阅读说明：本技术 一种t790m和c797s顺反式突变类型识别及计算方法 (T790M and C797S cis-trans mutation type identification and calculation method ) 是由 曹贵强 景奉香 义清文 张京红 谢中文 徐进 吴璋赟 侯琦芸 于 2021-08-11 设计创作，主要内容包括：本申请涉及一种T790M和C797S顺反式突变类型识别及计算方法。方法包括：确定反应单元内各信号通道的统计信息,统计信息中包括各信号通道的阳性信息和/或阴性信息；基于各信号通道的统计信息确定突变模板的阳性率,突变模板包括顺式突变和/或反式突变；基于突变模板的阳性率确定T790M和C797S顺反式突变的类型。采用本方法能够提高T790M和C797S顺反式突变类型识别及计算的准确性。(The application relates to a method for identifying and calculating cis-trans mutation types of T790M and C797S. The method comprises the following steps: determining statistical information of each signal channel in the reaction unit, wherein the statistical information comprises positive information and/or negative information of each signal channel; determining the positive rate of a mutation template based on the statistical information of each signal channel, wherein the mutation template comprises cis-mutation and/or trans-mutation; the types of cis-trans mutations of T790M and C797S were determined based on the positive rate of the mutant template. By adopting the method, the accuracy of the identification and calculation of the cis-trans mutation types of T790M and C797S can be improved.)

1. A T790M and C797S cis-trans mutation type identification and calculation method is characterized in that the method comprises the following steps:

determining statistical information of each signal channel in a reaction unit, wherein the statistical information comprises positive information and/or negative information of each signal channel;

determining a positive rate for a mutant template based on the statistical information for each of the signal channels, the mutant template comprising a cis-mutation and/or a trans-mutation;

determining the type of cis-trans mutation of T790M and C797S based on the positive rate of the mutant template.

2. The method of claim 1, wherein determining a positive rate of a mutated template based on the statistical information for each of the signal channels comprises:

determining the positive rate of each signal channel and the common positive rate of different signal channel combinations based on the statistical information of each signal channel;

determining an initial adjustment value based on the positive rate and the common positive rate for each of the signal channels;

and determining the positive rate of the mutant template based on the initial adjustment value, the positive rate of the signal channel and the common positive rate, wherein the positive rate of the mutant template and the basic information of the biological experiment are used for determining the copy number of the mutant template.

3. The method of claim 2, wherein determining the positive rate for each of the signal channels based on the statistical information for each of the signal channels and the common positive rate for different combinations of signal channels comprises at least one of:

determining a positive rate for the first signal channel based on a relationship of the number of positives for the first signal channel to the number of active reaction units;

determining a positive rate for the second signal channel based on a relationship of the number of positives for the second signal channel to the number of active reaction units;

determining a common positive rate for the first signal channel and the second signal channel based on a relationship between the number of first signal channel and second signal channel that are simultaneously positive and the number of active reaction units.

4. The method of claim 2, wherein determining an initial adjustment value based on the positive rate and the common positive rate for each of the signal channels comprises:

determining an initial adjustment value based on the positive rate of the first signal channel, the positive rate of the second signal channel, and the common positive rate;

said determining a positive rate for said mutant template based on said initial adjustment value, a positive rate for said signal channel, and said shared positive rate, comprising:

determining the positive rate of the mutant template in response to the positive rate of the first signal channel, the positive rate of the mutant template, the initial adjustment value and a preset threshold value meeting preset conditions, and the common positive rate, the positive rate of the mutant template and the initial adjustment value meeting preset conditions;

determining the positivity rate of the mutant template based on the updated initial adjustment value in response to the positivity rate of the first signal channel, the positivity rate of the mutant template and the initial adjustment value and the preset threshold value not meeting the preset condition, or the common positivity rate, the positivity rate of the mutant template and the initial adjustment value and the preset threshold value not meeting the preset condition, wherein updating the initial adjustment value comprises updating the initial adjustment value based on the positivity rate of the T790M mutant template in the trans-mutation of T790M and C797S, the positivity rate of the C797S mutant template in the trans-mutation of T790M and C797S, and the positivity rate of the cis-mutation.

5. The method of claim 4, wherein determining the positivity of the mutant template based on the initial adjustment value, the positivity of the signal channel, and the consensus positivity comprises:

determining the positive rate of the T790M mutant template in the trans mutation of T790M and C797S based on the positive rate of the first signal channel, the positive rate of the cis mutation, and the initial adjustment value;

determining the positive rate of the C797S mutant template in the trans mutation of T790M and C797S based on the positive rate of the second signal channel, the positive rate of the cis mutation, and the initial adjustment value;

determining a positive rate for the cis mutation based on a shared positive rate for the first signal channel and the second signal channel and the initial adjustment value, the positive rate for the first signal channel, the positive rate for the second signal channel, and the shared positive rate being determined based on the positive rate for the T790M mutant template in the trans mutation of T790M and C797S, the positive rate for the C797S mutant template in the trans mutation of T790M and C797S, and the positive rate for the cis mutation.

6. The method of claim 1, wherein determining the positive rate of a mutated template based on the statistical information for each of the signal channels comprises:

determining the positive rate and/or the negative rate of each signal channel and the positive rate and/or the negative rate of different signal channel combinations based on the statistical information of each signal channel;

and determining the positive rate of the mutant template based on the positive rate and/or the negative rate of each signal channel and the positive rate and/or the negative rate of different signal channel combinations.

7. The method of claim 6, wherein determining the positive rate and/or negative rate of each signal channel, and the positive rate and/or negative rate of different signal channel combinations based on the statistical information of each signal channel comprises at least one of:

determining the positive rate based on the relation between the number of two signal channels which are positive and negative in the reaction units and the number of effective reaction units;

determining a negative rate based on the relationship between the number of negative two signal channels in the reaction unit and the number of effective reaction units in the reaction unit;

determining the positive rate based on the relation between the number of three signal channels which are positive and one signal channel which is negative in the reaction units and the number of effective reaction units;

determining a negative rate based on a relationship between the number of negative signal channels in the reaction cells and the number of active reaction cells in the reaction cells;

determining the positive rate based on the relation between the number of the reaction units and the number of effective reaction units, wherein one signal channel is positive and three signal channels are negative;

the positive rate was determined based on the number of positive four signal channels in the reaction cell versus the number of effective reaction cells.

8. The method of claim 6, further comprising:

determining an abnormal reaction unit and removing the abnormal reaction unit;

determining the positive rate of the mutation template based on the positive rate and/or the negative rate of each signal channel of the effective reaction unit after the abnormal reaction unit is removed;

the determining an abnormal reaction unit includes at least one of:

determining a reaction unit with one positive signal channel and three negative signal channels as an abnormal reaction unit, and determining the abnormal reaction unit as an invalid reaction unit or converting positive information of the abnormal reaction unit into negative information;

determining a combination of two signal channels which are positive and two signal channels which are negative in the reaction units as an abnormal combination, determining the reaction unit corresponding to the abnormal combination as an abnormal reaction unit, and determining the abnormal reaction unit as an invalid reaction unit or converting positive information of the abnormal reaction unit into negative information.

9. The method of claim 6, wherein determining the positivity rate of the mutant template based on the positivity rate and/or the negativity rate of each signal channel and the positivity rate and/or the negativity rate of different signal channel combinations comprises:

determining the positive rate of the mutant template based on the statistical probability that two signal channels are negative in the reaction unit and the statistical probability that two signal channels are negative and the other two signal channels are positive in the reaction unit, wherein the positive rate of the mutant template comprises the positive rate of the T790M mutant template in the trans-mutation of T790M and C797S, the positive rate of the C797S mutant template in the trans-mutation of T790M and C797S and the positive rate of the cis-mutation.

