CN110951878A - Screening method, screening device and application of microsatellite loci related to genome stability - Google Patents
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Abstract
The invention provides a screening method and a screening device of microsatellite loci related to genome stability and application. The screening method comprises determining the state of the microsatellite loci of tumor tissues and blood cells of each of a plurality of samples and the state of the samples by a first method; verifying the sample states of the plurality of samples by adopting a second method; selecting a plurality of samples with consistent sample states determined by the first method and the second method, and dividing the plurality of samples with consistent sample states into a first type sample of the MSS and a second type sample of the MSI-H; respectively screening a plurality of microsatellite loci with highest correlation with the first type of samples to form a first locus set and a plurality of microsatellite loci with highest correlation with the second type of samples to form a second locus set; and taking the intersection of the first site set and the second site set to obtain the microsatellite sites related to the genome stability. The microsatellite loci screened by the method have high detection accuracy and high detection sensitivity and specificity.
Description
Technical Field
The invention relates to the field of gene sequencing data analysis, in particular to a screening method and a screening device of microsatellite loci related to genome stability and application of the microsatellite loci.
Background
The microsatellite locus is generally a short repetitive sequence with the length of about 1-6 bp and widely exists in a human genome. Microsatellite instability refers to any change in the length of a microsatellite in a tumor due to the insertion or deletion of repeat units, compared to normal tissue, with the appearance of new microsatellite alleles. The mechanism of occurrence mainly includes mismatches of 1 or more bases in the repetitive sequence caused by the sliding of DNA polymerase and deletion or insertion of base pairs caused by microsatellite recombination.
The traditional detection method of microsatellite instability mainly detects the stability of 5 microsatellite loci (NR-27, NR-24, NR-21, BAT-25 and BAT-26) of tumor tissues by MSI-PCR. MSI-H, i.e., highly microsatellite unstable, is considered if there are 2 or more sites unstable; if 1 bit is unstable, the MSI-L is considered as low-degree microsatellite instability; if no site instability is detected, it is denoted MSS, i.e., microsatellite stability. However, the conventional detection method based on MSI-PCR is often complex in experimental operation, limited in the number of microsatellite sites detected at one time, poor in sensitivity, low in flux and poor in result repeatability.
Compared with the traditional detection method, the method has the advantages that the positions indicating the microsatellite state are enriched by the liquid phase hybridization capture method through the NGS method, the sequencing data of a large number of samples are generated by high-throughput sequencing, and the stability states of a plurality of MSI positions are simultaneously evaluated through bioinformatics analysis, so that the microsatellite stability state of the samples is judged.
Colorectal Cancer (CRC), a common tumor of the digestive tract, occurs third after lung and stomach cancers; the mortality rate is 5 th after lung cancer, gastric cancer, liver cancer and esophageal cancer, the survival rate of the patients after radical excision is about 50 percent in 5 years, and postoperative recurrence and metastasis are important reasons for death. The occurrence of colorectal cancer is divided into hereditary and non-hereditary, wherein the colorectal cancer caused by hereditary factors comprises familial adenoadenomatosis sarcoma (FAP) and linch syndrome (Lynch syndrome, also known as hereditary non-polyposis colorectal cancer); non-hereditary bowel cancer is sporadic colorectal cancer. FAP and more than 85% of sporadic colorectal cancers occur primarily due to chromosomal instability; approximately 90% of the woodchuck syndrome and 10% to 15% of sporadic colorectal cancer occurrences are mainly determined by microsatellite instability (MSI).
However, in the conventional microsatellite state detection method, the flux of the MSI-PCR detection method is low, and the NGS detection method can judge the stability state of genome change (namely, the flux is high) by comprehensively judging the stability of a plurality of detected microsatellite loci, but the accuracy of a sample detection result is low, so that the microsatellite state of tumor tissues such as colorectal cancer is difficult to accurately detect.
Therefore, it is an urgent problem to find MSI sites that are highly correlated with the stability of genomic changes in tumor tissues such as colorectal cancer.
Disclosure of Invention
The invention mainly aims to provide a screening method, a screening device and application of microsatellite loci related to genome stability, so as to solve the problem of low accuracy of detection results in the prior art.
