet the preset condition, and the cycle number of the current cycle reaches the maximum cycle number value, it is considered that each channel template is successfully constructed; and / or,

[0115] If the maximum value and the minimum value of the base signal acquisition unit quantity of each channel template meet the preset condition, and the cycle number that meets the preset condition reaches the maximum cycle number value, it is considered that each channel template is successfully constructed.

[0116] A judgment criterion for judging whether the channel templates are successfully constructed is set according to whether a certain condition is met between the minimum value mix_temp and the maximum value max_temp of the number of base signal acquisition units in the channel templates of different channels, such as mix_temp>0.25*max_temp. If the judgment criterion is met, the construction of the channel templates is ended.

[0117] In addition, an upper limit of the number of cycles, such as a maximum cycle value max_cycle, is preset for the construction of the channel templates. Within the range of the maximum cycle value max_cycle, the number of base signal acquisition units in each channel template is counted for each cycle. If the judgment criterion for successfully constructed channel templates is met between the minimum value mix_temp and the maximum value max_temp, the construction of the channel templates is ended. If the judgment criterion for successfully constructed channel templates is not met between the minimum value mix_temp and the maximum value max_temp, the number of cycles for collecting channel images is increased to continue improving the construction of the channel templates, until the number of cycles accumulates to the maximum cycle value max_cycle. The setting of the maximum cycle value max_cycle can avoid the situation that the gene sequencer cannot be normally started due to unbalanced samples in a special scenario, and avoid the situation that the normal application of the gene sequencer is affected by a small probability event.

[0118] Optionally, in some embodiments, an upper limit of the number of cycles that meet the preset condition is also preset. The number of base signal acquisition units in each channel template is counted for each cycle. If the preset condition is met between the minimum value mix_temp and the maximum value max_temp, it is considered that the number of cycles that meet the preset condition is accumulated to the maximum cycle value. The number of cycles for collecting channel images is increased until the number of cycles that meet the preset condition accumulates to the maximum cycle value.

[0119] In the above embodiments, whether the minimum value of the number of base signal acquisition units in the channel templates is greater than the product of the maximum value and the preset proportion is determined to determine whether the preset condition is met, and the upper limit of the number of preset cycles is set to determine the judgment criterion for judging whether the channel templates are successfully constructed, thereby forming a floating strategy for the construction of the channel templates. That is, the channel templates constructed by collecting channel images under the condition that the sequencing samples are balanced samples can meet the requirement that the gene sequencer is started as soon as possible, and the requirement that the accuracy of the construction of the channel templates and the normal sequencing of the gene sequencer are greatly improved under the condition that the sequencing samples are unbalanced samples.

[0120] Please refer to Figure 9In another aspect of the present application, a gene sequencer is provided, comprising a memory 112 and a processor 111; the processor 111 has stored therein a computer program; the processor 111 is configured to execute the computer program to implement the template construction method for gene sequencing according to any of the embodiments of the present application and achieve the same technical effects. To avoid repetition, details are not described herein.

[0121] In another aspect of the present application, a computer readable storage medium is provided, having stored therein a computer program. The computer program, when executed by a processor, implements the processes of the template construction method for gene sequencing according to any of the embodiments described above and achieves the same technical effects. To avoid repetition, details are not described herein. The computer readable storage medium can be, for example, a Read-Only Memory (ROM), a Random Access Memory (RAM), a magnetic disk or an optical disk.

[0122] It should be noted that, in this document, the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusions, such that processes, methods, articles or apparatuses that include a series of elements not only include those elements, but also include other elements not explicitly listed, or inherent to such processes, methods, articles or apparatuses. Without more limitations, the element defined by the phrase "comprising a" does not exclude the presence of additional identical elements in the process, method, article or apparatus that includes the element.

[0123] From the above description of the embodiments, those skilled in the art can clearly understand the above-mentioned embodiment methods, which can be implemented by means of software and necessary general hardware platforms, or by hardware. However, the former is a better embodiment in many cases. Based on this understanding, the technical solutions of the present application, or the parts that contribute to the prior art, can be embodied in the form of a software product stored in a storage medium (such as a ROM / RAM, a magnetic disk, or an optical disk), which includes a number of instructions for causing a terminal (which can be a mobile phone, a computer, a server, or a network device) to execute the methods described in the various embodiments of the present application.

