阅读说明：本技术 筛选免疫组库测序生物标志物的方法、设备和存储介质 (Method, apparatus and storage medium for screening immunohistochemical library sequencing biomarkers ) 是由 范文涛 王勇斯 陈辉 李连凤 谢洪涛 温韵洁 于 2021-07-07 设计创作，主要内容包括：本公开涉及一种用于筛选免疫组库测序生物标志物的方法、计算设备和存储介质。该方法包括：确定样本中的每一种克隆的初始绝对丰度；基于样品内克隆基因的初始绝对丰度、样品内所有克隆的绝对丰度的总和,计算样品内克隆基因的第一相对丰度；响应于确定克隆基因的初始绝对丰度大于预定阈值,确定克隆基因的第一绝对丰度为1；基于样品内克隆基因的第一绝对丰度、样品内所有克隆的第一绝对丰度的总和,计算样品内克隆基因的第二相对丰度；针对候选克隆基因进行第一检验和第二检验,以便确定免疫组库测序生物标志物,第二检验不同于第一检验。本公开能够显著提高所筛选出的免疫组库的生物标志物的可靠性。(The present disclosure relates to a method, computing device, and storage medium for screening immune repertoire sequencing biomarkers. The method comprises the following steps: determining an initial absolute abundance of each clone in the sample; calculating a first relative abundance of the cloned genes in the sample based on the initial absolute abundance of the cloned genes in the sample and the sum of the absolute abundances of all clones in the sample; responsive to determining that the initial absolute abundance of the cloned gene is greater than a predetermined threshold, determining that the first absolute abundance of the cloned gene is 1; calculating a second relative abundance of the cloned genes in the sample based on the first absolute abundances of the cloned genes in the sample and the sum of the first absolute abundances of all clones in the sample; a first test and a second test are performed against the candidate cloned genes to determine an immune repertoire sequencing biomarker, the second test being different from the first test. The present disclosure can significantly improve the reliability of the biomarkers of the selected immune repertoire.)

1. A method for screening an immunohistochemical library sequencing biomarker comprising:

obtaining immunohistochemical library sequencing data on a sample for determining an initial absolute abundance of each clone in the sample;

calculating a first relative abundance of a cloned gene within the sample based on the initial absolute abundance of the cloned gene within the sample, the sum of the absolute abundances of all clones within the sample;

responsive to determining that the initial absolute abundance of a cloned gene is greater than a predetermined threshold, determining that the first absolute abundance of the cloned gene is 1;

calculating a second relative abundance of the cloned genes in the sample based on the first absolute abundance of the cloned genes in the sample, the sum of the first absolute abundances of all clones in the sample;

determining candidate cloned genes based on the first relative abundance and the second relative abundance of the cloned genes; and

performing a first test and a second test on the candidate cloned genes to determine an immunohistochemical library sequencing biomarker based on the verification result data, the second test being different from the first test.

2. The method of claim 1, wherein determining candidate cloned genes based on the first and second relative abundances of cloned genes comprises:

ranking the first relative abundances of the cloned genes so as to determine a plurality of cloned genes having a ranking order less than a first predetermined order threshold as first candidate cloned genes;

ranking the second relative abundances of the cloned genes to determine a plurality of cloned genes having a ranking order less than a second predetermined order threshold as second candidate cloned genes; and

and determining the candidate cloned genes based on the intersection or union of the first candidate cloned genes and the second candidate cloned genes.

3. The method of claim 1, wherein performing a first test and a second test on candidate cloned genes to determine immunohistochemical library sequencing biomarkers based on the verification result data comprises:

performing a first test and a second test, respectively, for the first relative abundance of the candidate cloned gene to generate first and second check result data, respectively;

performing a first test and a second test on the second relative abundance of the candidate cloned gene respectively to generate third test result data and fourth test result data; and

and determining an immune repertoire sequencing biomarker in the candidate cloned gene based on the first, second, third and fourth check result data.

4. The method of claim 3, wherein determining an immunohistochemical library sequencing biomarker in the candidate cloned gene based on the first validation result data, the second validation result data, the third validation result data, and the fourth validation result data comprises:

determining first significant difference data for the candidate cloned gene based on the comparison of the first check result data and the first threshold;

determining second significant difference data for the candidate cloned gene based on the comparison of the second check result data and the second threshold;

determining third significant difference data for the candidate cloned gene based on the comparison of the third check result data and the third threshold;

determining fourth significant difference data for the candidate cloned gene based on a comparison of the fourth check result data and a fourth threshold; and

determining an immune repertoire sequencing biomarker in the candidate clonal gene based on the first, second, third, and fourth significant difference data.

5. The method of claim 4, wherein determining second significant difference data for the candidate cloned gene based on the comparison of the second test result data and the second threshold comprises:

determining that second significant difference data for the candidate cloned genes in the first and second sets indicates that a significant difference exists in response to determining that the second check result data is greater than or equal to a second threshold; and

in response to determining that the second test result data is less than the second threshold, determining that second significant difference data for the candidate cloned genes in the first and second sets indicates that there is no significant difference.

6. The method of claim 1, further comprising:

in response to determining that the initial absolute abundance of the cloned gene is less than or equal to a predetermined threshold, determining that the first absolute abundance of the cloned gene is 0.

7. The method of claim 1, wherein the first test is a rank sum test and the second test is an analysis of variance test.

8. The method of claim 1, wherein the first test is a Wilcoxon test and the second test is an ANOVA test.

9. The method of claim 1, wherein obtaining immunohistochemical library sequencing data on a sample for determining an initial absolute abundance of each clone in the sample comprises:

obtaining immunohistochemical library sequencing data for the sample via second generation sequencing for the sample;

comparing the immunohistochemical library sequencing data to a predetermined database of clones to determine each clone in the sample; and

calculating an initial absolute abundance of each clone in the sample; and

normalization was performed for the initial absolute abundance of each clone.

10. A computing device, comprising:

at least one processing unit;

at least one memory coupled to the at least one processing unit and storing instructions for execution by the at least one processing unit, the instructions when executed by the at least one processing unit causing the computing device to perform the method of any of claims 1-9.

11. A computer readable storage medium having stored thereon machine executable instructions which, when executed, cause a machine to perform the method of any one of claims 1 to 9.

Technical Field

The present disclosure relates generally to bioinformatics processing, and in particular, to methods, devices, and storage media for screening immune repertoire sequencing biomarkers.