In order to achieve the above objects, according to one aspect of the present invention, there is provided a method for screening a microsatellite locus associated with genome stability, the method comprising: determining the state of the microsatellite loci of the tumor tissues and the blood cells of the plurality of samples and the state of the samples by adopting a first method; verifying the sample states of the plurality of samples using a second method different from the first method; selecting a plurality of samples with consistent sample states determined by the first method and the second method, and dividing the plurality of samples with consistent sample states into a first type sample of an MSS and a second type sample of an MSI-H according to the sample states of the samples; respectively screening a plurality of microsatellite loci with highest correlation with the first type of samples to form a first locus set and a plurality of microsatellite loci with highest correlation with the second type of samples to form a second locus set; and taking the intersection of the first site set and the second site set to obtain the microsatellite sites related to the genome stability.
Further, determining the status of the microsatellite loci of tumor tissue and blood cells in each of the plurality of samples using the first method and the status of the samples comprises: obtaining sequencing data of tumor tissues and blood cells of a plurality of samples and all microsatellite loci in a region covered by the sequencing data; counting the difference value delta d of the length d of the repeated sequences of the sequencing data of the tumor tissues and the blood cells of each sample at each microsatellite locus; and judging the state of the microsatellite loci according to the relation between the difference value delta d and the first threshold, and judging the sample state of each sample according to the relation between the mean value or the median of the difference values delta d of all the microsatellite loci of each sample and the second threshold.
Further, determining the state of the microsatellite locus according to the relationship between the difference Δ d and the first threshold includes: when the difference value delta d is larger than or equal to a first threshold value, judging that the state of the microsatellite locus is unstable; and when the difference value delta d is less than the first threshold value, judging that the state of the microsatellite locus is stable.
Further, the determining the sample state of each sample according to the relationship between the mean value or the median of the differences Δ d between all the microsatellite loci of each sample and the second threshold includes: when the mean value or median of the differences delta d of the sample at all the microsatellite loci is larger than or equal to a second threshold value, judging that the state of the sample is MSI-H; and when the mean value or median of the differences delta d of the samples at all the microsatellite loci is less than a second threshold value, judging that the state of the samples is MSS.
Further, the step of respectively screening out a plurality of microsatellite loci with the highest correlation with the first type of sample to form a first locus set and a plurality of microsatellite loci with the highest correlation with the second type of sample to form a second locus set comprises the following steps: merging the unstable microsatellite loci of each sample in the first class of samples, performing descending arrangement on the unstable microsatellite loci according to the difference value delta d, and selecting the first m-bit microsatellite loci as a first locus set; merging the stable-state microsatellite loci of all samples in the second type of samples, performing ascending arrangement on the microsatellite loci according to the difference value delta d, and selecting the microsatellite loci arranged at the first n positions as a second locus set; wherein m and n are each independently a natural number of 2 or more.
Further, the second method is a method of PCR, the method of PCR comprising: detecting the stability of each sample at five microsatellite loci of R-27, NR-24, NR-21, BAT-25 and BAT-26 by PCR, and judging the sample state of the sample to be MSI-H when the state of more than or equal to 2 microsatellite loci is unstable; when the status of the microsatellite locus is not detected to be unstable, the sample status of the sample is determined to be MSS.
Further, the sequencing data is sequencing data of a capture library; preferably, the length d of the repeat sequence at each microsatellite locus is calculated from the sequencing data of tumor tissue and blood cells as Euclidean distance.
Further, the tumor tissue is colorectal cancer tissue.
In order to achieve the above object, according to one aspect of the present invention, there is provided a tumor tissue marker microsatellite loci selected by any one of the above screening methods.
