CN114300052A - Method and device for evaluating capture specificity of nucleic acid probe - Google Patents
Method and device for evaluating capture specificity of nucleic acid probe Download PDFInfo
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Abstract
The invention discloses a method and a device for evaluating capture specificity of a nucleic acid probe. Wherein, the method comprises the following steps: s1, designing 2 auxiliary probes aiming at the reference sequence used in the design process of the probe to be evaluated; s2, comparing the 2 auxiliary probes obtained in the S1 and the probes to be evaluated with the reference sequence by using first comparison software to obtain a first classification result of each probe, and distinguishing unavailable probes from available probes according to the first classification result; and S3, comparing the available probes in the S2 with the reference sequence by using second comparison software to obtain a second scoring result of each probe, and further dividing the available probes into safety probes and risk probes according to the second scoring result, wherein the first comparison software is different from the second comparison software. By applying the technical scheme of the invention, the probes can be grouped according to the capture specificity of the probes, and the classification standards of unavailable probes, risk probes and safety probes in the probe design are defined.
Description
Technical Field
The invention relates to the technical field of biological information, in particular to a method and a device for evaluating capture specificity of a nucleic acid probe.
Background
The rapid development of NGS (second generation high throughput sequencing technology) has greatly reduced the sequencing cost of DNA and RNA, however, at present, the whole genome re-sequencing and transcriptome sequencing cost of a large number of samples are still very high, and the analysis speed is slow after mass data is obtained, and thus, the NGS cannot be applied in a large scale. The targeted sequencing technology can enrich the interested genome region for sequencing, a single sample has less sequencing data and high analysis speed, and can be applied to the fields of scientific research, clinical diagnosis, health screening and the like in a large scale.
Hybridization capture sequencing, which is one of the main technologies of targeted sequencing, is mainly applied to liquid phase hybridization capture sequencing at present, namely, a synthetic nucleic acid probe is designed based on the base complementary pairing principle, and sequencing is performed after a nucleic acid library is subjected to hybridization enrichment of a target region based on a liquid phase environment. The liquid phase hybridization capture sequencing relates to technical nodes such as probe sequence design, probe synthesis, liquid phase hybridization capture and the like, wherein the probe sequence design step directly influences the capture result and data analysis most deeply.
A probe designed aiming at a certain target needs to consider both specificity and sensitivity, namely, the probe can be subjected to base complementary pairing with a target region as much as possible, and simultaneously needs to be subjected to base complementary pairing with a non-target region as little as possible. At present, the above properties are considered in the probe design process, but individual low-specificity probes are often included in a final product, so that adverse effects such as poor capture specificity and poor capture uniformity are caused, the overall capture quality is further influenced, and the difficulty in subsequent data analysis is improved.
Therefore, it is necessary to develop a method for evaluating the capture specificity of a probe to distinguish between a low-specificity probe and a high-specificity probe.
Disclosure of Invention
The invention aims to provide a method and a device for evaluating capture specificity of a nucleic acid probe so as to distinguish a low-specificity probe from a high-specificity probe.
In order to achieve the above object, according to one aspect of the present invention, there is provided a method for evaluating capture specificity of a nucleic acid probe. The method comprises the following steps: s1, 2 auxiliary probes are designed aiming at the reference sequence used in the design process of the probe to be evaluated, wherein the length of the probe to be evaluated is N bp, the length of each auxiliary probe is N bp, one auxiliary probe comprises a sequence with a first preset length at the 5 'end of the probe to be evaluated and a sequence with a second preset length at the upstream of the near 5' end of the probe to be evaluated on the reference sequence, the other auxiliary probe comprises a sequence with a third preset length at the 3 'end of the probe to be evaluated and a sequence with a fourth preset length at the downstream of the near 3' end of the probe to be evaluated on the reference sequence, and the first preset length and the third preset length of the probe to be evaluated have coverage of more than 30 percent; s2, comparing the 2 auxiliary probes obtained in the S1 and the probes to be evaluated with the reference sequence by using first comparison software to obtain a first classification result of each probe, and distinguishing unavailable probes from available probes according to the first classification result; and S3, comparing the available probes in the S2 with the reference sequence by using second comparison software to obtain a second scoring result of each probe, and further dividing the available probes into safety probes and risk probes according to the second scoring result, wherein the first comparison software is different from the second comparison software.
Further, N is 90-160.
