Probe design method for improving detection performance of gene chip

Abstract

The invention provides a probe design method for improving the detection performance of a gene chip, and belongs to the technical field of DNA and RNA microarrays. Comprising the following steps: S1, downloading a genome sequence of a target detection species; s2, downloading a whole genome database of bacteria, fungi, animals, plants and viruses, and classifying according to species; s3, performing multi-sequence alignment on the whole genome data of each species; and S4, analyzing the comparison result of the genomic sequences of the to-be-detected species, and carrying out sequence conservative machine calculation to obtain the conservative properties of different genomic sequences of the same species. By optimizing the position relation of the probes on the chip, the detection performance of the DNA and RNA microarray can be remarkably improved, and the specific method comprises rectangular, linear and discrete layout of the probes, distribution of repeated probes and multi-probe design. The methods can improve the accuracy, sensitivity and reliability of detection, and provide data with higher quality for pathogenic microorganism identification.

Description

Technical Field

[0001] The invention relates to the technical field of DNA and RNA microarrays, and in particular to a probe design method for improving the detection performance of a gene chip. Background Art

[0002] DNA and RNA microarray technology has many similar technologies, which are also used in gene expression analysis, genome research and molecular biology research. Although DNA and RNA microarray technology has a wide range of applications in mutation, specific sequence qualitative detection, pathogenic microorganism detection, gene expression analysis and genome research, it also has some defects and limitations.

[0003] For example, the position arrangement of the probes on the chip is not ideal. The position relationship of the probes on the chip will directly affect the detection performance of DNA and RNA microarrays. Therefore, the present application provides a probe design method for improving the detection performance of gene chips to meet the needs. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a probe design method for improving the detection performance of gene chips to solve the existing problems.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] A probe design method for improving the detection performance of a gene chip comprises the following steps:

[0007] S1. Download the genome sequence of the target species;

[0008] S2, download the complete genome database of bacteria, fungi, plants, animals, and viruses and then classify them according to species;

[0009] S3, multiple sequence alignment of the whole genome data of each species;

[0010] S4. Analyze the results of genome sequence comparison of the species to be tested, perform sequence conservation calculation, and obtain the conservation of different genome sequences of the same species;

[0011] S5. Obtain species conservative sequences based on the conservation of different genome sequences of the same species;

[0012] S6, progressively cutting the conserved sequences obtained by aligning multiple genome sequences of the same species according to the length and step size to obtain multiple probes of the species;

[0013] S7. Generate rectangular, discrete, and linear designs for each probe of the species to be tested;

[0014] S8. For each probe layout, set probe repetition;

[0015] S9, repeat the setting of positive control and negative control internal reference probe clusters and probes;

[0016] S10, synthesizing and producing the chip, and designing PCR amplification primers;

[0017] S11. Construct a human negative baseline (excluding the species to be tested) and statistical distribution;

[0018] S12. Conduct positive sample testing and report the results.

[0019] In some examples, the downloading of the genome sequence of the target detection species in S1 is specifically: downloading the fasta sequence of the genome of the species to be detected from bv-brc according to the name of the species to be detected or the taxid, and the downloading needs to involve the pathogenic microorganism sequences of various time periods and regions around the world.

[0020] In some examples, the whole genome data of each species are subjected to multiple sequence alignment in S3, specifically: the downloaded fasta file sequences of the genome reference sequences of each species are grouped by pathogen type, and each group of pathogens obtained by grouping is subjected to multiple sequence alignment.

[0021] In some examples, S6 is specifically as follows: analyzing the multiple sequence alignment results of a species, setting a sliding window of x, and when the number of conserved base sequences in each window is greater than 90%, the region is considered to be a conserved region, and then progressive cutting is performed according to the length n and the step length m to obtain multiple probes for the species.

[0022] In some examples, the probe design of S7 is specifically as follows: first, the coordinates of each position on the chip are calculated.

[0023] Chip length x_range, width y_range

[0024] defcreat_coors(x_range,y_range):

[0025] x=np.arange(x_range[0],x_range[1])

[0026] y=np.arange(y_range[0],y_range[1])

[0027] X,Y = np.meshgrid(x,y)

[0028] coors=np.concatenate((X[:,:,None],Y[:,:,None]),axis=-1)

[0029] dfs=[pd.DataFrame(x)forx in coors]

[0030] df_coors=pd.concat(dfs,keys=range(len(dfs)))

[0031] df_coors=df_coors.reset_index()

[0032] df_coors.drop(['level_0','level_1'],axis=1,inplace=True)

[0033] df_coors.columns = ['x','y']

[0034] print(df_coors)

[0035] return df_coors

[0036] Then, the chip is cut into rectangular, discrete, and linear shapes according to the initialized coordinates to obtain the probe placement area under each layout;

[0037] The coordinates of each region were then matched to pre-designed species-specific probes;

[0038] This design method can effectively fill the empty positions in the probe cluster design and ensure the space utilization with maximum efficiency.