Background

In a biological individual, at any given time, the sum of all functionally diverse B and T cells in the circulatory system is called the Immune Repertoire (IR). T cells and B cells are used as main lymphocytes in organisms (such as human bodies) and are respectively responsible for cellular immunity and humoral immunity, a T Cell Receptor (TCR) and a B Cell Receptor (BCR) are composed of a plurality of peptide chains and have antigen binding specificity, the amino acid composition and arrangement sequence of a complementary determining region (CDR, also called hypervariable region) of each peptide chain are highly diversified, and a T cell receptor bank and a B cell receptor bank with huge capacity are formed.

The identification and accurate quantification of the immune repertoire are of great significance to the immune repertoire system of an individual. How to assess which of the dominant clones (or significantly different clones) in a sample is critical in exerting a major immune function, either resisting the disease or promoting recovery from the disease.

Traditional methods for screening immune repertoire sequencing biomarkers, such as the discrimination of disease detection, progression and prognosis, rely primarily on macroscopic clonal diversity, which is deficient in finding those dominant clones that perform the major immune function; in addition, the traditional method for screening sequencing biomarkers of immune repertoires also adopts t test, for example, and does not consider the influence of the types of cloned genes on significance difference; therefore, the reliability of the screening result is liable to be low.

In conclusion, the traditional method for screening the immune repertoire sequencing biomarker has the defect that the reliability of the screened immune repertoire biomarker is not high.

Disclosure of Invention

The present disclosure provides a method, computing device and computer storage medium for screening immune repertoire sequencing biomarkers that can significantly improve the reliability of the screened immune repertoire biomarkers.

According to a first aspect of the present disclosure, a method of screening an immune repertoire sequencing biomarker is provided. The method comprises the following steps: obtaining immunohistochemical library sequencing data on a sample for determining an initial absolute abundance of each clone in the sample; calculating a first relative abundance of a cloned gene within the sample based on the initial absolute abundance of the cloned gene within the sample, the sum of the absolute abundances of all clones within the sample; responsive to determining that the initial absolute abundance of a cloned gene is greater than a predetermined threshold, determining that the first absolute abundance of the cloned gene is 1; calculating a second relative abundance of the cloned genes in the sample based on the first absolute abundance of the cloned genes in the sample, the sum of the first absolute abundances of all clones in the sample; determining candidate cloned genes based on the first relative abundance and the second relative abundance of the cloned genes; and performing a first test and a second test on the candidate cloned genes to determine an immunohistochemical library sequencing biomarker based on the verification result data, the second test being different from the first test.

According to a second aspect of the present invention, there is also provided a computing device comprising: at least one processing unit; at least one memory coupled to the at least one processing unit and storing instructions for execution by the at least one processing unit, the instructions when executed by the at least one processing unit, cause the computing device to perform the method of the first aspect of the disclosure.

According to a third aspect of the present disclosure, there is also provided a computer-readable storage medium. The computer readable storage medium has stored thereon machine executable instructions which, when executed, cause a machine to perform the method of the first aspect of the disclosure.

In some embodiments, determining the candidate cloned gene based on the first and second relative abundances of the cloned gene comprises: ranking the first relative abundances of the cloned genes so as to determine a plurality of cloned genes having a ranking order less than a first predetermined order threshold as first candidate cloned genes; ranking the second relative abundances of the cloned genes to determine a plurality of cloned genes having a ranking order less than a second predetermined order threshold as second candidate cloned genes; and determining the candidate cloned gene based on the intersection or union of the first candidate cloned gene and the second candidate cloned gene.

In some embodiments, performing the first test and the second test on the candidate cloned genes to determine the immunohistochemical library sequencing biomarker based on the validation result data comprises: performing a first test and a second test, respectively, for the first relative abundance of the candidate cloned gene to generate first and second check result data, respectively; performing a first test and a second test on the second relative abundance of the candidate cloned gene respectively to generate third test result data and fourth test result data; and determining an immune repertoire sequencing biomarker in the candidate cloned gene based on the first, second, third, and fourth check result data.

In some embodiments, determining the immunohistochemical library sequencing biomarker in the candidate cloned gene based on the first validation result data, the second validation result data, the third validation result data, and the fourth validation result data comprises: determining first significant difference data for the candidate cloned gene based on the comparison of the first check result data and the first threshold; determining second significant difference data for the candidate cloned gene based on the comparison of the second check result data and the second threshold; determining third significant difference data for the candidate cloned gene based on the comparison of the third check result data and the third threshold; determining fourth significant difference data for the candidate cloned gene based on a comparison of the fourth check result data and a fourth threshold; and determining an immunohistochemical library sequencing biomarker in the candidate clonal gene based on the first, second, third, and fourth significance difference data.

In some embodiments, determining second significant difference data for the candidate cloned gene based on the comparison of the second check result data and the second threshold comprises: determining that second significant difference data for the candidate cloned genes in the first and second sets indicates that a significant difference exists in response to determining that the second check result data is greater than or equal to a second threshold; and in response to determining that the second test result data is less than the second threshold, determining that second significant difference data for the candidate cloned genes in the first and second sets indicates that there is no significant difference.

In some embodiments, the method of screening an immune repertoire sequencing biomarker further comprises: in response to determining that the initial absolute abundance of the cloned gene is less than or equal to a predetermined threshold, determining that the first absolute abundance of the cloned gene is 0.

In some embodiments, the first test is a rank sum test and the second test is an analysis of variance test.

In some embodiments, the first test is a Wilcoxon test and the second test is an ANOVA test.

In some embodiments, obtaining immunohistochemical library sequencing data on a sample for determining an initial absolute abundance of each clone in the sample comprises: obtaining immunohistochemical library sequencing data for the sample via second generation sequencing for the sample; comparing the immunohistochemical library sequencing data to a predetermined database of clones to determine each clone in the sample; calculating an initial absolute abundance of each clone in the sample; and normalization processing was performed for the initial absolute abundance of each clone.

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the disclosure, nor is it intended to be used to limit the scope of the disclosure.

Drawings

Fig. 1 shows a schematic diagram of a system for performing a method of screening an immune repertoire sequencing biomarker, according to an embodiment of the present disclosure.

Fig. 2 shows a flow diagram of a method for screening immune repertoire sequencing biomarkers according to an embodiment of the present disclosure.

FIG. 3 shows a flow diagram of a method for generating verification result data, in accordance with an embodiment of the present disclosure.

Fig. 4 shows a flow diagram of a method for screening immune repertoire sequencing biomarkers based on verification result data according to an embodiment of the present disclosure.

Fig. 5 shows a schematic of first verification result data generated via a first test for a first relative abundance of a candidate cloned gene, according to an embodiment of the disclosure.