Further, the first comparison software is Bowtie2, and the second comparison software is Blast; or the first comparison software is Blast and the second comparison software is Bowtie 2.
Further, in S2, when the first result of the scoring of the probe is higher than the first preset value, the probe is determined to belong to a usable probe, otherwise, the probe is determined to belong to a non-usable probe.
Further, in S3, when the second score of the probe is greater than the second preset value, the probe is determined to belong to the safety probe, otherwise, the probe is determined to belong to the risk probe.
According to another aspect of the present invention, there is provided an apparatus for evaluating the capture specificity of a nucleic acid probe. The device includes: the probe production unit is used for designing 2 auxiliary probes aiming at a reference sequence used in the design process of the probe to be evaluated, wherein the length of the probe to be evaluated is N bp, the length of each auxiliary probe is N bp, one auxiliary probe comprises a sequence with a first preset length at the 5 'end of the probe to be evaluated and a sequence with a second preset length at the upstream of the near 5' end of the probe to be evaluated on the reference sequence, the other auxiliary probe comprises a sequence with a third preset length at the 3 'end of the probe to be evaluated and a sequence with a fourth preset length at the downstream of the near 3' end of the probe to be evaluated on the reference sequence, and the first preset length and the third preset length of the probe to be evaluated have coverage of more than 30 percent; the first evaluation unit is used for comparing the 2 auxiliary probes obtained in the step S1 and the probes to be evaluated with the reference sequence by using first comparison software to obtain a first classification result of each probe, and distinguishing unavailable probes from available probes according to the first classification result; and the second evaluation unit is used for comparing the available probes in the S2 with the reference sequence by using second comparison software to obtain a second scoring result of each probe, and further dividing the available probes into safety probes and risk probes according to the second scoring result, wherein the first comparison software is different from the second comparison software.
Further, N is 90-160.
Further, the first comparison software is Bowtie2, and the second comparison software is Blast; or the first comparison software is Blast and the second comparison software is Bowtie 2.
Further, in the first evaluation unit, when the first result of the scoring of the probe is higher than the first preset value, the probe is determined to belong to a usable probe, otherwise, the probe is determined to belong to a non-usable probe.
Further, in the second evaluation unit, when the second scoring result of the probe is greater than the second preset value, the probe is judged to belong to the safety probe, otherwise, the probe is judged to belong to the risk probe.
By applying the technical scheme of the invention, the probes can be grouped according to the capture specificity of the probes, the classification standards of unavailable probes, risk probes and safety probes in the probe design are defined, and the probes classified according to the standards have good performance in the subsequent application process through verification, so that the evaluation level of the probes is obviously improved compared with that of the conventional probes.
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The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic view of FIG. 1 illustrating a method for designing an auxiliary probe according to the present invention
Detailed Description
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
The invention provides the following technical scheme aiming at the technical problems that in the prior art, individual low-specificity probes are often included in a final product in the design process of the probes, so that the adverse effects of poor capturing specificity, poor capturing uniformity and the like are caused, the overall capturing quality is further influenced, and the subsequent data analysis difficulty is increased.
The invention conception of the invention is as follows: the method comprises the steps of designing an upstream auxiliary probe and a downstream auxiliary probe for a DNA or RNA probe to be evaluated with the length of N bp, and comparing the probe to be evaluated and the auxiliary probe with a reference genome or transcriptome sequence by using two kinds of software; classifying the unavailable probes and the available probes by using the comparison result of the first comparison software; and classifying the available probes by using the comparison result of the second comparison software to obtain the safety probes and the risk probes.
According to an exemplary embodiment of the present invention, a method for assessing capture specificity of a nucleic acid probe. The method comprises the following steps: s1, designing 2 auxiliary probes for the probe to be evaluated with reference to the reference sequence used in the design process, wherein, as shown in fig. 1, the length of the probe to be evaluated is N bp, and each auxiliary probe is N bp, wherein one auxiliary probe (auxiliary probe 1) comprises a sequence with a first predetermined length (a bp) at the 5 'end of the probe to be evaluated and a sequence with a second predetermined length at the upstream of the near 5' end of the probe to be evaluated on the reference sequence, the other auxiliary probe (auxiliary probe 2) comprises a sequence with a third predetermined length (B bp) at the 3 'end of the probe to be evaluated and a sequence with a fourth predetermined length at the near 3' end of the probe to be evaluated on the reference sequence, and the first predetermined length and the third predetermined length both have a coverage of more than 30% for the probe to be evaluated; s2, comparing the 2 auxiliary probes obtained in the S1 and the probes to be evaluated with the reference sequence by using first comparison software to obtain a first classification result of each probe, and distinguishing unavailable probes from available probes according to the first classification result; and S3, comparing the available probes in the S2 with the reference sequence by using second comparison software to obtain a second scoring result of each probe, and further dividing the available probes into safety probes and risk probes according to the second scoring result, wherein the first comparison software is different from the second comparison software. It is to be understood that the first predetermined length + the second predetermined length is N, and the third predetermined length + the fourth predetermined length is N.