10. The method of claim 9, further comprising: checking the positive rate of the mutant template, and determining the copy number of the mutant template based on the checked positive rate; the checking the positive rate of the mutant template comprises the following steps:

verifying the positive rate of the mutation template based on the statistical probability that one signal channel in the reaction unit is negative, the statistical probability that one signal channel in the reaction unit is negative and the other three signal channels are positive;

and checking the positive rate of the mutant template based on the statistical probability that the four signal channels in the reaction unit are positive.

Technical Field

The application relates to the technical field of biological detection, in particular to a T790M and C797S cis-trans mutation type identification and calculation method.

Background

The DPCR (digital Polymerase Chain reaction) technology is an absolute nucleic acid molecule quantification technology, and can directly count the number of DNA molecules, and is an absolute quantification technology for a starting sample. In DPCR, it is crucial to identify cis and trans mutations and the number of cis and trans mutations (e.g., number, copy number or mutation rate) for the co-existence of mutations in T790M and C797S.

In the conventional technology, generally, the two signal channels are used for judging the positive and negative based on a fluorescence signal value, judging the cis-mutation type and the trans-mutation type of a sample based on the distribution condition, and calculating the occurrence probability of the cis-mutation type and the trans-mutation type in the sample.

However, the problem of co-chamber of the C797S mutant template in the trans mutation of T790M and C797S is not considered in the process of identifying the mutation type at present, so that the accuracy of identifying the obtained T790M and C797S cis-trans mutation types is low.

Disclosure of Invention

Based on this, it is necessary to provide a method, an apparatus, a device and a medium for identifying and calculating cis-trans mutation types of T790M and C797S, which can improve the accuracy of identifying and calculating cis-trans mutation types of T790M and C797S.

A T790M and C797S cis-trans mutation type identification and calculation method comprises the following steps: determining statistical information of each signal channel in the reaction unit, wherein the statistical information comprises positive information and/or negative information of each signal channel; determining the positive rate of a mutation template based on the statistical information of each signal channel, wherein the mutation template comprises cis-mutation and/or trans-mutation; the types of cis-trans mutations of T790M and C797S were determined based on the positive rate of the mutant template.

In some embodiments, the method further comprises determining the copy number of the mutated template based on the positivity rate of the mutated template and the basic information of the biological experiment.

In some embodiments, determining the positive rate of the mutant template based on the statistical information for each signal channel comprises: determining the positive rate of each signal channel and the common positive rate of different signal channel combinations based on the statistical information of each signal channel; determining an initial adjustment value based on the positive rate and the common positive rate of each signal channel; and determining the positive rate of the mutant template based on the initial adjustment value, the positive rate of the signal channel and the shared positive rate.

In some embodiments, determining the positive rate for each signal channel, and the common positive rate for different combinations of signal channels, based on the statistical information for each signal channel comprises at least one of: determining a positive rate for the first signal channel based on a relationship of the number of positives for the first signal channel to the number of active reaction units; determining a positive rate for the second signal channel based on a relationship of the number of positives for the second signal channel to the number of active reaction units; determining a common positive rate for the first signal channel and the second signal channel based on a relationship between the number of first signal channel and second signal channel that are simultaneously positive and the number of active reaction units.

In some embodiments, determining the initial adjustment value based on the positive rate and the common positive rate for each signal channel comprises: an initial adjustment value is determined based on the positive rate of the first signal channel, the positive rate of the second signal channel, and the shared positive rate.

In some embodiments, determining the positivity of the mutant template based on the initial adjustment value, the positivity rate of the signaling channel, and the consensus positivity rate comprises: determining the positive rate of the mutation template in response to the positive rate of the first signal channel, the positive rate of the mutation template and the initial adjustment value and the preset threshold value meeting preset conditions, and the common positive rate, the positive rate of the mutation template and the initial adjustment value meeting preset conditions; and determining the positive rate of the mutation template based on the updated initial adjustment value in response to the positive rate of the first signal channel, the positive rate of the mutation template, the initial adjustment value and the preset threshold value not meeting the preset condition, or the total positive rate, the positive rate of the mutation template, the initial adjustment value and the preset threshold value not meeting the preset condition.

In some embodiments, determining the positivity of the mutant template based on the initial adjustment value, the positivity rate of the signaling channel, and the consensus positivity rate comprises: determining the positive rate of the T790M mutant template in the trans mutation of T790M and C797S based on the positive rate of the first signal channel, the positive rate of the cis mutation, and the initial adjustment value; determining the positive rate of the C797S mutant template in the trans mutation of T790M and C797S based on the positive rate of the second signal channel, the positive rate of the cis mutation, and the initial adjustment value; the positive rates of cis-mutations of T790M and C797S were determined based on the common positive rate and initial adjustment value of the first signal channel and the second signal channel, and the positive rates of the first signal channel, the second signal channel and the common positive rate were determined based on the positive rate of the T790M mutant template in the trans-mutation of T790M and C797S, the positive rate of the C797S mutant template in the trans-mutation of T790M and C797S and the positive rate of the cis-mutation.

In some embodiments, the initial adjustment value is updated based on the positive rate of the T790M mutant template in the trans mutation of T790M and C797S, the positive rate of the C797S mutant template in the trans mutation of T790M and C797S, and the positive rate of the cis mutation.

In some embodiments, determining the positive rate of the mutant template based on the statistical information for each signal channel comprises: determining the positive rate and/or the negative rate of each signal channel and the positive rate and/or the negative rate of different signal channel combinations based on the statistical information of each signal channel; and determining the positive rate of the mutant template based on the positive rate and/or the negative rate of each signal channel and the positive rate and/or the negative rate of different signal channel combinations.

In some embodiments, determining the positive rate and/or negative rate of each signal channel, and the positive rate and/or negative rate of different signal channel combinations based on the statistical information of each signal channel comprises at least one of: determining the positive rate based on the relation between the number of two signal channels which are positive and negative in the reaction units and the number of effective reaction units; determining a negative rate based on the relationship between the number of negative two signal channels in the reaction unit and the number of effective reaction units in the reaction unit; determining the positive rate based on the relation between the number of three signal channels which are positive and one signal channel which is negative in the reaction units and the number of effective reaction units; determining a negative rate based on a relationship between the number of negative signal channels in the reaction cells and the number of active reaction cells in the reaction cells; determining the positive rate based on the relation between the number of the reaction units and the number of effective reaction units, wherein one signal channel is positive and three signal channels are negative; the positive rate was determined based on the number of positive four signal channels in the reaction cell versus the number of effective reaction cells.

In some embodiments, the method further comprises: determining an abnormal reaction unit and removing the abnormal reaction unit; and determining the positive rate of the mutant template based on the positive rate and/or the negative rate of each signal channel of the effective reaction unit after the abnormal reaction unit is removed.

In some embodiments, determining an anomalous reaction cell includes at least one of: determining a reaction unit with one positive signal channel and three negative signal channels as an abnormal reaction unit, and determining the abnormal reaction unit as an invalid reaction unit or converting positive information of the abnormal reaction unit into negative information; and determining a combination of two signal channels which are positive and two signal channels which are negative in the reaction units as an abnormal combination, determining the reaction unit corresponding to the abnormal combination as an abnormal reaction unit, and determining the abnormal reaction unit as an invalid reaction unit or converting the positive information of the abnormal reaction unit into negative information.

In some embodiments, determining the positive rate of a mutant template based on the positive rate and/or negative rate of each signal channel, and the positive rate and/or negative rate of different combinations of signal channels comprises: and determining the positive rate of the mutant template based on the statistical probability that two signal channels in the reaction unit are negative and the other two signal channels in the reaction unit are positive, wherein the positive rate of the mutant template comprises the positive rate of the T790M mutant template in the trans-mutation of T790M and C797S, the positive rate of the C797S mutant template in the trans-mutation of T790M and C797S and the positive rate of the cis-mutation.

In some embodiments, the method further comprises: and checking the positive rate of the mutant template, and determining the copy number of the mutant template based on the checked positive rate.

In some embodiments, the positive rate of the mutant template is checked, comprising: and checking the positive rate of the mutation template based on the statistical probability that one signal channel in the reaction unit is negative and the other three signal channels are positive.