Fig. 6 shows a schematic of second assay result data generated via a second assay for a first relative abundance of a candidate cloned gene, according to an embodiment of the disclosure.

Fig. 7 shows a schematic of third check result data generated via a first test for a second relative abundance of a candidate cloned gene, according to an embodiment of the disclosure.

Fig. 8 shows a schematic of fourth validation result data generated via a second test for a second relative abundance of a candidate cloned gene, according to an embodiment of the disclosure.

FIG. 9 schematically illustrates a block diagram of an electronic device suitable for use to implement embodiments of the present disclosure.

Like or corresponding reference characters designate like or corresponding parts throughout the several views.

Detailed Description

Preferred embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While the preferred embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

The term "include" and variations thereof as used herein is meant to be inclusive in an open-ended manner, i.e., "including but not limited to". Unless specifically stated otherwise, the term "or" means "and/or". The term "based on" means "based at least in part on". The terms "one example embodiment" and "one embodiment" mean "at least one example embodiment". The term "another embodiment" means "at least one additional embodiment". The terms "first," "second," and the like may refer to different or the same object.

As mentioned previously, traditional methods for screening immune repertoire sequencing biomarkers, such as discriminating disease detection, progression and prognosis, rely primarily on clonal diversity at the macroscopic level, and are deficient in finding those dominant clones that perform the primary immune function; in addition, the traditional method for screening sequencing biomarkers of immune repertoires also adopts t test, for example, and does not consider the influence of the types of cloned genes on significance difference; therefore, the reliability of the screening result is liable to be low.

To address, at least in part, one or more of the above problems, as well as other potential problems, example embodiments of the present disclosure propose a scheme for screening immune repertoire sequencing biomarkers. In this protocol, the initial absolute abundance of each clone in the sample is determined by sequencing data through the obtained immunohistochemical library; a first relative abundance of a cloned gene within the sample is then calculated based on the initial absolute abundance of the cloned gene and the sum of the absolute abundances of all clones within the sample. The present disclosure can take into account the absolute abundance of the clone in the quantification of the cloned gene by the first relative abundance, i.e., examine the importance of the clone in the sample as a whole. In addition, the present disclosure provides a method for determining that the first absolute abundance of the sample cloned gene is 1 when the initial absolute abundance of the clone is determined to be greater than a predetermined threshold; and calculating a second relative abundance of the cloned genes within the sample based on the first absolute abundance of the cloned genes and the sum of the first absolute abundances of all clones within the sample. The absolute abundance of the clone is not considered in the quantification of the cloned gene through the second relative abundance, namely, the V (D) J gene of the corresponding clone is respectively counted as 1, so that the cloned gene is quantified, and the importance of the clone in a sample can be further examined from the aspect of the clone type. Further, the present disclosure provides for determining a candidate dominant clone based on the first relative abundance and the second relative abundance; and performing a first test and a second test on the candidate clone genes to determine the sequencing biomarker of the immune repertoire based on the test result data, wherein the second test is different from the first test, the disclosure can simultaneously examine the importance of the clones from both the overall expression amount and the clone types of the clones by two quantitative methods, and finally screening out the clones with significant differences as the biomarkers of the immune repertoire through the two different tests, so that the disclosure can significantly improve the reliability of the screened biomarkers of the immune repertoire.

Fig. 1 shows a schematic diagram of a system 100 for implementing a method of screening immune repertoire sequencing biomarkers according to an embodiment of the present disclosure. As shown in fig. 1, system 100 includes, for example, a computing device 110, a sequencing device 130, a messaging server 150, and a network 140. The computing device 110 may interact with the sequencing device 130 and the messaging server 150 in a wired or wireless manner via the network 140.

The computing device 110 is used to screen the immune repertoire sequencing biomarkers. In particular, the computing device 110 is configured to determine whether the sample is a suitable sample based on obtaining immunohistochemical library sequencing data from the sequencing device 130 or the messaging server 150; determining an initial absolute abundance of each clone in the sample; and calculating a first relative abundance of the cloned genes within the sample. Computing device 110 is further configured to determine whether the calculated initial absolute abundance of each clone is greater than a predetermined threshold; if the initial absolute abundance of the cloned gene of the current species is determined to be greater than the predetermined threshold, determining that the first absolute abundance of the cloned gene of the current species is 1; and calculating a second relative abundance of the cloned genes within the sample. Computing device 110 is further for determining a candidate cloned gene based on the first and second relative abundances of the cloned gene; and performing a first test and a second test on the candidate cloned genes to determine an immune repertoire sequencing biomarker based on the verification result data. Computing device 110 may have one or more processing units, including special purpose processing units such as GPUs, FPGAs, ASICs, and the like, as well as general purpose processing units such as a CPU. In addition, one or more virtual machines may also be running on each computing device. The computing device 110 includes, for example, a clone initial absolute abundance computing unit 112, a first relative abundance computing unit 114, a first absolute abundance determining unit 116 of cloned genes, a second relative abundance computing unit 118, a candidate cloned gene determining unit 120, an immune repertoire sequencing biomarker determining unit 122. The clone initial absolute abundance calculating unit 112, the first relative abundance calculating unit 114, the first absolute abundance determining unit 116 of the clone gene, the second relative abundance calculating unit 118, the candidate clone gene determining unit 120, and the immune repertoire sequencing biomarker determining unit 122. May be configured on one or more computing devices 110.

An initial absolute abundance for clone calculation unit 112 for obtaining immunohistochemical library sequencing data for a sample for determining an initial absolute abundance of each clone in the sample.

A first relative abundance calculating unit 114 for calculating a first relative abundance of a cloned gene within the sample based on the initial absolute abundance of the cloned gene within the sample, the sum of the absolute abundances of all clones within the sample.

A first absolute abundance determining unit 116 for determining whether the initial absolute abundance of each of the cloned genes is greater than a predetermined threshold; and determining the first absolute abundance of the cloned gene to be 1 if the initial absolute abundance of the cloned gene is determined to be greater than a predetermined threshold.

A second relative abundance calculating unit 118 for calculating a second relative abundance of the cloned genes in the sample based on the first absolute abundance of the cloned genes in the sample, the sum of the first absolute abundances of all clones in the sample.

With respect to the candidate cloned gene determination unit 120, it is used to determine a candidate cloned gene based on the first relative abundance and the second relative abundance of the cloned gene.

With respect to, the immunohistochemical library sequencing biomarker determination unit 122 is used to perform a first test and a second test for the candidate cloned gene in order to determine the immunohistochemical library sequencing biomarker based on the verification result data, the second test being different from the first test.