By applying the technical scheme of the invention, the probes can be grouped according to the capture specificity of the probes, the classification standards of unavailable probes, risk probes and safety probes in the probe design are defined, and the probes classified according to the standards have good performance in the subsequent application process through verification, so that the evaluation level of the probes is obviously improved compared with that of the conventional probes.
In one embodiment of the present invention, N is 90 to 160.
According to an exemplary embodiment of the present invention, the first alignment software is Bowtie2, and the second alignment software is Blast; or the first comparison software is Blast and the second comparison software is Bowtie 2. The parameters used by the two kinds of software can change along with the change of species of actual research and the length N of the probe to be evaluated, and the optimal parameters can be determined after overall evaluation according to a large number of actual probe trial capture effects. The setting of relevant parameters can be carried out by those skilled in the art according to the practical situation under the teaching of the inventive idea. In S2, when the first result of the scoring of the probe is higher than the first preset value, the probe is determined to belong to a usable probe, otherwise, the probe is determined to belong to an unusable probe. Similarly, the first preset value may vary with the species actually studied and the length N of the probe to be evaluated, and the optimal value may be determined after overall evaluation according to a large number of actual probe capture effects. And in S3, when the second scoring result of the probe is larger than the second preset value, the probe is judged to belong to the safety probe, otherwise, the probe is judged to belong to the risk probe. Similarly, the second preset value can be changed along with the change of the species actually researched and the length N of the probe to be evaluated, and the optimal value can be determined after overall evaluation according to a large number of actual probe trial capture effects.
According to an exemplary embodiment of the present invention, an apparatus for evaluating the capture specificity of a nucleic acid probe is provided. The device includes: the probe production unit is used for designing 2 auxiliary probes aiming at a reference sequence used in the design process of the probe to be evaluated, wherein the length of the probe to be evaluated is N bp, the length of each auxiliary probe is N bp, one auxiliary probe comprises a sequence with a first preset length at the 5 'end of the probe to be evaluated and a sequence with a second preset length at the upstream of the near 5' end of the probe to be evaluated on the reference sequence, the other auxiliary probe comprises a sequence with a third preset length at the 3 'end of the probe to be evaluated and a sequence with a fourth preset length at the downstream of the near 3' end of the probe to be evaluated on the reference sequence, and the first preset length and the third preset length of the probe to be evaluated have coverage of more than 30 percent; the first evaluation unit is used for comparing the 2 auxiliary probes obtained in the step S1 and the probes to be evaluated with the reference sequence by using first comparison software to obtain a first classification result of each probe, and distinguishing unavailable probes from available probes according to the first classification result; and the second evaluation unit is used for comparing the available probes in the S2 with the reference sequence by using second comparison software to obtain a second scoring result of each probe, and further dividing the available probes into safety probes and risk probes according to the second scoring result, wherein the first comparison software is different from the second comparison software.
In one embodiment of the present invention, N is 90 to 160.
According to an exemplary embodiment of the present invention, the first alignment software is Bowtie2, and the second alignment software is Blast; or the first comparison software is Blast and the second comparison software is Bowtie 2. In the first evaluation unit, when the first scoring result of the probe is higher than the first preset value, the probe is judged to belong to the usable probe, otherwise, the probe is judged to belong to the unusable probe. In the second evaluation unit, when the second scoring result of the probe is larger than a second preset value, the probe is judged to belong to the safety probe, otherwise, the probe is judged to belong to the risk probe.
In one embodiment of the present invention, a method for evaluating nucleic acid probe capture specificity by constructing an evaluation model with double-alignment software comprises the following steps:
s1, aiming at the 120bp probe to be evaluated, referring to the reference sequence used in the design process, designing 2 120bp auxiliary probes, wherein one auxiliary probe comprises the upstream 60bp sequence of the 5 'end of the probe to be evaluated and the near 5' end 60bp sequence of the probe to be evaluated, and the other auxiliary probe comprises the downstream 60bp sequence of the 3 'end of the probe to be evaluated and the near 3' end 60bp sequence of the probe to be evaluated.