In some embodiments, the positive rate of the mutant template is checked, comprising: and checking the positive rate of the mutation template based on the statistical probability that the four signal channels in the reaction unit are positive.

A T790M and C797S cis-trans mutation type identification and calculation device, the device includes: the statistical information determining module is used for determining the statistical information of each signal channel in the reaction unit, and the statistical information comprises the positive information and/or the negative information of each signal channel; the positive rate calculation module is used for determining the positive rate of a mutation template based on the statistical information of each signal channel, and the mutation template comprises cis-mutation and/or trans-mutation; a type recognition module for determining the type of cis-trans mutation of T790M and C797S based on the positive rate of the mutation template.

A computer device comprising a memory and a processor, the memory storing a computer program, the processor implementing the following steps when executing the computer program: determining statistical information of each signal channel in the reaction unit, wherein the statistical information comprises positive information and/or negative information of each signal channel; determining the positive rate of a mutation template based on the statistical information of each signal channel, wherein the mutation template comprises cis-mutation and/or trans-mutation; the types of cis-trans mutations of T790M and C797S were determined based on the positive rate of the mutant template.

A computer-readable storage medium, on which a computer program is stored which, when executed by a processor, carries out the steps of: determining statistical information of each signal channel in the reaction unit, wherein the statistical information comprises positive information and/or negative information of each signal channel; determining the positive rate of a mutation template based on the statistical information of each signal channel, wherein the mutation template comprises cis-mutation and/or trans-mutation; the types of cis-trans mutations of T790M and C797S were determined based on the positive rate of the mutant template.

The method, the device, the equipment and the medium for identifying and calculating the cis-trans mutation types of the T790M and the C797S calculate the positive rate of the cis-mutation and/or the trans-mutation based on the positive information and/or the negative information of each signal channel in the reaction unit, so as to determine the cis-trans mutation types of the T790M and the C797S according to the positive rate of the mutation template, and determine the corresponding medical medication types according to the cis-trans mutation types of the T790M and the C797S. In the process of calculating the cis-trans mutation types of T790M and C797S, the problem that a T790M mutation template in the trans mutation of T790M and C797S and a C797S mutation template in the trans mutation of T790M and C797S are in a co-chamber mode is considered, the accuracy of identification and calculation of the cis-trans mutation types of T790M and C797S is improved, and the accuracy of selection of the medical drug types is further improved.

Drawings

FIG. 1 is a diagram showing an example of an application environment of the cis-trans mutation type identification and calculation method using T790M and C797S;

FIG. 2 is a schematic flow chart of a method for identifying and calculating cis-trans mutation types of T790M and C797S in one embodiment;

FIG. 3 is a schematic flow chart of a method for determining the positivity of a mutated template provided in some embodiments;

FIG. 4 is a schematic diagram of a process for calculating the copy number of a mutated template based on two signal channels as provided in one embodiment;

FIG. 5 is a schematic flow chart of the method provided in some embodiments for determining the positive rate of a mutated template;

FIG. 6 is a schematic flow chart for calculating the copy number of a mutated template based on four signal channels as provided in one embodiment;

FIG. 7 is a graph of the results provided in one example for identifying cis and trans mutations based on two signal channels;

FIG. 8 is a graph of the results provided in one example for identifying cis and trans mutations based on four signal channels;

FIG. 9 is a graph of the results provided in one example to simulate the results of four signal channels at low positive rates identifying cis and trans mutations;

FIG. 10 is a graph of the results provided in one example to simulate the results of four signal channels at high positive rates identifying cis-and trans-mutations;

FIG. 11 is a block diagram of a device for cis-trans mutation type identification and calculation of T790M and C797S in one embodiment;

FIG. 12 is a diagram illustrating an internal structure of a computer device according to an embodiment.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

The T790M and C797S cis-trans mutation type identification and calculation method provided by the application can be applied to the application environment shown in FIG. 1. Wherein the terminal 102 communicates with the server 104 via a network. The server 104 acquires statistical information of each signal channel in the reaction unit, wherein the statistical information comprises positive information and/or negative information of the signal channel; determining the positive rate of a mutation template based on the statistical information of each signal channel, wherein the mutation template comprises cis-mutation and/or trans-mutation; the types of cis-trans mutations of T790M and C797S were determined based on the positive rate of the mutant template. Further, the types of cis-trans mutations of T790M and C797S may also be sent to the terminal 102 for display. The terminal 102 may be, but not limited to, various personal computers, notebook computers, smart phones, tablet computers, and portable wearable devices, and the server 104 may be implemented by an independent server or a server cluster formed by a plurality of servers.

In one embodiment, as shown in fig. 2, a flow chart of a T790M and C797S cis-trans mutation type identification and calculation method is provided, which is illustrated by taking the method as an example applied to the server 104 in fig. 1, and includes the following steps:

in step 202, statistical information of each signal channel in the reaction unit may be determined, and the statistical information includes positive information and/or negative information of the signal channel.

In some embodiments, two signal channels may be included in the reaction unit, for example, an a signal channel and a B signal channel. The A signal channel can be a T790M mutation site corresponding channel, and the B signal channel can be a C797S mutation site corresponding channel. In some embodiments, the a and B signal channels may be labeled with different fluorescence, which may be detected by different fluorescence channels, such as FAM and ROX. Thereby realizing that the signal channel can judge the negativity or the positivity of the signal channel based on the fluorescence signal value. In other embodiments, a general signal channel E may be added, for example, a general probe E channel, and the abnormal reaction unit is identified according to the general probe E channel.

In some embodiments, an analysis result of the DPCR may be obtained, the analysis result including statistical information of each signal channel, a positive rate of the signal channel may be determined based on positive information in the statistical information, and a negative rate of each signal channel may be determined based on negative information in the statistical information.

In some embodiments, after obtaining the analysis result of the DPCR, determining whether the reaction unit includes the a signal channel and the B signal channel based on the analysis result is further included. And if the A signal channel and the B signal channel are judged to be contained in the reaction unit based on the analysis result, counting the positive information of each signal channel in the reaction unit, and calculating the positive rate of the signal channel based on the positive information. And if the reaction unit does not contain the signal channel A and the signal channel B based on the analysis result, exiting and prompting that the analysis is unsuccessful, and giving prompt information. The prompt message includes a flag indicating whether the reaction unit is a valid reaction unit, and also includes information indicating whether the signal channel A and the signal channel B corresponding to the reaction unit are positive.

In some embodiments, it may be further determined whether an E signal channel exists based on the analysis result, if the E signal channel exists, the result needs to be calibrated, the result of the a signal channel and the result of the B signal channel are calibrated by using the result of the E signal channel, and when the a signal channel is positive or the B signal channel is positive, the E signal channel in the corresponding reaction unit is also positive.

In some embodiments, the types of mutation templates include cis-mutations and trans-mutations. The trans mutation comprises a T790M mutant template in the trans mutation of T790M and C797S, a C797S mutant template in the trans mutation of T790M and C797S, and the T790M and C797S trans mutations are embodied as follows: both trans mutations occur simultaneously and are located on different alleles. For example, the T790M and C797S cis mutations: T790M and C797S occur simultaneously and are located on the same allele; specifically, the T790M mutation site corresponding to the A signal channel on one gene is positive, the C797S mutation site corresponding to the B signal channel is positive, and the positive rate of cis-mutation can be recorded as T_AB. For example, in the trans-mutation of T790M and C797S, the T790M mutant template in the T790M and C797S refers to that the mutation site corresponding to the A signal channel in one gene is positive, the C797S mutant template site in the T790M and C797S in the trans-mutation of the B signal channel is not positive, and the positive rate of the T790M mutant template in the T790M and C797S in the trans-mutation can be recorded as the positive rate of the T790M mutant template_A. The C797S mutant template in the trans-mutation of T790M and C797S means that the mutant site corresponding to the B signal channel on one gene is positive, the T790M mutant site corresponding to the A signal channel is not positive, and the positive rate of the C797S mutant template in the trans-mutation of T790M and C797S can be recorded as T_B。

Figure 3 is a schematic flow diagram of a method for determining the positive rate of a mutated template provided in some embodiments. Referring to fig. 3, in some embodiments, determining the positive rate of a mutated template based on the statistical information for each signal channel comprises:

in step 302, the positive rate of each signal channel and the common positive rate of different signal channel combinations can be determined based on the statistical information of each signal channel.