Further, the microsatellite loci are those for detecting the genomic stability status of the colorectal cancer sample, preferably the microsatellite loci include one or more of the loci shown in the following table:
| chromosome | Starting position | End position | (base sequence) number of repetitions |
| chr1 | 161309335 | 161309346 | (T)11 |
| chr2 | 39240584 | 39240595 | (A)11 |
| chr3 | 52696310 | 52696321 | (A)11 |
| chr3 | 169992975 | 169992993 | (T)18 |
| chr6 | 32166160 | 32166173 | (T)13 |
| chr11 | 118353037 | 118353053 | (T)16 |
| chr19 | 50911947 | 50911959 | (T)12 |
| chr22 | 41545024 | 41545038 | (T)14 |
| chrX | 44949951 | 44949962 | (T)11 |
。
In order to achieve the above object, according to a third aspect of the present invention, there is provided a kit for detecting the stability state of a genome, the kit comprising microsatellite loci, the microsatellite loci being one or more of the microsatellite loci provided above.
According to a fourth aspect of the present invention, there is provided an apparatus for detecting the genome stability state, the apparatus detecting the genome stability state of a test sample using the stability state of microsatellite loci, the microsatellite loci being one or more of the provided microsatellite loci.
According to a fifth aspect of the present invention, there is provided a screening apparatus for microsatellite loci associated with genome stability, the screening apparatus comprising: a measuring module for measuring the state of the microsatellite loci of the tumor tissue and the blood cells of each of the plurality of samples and the state of the sample by using a first method; a verification module for verifying the sample states of the plurality of samples using a second method different from the first method; the selecting and classifying module is used for selecting a plurality of samples with consistent sample states determined by the first method and the second method, and dividing the plurality of samples with consistent sample states into a first type sample of an MSS and a second type sample of the MSI-H according to the sample states of the samples; the site screening module is used for respectively screening a plurality of microsatellite sites with highest correlation with the first type of samples to form a first site set and a plurality of microsatellite sites with highest correlation with the second type of samples to form a second site set; and the intersection acquisition module is used for acquiring the intersection of the first site set and the second site set to obtain the microsatellite sites related to the genome stability.
Further, the assay module comprises: the first acquisition unit is used for acquiring sequencing data of tumor tissues and blood cells of a plurality of samples and all microsatellite loci in an area covered by the sequencing data; the statistical unit is used for counting the difference value delta d of the length d of the repeated sequences of the sequencing data of the tumor tissues and the blood cells of each sample at each microsatellite locus; and the judging unit is used for judging the state of the microsatellite loci according to the relation between the difference value delta d and the first threshold value, and judging the sample state of each sample according to the relation between the mean value or the median of the difference values delta d of all the microsatellite loci of each sample and the second threshold value.
Further, the judging unit includes: the first judgment submodule is used for judging the state of the microsatellite locus to be unstable when the difference value delta d is larger than or equal to a first threshold value; and the second judgment submodule is used for judging the state of the microsatellite locus to be stable when the difference value delta d is less than the first threshold value.
Further, the judging unit further includes: the third judgment submodule is used for judging the state of the sample to be MSI-H when the mean value or the median of the difference values delta d of the sample at all the microsatellite loci is larger than or equal to a second threshold value; and the fourth judgment submodule is used for judging the state of the sample to be MSS when the mean value or the median of the difference values delta d of the sample at all the microsatellite loci is less than the second threshold value.
Further, the site screening module comprises: the first screening submodule is used for taking a union set of the unstable-state microsatellite loci of each sample in the first class of samples, performing descending order arrangement on the unstable-state microsatellite loci according to the difference value delta d, and selecting the upper m-position microsatellite loci as a first locus set; the second screening submodule is used for taking a union set of the stable-state microsatellite loci of each sample in the second type of sample, performing ascending arrangement on the microsatellite loci according to the difference value delta d, and selecting the microsatellite loci arranged at the front n positions as a second locus set; wherein m and n are each independently a natural number of 2 or more.
Further, the verification module is a PCR module, and the PCR module includes: the detection submodule is used for detecting the stability of each sample at five microsatellite loci of R-27, NR-24, NR-21, BAT-25 and BAT-26 through PCR, and the sixth judgment submodule is used for judging the sample state of the sample to be MSI-H when the state of more than or equal to 2 microsatellite loci is unstable; and the seventh judging submodule is used for judging the sample state of the sample to be MSS when the state of the microsatellite locus is not detected to be unstable.
Further, the sequencing data is sequencing data of a capture library; preferably, the length d of the repeat sequence at each microsatellite locus is calculated from the sequencing data of tumor tissue and blood cells as Euclidean distance.