[0124] The above is merely a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art can easily think of changes or replacements within the technical scope disclosed in the present application, which should be covered by the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A method for template construction for genetic sequencing, characterized by, The method comprises the following steps: acquiring multiple channel images corresponding to the responses of the sequencing signals of different base types respectively, which are collected in the current cycle of gene sequencing, and constructing channel templates corresponding to the channels of different base types according to the channel images; acquiring the number of base signal acquisition units included in each channel template; determining whether each channel template has been successfully constructed according to whether the maximum and minimum values of the number of base signal acquisition units of each channel template satisfy a preset condition and the maximum number of cycles, including: if the maximum and minimum values of the number of base signal acquisition units of each channel template satisfy the preset condition, it is considered that each channel template has been successfully constructed; and / or, if the maximum and minimum values of the number of base signal acquisition units of each channel template do not satisfy the preset condition and the cycle number of the current cycle reaches the maximum number of cycles, it is considered that each channel template has been successfully constructed; and / or, if the maximum and minimum values of the number of base signal acquisition units of each channel template satisfy the preset condition and the cycle number of the cycle satisfying the preset condition reaches the maximum number of cycles, it is considered that each channel template has been successfully constructed; if each channel template has been successfully constructed, merging each channel template to obtain a sequencing template.

2. The method of claim 1, wherein, Before the step of merging each channel template to obtain a sequencing template, the method comprises the following steps: determining a reference channel, and constructing a channel template of the reference channel according to the channel image of the reference channel; for each other channel, respectively constructing a corresponding channel template, determining a correction parameter between the channel template of the other channel and the channel template of the reference channel, and correcting the channel template of the other channel through the correction parameter; merging each channel template to obtain a sequencing template according to the channel templates of the reference channel and the other channels after correction.

3. The method of claim 2, wherein the template is a double-stranded DNA template. The step of, for each other channel, respectively constructing a corresponding channel template, determining a correction parameter between the channel template of the other channel and the channel template of the reference channel, comprises the following steps: determining m groups of adjacent signal units in the channel template of the reference channel; wherein each group of adjacent signal units includes n base signal acquisition units; respectively taking each other channel as a target channel to be corrected and each group of adjacent signal units as a matching object, determining whether the channel template of the target channel to be corrected contains qualified base signal acquisition units corresponding to the current group of adjacent signal units, determining a set of candidate offset vectors corresponding to the current group of adjacent signal units according to the qualified base signal acquisition units, and determining an offset correction parameter corresponding to the channel template of the target channel to be corrected according to the set of candidate offset vectors of the m groups of adjacent signal units.

4. The method of claim 3, wherein the template is a double-stranded DNA template. The step of, respectively taking each other channel as a target channel to be corrected and each group of adjacent signal units as a matching object, determining whether the channel template of the target channel to be corrected contains qualified base signal acquisition units corresponding to the current group of adjacent signal units, comprises the following steps: Respectively taking each neighbor signal unit group as a matching object, sequentially taking each base signal acquisition unit in the neighbor signal unit group as a base point, n-1 base signal acquisition units as control points, judging whether each control point after being offset by the distance value exists a required matching point in the channel template of the target channel to be corrected based on the distance value between the base point and any base signal acquisition unit to be matched in the target channel to be corrected; If the control points associated with the base point all exist a required matching point, the current base signal acquisition unit to be matched is confirmed as a qualified base signal acquisition unit corresponding to the neighbor signal unit group.

5. The method of claim 4, wherein the template is a double-stranded DNA template. The judgment whether each control point after being offset by the distance value exists a required matching point in the channel template of the target channel to be corrected comprises: ​ A control point is selected, and it is judged whether a required matching point exists in the channel template of the target channel to be corrected by taking the coordinates of the control point as the center and offsetting the distance value. If a required matching point exists, the next control point is selected, and the step of judging whether a required matching point exists in the channel template of the target channel to be corrected by taking the coordinates of the control point as the center and offsetting the distance value is executed again. If a required matching point does not exist, the matching search of other control points associated with the base point is ended.

6. The method of claim 3, wherein the template is a double-stranded DNA template. The determination of the offset correction parameter corresponding to the channel template of the target channel to be corrected according to the set of candidate offset vectors of the m neighbor signal unit groups comprises: The channel template of the target channel to be corrected is divided into a plurality of image regions. The offset value corresponding to each image region is determined according to the set of candidate offset vectors of the neighbor signal unit groups in the image region. The offset correction parameter corresponding to the channel template of the target channel to be corrected is determined according to the offset values of the image regions.

7. The method of claim 1 to 6, wherein, The judgment whether each channel template has been successfully constructed according to whether the maximum value and the minimum value in the number of base signal acquisition units of each channel template satisfy a preset condition and a maximum number of rounds comprises: The judgment whether each channel template has been successfully constructed according to whether the difference between the product of the minimum value and the maximum value in the number of base signal acquisition units of each channel template and a preset ratio satisfies a preset condition and whether the number of cycles in the current cycle or the number of cycles that satisfy the preset condition in the current cycle reaches the maximum number of rounds.

8. A genetic sequencer, characterized by, The device comprises a memory and a processor. The processor stores a computer program. The processor is configured to execute the computer program to implement the template construction method for gene sequencing.

9. A computer program product comprising a computer program, characterized in that, The computer program is executed by the processor to implement the template construction method for gene sequencing.

10. A computer-readable storage medium, characterized in that, The computer program is stored on the computer readable storage medium and is executed by the processor to implement the template construction method for gene sequencing.

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