A method for screening immune repertoire sequencing biomarkers according to an embodiment of the present disclosure will be described below with reference to fig. 2. Fig. 2 shows a flow diagram of a method 200 for screening immune repertoire sequencing biomarkers according to an embodiment of the present disclosure. It should be understood that the method 200 may be performed, for example, at the electronic device 900 depicted in fig. 9. May also be executed at the computing device 110 depicted in fig. 1. It should be understood that method 200 may also include additional acts not shown and/or may omit acts shown, as the scope of the disclosure is not limited in this respect.

At step 202, the computing device 110 obtains immunohistochemical library sequencing data for a sample for determining an initial absolute abundance of each clone in the sample.

For the immunohistorian sequencing data, it is, for example, sequencing sequence data for a sample obtained by bidirectional sequencing of the supramachine library molecules using the Illumina high throughput sequencing platform.

Methods for determining the initial absolute abundance of each clone in a sample include, for example: based on the filtered immunohistochemical library sequencing data (e.g., base sequence data) for the sample, clones are generated, and then normalized after obtaining an initial absolute abundance of each clone via identification and quantification of the clones, so as to generate a normalized (or normalized) initial absolute abundance of each clone. Specifically, methods for determining the initial absolute abundance of each clone in a sample include, for example: obtaining immunohistochemical library sequencing data generated via an Illumina high-throughput sequencing platform for a sample; filtering out read lengths whose sequencing quality does not meet predetermined conditions; clustering for consensus sequences in the filtered sequencing data; assembling for pairwise consensus sequences to generate clones; comparing the generated clones to determine the same clone as the same clone species; the number of clones contained in each clonal species was calculated in order to calculate the initial absolute abundance of each clone in the sample.

In some embodiments, the initial absolute abundance is via a normalization process, for example. For example, the normalization process is performed on the calculated initial absolute abundance of each clone to obtain the normalized initial absolute abundance of each clone. By carrying out normalization processing on the initial absolute abundance of each clone, the calculated abundance value can not be influenced by the difference of the processes of extracting sample RNA or preparing a receptor library at the early stage, and further the influence on the clone significance evaluation result is avoided.

The algorithm used to calculate the initial absolute abundance of each clone after normalization is described below in conjunction with expression (1).

In the above expression (1), i represents the ith clone in the sample. n represents the total number of clonal species. relateAb (i) represents the normalized absolute abundance of the ith clone in the sample. Ab (i) represents the initial absolute abundance of the ith clone in the sample.Represents the sum of the absolute abundance of all clones within the sample.

For example, clones of n different clonal species (clone1 through clone) contained in the sample. Table 1 below schematically shows the initial absolute abundance of n clones in a sample. For example, the initial absolute abundance of clone1 was 50, clone2 was 100, clone3 was 150, and clone en was 120.

TABLE 1

Cloning	Absolute abundance
		clone1	50
clone2	100
		clone3	150
....	....
		clonen	120

The normalized initial absolute abundance of Clone1 can be calculated as relateAb (Clone1) 50/(50+100+ 150. +120) × 100 according to expression (1) above.

At step 204, computing device 110 calculates a first relative abundance of the cloned genes within the sample based on the initial absolute abundance of the cloned genes within the sample, the sum of the absolute abundances of all clones within the sample. The first relative abundance is generated, for example, via Total clone normalization. By adopting the above means, the cloned genes are quantified in the first relative abundance mode, and the absolute expression quantity of the clones is considered, so that the importance of the clones in the sample can be examined from the whole. That is, the computing device 110 calculates based on the absolute abundance of a gene representing a certain V, D, J clone within a sample and the sum of the absolute abundances of all clones within the sample. The first relative abundance of the cloned genes (e.g., the first relative abundance of the V cloned genes) is calculated taking into account the amount of expression of all clones within the sample.

An algorithm for calculating the first relative abundance of cloned genes in the sample is described below in conjunction with expression (2), using the V cloned gene as an example.

In the above expression (2), j represents an V, D, J-cloned gene in the sample. n represents the total number of clonal species. V (j) represents the first relative abundance of a certain V, D, J cloned gene within the sample. V clone (j) represents the initial absolute abundance of a certain V, D, J cloned gene within the sample.Represents the sum of the absolute abundance of all clones within the sample.

For example, clone1 consists of TRBV2, TRBD2, TRBJ2-7, clone2 consists of TRBV2, TRBD2, TRBJ2-7, clone3 consists of TRBV5-1, TRBD2, TRBJ2-7, … clone consists of TRBV7-2, RBD1 TRBJ 1-1. For example, the initial absolute abundance of clone genes of clone1 … clonen is 50, 100, 0.. 0, then the first relative abundance V (TRBV2) for clone gene TRBV2 is (50+100+ 0. +0)/(50+100+ 150. +120) × 100.

At step 206, the computing device 110 determines whether the initial absolute abundance of the cloned gene is greater than a predetermined threshold. In some embodiments, the predetermined threshold is, for example and without limitation, 0.

At step 208, if the computing device 110 determines that the initial absolute abundance of the cloned gene is greater than the predetermined threshold, it determines that the first absolute abundance of the cloned gene is 1. At step 210, if the computing device 110 determines that the initial absolute abundance of the cloned gene is less than or equal to a predetermined threshold (e.g., is 0), the first absolute abundance of the cloned gene is determined to be 0. For another example, if the initial absolute abundance of a clone of the current species is greater than zero, i.e., the clone of the species is present in the sample, the first absolute abundance of the clone of the current species is determined to be 1 regardless of the expression level of the clone of the current species.

At step 212, the computing device 110 calculates a second relative abundance of the cloned genes within the sample based on the first absolute abundance of the cloned genes within the sample, the sum of the first absolute abundances of all clones within the sample. For example, the computing device 110 generates three second relative abundances for V, D, J cloned genes, respectively.

The second relative abundance is generated, for example, via normalization of the Unique clone. For example, the computing device 110 calculates a second relative abundance of a cloned gene based on a first absolute abundance representing a certain V, D, J cloned gene within the sample and a sum of absolute abundances of all clones within the sample.

The algorithm for calculating the second relative abundance of cloned genes in a sample is described below in conjunction with expression (3), using the V cloned gene as an example.

In the above expression (3), k represents a cloned gene of a certain kind within a sample, for example, V, D, J gene. n represents the total number of clonal species. Vu (k) represents the second relative abundance of a cloned gene within the sample. Vuclone (k) represents the first absolute abundance of the k species cloned genes within the sample. uclone (k) represents the first absolute abundance of a certain clone within the sample.Represents the sum of the first absolute abundances of all clones within the sample.