In some embodiments, determining the positive rate for each signal channel based on the statistical information for each signal channel comprises: the positive rate of the first signal channel is determined based on the relationship of the number of positives of the first signal channel to the number of active reaction units in the reaction unit. For example, the number of effective reaction units in the reaction units is counted by DPCR analysis resultsQuantity N, the number of positives N of a first signal channel (e.g., A signal channel) is counted on an effective reaction unit basis_ACalculating the positive rate of the A signal channel as P_A＝N_A/N。

In some embodiments, determining the positive rate for each signal channel based on the statistical information for each signal channel comprises: the positive rate of the second signal channel is determined based on the relationship of the number of positives of the second signal channel to the number of active reaction units in the reaction unit. For example, the number N of effective reaction units in the reaction unit is counted, and the number N of positives of the second signal channel (e.g., B signal channel) is counted on the basis of the effective reaction units by DPCR analysis results_BAnd calculating the positive rate of the B signal channel as P_B＝N_B/N。

In some embodiments, determining a common positive rate for different combinations of signal channels based on statistics for each signal channel comprises: and determining the common positive rate of the first signal channel and the second signal channel based on the relation between the number of the first signal channel and the second signal channel which are simultaneously positive and the number of the effective reaction units in the reaction units. For example, the number N of effective reaction units in a reaction unit is counted by the DPCR analysis result, and the number N of positive A signal channel and positive B signal channel is counted on the basis of the effective reaction units_ABCalculating the common positive rate of the A signal channel and the B signal channel in the same reaction unit as P_AB＝N_AB/N。

At step 304, an initial adjustment value may be determined based on the positive rate and the common positive rate of each signal channel. In some embodiments, the initial adjustment value may be determined based on a positive rate of the first signal channel, a positive rate of the second signal channel, and a common positive rate of the first signal channel and the second signal channel. For example, the initial adjustment value tmp, tmp ═ P_A*P_B(1-P_AB)。

At step 204, the positivity of the mutated template may be determined based on the statistical information of each signal channel. For example, at step 306, the positivity rate of the mutant template can be determined based on the initial adjustment value and the positivity rate of each signal channel.

In some embodiments, the positive rate of the mutated template is determined in response to the positive rate of the first signal channel, the positive rate of the mutated template, and the initial adjustment value satisfying a preset condition with a preset threshold, and the common positive rate, the positive rate of the mutated template, and the initial adjustment value satisfying the preset condition. For example, in response to the positive rate of the first signal channel, the template positive rate of the T790M mutant template in the trans-mutation of T790M and C797S, the template positive rate and the initial adjustment value of the cis-mutation and the preset threshold satisfying the preset conditions, and the common positive rate of the first signal channel and the second signal channel, the template positive rate and the initial adjustment value of the cis-mutation and the preset threshold satisfying the preset conditions, the positive rate of the mutant template is determined. For example, minErr is a preset threshold (e.g., an expected target demand value). When P is present_A–(T_A-T_A*T_AB-tmp)<minErr&&P_AB-(T_AB+tmp)<And (5) judging that the predetermined condition is met during minErr, and further obtaining the positive rate of the mutation template.

In some embodiments, in response to the positive rate of the first signal channel, the positive rate of the mutated template, and the initial adjustment value and the preset threshold not satisfying the preset condition, or the common positive rate, the positive rate of the mutated template, and the initial adjustment value and the preset threshold not satisfying the preset condition, the positive rate of the mutated template is determined based on the updated initial adjustment value. For example, in response to the positive rate of the first signal channel, the template positive rate of the T790M mutant template in the trans mutation of T790M and C797S, the template positive rate of the cis mutation, and the initial adjustment value and the preset threshold value not satisfying the preset conditions, or the common positive rate of the first signal channel and the second signal channel, the template positive rate of the cis mutation, and the initial adjustment value and the preset threshold value not satisfying the preset conditions, the initial adjustment value is updated, the calculation is continued based on the updated initial adjustment value until the positive rate determined based on the updated initial adjustment value satisfies the preset conditions, at which time the positive rate of the mutant template is determined based on the updated initial adjustment value.

In some embodiments, the positive rate of the T790M mutant template in the trans mutation of T790M and C797S, and the positive rate of the C797S mutant template in the trans mutation of T790M and C797S may be based onAnd updating the initial adjustment value by the rate and the positive rate of the cis-mutation, and determining the positive rate of the mutation template based on the updated initial adjustment value. For example, when P is not satisfied_A–(T_A-T_A*T_AB-tmp)<minErr&&P_AB-(T_AB+tmp)<minErr, the initial adjustment value tmp ═ T is updated_AT_B-T_AT_BT_ABCalculating T based on the updated initial adjustment value_A、T_B、T_AB、P_A、P_B、P_ABUp to P_A–(T_A-T_A*T_AB-tmp)<minErr&&P_AB-(T_AB+tmp)<And obtaining the positive rate of the mutant template in minErr.

In some embodiments, the positive rate of the first signal channel, the positive rate of the second signal channel, and the shared positive rate are determined based on the positive rate of the T790M mutant template in the trans mutation of T790M and C797S, the positive rate of the C797S mutant template in the trans mutation of T790M and C797S, and the positive rate of the cis mutation. And determining the positive rate of the mutant template based on the initial adjustment value and the positive rate of each signal channel, wherein the positive rate of the cis-mutant template and the positive rate of the trans-mutant template are determined.

In some embodiments, the positive rate of the T790M mutant template in the trans mutation of T790M and C797S may be determined based on the positive rate of the first signal channel, the positive rate of the cis mutation, and the initial adjustment value. For example, the T790M template positivity in the T790M and C797S trans mutations_A＝(P_A+tmp)/(1-T_AB). And, the positive rate P of the first signal channel_ACan be expressed as P_A＝T_A(1-T_B-T_AB+T_BT_AB)。

In some embodiments, the positive rate of the C797S mutant template in the trans mutation of T790M and C797S may be determined based on the positive rate of the second signal channel, the positive rate of the cis mutation, and the initial adjustment value. For example, the positive rate T of the C797S mutant template in the trans mutation of T790M and C797S_B＝(P_B+tmp)/(1-T_AB). And, the positive rate P of the second signal channel_BCan be used forIs expressed as P_B＝T_B(1-T_A-T_AB+T_AT_AB)。

In some embodiments, the positive rate of cis-mutations can be determined based on the shared positive rate of the first signal channel and the second signal channel and the initial adjustment value. For example, positive rate T of cis-mutation_AB＝P_AB-tmp. And, the common positive rate P of the first signal channel and the second signal channel_ABCan be expressed as P_AB＝T_AB+(T_AT_B-T_AT_BT_AB)。

In one embodiment, the step of calculating the positive rate of the mutant template is as follows:

step 1: tmp ═ P_A*P_B(1-P_AB)。

Step 2: p_A＝T_A(1-T_B-T_AB+T_BT_AB)，T_A＝(P_A+tmp)/(1-T_AB)。

And step 3: p_B＝T_B(1-T_A-T_AB+T_AT_AB)，TB＝(P_B+tmp)/(1-T_AB)。

And 4, step 4: p_AB＝T_AB+(T_AT_B-T_AT_BT_AB)，T_AB＝P_AB–tmp，tmp＝T_AT_B-T_AT_BT_AB。

And 5: p_A–(T_A-T_A*T_AB-tmp)<minErr&&P_AB-(T_AB+tmp)<minErr。

Wherein: minErr is a preset threshold, which can be understood as the expected target demand value. Step 1 is to initialize the initial adjustment value, and then to loop step 2 to step 4 until the condition of step 5 is met. And calculating to obtain the positive rate of the mutant template.