Further, the tumor tissue is colorectal cancer tissue.
According to a third aspect of the present invention, there is provided a storage medium having a computer program stored therein, wherein the computer program is arranged to perform any of the above-mentioned screening methods when executed.
According to a third aspect of the present invention, there is provided an electronic device comprising a memory having a computer program stored therein and a processor configured to execute the computer program to perform any of the above-described screening methods.
By applying the technical scheme of the invention, the stability state of the sample is detected by adopting two different methods, the two samples with consistent verification states are taken as objects, the intersection of the most relevant multiple microsatellite loci in the highly unstable state sample and the most relevant multiple microsatellite loci in the stable state sample is further obtained, so that the microsatellite loci with obvious differences in the two types of samples with highly unstable state temperature and state are obtained, and the microsatellite loci can be used as the tumor tissue specific or symbolic microsatellite loci. The microsatellite loci screened by the screening method are highly related to the unstable state of the genome, so that the microsatellite loci screened by the screening method are high in accuracy, high in detection sensitivity and specificity and more beneficial to accurately judging the stable state of the genome microsatellite of the sample.
Drawings
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 shows a schematic flow diagram of a method for screening for microsatellite loci associated with genome stability according to a preferred embodiment of the present application;
fig. 2 shows a diagram of the results of the detection classification of 43 samples according to a preferred embodiment of the present application.
FIG. 3 shows a schematic structural diagram of a screening apparatus for microsatellite loci associated with genome stability according to a preferred embodiment of the present application;
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 with reference to examples.
MSI: microsatellite instability.
MSI-H: microsatellite is highly unstable.
MSS: micro satellite stability.
And (3) NGS: next-generation sequencing, second generation high throughput sequencing.
Euclidean distance: also known as the euclidean metric, is a distance definition that refers to the true distance between two points in an m-dimensional space or the natural length of a vector (i.e., the distance of the point from the origin). The euclidean distance in two and three dimensions is the actual distance between two points.
Microsatellites: microsatellites are short tandem repeats distributed throughout the human genome with repeats of single, double or higher nucleotides, 10-50 times.
Microsatellite instability: microsatellites result in a change in the length of the microsatellite due to the insertion or deletion of repeat units.
Example 1
In a preferred embodiment of the present application, a method for screening for microsatellite loci associated with genomic stability is provided, and FIG. 1 is a flow chart of a method according to an embodiment of the present invention. As shown, the screening method includes:
step S101, measuring the states of the microsatellite loci of the tumor tissues and the blood cells of a plurality of samples and the states of the samples by adopting a first method;
step S102, verifying the sample states of a plurality of samples by adopting a second method different from the first method;
step S103, selecting a plurality of samples with consistent sample states determined by the first method and the second method, and dividing the plurality of samples with consistent sample states into a first type sample of MSS (namely MSS) and a second type sample of MSI-H (MSI-H) according to the sample states of the samples;
step S104, respectively screening a plurality of microsatellite loci with highest correlation with the first type of samples to form a first locus set and a plurality of microsatellite loci with highest correlation with the second type of samples to form a second locus set;
and S105, taking the intersection of the first site set and the second site set to obtain the microsatellite sites related to the genome stability.
According to the screening method, the stability states of the samples are detected by adopting two different methods, the samples with the same verification states are taken as objects, the intersection of the most relevant multiple microsatellite loci in the samples in the highly unstable state and the most relevant multiple microsatellite loci in the samples in the stable state is further obtained, so that the microsatellite loci which are obviously different in the two types of samples in the highly unstable state and the highly unstable state are obtained, and the microsatellite loci can be used as the microsatellite loci with the specificity or the sign of the tumor tissue. The microsatellite loci screened by the screening method are highly related to the unstable state of the genome, so that the detection of the microsatellite loci screened by the method is high in accuracy, high in detection sensitivity and specificity and more beneficial to accurately judging the stable state of the genome of a sample.
