CN112322714A - Method for detecting gene editing efficiency and gene editing mode and application thereof - Google Patents
Method for detecting gene editing efficiency and gene editing mode and application thereof Download PDFInfo
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- CN112322714A CN112322714A CN202011218400.1A CN202011218400A CN112322714A CN 112322714 A CN112322714 A CN 112322714A CN 202011218400 A CN202011218400 A CN 202011218400A CN 112322714 A CN112322714 A CN 112322714A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6869—Methods for sequencing
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- G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
- G16B30/10—Sequence alignment; Homology search
Abstract
The invention relates to a method for detecting gene editing efficiency and a gene editing mode and application thereof, belonging to the technical field of gene detection. The method comprises the following steps: s1, extracting sample genome DNA, designing a primer for a region to be detected and amplifying; s2, constructing a library to perform high-throughput sequencing, and filtering, splitting and splicing data to obtain a sequence of a region to be detected; s3, comparing the sequence of the region to be detected with the genome reference sequence to obtain the gene editing efficiency and mode of each sample; and S4, performing significance analysis on the gene editing efficiency of the treatment group sample and the control group sample and outputting a corrected result. Compared with the prior art, the method simplifies the construction process of the gene library, accelerates the speed of data analysis and processing, improves the economy, sensitivity, specificity and accuracy of gene editing efficiency detection, and also provides multi-dimensional evaluation on the gene editing mode.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a method for detecting gene editing efficiency and a gene editing mode and application thereof.
Background
Gene editing techniques have been widely used and have shown great potential in gene research, gene therapy, and genetic improvement. After gene editing, analysis of the target DNA of the gene editing mutant is required to understand the efficiency and pattern of gene editing. In the technical field of gene detection, the gene editing efficiency is estimated by using a T7 endonuclease digestion method and a TA cloning Sanger sequencing method which are used in the early stage, the detection flux of the two methods is very low, the detection process is time-consuming and labor-consuming, the price is high, and the identification result is not very accurate. Currently, more advanced gene detection methods are all based on high-throughput sequencing. The method has high flux and better sensitivity and specificity than the old method.
However, there are several major drawbacks to the high-throughput sequencing-based gene editing analysis platform: 1) the preparation process of the high-throughput sequencing library is complicated, and the time cost and the economic cost of platform operation are increased; 2) the high-throughput sequencing data volume is large, the flow of data processing and analysis is complex, the equipment requirement is high, and the time consumption is long; 3) because the high-throughput sequencing platform has extremely high sensitivity, the false positive rate is also high, and the traditional analysis and treatment process cannot well eliminate false positive samples; 4) traditional analysis methods can only output gene editing efficiency, but cannot well characterize gene editing patterns.
Disclosure of Invention
The invention aims to provide a method for detecting gene editing efficiency and a gene editing mode and application thereof.
The method disclosed by the invention utilizes the advantages of high-throughput sequencing, is low in cost, rapid and accurate, and can determine the gene editing efficiency and the gene editing mode.
The method can simplify the construction process of the gene library, accelerate the speed of data analysis and processing, improve the economy, sensitivity, specificity and accuracy of gene editing efficiency detection, and provide multi-dimensional evaluation on a gene editing mode.
The purpose of the invention can be realized by the following technical scheme:
the invention provides a method for detecting gene editing efficiency and a gene editing mode, which comprises the following steps:
s1, extracting sample genome DNA, designing a primer for a region to be detected and amplifying;
s2, constructing a library to perform high-throughput sequencing, and filtering, splitting and splicing data to obtain a sequence of a region to be detected;
s3, comparing the sequence of the region to be detected with the genome reference sequence to obtain the gene editing efficiency and the gene editing mode of each sample;
and S4, performing significance analysis on the gene editing efficiency of the treatment group sample and the control group sample and outputting a corrected result.
In one embodiment of the present invention, in the step S1, in the process of designing primers for a region to be tested, the designed primers are amplification primers with different adaptor sequences added, so that each sample can be distinguished in the multi-sample mixed sequencing.
In an embodiment of the invention, in the process of designing the primer for the region to be detected in S1, the sequencing overlapping region at the left end and the right end of the designed primer is greater than 10bp, and can splice double-ended sequences.
In one embodiment of the present invention, the constructing the library in step S2 for high throughput sequencing, and the filtering, splitting and splicing the data comprises the following steps:
s21, high-throughput sequencing of the amplicon library using Illumina high-throughput sequencing platform;
s22, performing quality filtration on the double-ended sequence obtained by sequencing, removing reads with the length less than 36bp, and pruning reads with the quality value less than 30;
s23, splitting the sequence of the double-end sequence obtained by the quality filtration according to the amplification primer sequences of different adaptor sequences, and splicing the front-end sequence and the rear-end sequence.