[0039] In some examples, the specific steps of S10 are: selecting a chip size of appropriate specifications to accommodate the above design, performing probe synthesis and production, and designing primers using the conserved regions in the species conservation analysis results.

[0040] In some examples, the specific steps of S11 are: collecting oral swabs from 50 healthy people, performing PCR amplification, hybridizing the amplification products with the chip and performing fluorescence detection, and for the probe cluster design, taking the cluster as the unit, obtaining the fluorescence mean of each cluster of each sample and calculating the variance between samples; for the probe distributed design, taking each detection unit as the unit, obtaining the fluorescence mean of each point of each sample and calculating the variance between samples.

[0041] In some examples, the specific steps of S12 are: for a probe cluster, if the positive sample to be detected exceeds the mean plus twice the variance, the species is considered to be detected. In the probe distribution design, if the positive sample to be detected exceeds the mean plus twice the variance, the probe is determined to be positive. If multiple similarity probes are positive, the one with the largest (value-mean) / std is taken as the basis, which is used for specific clinical applications such as pathogen detection.

[0042] Compared with the prior art, the present invention has at least the following beneficial effects:

[0043] In the above scheme, the detection performance of DNA and RNA microarrays can be significantly improved by optimizing the position relationship of probes on the chip. Specific methods include rectangular, linear, discrete layout of probes, distribution of repeated probes, and multiple probe design. These methods can improve the accuracy, sensitivity, and reliability of detection and provide higher quality data for pathogen identification. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, further serve to explain the principles of the present disclosure and to enable those skilled in the relevant art to make and use the present disclosure.

[0045] Figure 1 A method flow chart of a probe design method for improving the detection performance of a gene chip;

[0046] Figure 2 A spatial layout diagram of a chip in a probe design method for improving the detection performance of a gene chip.

[0047] As shown in the figure, in order to clearly implement the structure of the embodiment of the present invention, specific structures and devices are marked in the figure, but this is only for illustrative purposes and is not intended to limit the present invention to the specific structure, device and environment. According to specific needs, ordinary technicians in this field can adjust or modify these devices and environments, and the adjustments or modifications made are still included in the scope of the attached claims. DETAILED DESCRIPTION

[0048] The following is a detailed description of a probe design method for improving the detection performance of a gene chip provided by the present invention in conjunction with the accompanying drawings and specific embodiments. At the same time, it is explained here that in order to make the embodiments more detailed, the following embodiments are listed as the best and preferred embodiments, and other alternatives can also be used by technicians in some known technical fields; and the accompanying drawings are only for more specific description of the embodiments, and are not intended to specifically limit the present invention.

[0049] It should be noted that the references to "one embodiment", "embodiment", "exemplary embodiment", "some embodiments" and the like in the specification indicate that the embodiments described may include specific features, structures or characteristics, but not every embodiment may include the specific features, structures or characteristics. In addition, when a specific feature, structure or characteristic is described in conjunction with an embodiment, it should be within the knowledge of a person skilled in the art to implement such feature, structure or characteristic in conjunction with other embodiments (whether or not explicitly described).

[0050] In general, a term can be understood, at least in part, from its use in context. For example, depending, at least in part, on the context, the term "one or more" as used herein can be used to describe any feature, structure, or characteristic in the singular sense, or can be used to describe a combination of features, structures, or characteristics in the plural sense. Additionally, the term "based on" can be understood as not necessarily intended to convey an exclusive set of factors, but can instead, depending, at least in part, on the context, allow for the presence of other factors that are not necessarily explicitly described.

[0051] It will be understood that the meaning of “on,” “over,” and “above” in this disclosure should be interpreted in the broadest manner, so that “on” not only means “directly on” something, but also includes the meaning of being “on” something with intervening features or layers therebetween, and “on” or “over” not only means “on” or “above” something, but also includes the meaning of being “on” or “above” something with no intervening features or layers therebetween.

[0052] Additionally, spatially relative terms such as "under," "beneath," "lower," "above," "upper," and the like may be used herein for descriptive convenience to describe the relationship of one element or feature to another element or features, as shown in the accompanying drawings. Spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the accompanying drawings. The device may be oriented in other ways, and the spatially relative descriptors used herein may be similarly interpreted accordingly.