In the above embodiment, in the digital PCR instrument, mutations are represented by two signal channels (or one channel using a universal probe is added), and on the basis of the positive rate of each signal channel, the more accurate positive rates and copy numbers of the trans-mutation and the cis-mutation are estimated by using an iterative algorithm, so that the identification accuracy of the mutant template and the calculation accuracy of the positive rate and copy number of the mutant template are improved.

At step 206, the type of cis-trans mutation of T790M and C797S may be determined based on the positive rate of the mutant template. The magnitude of the positive rate indicates the magnitude of the probability, so that the types of cis-trans mutations of T790M and C797S can be determined by the magnitude of the positive rate. For example, when the corresponding probability is 0, it indicates that the T790M and C797S cis-trans mutations are not present.

In some embodiments, determining the copy number of the mutant template based on the positive rate of the mutant template comprises: and calculating the copy number of the mutant template based on the positive rate of the signal channel, and calculating the copy number of the mutant template through the positive rate distribution of the mutant template in the reaction unit and the basic information of the biological experiment. And guiding the medication in the actual scene according to the calculated copy number. The basic information of the biological experiment can include dilution times of biological reagents, reaction unit volumes, poisson distribution and the like.

As shown in fig. 4, fig. 4 is a schematic flow chart for calculating the copy number of the mutated template based on two signal channels provided in one embodiment. The method specifically comprises the following steps:

step 402, obtaining DPCR analysis results.

And step 404, calculating the positive rate of the A signal channel and the B signal channel and the common positive rate of the A signal channel and the B signal channel based on the analysis result.

And step 406, calculating the positive rate of the mutation template based on the positive rate of each signal channel and the shared positive rate.

Step 408, calculating the copy number of the mutated template based on the positive rate of each mutated template.

In the process of calculating the positive rate of the mutation template, the common positive rate of different signal channel combinations is considered, and the accuracy of calculating the positive rate of the mutation model is further improved.

In some embodiments, four signal channels may be included in a reaction unit, for example two mutant signal channels and two wild signal channels may be included. The two abrupt signal channels are an A signal channel and a B signal channel respectively. For example, the a signaling channel may be the T790M mutation site-corresponding channel and the B signaling channel may be the C797S mutation site-corresponding channel. The two wild signal channels may be a C signal channel and a D signal channel, respectively. For example, the C wild-signal channel may be the channel corresponding to the T790M wild-signal, and the D wild-signal channel may be the channel corresponding to the C797S wild-signal. In other embodiments, a general signal channel E may be added, for example, a general probe E channel, and the abnormal reaction unit is identified according to the general probe E channel.

In some embodiments, the analysis result of the digital PCR may be obtained, and it is determined whether there are four signal channels based on the analysis result (e.g., the a signal channel is the T790M mutant signal corresponding channel, the B signal channel is the C797S mutant signal corresponding channel, the C signal channel is the T790M wild signal corresponding channel, and the D signal channel is the C797S wild signal corresponding channel). If the result data of the four signal channels exist, the result data of the four signal channels are extracted, the result data correspond to each reaction unit, namely, each reaction unit corresponds to the state of the four signal channels, each state is divided into a positive state and a negative state, a positive state indicates that the current signal channel has a signal, and the positive state and the negative state are mainly distinguished through the brightness in the reaction units.

And judging whether an E signal channel exists or not based on the analysis result data, if so, calibrating the result, and checking A, B, C and D signal channel results based on the channel result, wherein when an A or B or C or D signal channel is positive, the E signal channel in the corresponding reaction unit is also positive.

Figure 5 is a schematic flow chart of the method provided in some embodiments for determining the positive rate of a mutated template. As shown in fig. 5, in some embodiments, determining the positive rate of the mutated template, and the positive rate and/or negative rate of different signal channel combinations based on the statistical information of each signal channel comprises:

step 502, the positive rate and/or negative rate of each signal channel and the different signal channels can be determined based on the statistical information of each signal channelPositive and/or negative rates of the combinations. The method comprises the following steps: the positive rate is determined based on the number of two signal channels in the reaction unit that are positive and two signal channels that are negative versus the number of active reaction units in the reaction unit. For example, the valid reaction unit index and the number of valid reaction units are counted based on the label of each reaction unit (e.g., whether it is a label of a valid reaction unit). On the basis of the index of the effective reaction unit, counting the information that only two signal channels in the effective reaction unit are positive, wherein the detailed statistical information comprises the following information: the positive rate that the A signal channel and the D signal channel are positive and the B signal channel and the C signal channel are negative in the reaction unitThe positive rate that the B signal channel and the C signal channel in the reaction unit are positive and the A signal channel and the D signal channel in the reaction unit are negativeThe positive rate that the A signal channel and the B signal channel are positive and the C signal channel and the D signal channel are negative in the reaction unitThe positive rate that the C signal channel and the D signal channel in the reaction unit are positive and the A signal channel and the B signal channel are negativePositive rate and index of A signal channel and C signal channel in reaction unit, and negative rate of B signal channel and D signal channel; and the positive rate and the index of the B signal channel and the D signal channel in the reaction unit are positive, and the positive rate and the index of the A signal channel and the C signal channel are negative.

In some embodiments, determining the positive rate and/or negative rate of each signal channel, and the positive rate and/or negative rate of different signal channel combinations based on the statistical information of each signal channel comprises: the negative rate is determined based on the relationship between the number of negative two signal channels in the reaction cell and the number of effective reaction cells in the reaction cell. For exampleOn the basis of the effective reaction unit index, counting the negative information of the two signal channels, wherein the detailed statistical information comprises the following steps: the number ratio of the number of negative B signal channels and the number of negative C signal channels to the number of effective N channels in the reaction unitThe ratio of the number of negative A signal channels and the number of negative D signal channels to the number of the effective number N in the reaction unitThe number ratio of the number of negative C-signal channels and the number of negative D-signal channels to the number of the effective number N in the reaction unitThe ratio of the number of negative A signal channels and the number of negative B signal channels to the number of effective N channels in the reaction unit

In some embodiments, determining the positive rate and/or negative rate of each signal channel, and the positive rate and/or negative rate of different signal channel combinations based on the statistical information of each signal channel comprises: the positive rate was determined based on the number of three signal channels positive and one signal channel negative in the reaction unit versus the number of active reaction units in the reaction unit. For example, on the basis of the effective reaction unit index, the positive rate that three signal channels are positive and one signal channel is negative is counted, and specific statistical information includes: positive rate of A signal channel, B signal channel and C signal channel being positive and D signal channel being negative in reaction unitPositive rate of A signal channel, B signal channel and D signal channel being positive and C signal channel being negative in reaction unitIn the reaction unit APositive rate of signal channel, C signal channel, D signal channel being positive and B signal channel being negativePositive rate of B signal channel, C signal channel, D signal channel being positive and A signal channel being negative in reaction unit

In some embodiments, determining the positive rate and/or negative rate of each signal channel, and the positive rate and/or negative rate of different signal channel combinations based on the statistical information of each signal channel comprises: the negative rate is determined based on the relationship between the number of negative signal channels in the reaction unit and the number of effective reaction units in the reaction unit. For example, on the basis of the index of the effective reaction units, the positive rate that a signal channel is negative is counted, and specific statistical information includes: positive rate of A signal channel in reaction unit being negativePositive rate in reaction cell in which B is negativePositive rate of C signal channel in reaction cell being negativePositive rate of negative D signal in reaction cell

In some embodiments, determining the positive rate and/or negative rate of each signal channel, and the positive rate and/or negative rate of different signal channel combinations based on the statistical information of each signal channel comprises: the positive rate is determined based on the number of positive signal channels and negative signal channels in the reaction unit in relation to the number of active reaction units in the reaction unit. For example, on the basis of the index of effective reaction units, the condition that one channel is positive appears in the median of each effective reaction unit is counted as follows: positive rate and position index of only A signal channel in the reaction unit as positive; positive rate and position index of only B signal channel in reaction unit; positive rate and position index of only C signal channel in reaction unit; positive rate and position index where only the D signal channel in the reaction unit is positive. It can be understood that the corresponding position can be determined through the position index, and thus accurate positioning of the corresponding information can be realized.