In order to further improve the correlation between the selected site and the genomic stability, in a preferred embodiment of the present application, the first method for determining the status of the microsatellite sites of tumor tissues and blood cells of each of a plurality of samples and the status of the sample includes: obtaining sequencing data of tumor tissues and blood cells of a plurality of samples and all microsatellite loci in a region covered by the sequencing data; counting the difference value delta d of the length d of the repeated sequences of the sequencing data of the tumor tissues and the blood cells of each sample at each microsatellite locus; and judging the state of the microsatellite loci according to the relation between the difference value delta d and the first threshold, and judging the sample state of each sample according to the relation between the mean value or the median of the difference values delta d of all the microsatellite loci of each sample and the second threshold.
In a preferred embodiment, determining the state of the microsatellite locus based on the relationship between the difference Δ d and the first threshold comprises: when the difference value delta d is larger than or equal to a first threshold value, judging that the state of the microsatellite locus is unstable; and when the difference value delta d is less than the first threshold value, judging that the state of the microsatellite locus is stable.
In a preferred embodiment, the determining the sample state of each sample according to the relationship between the mean or median of the differences Δ d between all the microsatellite loci of each sample and the second threshold comprises: when the mean value or median of the differences delta d of the sample at all the microsatellite loci is larger than or equal to a second threshold value, judging that the state of the sample is MSI-H; and when the mean value or median of the differences delta d of the samples at all the microsatellite loci is less than a second threshold value, judging that the state of the samples is MSS.
In the above preferred embodiment, the length d of the repeat sequence at each microsatellite locus in the sequencing data of tumor tissue and blood cells is preferably calculated using the Euclidean distance. And euclidean distance refers to the true distance between two points in m-dimensional space or the natural length of the vector (i.e., the distance of the point from the origin). The euclidean distance in two and three dimensions is the actual distance between two points. The calculation of the length d of the repeated sequences in the tumor tissue and the blood cell tissue by using the Euclidean distance is not influenced by the statistical distribution of the sample, so that the statistical result is more accurate compared with other methods for counting difference (such as p test and the like, the data are assumed to be in certain statistical distribution).
In the above preferred embodiment, the specific values of the first threshold and the second threshold can be obtained according to a large number of sample statistics. In the present application, the first threshold value is preferably 0.2, and the second threshold value is preferably 0.2.
The genome stability states of the samples with consistent states verified by the two methods are considered to be accurate, so that the MSI-H state samples and the MSS state samples are randomly selected from the MSI-H state samples, the microsatellite loci appearing in the samples are subjected to correlation detection, the microsatellite loci with high correlation with a certain state can be obtained, and the microsatellite loci with high correlation with the two states can be used as marker loci for detecting the genome stability of the samples. The specific correlation site can be obtained by screening using various methods, for example, the correlation analysis can be performed by using SPSS software, and the specific method is not particularly limited in this application.
In a preferred embodiment, the step of selecting the plurality of microsatellite loci with the highest correlation with the first type of sample to form a first locus set and the plurality of microsatellite loci with the highest correlation with the second type of sample to form a second locus set comprises: merging the unstable microsatellite loci of each sample in the first class of samples, performing descending arrangement on the unstable microsatellite loci according to the difference value delta d, and selecting the first m-bit microsatellite loci as a first locus set; merging the stable-state microsatellite loci of all samples in the second type of samples, performing ascending arrangement on the microsatellite loci according to the difference value delta d, and selecting the microsatellite loci arranged at the first n positions as a second locus set; wherein m and n are each independently a natural number of 2 or more.
The positions which are highly related to the MSI-H state and the MSS state are screened by the preferred method, and the difference value delta d of the positions in two different states is obviously different, so that the positions are used for detecting a sample in an unknown state, the state of the sample can be accurately characterized, and the detection result is more accurate. And the correlation of the sites and the state is the highest, so that the detection sensitivity and specificity are higher. The specific values of m and n can be determined reasonably according to the actually selected sample size, and the two values can be the same or different. For example, the values of m and n can be both 50 or 60 and 40; alternatively, m may be 40 and n may be 70. The greater the specific values of both, the greater the probability that the number of sites in common in both corresponding sets will also be.