For example, as described earlier, clone1 consists of TRBV2, TRBD2, TRBJ2-7, clone2 consists of TRBV2, TRBD2, TRBJ2-7, clone3 consists of TRBV5-1, TRBD2, TRBJ2-7 to form … clone, e.g., TRBV7-2, RBD1 TRBJ 1-1. For example, the initial absolute abundance of the cloned gene of clone1 … clonen is 50, 100, 0.. 0, and the first absolute abundance of the cloned gene TRBV2 in the sample is 1, 0.. 0, respectively. The first absolute abundance of all clones within the sample is, for example, 1.. 1, respectively. The second relative abundance Vu (TRBV2) of the cloned gene TRBV2 is (1+1+0+. +0)/(1+1+1+. +1) × 100.

At step 212, the computing device 110 determines candidate cloned genes based on the first and second relative abundances of the cloned genes.

Regarding the method for determining a candidate cloned gene, it includes, for example: ranking the first relative abundances of the cloned genes so as to determine a plurality of cloned genes having a ranking order less than a first predetermined order threshold as first candidate cloned genes; ranking the second relative abundances of the cloned genes to determine a plurality of cloned genes having a ranking order less than a second predetermined order threshold as second candidate cloned genes; and determining the candidate cloned gene based on the intersection or union of the first candidate cloned gene and the second candidate cloned gene. The first predetermined order threshold and the second predetermined order threshold may be the same or different. For example, the top 20 clones of the first relative abundance and the second relative abundance were selected as candidate cloned genes.

It will be appreciated that the first relative abundance is calculated based on the initial absolute abundance of the cloned genes in the sample and the absolute abundance of all clones, and therefore the first relative abundance takes into account the amount of expression of all clones within the sample and therefore contributes to the overall impact of the clones. The second relative abundance is determined based on, for example, the sum of the first absolute abundances of the cloned genes (taking the value of "0" or "1") and the first absolute abundances of all clones, and therefore, the second relative abundance does not take into account the expression amount of the same type of clone, but takes into account the overall influence of the clone species. Thus, the present disclosure facilitates more accurate and reliable determination of dominant clones by determining an immunohistochemical library sequencing biomarker based on a first relative abundance of the overall effect of the heavy clone and a second relative abundance of the effect of the heavy clone species.

At step 214, the computing device 110 performs a first test and a second test for the candidate cloned genes to determine an immunohistochemical library sequencing biomarker based on the verification result data, the second test being different from the first test.

Regarding methods for determining sequencing biomarkers of an immunohistochemical library based on the verification result data, for example, it includes: performing a first test and a second test, respectively, for the first relative abundance of the candidate cloned gene to generate first and second check result data, respectively; performing a first test and a second test on the second relative abundance of the candidate cloned gene respectively to generate third test result data and fourth test result data; and determining an immune repertoire sequencing biomarker in the candidate cloned gene based on the first, second, third, and fourth check result data. Regarding the method for generating the verification result data, the present disclosure will be described with reference to fig. 3, and will not be described herein.

With respect to the first test, in some embodiments, it is, for example, a rank sum test that does not reduce dimensions. By taking rank sum test (rank sum test) as the first test, the verification result of the present disclosure is made independent of the specific form of the overall distribution. It should be understood that since the data type of the candidate cloned gene does not fit a normal distribution, the rank-sum test is more suitable for the significant difference analysis of the candidate cloned gene than the t-test. In some embodiments, the first test is, for example, a Wilcoxon test, and the first test is performed by using a Wilcoxon rank sum test, so that the test result can take the direction of the difference and the magnitude of the difference into consideration, and thus the test is more effective.

As to the second test, it is, for example, an analysis of variance test or a PCA dimension reduction one-factor test. The second test is for example and without limitation an ANOVA test. It should be understood that the first check and the second check may be other suitable check manners.

A method for determining an immunohistochemical library sequencing biomarker in a cloned gene within the sample, comprising, for example: determining first significant difference data for the candidate cloned gene based on the comparison of the first check result data and the first threshold; determining second significant difference data for the candidate cloned gene based on the comparison of the second check result data and the second threshold; determining third significant difference data for the candidate cloned gene based on the comparison of the third check result data and the third threshold; determining fourth significant difference data for the candidate cloned gene based on a comparison of the fourth check result data and a fourth threshold; and determining an immunohistochemical library sequencing biomarker in the candidate clonal gene based on the first, second, third, and fourth significance difference data.

The effect of the method of screening immunohistochemical library sequencing biomarkers of the present disclosure is illustrated below with reference to fig. 5 to 8. Fig. 5 shows a schematic of first verification result data generated via a first test for a first relative abundance of a candidate cloned gene, according to an embodiment of the disclosure. Fig. 6 shows a schematic of second assay result data generated via a second assay for a first relative abundance of a candidate cloned gene, according to an embodiment of the disclosure. Fig. 7 shows a schematic of third check result data generated via a first test for a second relative abundance of a candidate cloned gene, according to an embodiment of the disclosure. Fig. 8 shows a schematic of fourth validation result data generated via a second test for a second relative abundance of a candidate cloned gene, according to an embodiment of the disclosure.

The four test results data shown in figures 5 to 8 indicate that the TRBV20 family is significantly upregulated in patients, that the first relative abundance of the cloned gene TRBV13 is significantly downregulated in the first test (e.g., Wilcoxon test) and the second test (e.g., ANOVA test), and that the second relative abundance of the cloned gene TRBV2 is significantly downregulated in the first test (e.g., Wilcoxon test) and the second test (e.g., ANOVA test), and thus TRBV20, TRBV13, TRBV2 may serve as biomarkers for the disease. By considering the total clone amount and the clone species respectively based on two quantitative methods of the first relative abundance and the second relative abundance, the TRBV20, the TRBV13 and the TRBV2 are determined to be the biomarkers of the immune repertoire, thereby avoiding only obtaining the TRBV13 as the biomarkers from the viewpoint of clone expression amount.