In some embodiments, determining the positive rate and/or negative rate of each signal channel, and the positive rate and/or negative rate of different signal channel combinations based on the statistical information of each signal channel comprises: the positive rate was determined based on the number of positive four signal channels in the reaction cell versus the number of active reaction cells in the reaction cell. For example, on the basis of the effective reaction unit index, the positive rate P (ABCD) of positive signals of the A signal channel, the B signal channel, the C signal channel and the D signal channel, all four signal channels are counted.

In some embodiments, the method further comprises: determining an abnormal reaction unit and removing the abnormal reaction unit; and determining the positive rate of the mutant template based on the positive rate and/or the negative rate of each signal channel of the effective reaction unit after the abnormal reaction unit is removed. For example, removing outliers specifically includes: and carrying out abnormal value removing processing on the reaction unit with one signal channel as positive, and carrying out abnormal value removing processing on the reaction unit with two signal channels as positive.

In some embodiments, the abnormal reaction unit specifically includes: and determining the reaction unit with one positive signal channel and three negative signal channels as an abnormal reaction unit, and determining the abnormal reaction unit as an invalid reaction unit or converting the positive information of the abnormal reaction unit into the negative information. For example, the way of removing outliers for a reaction unit that has one signal channel positive includes: and determining the reaction unit as an invalid reaction unit, and updating the number N of the valid reaction units and all the counted positive rates. Alternatively, the reaction unit is used as an effective reaction unit, and information of a signal channel in the reaction unit is converted into negative information.

In some embodiments, the abnormal reaction unit specifically includes: and determining an abnormal combination in a reaction unit of which two signal channels are positive and two signal channels are negative, determining a reaction unit corresponding to the abnormal combination as an abnormal reaction unit, and determining the abnormal reaction unit as an invalid reaction unit or converting positive information of the abnormal reaction unit into negative information. For example, the reaction unit with two signal channels positive is processed, specifically includes processing the combination which does not occur, and specifically, all the positive channels in the reaction unit with abnormal combination can be set as negative. Or setting the reaction units with abnormal combination as invalid reaction units, and updating the number N of the valid reaction units and the positive rate of all statistics. In some embodiments, the combined case specific details that do not occur include: in the reaction unit, the signal channel A and the signal channel C are positive, and the signal channel B and the signal channel D are negative. Alternatively, the B signal channel and the D signal channel in the reaction unit are positive, and the A signal channel and the C signal channel are negative.

And step 504, determining the positive rate of the mutation template based on the positive rate and/or the negative rate of each signal channel and the positive rate and/or the negative rate of different signal channel combinations. In some embodiments, determining the positive rate of a mutant template based on the positive rate and/or negative rate of each signal channel, and the positive rate and/or negative rate of different combinations of signal channels comprises: and determining the positive rate of the mutant template based on the statistical probability that two signal channels in the reaction unit are negative and the other two signal channels in the reaction unit are positive, wherein the positive rate of the mutant template comprises the positive rate of the T790M mutant template in the trans-mutation of T790M and C797S, the positive rate of the C797S mutant template in the trans-mutation of T790M and C797S and the positive rate of the cis-mutation.

For example, the positive rate of a mutated template can be calculated from statistical information that only two signal channels in a reaction unit are positiveThe specific calculation comprises: wherein ai is the positive rate of the mutant template. a1 is the positive rate of T790M mutant template in the trans mutation of T790M and C797S, a2 is the positive rate of C797S mutant template in the trans mutation of T790M and C797S, a3 is the positive rate of cis-mutant template, and a4 is the positive rate of pure wild template. Ai can be calculated through the statistical information of each signal channel. P1i is the positive rate of only a single template in the mutant template in the reaction unit.

In some embodiments, the method further comprises verifying the positive rate of the mutated template to determine whether the positive rate of the mutated template is as expected. In some embodiments, the method further comprises: and checking the positive rate of the mutant template, and determining the copy number of the mutant template based on the checked positive rate.

In some embodiments, the positive rate of the mutant template is checked, comprising: and checking the positive rate of the mutation template based on the statistical probability that one signal channel in the reaction unit is negative and the other three signal channels are positive. For example, whether the probability distribution is satisfied is determined based on a combination of three signal channels being positive and one signal channel being negative, and one signal channel being negative. The specific embodiment is as follows: wherein di is U sqrt (Pi/N), a is in [ P-di, P + di]U is a proportionality coefficient, N is the number of effective reaction units, Pi is the probability of the calculation result,a is the positive rate of the signal channel. By the above formula, it can be verified thatThree signal channels in one reaction unit are positive and whether the probability of two signal channels in one reaction unit meets the probability formula. If the template is matched, the positive rate result of the mutation template obtained by the initial calculation is used for accurate calculation. If not, further calculation is performed, for example, the initial positive rate can be determined based on the sum of the probability of two signal channels of a mutation template in the same reaction unit, the probability of three signal channels in the same reaction unit, and the probability of the occurrence of the mutation template in four signal channels (based on the positive rate of the initial template).

In some embodiments, the positive rate of the mutant template is checked, comprising: and checking the positive rate of the mutation template based on the statistical probability that the four signal channels in the reaction unit are positive and the positive rate of the mutation template. For example, the preliminary positive rate results are checked based on four signal channels to determine whether the probability distribution is satisfied, p ═ a3 × a4+ a1 × a2-a1 × a2 × a3 × a4, di ═ U2 × sqrt (Pi/N), p (abcd) epsilon [ p-di, p + di ]. Wherein U2 is a proportionality coefficient, if the formula P (ABCD) epsilon [ p-di, p + di ] is not satisfied, a prompt needs to be given, and then further verification is carried out, if the formula P (ABCD) epsilon [ p-di, p + di ] is satisfied, the next step is directly carried out.

In some embodiments, the copy number of each mutated template is determined based on the distribution of the positive rates of template occurrences within the reaction cell, the basic information of the biological experiment, and the positive rates of the mutated templates.

In some embodiments, when two signaling pathways are in one reaction unit, there is a positive rate of only one mutated template, P1 i.

In some embodiments, when three signal channels are in one reaction unit, there are two instances of mutated template. For example, there are two cases including P1i, and the positive rates are P21i and P22i, respectively. For example, the mutation template 1 corresponds to two cases, respectivelyAnd

in some embodiments, when four signal channels are in one reaction unit, two cases can be distinguished. In the case of two mutant templates in one reaction unit, two trans mutant templates in one reaction unit or a cis mutant template and a wild template in one reaction unit, P31i can be calculated. In another case, a certain mutant template and the first type without the mutant template in a reaction unit can be used to calculate the positive rate P32i, P31i aj ak, and P32i ai aj ak. Wherein, the calculation of P31i shows that two mutation templates are in one reaction unit, two cases are mainly used, and when P31i represents the positive rate of a certain trans mutation template in the case, the result is the joint probability of the two trans mutation templates. If it is a cis-mutation or a wild-signal template, it is the combined probability of the cis-mutation and the wild-signal template. P32i shows that three mutant templates are in the same reaction unit, and the following cases mainly exist: a certain trans mutation, cis mutation and wild signal are in the same reaction unit, or a cis mutation or wild signal and two trans mutations are in the same reaction unit.