In one embodiment of the invention, the sequencing depth of the high-throughput sequencing of the amplicon library by using the Illumina high-throughput sequencing platform is 30000-60000 ×.
In one embodiment of the present invention, the splicing condition in step S2 is that the minimum overlapping region is 10bp, and the ratio of mismatched bases in the overlapping region is less than 10%.
In one embodiment of the present invention, the method for aligning the sequence of the test region with the genomic reference sequence in step S3 comprises: comparing the sequence of the region to be detected with the genome reference sequence by using a script (https:// githu. com/rexhancx/CRISPRpic) modified based on CRISPRpic (Lee H. et al, 2020) to obtain a sequence comparison result of each sample; and counting the sequence comparison results to obtain the gene editing efficiency, the gene editing efficiency composition pattern diagram, the base deletion site pattern diagram, the base deletion length pattern diagram, the base insertion length pattern diagram and the base frame shift mutation pattern diagram of each sample.
In one embodiment of the present invention, the significance analysis in step S4 includes: and performing one-factor variance analysis on the gene editing efficiency of the processing group sample and the control group sample, if the p-value is less than 0.05, considering that the gene editing efficiency has statistical significance, filtering the background noise of the control group sample from the processing group sample, outputting a corrected result, and otherwise, not outputting the corrected result and prompting.
The invention also provides application of the method for detecting the gene editing efficiency and the gene editing mode in screening gene editing mutants.
In one embodiment of the invention, the gene editing mutants include animal gene editing mutants, plant gene editing mutants, fungal gene editing mutants and prokaryotic gene editing mutants.
Compared with the prior art, the invention has the beneficial effects that:
1) the cost is low: the preparation method of the gene library is simplified, the sequencing depth of high-throughput sequencing is optimized, and a plurality of samples are mixed for simultaneous sequencing by self-defining a joint primer, so that the cost is reduced to 20 yuan per 1 ten thousand sequences;
2) fast and accurate: optimizing a statistical analysis flow, developing a script modified based on CRISPRpic (Lee H. et al, 2020), and processing 1GB sequence data in 30 seconds by using only a common office computer (1.8GHz dual-core i5-5350U) without using a server or a high-performance processor;
3) post-examination: a single-factor variance analysis method is adopted to carry out statistical significance test on the treatment group samples and the control group samples and correct the treatment group sample results, thereby solving the problem of false positive of high-throughput sequencing detection;
4) and (3) multidimensional evaluation: the method has the advantages that the potential of high-throughput sequencing detection is fully excavated, the efficiency value of gene editing is obtained, various gene editing mode diagrams are provided, and multidimensional reference is provided for the gene editing effect.
Drawings
FIG. 1 shows a schematic flow diagram of the method of the present invention;
FIG. 2 shows a schematic diagram of the gene editing efficiency composition of the method of the present invention;
FIG. 3 shows a pattern of base deletion sites for the method of the present invention;
FIG. 4 is a diagram showing a pattern of the length of base deletion according to the method of the present invention;
FIG. 5 is a schematic diagram showing the base insertion length pattern of the method of the present invention;
FIG. 6 shows a base-shift pattern of mutation in the method of the present invention;
figure 7 shows a graph of the results of the significance test of the method of the invention.
Detailed Description
The invention is described in detail below with reference to the figures and specific embodiments. The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the invention and not to limit the invention.
Example 1
The method for detecting the gene editing efficiency and the gene editing mode comprises the following steps:
s1, extracting sample genome DNA, designing a primer for a region to be detected and amplifying;
s2, constructing a library to perform high-throughput sequencing, and filtering, splitting and splicing data to obtain a sequence of a region to be detected;
s3, comparing the sequence of the region to be detected with the genome reference sequence to obtain the gene editing efficiency and the gene editing mode of each sample;
and S4, performing significance analysis on the gene editing efficiency of the treatment group sample and the control group sample and outputting a corrected result.
In the step S1, in the process of designing primers for a region to be detected, the designed primers are amplification primers added with different adaptor sequences, so that each sample can be distinguished in multi-sample mixed sequencing, sequencing overlapping regions at the left and right ends of the designed primers are greater than 10bp, and double-ended sequences can be spliced.