In the above protocol, the initial absolute abundance of each clone in the sample is determined by sequencing data through the obtained immunohistochemical library; a first relative abundance of a cloned gene within the sample is then calculated based on the initial absolute abundance of the cloned gene and the sum of the absolute abundances of all clones within the sample. The present disclosure can take into account the absolute abundance of the clone in the quantification of the cloned gene by the first relative abundance, i.e., examine the importance of the clone in the sample as a whole. In addition, the present disclosure provides a method for determining that the first absolute abundance of the sample cloned gene is 1 when the initial absolute abundance of the clone is determined to be greater than a predetermined threshold; and calculating a second relative abundance of the cloned genes within the sample based on the first absolute abundance of the cloned genes and the sum of the first absolute abundances of all clones within the sample. The absolute abundance of the clone is not considered in the quantification of the cloned gene through the second relative abundance, namely, the V (D) J gene of the corresponding clone is respectively counted as 1, so that the cloned gene is quantified, and the importance of the clone in a sample can be further examined from the aspect of the clone type. Further, the present disclosure provides for determining a candidate dominant clone based on the first relative abundance and the second relative abundance; and performing a first test and a second test on the candidate clone genes to determine the sequencing biomarker of the immune repertoire based on the test result data, wherein the second test is different from the first test, the disclosure can simultaneously examine the importance of the clones from both the overall expression amount and the clone types of the clones by two quantitative methods, and finally screening out the clones with significant differences as the biomarkers of the immune repertoire through the two different tests, so that the disclosure can significantly improve the reliability of the screened biomarkers of the immune repertoire.

FIG. 3 shows a flow diagram of a method 300 for generating verification result data, in accordance with an embodiment of the present disclosure. It should be understood that the method 300 may be performed, for example, at the electronic device 900 depicted in fig. 9. May also be executed at the computing device 110 depicted in fig. 1. It should be understood that method 300 may also include additional acts not shown and/or may omit acts shown, as the scope of the disclosure is not limited in this respect.

At step 302, computing device 110 performs a first test and a second test, respectively, for a first relative abundance of a candidate cloned gene to generate first and second test result data, respectively.

Performing a first test (e.g., Wilcoxon test) for a first relative abundance of the cloned gene to generate first test result data (e.g., Wilcoxon test result data) is described below in conjunction with expression (4).

In the above expression (4), z1 represents Wilcoxon test result data. Wx represents the rank sum of a cloned gene of the first group (e.g., the Case group). n1 represents the number of samples of the first group. n2 represents the number of samples in the second set (e.g., Control set). Plus 0.5 or minus 0.5 in + -0.5 represents a correction to the discrete variable, where for Wx- μ greater than 0, minus 0.5 correction, and for Wx- μ less than or equal to 0, plus 0.5 correction. t represents the number of samples exhibiting the same abundance.

Table 2 below shows the rank and statistics of the first relative abundance of the cloned gene TRBV13 in different samples in the first and second sets.

TABLE 2

In table 2 above, Wx1 represents the rank sum of TRBV13 gene of the first 16 samples, Wx1 ═ 286. Wx2 represents the rank sum of TRBV13 gene of the first 11 samples, Wx2 ═ 92. The first group has a number of samples n1 of 16. The number of samples n2 of the second group is 11. The rank for each sample in the rank sum test is determined by: by ranking the first relative abundance values of TRBV13 in order from small to large for all samples, the first relative abundance of each TRBV13 is numbered in order, which is the rank (or rank). The same abundance did not occur for the first and second groups, i.e. t was 0.

The calculation of the first set of Wilcoxon test result data z1 is exemplified below in conjunction with expression (5).

As can be seen from the foregoing expression (5), the Wilcoxon test result data z1 ═ 3.034 of the Wilcoxon test was performed for the first relative abundance of the cloned gene TRBV13 (i.e., the first test result data was 3.034).

The following describes, in conjunction with expression (6), performing a second test (e.g., ANOVA test) on the first relative abundance of the cloned gene to generate second test result data (e.g., ANOVA test result data).

In expression (6) above, F1 represents ANOVA test result data, i.e., second test result data. "MS groups" represents the mean square of variation between groups. Mean square of variation within the "MS group". k represents k groups. ni represents the number of samples representing the i-th group. a represents that the number of packets is equal to k.Represents the mean relative abundance of the i-th group of cloned genes.Represents the mean relative abundance of a cloned gene in the population. Xij represents the first relative abundance of a cloned gene from the jth sample in group i. N represents the total number of samples.

Table 3 below shows the mean statistics of the first relative abundance expression of the cloned gene TRBV13 in different samples in the first and second groups.

TABLE 3

According to the table3, the mean relative abundance of the cloned gene TRBV13 in the first groupIs 4.9539. Mean relative abundance of the second set of cloned genes TRBV13Is 1.9928.Represents the mean relative abundance of a cloned gene in the population. Mean relative abundance of a cloned gene in the population

The manner in which the first set of ANOVA test result data F1 was calculated is exemplified below in connection with expression (7).

As can be seen from the foregoing expression (7), ANOVA test result data F1 ═ 11.070 for the ANOVA test on the first relative abundance of the cloned gene TRBV13 (i.e., second test result data F2 was 11.070).

At step 304, the computing device 110 performs a first test and a second test, respectively, for a second relative abundance of the candidate cloned gene to generate third and fourth test result data.

A method of performing a first test (e.g., Wilcoxon test) on the second relative abundance of the cloned gene to generate third check result data is described below, taking the cloned gene TRBV13 as an example, in conjunction with expression (8).

In the above expression (8), z2 represents the third check result data of the first check (for example, Wilcoxon test) against the second relative abundance of the cloned gene TRBV 13. Wx represents the rank sum of a certain cloned gene of the first group. n1 represents the number of samples of the first group. n2 represents the number of samples of the second group. Plus 0.5 or minus 0.5 in + -0.5 represents a correction to the discrete variable, where for Wx- μ greater than 0, minus 0.5 correction, and for Wx- μ less than or equal to 0, plus 0.5 correction. t represents the number of samples exhibiting the same abundance.

TABLE 4

In table 4 above, Wx1 represents the rank sum of TRBV13 gene of the first 16 samples, and Wx1 is 241. Wx2 represents the rank sum of TRBV13 gene for the second set of 11 samples, Wx2 ═ 137. The first group has a number of samples n1 of 16. The number of samples n2 of the second group is 11. The same abundance did not occur for the first and second groups, i.e. t was 0.

The calculation of Wilcoxon test result data z2 generated by Wilcoxon test on the second relative abundance of the cloned gene is exemplified below by the cloned gene TRBV13 in combination with expression (9).

As can be seen from the foregoing expression (9), the Wilcoxon test result data z2 of the Wilcoxon test was 0.814 (i.e., the third test result data F2 was 0.814) for the second relative abundance of the cloned gene TRBV 13.