In some embodiments, further comprising: and calculating the positive rate Ri, Ri ═ P1i + P21i + P22i + P31i + P32i of the mutant template. When P31i is greater than P (abcd), it is set to P (abcd). And the value of P31i needs to be comprehensively judged subsequently. After the positive rate Ri of the mutant template is obtained, calculating the copy number of each mutant template according to the distribution condition of the positive rate of the mutant template appearing in the reaction unit and the basic information of the biological experiment.

In some embodiments, the method further comprises a verification step. Defining di as U3 sqrt (Ri/N), wherein U3 is a proportionality coefficient, if ai e [ Ri-di, Ri + di ], the result is judged to be reasonable, otherwise, the result needs to be re-evaluated (for example, when the data result is abnormal, or the local data does not accord with the probability distribution, the phenomenon occurs). Specific evaluation embodiments include:

(1) and judging whether the number of all positive signals in the four signal channels is less, if so, replacing ai with Ri, recalculating the positive rate of the mutation template, and outputting the result.

(2) When the number of all positive signals in the signal channel is more, the re-evaluation is needed, and the specific implementation scheme is as follows: and (4) calculating positive rates PA, PB and PAB of cis-mutation and trans-mutation by using a scheme of an A signal channel and a scheme of a B signal channel. And calculating the positive rate PCD of the wild signal according to the two signal channel schemes of the C signal channel and the D signal channel. ③ PA replaces a1, PB replaces a2, PAB replaces a3, PCD replaces a4, and then the calculation of the positive rate of the mutation template is repeated, if the positive rate of the mutation template is abnormal, abnormal information is given and the termination is performed, or other alternative schemes are used, such as a1, a2, a3 and a4 are calculated by using the relations that three signal channels are positive and one signal channel is negative.

FIG. 6 is a schematic flow chart of calculating the copy number of the mutated template based on four signal channels, as shown in FIG. 6, comprising:

step 602, obtaining the DPCR analysis result.

And step 604, determining the statistics of the positive information of two signal channels in the reaction unit, the statistics of the positive information of three signal channels in the reaction unit, the statistics of the positive information of one signal channel in the reaction unit and the statistics of the positive information of four signal channels in the reaction unit based on the obtained DPCR analysis result.

Step 606, the number of mutated templates is determined based on the positive information of the signal channels within the reaction unit.

Step 608, determining the copy number of the mutated template based on the number calculation result of the mutated template.

In the above example, the results calculated based on the two signal channels were good for identifying the types of cis-trans mutations of T790M and C797S. However, when two trans-mutation signals are present in the same reaction unit, there is a case where a cis-mutation cannot be accurately identified. The low positive rate is 1% or less, and the high positive rate is [ 1% -15% ]. The specific recognition accuracy is shown in fig. 7 to 10. Fig. 7 and 8 are used to show the recognition situation, and fig. 7 is directed to the correct rate of recognition of cis-and trans-mutations in the case of various positive rates of results for both signaling channels. Figure 8 identifies the correct rates of the cis-mutation and trans-mutation for various positive rates of the four signal channel results. Fig. 9 and 10 are used for analysis of the accuracy of identification of cis-and trans-mutations of four signal channels. As can be seen by analysis, the number error mainly comes from the number of two trans-mutations in one reaction unit, cis-mutation and pure wild-type in one reaction unit. But this error is smaller than the error of the two signal path results.

FIG. 9 shows that the positive rate of the C797S mutant template in the trans mutation based on T790M and C797S was 2%, the positive rate of the T790M mutant template in the trans mutation based on T790M and C797S was 1.5%, the positive rate of the homeopathic mutation signal was 0.03%, and the positive rate of the pure wild signal was 14%. On the basis, when the positive rate of only one signal channel is changed (for example, when the positive rate of the T790M mutant template signal in the T790M and C797S trans-mutation is 0.15 percent, the experiment is simulated for a plurality of times, the result of the experiment is analyzed, and the concentration or other templates can be changed in the same way), and the amplitude of the error condition is taken as a basis. The positive rate of the C797S mutant template in the trans mutation of T790M and C797S in FIG. 10 was 7.7%, the positive rate of the T790M mutant template in the trans mutation of T790M and C797S was 6.9%, the positive rate of the homeopathic mutation signal was 0.1%, and the positive rate of the pure wild signal was 14%, and the error cases were counted using the same method. At this reference concentration, when the cis mutation concentration is 0, the recognition rate of two signal channels is less than 100%, so that the four signal channels have better recognition results than two signal channels. The simulation experiment result, the actual experiment result and the experiment result are subjected to t distribution inspection and belong to the same sample. The error relative proportion is a known number compared with the average difference of the identified cis-mutation positive number and the known number; the results of the four signal channels are more accurate than the results of the two signal channels in the aspect of results, and when the positive rate of the trans-mutation is higher and the positive rate of the cis-mutation is lower, the two signal channels are mistakenly identified; the cis-trans mutation types can be well identified by the two signal channels, but errors exist in extreme cases, for example, in fig. 10, when the positive rate of cis-mutation is 0, the error ratio of the two signal channels is greater than 0, which indicates that a false identification condition exists.

In the above embodiment, in order to identify the number of cis-mutations and trans-mutations more accurately, it is proposed to calculate the number of cis-mutations and trans-mutations based on that four signal channels (or one signal channel using a general probe is added) represent the mutation condition, and the calculated result is better, and the calculated result is checked and verified to ensure that the obtained results meet the respective probability ratios, thereby improving the accuracy of the calculated result.

In the above examples, a universal signaling channel (e.g., universal probe) is added to verify the presence of a designated mutant template (e.g., cis-mutation, trans-mutation, wild-type template). If the template universal channel is positive, the interference of false positive signals is eliminated, and the results of cis-mutation and trans-mutation can be better identified.

In the above embodiments, the reason why the use of four signal channels is more reasonable and accurate than the use of two signal channels includes, but is not limited to: 1. the number of the trans mutation template and the cis mutation template in the same reaction unit can be accurately identified by four signal channels, and is known, while two signal channels are unknown. 2. Only the number of cis-mutations in the reaction unit is known, and the two signal channels are unknown. 3. The result calculation of the four signal channels is simpler, and the result detection has diversity.

It should be understood that although the various steps in the flow charts of fig. 2-6 are shown in order as indicated by the arrows, the steps are not necessarily performed in order as indicated by the arrows. The steps are not performed in the exact order shown and described, and may be performed in other orders, unless explicitly stated otherwise. Moreover, at least some of the steps in fig. 2-6 may include multiple steps or multiple stages, which are not necessarily performed at the same time, but may be performed at different times, which are not necessarily performed in sequence, but may be performed in turn or alternately with other steps or at least some of the other steps.

In one embodiment, as shown in fig. 11, there is provided a T790M and C797S cis-trans mutation type identification and calculation apparatus, which may be a software module or a hardware module, or a combination of both, as a part of a computer device, and specifically includes:

a statistical information determining module 1102, configured to determine statistical information of each signal channel in the reaction unit, where the statistical information includes positive information and/or negative information of each signal channel.

And a positive rate calculation module 1104 for determining a positive rate of the mutation template based on the statistical information of each signal channel, wherein the mutation template comprises cis-mutation and/or trans-mutation.

A type identification module 1106 for determining the type of cis-trans mutation of T790M and C797S based on the positive rate of the mutation template.

In some embodiments, the apparatus further comprises a copy number determination module for determining the copy number of the mutated template based on the positivity of the mutated template and the basic information of the biological experiment.

In some embodiments, the positivity rate calculation module 1104 is further configured to determine a positivity rate of each signal channel based on the statistical information of each signal channel, and a common positivity rate of different signal channel combinations; determining an initial adjustment value based on the positive rate and the common positive rate of each signal channel; and determining the positive rate of the mutant template based on the initial adjustment value, the positive rate of the signal channel and the shared positive rate.

In some embodiments, the positivity rate calculation module 1104 is further configured to determine a positivity rate of the first signal channel based on a relationship of the number of positivity of the first signal channel to the number of effective reaction units; determining a positive rate for the second signal channel based on a relationship of the number of positives for the second signal channel to the number of active reaction units; determining a common positive rate for the first signal channel and the second signal channel based on a relationship between the number of first signal channel and second signal channel that are simultaneously positive and the number of active reaction units.