[0053] like Figure 1 and Figure 2 As shown, an embodiment of the present invention provides a probe design method for improving the detection performance of a gene chip, comprising the following steps:

[0054] S1. Download the genome sequence of the target species to be detected; download the fasta sequence of the genome of the species to be detected from bv-brc according to the species name or taxid to be detected. The downloading process needs to involve the sequences of pathogenic microorganisms in various time periods and regions around the world;

[0055] S2, download the complete genome database of bacteria, fungi, plants, animals, and viruses and then classify them according to species;

[0056] S3. Perform multiple sequence alignment on the whole genome data of each species; group the downloaded FASTA file sequences of the genome reference sequences of each species by pathogen type, and perform multiple sequence alignment on each group of pathogens obtained by grouping;

[0057] S4. Analyze the results of genome sequence comparison of the species to be tested, perform sequence conservation calculation, and obtain the conservation of different genome sequences of the same species;

[0058] S5. Obtain species conservative sequences based on the conservation of different genome sequences of the same species;

[0059] S6. The conserved sequences obtained by aligning multiple genome sequences of the same species are progressively cut according to the length and step size to obtain multiple probes of the species; the multiple sequence alignment results of a species are analyzed, and a sliding window of x is set. When the number of conserved base sequences in each window is greater than 90%, the region is considered to be a conserved region, and then progressive cutting is performed according to the length n and step size m to obtain multiple probes of the species;

[0060] S7. Generate rectangular, discrete, and linear designs for each probe of the species to be tested;

[0061] S8. For each probe layout, set probe repetition;

[0062] S9, repeat the setting of positive control and negative control internal reference probe clusters and probes;

[0063] S10, synthesizing and producing the chip, and designing PCR amplification primers;

[0064] Select a chip size of appropriate specifications that can accommodate the above design, perform probe synthesis and production, and use the conserved regions in the species conservation analysis results to design primers;

[0065] S11. Construct a human negative baseline (excluding the species to be tested) and statistical distribution;

[0066] S12. Conduct positive sample testing and report the results.

[0067] It should be further explained in this embodiment that the probe design of S7 is specifically as follows: firstly, the coordinates of each position on the chip are calculated.

[0068] Chip length x_range, width y_range

[0069] defcreat_coors(x_range,y_range):

[0070] x=np.arange(x_range[0],x_range[1])

[0071] y=np.arange(y_range[0],y_range[1])

[0072] X,Y = np.meshgrid(x,y)

[0073] coors=np.concatenate((X[:,:,None],Y[:,:,None]),axis=-1)

[0074] dfs=[pd.DataFrame(x)forx in coors]

[0075] df_coors=pd.concat(dfs,keys=range(len(dfs)))

[0076] df_coors=df_coors.reset_index()

[0077] df_coors.drop(['level_0','level_1'],axis=1,inplace=True)

[0078] df_coors.columns = ['x','y']

[0079] print(df_coors)

[0080] return df_coors

[0081] Then, the chip is cut into rectangular, discrete, and linear shapes according to the initialized coordinates to obtain the probe placement area under each layout;

[0082] The coordinates of each region were then matched to pre-designed species-specific probes;

[0083] This design method can effectively fill the empty positions in the probe cluster design and ensure the space utilization with maximum efficiency.

[0084] It should be further explained in this embodiment that the specific steps of S11 are: collecting oral swabs from 50 healthy people, performing PCR amplification, hybridizing the amplification products with the chip and performing fluorescence detection, and for the probe cluster design, taking the cluster as the unit, obtaining the fluorescence mean of each cluster of each sample and calculating the variance between samples, and for the probe distributed design, taking each detection unit as the unit, obtaining the fluorescence mean of each point of each sample and calculating the variance between samples.

[0085] It should be further explained in this embodiment that the specific steps of S12 are: for the probe cluster, if the positive sample to be detected exceeds the mean plus twice the variance, the species is considered to be detected. In the probe distribution design, if the positive sample to be detected exceeds the mean plus twice the variance, the probe is determined to be positive. If multiple similarity probes are positive, the one with the largest (value-mean) / std is taken as the basis, which is used for specific clinical applications such as pathogen detection.

[0086] The technical solution provided by the present invention establishes the influence of spatial layout on the detection performance of DNA and RNA microarray chips, avoiding the difficult problem that DNA and RNA microarray chip detection is affected by signal intensity. Under the guidance of this theory, we can improve the detection performance with a more effective spatial layout method.

[0087] The present invention covers any substitution, modification, equivalent method and scheme made on the essence and scope of the present invention. In order to make the public have a thorough understanding of the present invention, specific details are described in detail in the following preferred embodiments of the present invention, but those skilled in the art can fully understand the present invention without the description of these details. In addition, in order to avoid unnecessary confusion about the essence of the present invention, well-known methods, processes, procedures, components and circuits are not described in detail.