The method of performing a second test (e.g., ANOVA test) on the second relative abundance of the cloned gene to generate fourth check result data is described below, using the cloned gene TRBV13 as an example, in conjunction with expression (10).

In expression (10) above, F2 represents ANOVA verification result data, i.e., fourth verification result data. "MS groups" represents the mean square of variation between groups. Mean square of variation within the "MS group". k represents k groups. ni represents the number of samples representing the i-th group. a represents that the number of packets is equal to k.Represents the mean relative abundance of the i-th group of cloned genes.Represents the mean relative abundance of a cloned gene in the population. Xij represents the second relative abundance of a cloned gene from the jth sample in group i. N represents the total number of samples.

Table 5 below shows the second relative abundance expression mean statistics of the cloned gene TRBV13 in different samples in the first and second groups.

TABLE 5

As can be seen from Table 5, the mean relative abundance of the cloned gene TRBV13 in the first groupIs 1.1040. Mean relative abundance of the second set of cloned genes TRBV13Is 0.9952.Represents the totalMean relative abundance of a cloned gene in vivo. Mean relative abundance of a cloned gene in the population

The following calculation of ANOVA test result data F2 generated by ANOVA test for the second relative abundance of the cloned gene TRBV13 is exemplified in connection with expression (11).

As can be seen from the above expression (11), ANOVA test result data F2 generated by ANOVA test on the second relative abundance of the cloned gene TRBV13 was 0.360 (i.e., fourth test result data F2 was 0.360).

At step 306, the computing device 110 determines an immunohistochemical library sequencing biomarker in the candidate cloned gene based on the first, second, third, and fourth verification result data.

A method for screening sequencing biomarkers of an immunohistochemical library based on the verification result data, comprising, for example: determining first significant difference data for the candidate cloned gene based on the comparison of the first check result data and the first threshold; determining second significant difference data for the candidate cloned gene based on the comparison of the second check result data and the second threshold; determining third significant difference data for the candidate cloned gene based on the comparison of the third check result data and the third threshold; determining fourth significant difference data for the candidate cloned gene based on a comparison of the fourth check result data and a fourth threshold; and determining an immunohistochemical library sequencing biomarker in the candidate clonal gene based on the first, second, third, and fourth significance difference data. The method for screening the sequencing biomarkers of the immune repertoire based on the verification result data will be described in detail below with reference to fig. 4.

Fig. 4 shows a flow diagram of a method 400 for screening immune repertoire sequencing biomarkers based on verification result data, according to an embodiment of the present disclosure. It should be understood that method 400 may be performed, for example, at electronic device 900 depicted in fig. 9. May also be executed at the computing device 110 depicted in fig. 1. It should be understood that method 400 may also include additional acts not shown and/or may omit acts shown, as the scope of the disclosure is not limited in this respect.

At step 402, computing device 110 determines first significant difference data for the candidate cloned gene based on a comparison of the first verification result data and a first threshold.

For example, for the candidate clone gene TRBV13, the first test result data z1 is 3.034 according to the expression (5) above. Setting the significance level as 0.05, when the standard normal distribution is at 0.05 significance level, the first threshold (e.g., the upper critical value) is 1.645, and since 3.034>1.645, that is, the first test result data is greater than the first threshold, the predetermined condition for significance is satisfied, and the null hypothesis is rejected. Thus, the first significant difference data indicates that: the difference in expression of the first relative abundance of the candidate cloned gene TRBV13 in the first and second sets reached a significant level.

At step 404, computing device 110 determines second significant difference data for the candidate cloned gene based on a comparison of the second check result data and a second threshold.

As can be seen from expression (7), the second verification result data F1 is 11.070. The significance level a is set to 0.05, and the second threshold F0.05(1,15) is, for example, 4.543 when the standard normal distribution is at the significance level of 0.05. Since the second test result data F1> F0.05, p <0.05, the null hypothesis is rejected, i.e. the second test result data is greater than the second threshold, and the corresponding significance predetermined condition is satisfied. Thus, the second significant difference data indicates that: the difference in expression of the first relative abundance of TRBV13 in the first and second groups reached a significant level via a second test (e.g., an ANOVA test).

At step 406, the computing device 110 determines third significant difference data for the candidate cloned gene based on a comparison of the third verification result data and a third threshold.

As can be seen from expression (9), the third verification result data z2 is 0.814. Setting the significance level as 0.05, setting the third threshold (e.g., the upper threshold) to be 1.645 when the standard normal distribution is at the significance level of 0.05, and failing to satisfy the predetermined condition for the corresponding significance since the third verification result data z2 is less than 1.645, that is, the third verification result data is less than the third threshold. The null hypothesis cannot be rejected, i.e., the expression of TRBV13 in the second relative abundance was not significantly different between the first and second groups. Thus, the third significant difference data indicates that: the expression of TRBV13 in the second relative abundance was not significantly different in the first and second sets via the first test (e.g., Wilcoxon test).

At step 408, the computing device 110 determines fourth significant difference data for the candidate cloned gene based on a comparison of the fourth verification result data and a fourth threshold.

For example, as can be seen from expression (11), the fourth verification result data F2 is 0.360. The significance level a is set to 0.05, and the fourth threshold F0.05(1,15) is, for example, 4.543 when the standard normal distribution is at the significance level of 0.05. Since the fourth test result data F2<4.543, p <0.05, the null hypothesis cannot be rejected, i.e., the fourth test result data F2 is smaller than the fourth threshold value, the predetermined condition for correspondence significance is not satisfied. Thus, the fourth significant difference data indicates that: the expression of TRBV13 in the second relative abundance was not significantly different in the first and second groups via a second check (e.g., ANOVA test).

At step 410, computing device 110 determines an immune repertoire sequencing biomarker in the candidate clonal genes based on the first, second, third, and fourth significant difference data.

For example, the first relative abundance of TRBV13 was significantly different in a first check (e.g., Wilcoxon check) and a second check (e.g., ANOVA test) based on the first significant difference data and the second significant difference data. The second relative abundance of TRBV13 did not significantly differ in the first check (e.g., Wilcoxon check) and the second check (e.g., ANOVA test) based on the third significance difference data and the fourth significance difference data. By considering the total amount of clones and the clone species respectively based on two quantitative methods of the first relative abundance and the second relative abundance, the TRBV13 is determined as the biomarker of the immune repertoire, and the TRBV13 is prevented from being omitted as the biomarker only from the viewpoint of the clone species. In the above protocol, the present disclosure enables accurate screening of immune repertoires for biomarkers by combining two methods of quantifying a first relative abundance and a second relative abundance, with Wilcoxon and ANOVA tests.