Wherein, the library constructed in the step S2 is subjected to high-throughput sequencing, and the steps of filtering, splitting and splicing data comprise the following steps:
s21, using an Illumina high-throughput sequencing platform to perform high-throughput sequencing on the amplicon library with the sequencing depth of 30000-60000X;
s22, performing quality filtration on the double-ended sequence obtained by sequencing, removing reads with the length less than 36bp, and pruning reads with the quality value less than 30;
s23, splitting the sequence of the double-end sequence obtained by the quality filtration according to the amplification primer sequences of different adaptor sequences, and splicing the front-end sequence and the rear-end sequence. The splicing condition is that the minimum overlapping area is 10bp, and the mismatching base ratio of the overlapping area is less than 10 percent.
Wherein, the method for comparing the sequence of the region to be detected with the genome reference sequence in the step S3 comprises: comparing the sequence of the region to be detected with the genome reference sequence by using a script modified based on CRISPRpic (Lee H, et al, 2020) to obtain a sequence comparison result of each sample; and counting the sequence comparison results to obtain the gene editing efficiency, the gene editing efficiency composition pattern diagram, the base deletion site pattern diagram, the base deletion length pattern diagram, the base insertion length pattern diagram and the base frame shift mutation pattern diagram of each sample.
Wherein the significance analysis in step S4 includes: and performing one-factor variance analysis on the gene editing efficiency of the processing group sample and the control group sample, if the p-value is less than 0.05, considering that the gene editing efficiency has statistical significance, filtering the background noise of the control group sample from the processing group sample, outputting a corrected result, and otherwise, not outputting the corrected result and prompting.
The flow chart of the above method is shown in FIG. 1.
In this example, the samples to be tested were liver tissues of animal gene editing chimeras (prepared in the laboratory of the inventors), and three samples were taken from the treatment group and three samples from the control group.
The specific procedure of this example is as follows (all reagents are purchased from Nanjing NuoZan Biotechnology Ltd or Omega Bio-tek, Inc, and primers are synthesized by Shanghai Senno Biotechnology Ltd.).
1) Extracting genome DNA of a sample to be detected, carrying out quantification and quality inspection by using an ultraviolet spectrophotometer (Saimer Feishell Nanodrop Lite), and diluting with ultrapure water to ensure that the concentration of dsDNA is 100 ng/mu L, and A260/A280 is between 1.6 and 2.0.
2) The amplification primers containing different linkers are used to amplify the region to be detected by using the PCR technology, and the PCR reaction system is shown in Table 1.
TABLE 1
| Reagent | Volume of |
| High-fidelity PCR amplification reaction premix solution | 25μL |
| Primer mixture | 4μL |
| Genomic DNA | 1μL |
| Ultrapure water | 20μL |
Wherein, the specific sequence of the amplification primer containing the adaptor is shown in Table 2.
TABLE 2
The PCR reaction conditions are shown in Table 3.
TABLE 3
3) The library was quality checked on a LabChip. After the qualified sequencing libraries on the computers are subjected to gradient dilution, the sequencing libraries are mixed according to the required sequencing quantity in equal proportion, the PE300 sequencing is completed on an Illumina Miseq high-throughput sequencer, and all base sequence sequencing results are stored in fastq format files. The results showed that the average sequencing depth was 90814 ×, the total data size of the next machine was 660MB, and the length of the next machine sequence was 5.45e7 bp.
4) Using trimmatic v0.39(Bolger a.m. et al, 2014) software (parameters: -phred33 mutation: 20 mutation: 25 SLIDINGWINDOW:4:25 MINLEN:36) mass-filtering the sequenced double-ended sequence to remove reads with length less than 36bp and trim reads with mass value less than 30; using Cutadapt v2.10(Martin M. et al, 2011) software (parameter: -e 0.1-O5-m 50-n 2-j 2-q 20) splits the double-ended sequence after mass filtration according to the amplification primer sequence joints of different joint sequences and removes the joint primer sequence; using FLASH2(
T, et al, 2011) software (parameters: -m 20-t 4) splicing the paired sequences to form a complete sequence. Similarly, AdapterRemoval v2.3.0(Schubert m. et al, 2016) software and fastp v0.20.1(Chen s.f. et al, 2018) software may be used to complete the above data processing procedure, which is not described herein again.
5) Using a script modified based on CRISPRpic (Lee h. et al, 2020) (parameters: -w15-s 8), and outputting a gene editing efficiency composition diagram, a base deletion site pattern diagram, a base deletion length pattern diagram, a base insertion length pattern diagram, and a base shift mutation pattern diagram.
The calculated gene editing efficiencies are shown in table 4.
TABLE 4
FIG. 2, FIG. 3, FIG. 4, FIG. 5 and FIG. 6 show a schematic diagram of gene editing efficiency, a base deletion site pattern, a base deletion length pattern, a base insertion length pattern and a base shift mutation pattern of the sample "treatment group 1", respectively.