[0088] A person skilled in the art will appreciate that all or part of the steps in the above-mentioned embodiment method can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium, such as ROM / RAM, a disk, an optical disk, etc.

[0089] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A probe design method for improving the detection performance of a gene chip, characterized in that: The following steps are involved: S1. Download the genome sequence of the target species; S2, download the complete genome database of bacteria, fungi, plants, animals, and viruses and then classify them according to species; S3, multiple sequence alignment of the whole genome data of each species; S4. Analyze the results of genome sequence alignment of the species to be tested, perform sequence conservation calculation, and obtain the conservation of different genome sequences of the same species; S5. Obtain species conservative sequences based on the conservation of different genome sequences of the same species; S6, progressively cutting the conserved sequences obtained by aligning multiple genome sequences of the same species according to the length and step size to obtain multiple probes for the species; S7. Generate rectangular, discrete, and linear designs for each probe of the species to be tested; S8. For each probe layout, set probe repetition; S9, repeat the setting of positive control and negative control internal reference probe clusters and probes; S10, synthesizing and producing the chip, and designing PCR amplification primers; S11. Construct human negative baseline and statistical distribution; S12. Conduct positive sample testing and report the results.

2. A probe design method for improving the detection performance of a gene chip according to claim 1, characterized in that: The downloading of the genome sequence of the target detection species in S1 is specifically as follows: downloading the fasta sequence of the genome of the species to be detected from bv-brc according to the name of the species to be detected or the taxid, and the downloading needs to involve the sequences of pathogenic microorganisms in various time periods and regions around the world.

3. A probe design method for improving the detection performance of a gene chip according to claim 1, characterized in that: In S3, the whole genome data of each species are subjected to multiple sequence alignment, specifically: the downloaded fasta file sequences of the genome reference sequences of each species are grouped by pathogen type, and each group of pathogens obtained by grouping is subjected to multiple sequence alignment.

4. A probe design method for improving the detection performance of a gene chip according to claim 1, characterized in that: The S6 is specifically as follows: analyzing the multiple sequence alignment results of a species, setting a sliding window of x, and when the number of conservative base sequences in each window is greater than 90%, the region is considered to be a conservative region, and then progressive cutting is performed according to the length n and the step length m to obtain multiple probes of the species.

5. A probe design method for improving the detection performance of a gene chip according to claim 1, characterized in that: The probe design of S7 is as follows: First, the coordinates of each position on the chip are calculated: the length x_range, the width y_range defcreat_coors(x_range,y_range): x=np.arange(x_range[0],x_range[1]) y=np.arange(y_range[0],y_range[1]) X,Y = np.meshgrid(x,y) coors=np.concatenate((X[:,:,None],Y[:,:,None]),axis=-1) dfs=[pd.DataFrame(x)forx in coors] df_coors=pd.concat(dfs,keys=range(len(dfs))) df_coors=df_coors.reset_index() df_coors.drop(['level_0','level_1'],axis=1,inplace=True) df_coors.columns = ['x','y'] print(df_coors) return df_coors Then, the chip is cut into rectangular, discrete, and linear shapes according to the initialized coordinates to obtain the probe placement area under each layout; The coordinates of each region were then matched to pre-designed species-specific probes.

6. A probe design method for improving the detection performance of a gene chip according to claim 1, characterized in that: The specific steps of S10 are: selecting a chip size of appropriate specifications to accommodate the above design, performing probe synthesis and production, and designing primers using the conserved regions in the species conservation analysis results.

7. A probe design method for improving the detection performance of a gene chip according to claim 1, characterized in that: The specific steps of S11 are: collecting oral swabs from 50 healthy people, performing PCR amplification, hybridizing the amplification products with the chip and performing fluorescence detection, and taking clusters as units for probe cluster design to obtain the fluorescence mean of each cluster of each sample and calculate the variance between samples; and taking each detection unit as a unit for probe distributed design to obtain the fluorescence mean of each point of each sample and calculate the variance between samples.

8. A probe design method for improving the detection performance of a gene chip according to claim 1, characterized in that: The specific steps of S12 are: for the probe cluster, if the positive sample to be detected exceeds the mean plus twice the variance, the species is considered to be detected. In the probe distribution design, if the positive sample to be detected exceeds the mean plus twice the variance, the probe is determined to be positive. If multiple similarity probes are positive, the one with the largest (value-mean) / std is taken as the basis, which is used for specific clinical applications such as pathogen detection.

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