S2, aligning the 2 auxiliary probes obtained in S1 with the probe to be evaluated by using Bowtie2 and a reference sequence. Bowtie2 scores are obtained for each probe, and unusable probes and usable probes are distinguished according to the score, wherein a probe belongs to a usable probe when the score of the probe is higher than a first preset value, and a probe belongs to a unusable probe otherwise.
S3, aligning the available probes in S2 with the reference sequence using Blast software. And obtaining a comparison result of each probe, further dividing the available probes into safety probes and risk probes according to hits values, wherein the probes belong to the safety probes when the hits results of the probes are larger than a second preset value, and belong to the risk probes when the hits results of the probes are lower than a certain value.
The following examples are provided to further illustrate the advantageous effects of the present invention.
Example 1
The probe to be evaluated was assumed to be a 120bp long DNA probe designed for the gene on hg19 genome.
The specific evaluation of the probe to be evaluated comprises the following steps:
s1, aiming at the probe to be evaluated, referring to hg19 reference genome, designing auxiliary probes thereof, wherein one auxiliary probe comprises an upstream 60bp sequence at the 5 'end of the probe to be evaluated and a near 5' end 60bp sequence of the probe to be evaluated, and the other auxiliary probe comprises a downstream 60bp sequence at the 3 'end of the probe to be evaluated and a near 3' end 60bp sequence of the probe to be evaluated.
And S2, comparing the two auxiliary probes obtained in the step S1 with the probe to be evaluated by using Bowtie2 and hg19 reference genome to obtain the score value of each probe. Wherein probes with score less than a certain value are considered unusable probes and probes greater than a certain value are considered usable probes.
S3, comparing the available probes obtained in the step S2 with the hg19 reference genome by using Blast to obtain hits value of each probe. Probes with hits values less than a certain value are considered risk probes, and probes with hits values above a certain value are considered safety probes.
For the case of multiple probes to be evaluated, the steps S1-S3 are repeated for each probe to be evaluated, and the probes are grouped into different sets according to the classification result after each step of classification. And finally obtaining an unavailable probe set, a risk probe set and a safety probe set.
When the probe to be evaluated is an RNA probe, the hg19 reference genome is replaced with the hg19 reference transcript set in steps S1-S3.
According to the steps, 10 ten thousand DNA probes to be evaluated are evaluated, and actual capture verification is carried out. The actual capture performance of the probe is compared with the evaluation record of the probe, the actual capture performance is considered to be accurate if the actual capture performance is consistent with the evaluation record, and the result shows that the accuracy of the invention in evaluating 10 ten thousand probes to be evaluated can reach 99.5%.
Example 2
An apparatus for evaluating the capture specificity of a nucleic acid probe. The device includes: the probe production unit is used for designing 2 auxiliary probes aiming at a reference sequence used in the design process of the probe to be evaluated, wherein the length of the probe to be evaluated is N bp, the length of each auxiliary probe is N bp, one auxiliary probe comprises a sequence with a first preset length at the 5 'end of the probe to be evaluated and a sequence with a second preset length at the upstream of the near 5' end of the probe to be evaluated on the reference sequence, the other auxiliary probe comprises a sequence with a third preset length at the 3 'end of the probe to be evaluated and a sequence with a fourth preset length at the downstream of the near 3' end of the probe to be evaluated on the reference sequence, and the first preset length and the third preset length of the probe to be evaluated have coverage of more than 30 percent; the first evaluation unit is used for comparing the 2 auxiliary probes obtained in the step S1 and the probes to be evaluated with the reference sequence by using first comparison software to obtain a first classification result of each probe, and distinguishing unavailable probes from available probes according to the first classification result; and the second evaluation unit is used for comparing the available probes in the S2 with the reference sequence by using second comparison software to obtain a second scoring result of each probe, and further dividing the available probes into safety probes and risk probes according to the second scoring result, wherein the first comparison software is different from the second comparison software.
Further, N is 90-160.
Further, the first comparison software is Bowtie2, and the second comparison software is Blast; or the first comparison software is Blast and the second comparison software is Bowtie 2.
Further, in the first evaluation unit, when the first result of the scoring of the probe is higher than the first preset value, the probe is determined to belong to a usable probe, otherwise, the probe is determined to belong to a non-usable probe.