FIG. 9 schematically illustrates a block diagram of an electronic device 900 suitable for use in implementing embodiments of the present disclosure. The device 900 may be a device for implementing the methods 200, 300, and 400 shown in fig. 2, 3, and 4. As shown in fig. 7, device 900 includes a Central Processing Unit (CPU)901 that can perform various appropriate actions and processes in accordance with computer program instructions stored in a Read Only Memory (ROM)902 or loaded from a storage unit 908 into a Random Access Memory (RAM) 903. In the RAM, various programs and data required for the operation of the device 900 may also be stored. The CPU, ROM, and RAM are connected to each other via a bus 904. An input/output (I/O) interface 905 is also connected to bus 904.

A number of components in the device 900 are connected to the I/O interface 905, including: an input unit 906, an output unit 907, a storage unit 908, a central processing unit 901 performs the various methods and processes described above, such as performing the methods 200, 300 and 400. For example, in some embodiments, methods 200, 300, and 400 may be implemented as a computer software program stored on a machine-readable medium, such as storage unit 908. In some embodiments, part or all of the computer program may be loaded and/or installed onto device 900 via ROM and/or communications unit 909. When loaded into RAM and executed by a CPU, the computer program may perform one or more of the operations of methods 200, 300 and 400 described above. Alternatively, in other embodiments, the CPU may be configured by any other suitable means (e.g., by way of firmware) to perform one or more of the acts of methods 200, 300, and 400.

It should be further appreciated that the present disclosure may be embodied as methods, apparatus, systems, and/or computer program products. The computer program product may include a computer-readable storage medium having computer-readable program instructions embodied thereon for carrying out various aspects of the present disclosure.

The computer readable storage medium may be a tangible device that can hold and store the instructions for use by the instruction execution device. The computer readable storage medium may be, for example, but not limited to, an electronic memory device, a magnetic memory device, an optical memory device, an electromagnetic memory device, a semiconductor memory device, or any suitable combination of the foregoing. More specific examples (a non-exhaustive list) of the computer readable storage medium would include the following: a portable computer diskette, a hard disk, a Random Access Memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), a Static Random Access Memory (SRAM), a portable compact disc read-only memory (CD-ROM), a Digital Versatile Disc (DVD), a memory stick, a floppy disk, a mechanical coding device, such as punch cards or in-groove projection structures having instructions stored thereon, and any suitable combination of the foregoing. Computer-readable storage media as used herein is not to be construed as transitory signals per se, such as radio waves or other freely propagating electromagnetic waves, electromagnetic waves propagating through a waveguide or other transmission medium (e.g., optical pulses through a fiber optic cable), or electrical signals transmitted through electrical wires.

The computer-readable program instructions described herein may be downloaded from a computer-readable storage medium to a respective computing/processing device, or to an external computer or external storage device via a network, such as the internet, a local area network, a wide area network, and/or a wireless network. The network may include copper transmission cables, fiber optic transmission, wireless transmission, routers, firewalls, switches, gateway computers and/or edge servers. The network adapter card or network interface in each computing/processing device receives computer-readable program instructions from the network and forwards the computer-readable program instructions for storage in a computer-readable storage medium in the respective computing/processing device.

The computer program instructions for carrying out operations of the present disclosure may be assembler instructions, Instruction Set Architecture (ISA) instructions, machine-related instructions, microcode, firmware instructions, state setting data, or source or object code written in any combination of one or more programming languages, including an object oriented programming language such as Smalltalk, C + + or the like and conventional procedural programming languages, such as the "C" programming language or similar programming languages. The computer-readable program instructions may execute entirely on the user's computer, partly on the user's computer, as a stand-alone software package, partly on the user's computer and partly on a remote computer or entirely on the remote computer or server. In the case of a remote computer, the remote computer may be connected to the user's computer through any type of network, including a Local Area Network (LAN) or a Wide Area Network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet service provider). In some embodiments, the electronic circuitry that can execute the computer-readable program instructions implements aspects of the present disclosure by utilizing the state information of the computer-readable program instructions to personalize the electronic circuitry, such as a programmable logic circuit, a Field Programmable Gate Array (FPGA), or a Programmable Logic Array (PLA).

Various aspects of the present disclosure are described herein with reference to flowchart illustrations and/or block diagrams of methods, apparatus (systems), and computer program products according to embodiments of the disclosure. It will be understood that each block of the flowchart illustrations and/or block diagrams, and combinations of blocks in the flowchart illustrations and/or block diagrams, can be implemented by computer-readable program instructions.

These computer-readable program instructions may be provided to a processor in a voice interaction device, a processing unit of a general purpose computer, special purpose computer, or other programmable data processing apparatus to produce a machine, such that the instructions, which execute via the processing unit of the computer or other programmable data processing apparatus, create means for implementing the functions/acts specified in the flowchart and/or block diagram block or blocks. These computer-readable program instructions may also be stored in a computer-readable storage medium that can direct a computer, programmable data processing apparatus, and/or other devices to function in a particular manner, such that the computer-readable medium storing the instructions comprises an article of manufacture including instructions which implement the function/act specified in the flowchart and/or block diagram block or blocks.

The computer readable program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other devices to cause a series of operational steps to be performed on the computer, other programmable apparatus or other devices to produce a computer implemented process such that the instructions which execute on the computer, other programmable apparatus or other devices implement the functions/acts specified in the flowchart and/or block diagram block or blocks.

The flowchart and block diagrams in the figures illustrate the architecture, functionality, and operation of possible implementations of apparatus, methods and computer program products according to various embodiments of the present disclosure. In this regard, each block in the flowchart or block diagrams may represent a module, segment, or portion of instructions, which comprises one or more executable instructions for implementing the specified logical function(s). In some alternative implementations, the functions noted in the block may occur out of the order noted in the figures. For example, two blocks shown in succession may, in fact, be executed substantially concurrently, or the blocks may sometimes be executed in the reverse order, depending upon the functionality involved. It will also be noted that each block of the block diagrams and/or flowchart illustration, and combinations of blocks in the block diagrams and/or flowchart illustration, can be implemented by special purpose hardware-based systems which perform the specified functions or acts, or combinations of special purpose hardware and computer instructions.

Having described embodiments of the present disclosure, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen in order to best explain the principles of the embodiments, the practical application, or improvements made to the technology in the marketplace, or to enable others of ordinary skill in the art to understand the embodiments disclosed herein.

The above are only alternative embodiments of the present disclosure and are not intended to limit the present disclosure, which may be modified and varied by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.