In some embodiments, the positivity rate calculation module 1104 is further configured to determine an initial adjustment value based on the positivity rate of the first signal channel, the positivity rate of the second signal channel, and the common positivity rate.

In some embodiments, the positive rate calculation module 1104 is further configured to determine the positive rate of the mutated template in response to the positive rate of the first signal channel, the positive rate of the mutated template, and the initial adjustment value and the preset threshold satisfying the preset condition, and the common positive rate, the positive rate of the mutated template, and the initial adjustment value satisfying the preset condition; and determining the positive rate of the mutation template based on the updated initial adjustment value in response to the positive rate of the first signal channel, the positive rate of the mutation template, the initial adjustment value and the preset threshold value not meeting the preset condition, or the total positive rate, the positive rate of the mutation template, the initial adjustment value and the preset threshold value not meeting the preset condition.

In some embodiments, the positivity rate calculation module 1104 is further configured to determine the positivity rate of the T790M mutation template in the trans mutation of T790M and C797S based on the positivity rate of the first signal channel, the positivity rate of the cis mutation, and the initial adjustment value; determining the positive rate of the C797S mutant template in the trans mutation of T790M and C797S based on the positive rate of the second signal channel, the positive rate of the cis mutation, and the initial adjustment value; and determining the positive rate of the cis-mutation based on the common positive rate and the initial adjustment value of the first signal channel and the second signal channel, wherein the positive rate of the first signal channel, the positive rate of the second signal channel and the common positive rate are determined based on the positive rate of the T790M mutant template in the trans-mutation of T790M and C797S, the positive rate of the C797S mutant template in the trans-mutation of T790M and C797S and the positive rate of the cis-mutation.

In some embodiments, the apparatus further comprises an update module for updating the initial adjustment value based on the positive rate of the T790M mutant template in the trans mutation of T790M and C797S, the positive rate of the C797S mutant template in the trans mutation of T790M and C797S, and the positive rate of the cis mutation.

In some embodiments, the positive rate calculation module 1104 is further configured to determine a positive rate and/or a negative rate of each signal channel, and a positive rate and/or a negative rate of different signal channel combinations based on the statistical information of each signal channel; and determining the positive rate of the mutant template based on the positive rate and/or the negative rate of each signal channel and the positive rate and/or the negative rate of different signal channel combinations.

In some embodiments, the positivity rate calculation module 1104 is further configured to determine a positivity rate based on a number of reaction units in which two signal channels are positive and two signal channels are negative versus a number of active reaction units; determining a negative rate based on the relationship between the number of negative two signal channels in the reaction unit and the number of effective reaction units in the reaction unit; determining the positive rate based on the relation between the number of three signal channels which are positive and one signal channel which is negative in the reaction units and the number of effective reaction units; determining a negative rate based on a relationship between the number of negative signal channels in the reaction cells and the number of active reaction cells in the reaction cells; determining the positive rate based on the relation between the number of the reaction units and the number of effective reaction units, wherein one signal channel is positive and three signal channels are negative; the positive rate was determined based on the number of positive four signal channels in the reaction cell versus the number of effective reaction cells.

In some embodiments, the apparatus further comprises an exception removal module, configured to determine an exception reaction unit and remove the exception reaction unit; and determining the positive rate of the mutant template based on the positive rate and/or the negative rate of each signal channel of the effective reaction unit after the abnormal reaction unit is removed.

In some embodiments, the abnormality removing module is further configured to determine a reaction unit in which one signal channel is positive and three signal channels are negative as an abnormal reaction unit, determine the abnormal reaction unit as an invalid reaction unit, or convert positive information of the abnormal reaction unit into negative information; and determining a combination of two signal channels which are positive and two signal channels which are negative in the reaction units as an abnormal combination, determining the reaction unit corresponding to the abnormal combination as an abnormal reaction unit, and determining the abnormal reaction unit as an invalid reaction unit or converting the positive information of the abnormal reaction unit into negative information.

In some embodiments, the positive rate calculation module 1104 is further configured to determine the positive rate of the mutant template based on the statistical probability of two signal channels being negative in the reaction unit and the statistical probability of two signal channels being negative and two other signal channels being positive in the reaction unit, wherein the positive rate of the mutant template comprises the positive rate of the T790M mutant template in the trans mutation of T790M and C797S, the positive rate of the C797S mutant template in the trans mutation of T790M and C797S, and the positive rate of the cis mutation.

In some embodiments, the apparatus further comprises a verification module for verifying a positive rate of the mutated template, and determining the copy number of the mutated template based on the verified positive rate.

In some embodiments, the verification module is further configured to verify the positive rate of the mutant template based on the statistical probability that one signal channel in the reaction unit is negative, and the other three signal channels are positive.

In some embodiments, the verification module is further configured to verify the positivity of the mutant template based on the statistical probability that the four signal channels in the reaction unit are positive.

For specific limitations of the T790M and C797S cis-trans mutation type identification and calculation devices, see the above limitations for the T790M and C797S cis-trans mutation type identification and calculation methods, and are not described herein again. The various modules in the T790M and C797S cis-trans mutation type identification and computing devices described above may be implemented in whole or in part by software, hardware, and combinations thereof. The modules can be embedded in a hardware form or independent from a processor in the computer device, and can also be stored in a memory in the computer device in a software form, so that the processor can call and execute operations corresponding to the modules.

In one embodiment, a computer device is provided, which may be a server, and its internal structure diagram may be as shown in fig. 12. The computer device includes a processor, a memory, and a network interface connected by a system bus. Wherein the processor of the computer device is configured to provide computing and control capabilities. The memory of the computer device comprises a nonvolatile storage medium and an internal memory. The non-volatile storage medium stores an operating system, a computer program, and a database. The internal memory provides an environment for the operation of an operating system and computer programs in the non-volatile storage medium. The database of the computer device is used for storing statistical information data. The network interface of the computer device is used for communicating with an external terminal through a network connection. The computer program is executed by a processor to realize a T790M and C797S cis-trans mutation type identification and calculation method.

Those skilled in the art will appreciate that the architecture shown in fig. 12 is merely a block diagram of some of the structures associated with the disclosed aspects and is not intended to limit the computing devices to which the disclosed aspects apply, as particular computing devices may include more or less components than those shown, or may combine certain components, or have a different arrangement of components.

In one embodiment, a computer device is further provided, which includes a memory and a processor, the memory stores a computer program, and the processor implements the steps of the above method embodiments when executing the computer program.

In an embodiment, a computer-readable storage medium is provided, in which a computer program is stored which, when being executed by a processor, carries out the steps of the above-mentioned method embodiments.

In one embodiment, a computer program product or computer program is provided that includes computer instructions stored in a computer-readable storage medium. The computer instructions are read by a processor of a computer device from a computer-readable storage medium, and the computer instructions are executed by the processor to cause the computer device to perform the steps in the above-mentioned method embodiments.

It will be understood by those skilled in the art that all or part of the processes of the methods of the embodiments described above can be implemented by hardware related to instructions of a computer program, which can be stored in a non-volatile computer-readable storage medium, and when executed, can include the processes of the embodiments of the methods described above. Any reference to memory, storage, database or other medium used in the embodiments provided herein can include at least one of non-volatile and volatile memory. Non-volatile Memory may include Read-Only Memory (ROM), magnetic tape, floppy disk, flash Memory, optical storage, or the like. Volatile Memory can include Random Access Memory (RAM) or external cache Memory. By way of illustration and not limitation, RAM can take many forms, such as Static Random Access Memory (SRAM) or Dynamic Random Access Memory (DRAM), among others.

The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, but should be considered as the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above examples only express several embodiments of the present application, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the concept of the present application, which falls within the scope of protection of the present application. Therefore, the protection scope of the present patent shall be subject to the appended claims.