Further, in the second evaluation unit, when the second scoring result of the probe is greater than a second preset value, the probe is judged to belong to the safety probe, otherwise, the probe is judged to belong to the risk probe.
The method of example 1 above may be performed by the apparatus of example 2.
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the accuracy of evaluating the capture specificity of the probes to be evaluated can reach 99.5%, the accurate classification of the capture specificity of the probes is realized, and the probes in the probe set to be evaluated can be accurately classified into usable probes, risk probes and unusable probes.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method of assessing the capture specificity of a nucleic acid probe, comprising the steps of:
s1, 2 auxiliary probes are designed aiming at a probe to be evaluated and referring to a reference sequence used in the design process of the probe to be evaluated, wherein the length of the probe to be evaluated is N bp, each auxiliary probe is N bp, one auxiliary probe comprises a sequence with a first preset length at the 5 'end of the probe to be evaluated and a sequence with a second preset length at the upstream of the 5' end of the probe to be evaluated on the reference sequence, the other auxiliary probe comprises a sequence with a third preset length at the 3 'end of the probe to be evaluated and a sequence with a fourth preset length at the downstream of the 3' end of the probe to be evaluated on the reference sequence, and the first preset length and the third preset length both have a coverage degree of more than 30% on the probe to be evaluated;
s2, comparing the 2 auxiliary probes obtained in the S1 and the probe to be evaluated with the reference sequence by using first comparison software to obtain a first classification result of each probe, and distinguishing an unavailable probe from an available probe according to the first classification result;
s3, using second alignment software to align the available probes in S2 with the reference sequence to obtain a second scoring result of each probe, further dividing the available probes into safety probes and risk probes according to the second scoring result,
wherein the first comparison software is different from the second comparison software.
2. The method of claim 1, wherein N is 90-160.
3. The method of claim 1, wherein the first alignment software is Bowtie2 and the second alignment software is Blast; or the first comparison software is Blast and the second comparison software is Bowtie 2.
4. The method according to any one of claims 1 to 3, wherein in S2, when the first score of a probe is higher than a first preset value, the probe is determined to belong to a usable probe, otherwise, the probe is determined to belong to a non-usable probe.
5. The method according to any one of claims 1 to 3, wherein in S3, when the second score of a probe is greater than a second preset value, the probe is determined to belong to a safety probe, otherwise, the probe is determined to belong to a risk probe.
6. An apparatus for evaluating capture specificity of a nucleic acid probe, comprising:
the probe production unit is used for designing 2 auxiliary probes by referring to a reference sequence used in the design process of the probe to be evaluated, wherein the length of the probe to be evaluated is N bp, each auxiliary probe is N bp, one auxiliary probe comprises a sequence with a first preset length at the 5 'end of the probe to be evaluated and a sequence with a second preset length at the upstream of the near 5' end of the probe to be evaluated on the reference sequence, the other auxiliary probe comprises a sequence with a third preset length at the 3 'end of the probe to be evaluated and a sequence with a fourth preset length at the downstream of the near 3' end of the probe to be evaluated on the reference sequence, and the first preset length and the third preset length both have coverage of more than 30% on the probe to be evaluated;
a first evaluation unit, configured to compare the 2 auxiliary probes obtained in S1 with the probe to be evaluated with the reference sequence using first comparison software, to obtain a first scoring result for each probe, and distinguish an unavailable probe from an available probe according to the first scoring result;
a second evaluation unit configured to compare the available probes in S2 with the reference sequence by using second comparison software to obtain a second scoring result of each probe, further divide the available probes into safety probes and risk probes according to the second scoring result,
wherein the first comparison software is different from the second comparison software.
7. The device of claim 6, wherein N is 90-160.
8. The apparatus of claim 6, wherein the first alignment software is Bowtie2 and the second alignment software is Blast; or the first comparison software is Blast and the second comparison software is Bowtie 2.
9. The apparatus according to any one of claims 6 to 8, wherein in the first evaluation unit, when the first result of scoring of a probe is higher than a first preset value, the probe is determined to belong to a usable probe, otherwise, the probe is determined to belong to a non-usable probe.
10. The apparatus according to any one of claims 6 to 8, wherein in the second evaluation unit, when the second score of the probe is greater than a second preset value, the predetermined probe is determined to belong to a safety probe, otherwise, the predetermined probe is determined to belong to a risk probe.
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