6) The results of significance tests on the gene editing efficiency of the treatment group samples and the control group samples show that the gene editing efficiency of the treatment group samples and the gene editing efficiency of the control group samples are extremely different from each other statistically, and the p-value is 5.78e-4< 0.05. The average gene editing efficiency of the control group was subtracted from the gene editing efficiency of the treatment group, and a corrected gene editing efficiency value was output.
The significance test results are shown in fig. 7.
The corrected gene editing efficiency values are shown in table 5.
TABLE 5
| Sample numbering | Corrected gene editing |
| Treatment group | |
| 1 | 0.273 |
| |
0.335 |
| Treatment group 3 | 0.237 |
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (10)
1. A method for detecting gene editing efficiency and gene editing mode, comprising the steps of:
s1, extracting sample genome DNA, designing a primer for a region to be detected and amplifying;
s2, constructing a library to perform high-throughput sequencing, and filtering, splitting and splicing data to obtain a sequence of a region to be detected;
s3, comparing the sequence of the region to be detected with the genome reference sequence to obtain the gene editing efficiency and the gene editing mode of each sample;
and S4, performing significance analysis on the gene editing efficiency of the treatment group sample and the control group sample and outputting a corrected result.
2. The method of claim 1, wherein in the step S1, the primers are designed to be amplification primers with different adaptor sequences added thereto, so that each sample can be distinguished during the multi-sample mixed sequencing.
3. The method for detecting gene editing efficiency and gene editing mode of claim 1, wherein in the process of designing primers for the region to be detected in S1, the sequencing overlapping regions at the left and right ends of the designed primers are greater than 10bp, and can splice double-ended sequences.
4. The method for detecting gene editing efficiency and gene editing pattern as claimed in claim 1, wherein the step S2 of constructing the library for high throughput sequencing, and the step of filtering, splitting and splicing the data comprises the following steps:
s21, high-throughput sequencing of the amplicon library using Illumina high-throughput sequencing platform;
s22, performing quality filtration on the double-ended sequence obtained by sequencing, removing reads with the length less than 36bp, and pruning reads with the quality value less than 30;
s23, splitting the sequence of the double-end sequence obtained by the quality filtration according to the amplification primer sequences of different adaptor sequences, and splicing the front-end sequence and the rear-end sequence.
5. The method for detecting gene editing efficiency and gene editing pattern as claimed in claim 4, wherein the sequencing depth of the Illumina high-throughput sequencing platform for high-throughput sequencing of the amplicon library is 30000-60000 x.
6. The method of claim 4, wherein the splicing condition in step S2 is 10bp of minimum overlap region, and the mismatch base ratio in the overlap region is less than 10%.
7. The method of claim 1, wherein the step S3 of aligning the sequence of the test region with the genomic reference sequence comprises:
comparing the sequence of the region to be detected with the genome reference sequence by using a CRISPRpic modification-based script to obtain a sequence comparison result of each sample; and counting the sequence comparison results to obtain the gene editing efficiency, the gene editing efficiency composition pattern diagram, the base deletion site pattern diagram, the base deletion length pattern diagram, the base insertion length pattern diagram and the base frame shift mutation pattern diagram of each sample.
8. The method for detecting gene editing efficiency and gene editing mode as claimed in claim 1, wherein the significance analysis in step S4 includes:
and performing one-factor variance analysis on the gene editing efficiency of the processing group sample and the control group sample, if the p-value is less than 0.05, considering that the gene editing efficiency has statistical significance, filtering the background noise of the control group sample from the processing group sample, outputting a corrected result, and otherwise, not outputting the corrected result and prompting.
9. Use of the method of claim 1 for detecting gene editing efficiency and gene editing pattern in screening gene editing mutants.
10. The use of claim 9, wherein the gene-editing mutants comprise animal gene-editing mutants, plant gene-editing mutants, fungal gene-editing mutants, and prokaryotic gene-editing mutants.
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| CN113969311A (en) * | 2021-10-20 | 2022-01-25 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Method for detecting mutation after gene editing |
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| CN111676276A (en) * | 2020-07-13 | 2020-09-18 | 湖北伯远合成生物科技有限公司 | Method for rapidly and accurately determining gene editing mutation condition and application thereof |
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| CN111676276A (en) * | 2020-07-13 | 2020-09-18 | 湖北伯远合成生物科技有限公司 | Method for rapidly and accurately determining gene editing mutation condition and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113969311A (en) * | 2021-10-20 | 2022-01-25 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Method for detecting mutation after gene editing |
| CN113969311B (en) * | 2021-10-20 | 2024-02-23 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Method for detecting mutation after gene editing |
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