In the second method, the verification may be performed by using an existing method. In a preferred embodiment, the second method is a method of PCR, the method of PCR comprising: detecting the stability of each sample at five microsatellite loci of R-27, NR-24, NR-21, BAT-25 and BAT-26 by PCR, and judging the sample state of the sample to be MSI-H when the state of more than or equal to 2 microsatellite loci is unstable; when the status of the microsatellite locus is not detected to be unstable, the sample status of the sample is determined to be MSS.
The sequencing data of the tumor tissue and the blood cells are preferably sequencing data of the capture library from the viewpoint of detection efficiency.
The above screening method is applicable to any tumor tissue associated with genomic instability, and is particularly applicable to colorectal cancer tissue. In a preferred embodiment, the tumor tissue is colorectal cancer tissue.
Example 2
This example provides a method for screening for microsatellite loci specific to colorectal cancer tissue and using the method to test the status of samples therein. The method comprises the following specific steps:
1. obtaining tumor tissues and leucocyte DNA of a plurality of colorectal cancer patients.
2. The genome sequences of the tumor of the patient sample and the leucocyte control sample are obtained by using a high-throughput chip capture method.
3. The sequenced sequences were aligned to the human hg19 reference genomic sequence, and the alignment removed duplicate sequences and aligned to multiple positions.
4. The MSIsensor tool was used to scan the human hg19 reference genomic sequence to obtain a distribution of microsatellite loci throughout the human genome.
5. All the microsatellite loci covered in a chip capturing interval are selected from the microsatellite loci distributed in the whole human genome.
6. And respectively counting the length of the repetitive sequences of the sequencing reads at different microsatellite sites in the NGS data of the tumor sample and the leucocyte control sample of each sample.
7. Using the euclidean distance, calculating the length difference value of each microsatellite locus in each sample in the tumor sample and the control sample thereof, and if the length difference value at the locus is higher than a set first threshold (for example, can be 0.2), the locus is considered as an instability locus, otherwise, the locus is considered as a stability locus. Then, the average value of the length difference values of each sample at all selected microsatellite loci is calculated, if the average value is higher than a set second threshold (for example, the average value can be 0.2), the sample is considered as being highly unstable, otherwise, the sample is considered as being stable.
8. A plurality of samples of microsatellite stability (MSS) and high instability (MSI-H) with the judgment results of NGS data consistent with the verification results of MSI-PCR are randomly selected, and the microsatellite loci most relevant to the microsatellite state judgment of the sample genome are selected by using the following locus screening method.
The site screening method is as follows:
1) extracting a union set of all unstable microsatellite loci from all selected unstable samples (MSI-H), performing descending order arrangement according to the length difference value of each locus, and taking out 50 loci arranged at the top;
2) extracting a union set of all stability microsatellite loci from all selected stability samples, arranging the stability microsatellite loci in an ascending order according to the Euclidean distance difference of each point, and taking out the 50 loci arranged at the most front;
3) and finally, taking intersection of all the sites obtained in the steps 1) and 2) to obtain 9 microsatellite sites which are strongly related to the MSI state judgment of the sample and are shown in the table 1.
Table 1: nine bit information:
| chromosome | Starting position | End position | (base sequence) number of repetitions |
| chr1 | 161309335 | 161309346 | (T)11 |
| chr2 | 39240584 | 39240595 | (A)11 |
| chr3 | 52696310 | 52696321 | (A)11 |
| chr3 | 169992975 | 169992993 | (T)18 |
| chr6 | 32166160 | 32166173 | (T)13 |
| chr11 | 118353037 | 118353053 | (T)16 |
| chr19 | 50911947 | 50911959 | (T)12 |
| chr22 | 41545024 | 41545038 | (T)14 |
| chrX | 44949951 | 44949962 | (T)11 |
。
9. And (3) testing the newly selected 9 microsatellite loci in a plurality of samples by using the method in 8, and judging the final state of the genome microsatellite of the sample according to the sample genome microsatellite state judgment standard mentioned in 7.
Example 3
In this example, a microsatellite locus for testing genome stability is provided, which is obtained by screening using any one of the screening methods described above. The microsatellite loci obtained by screening by the method are highly related to the unstable state of the microsatellite, so that the stability state of a sample to be detected can be accurately distinguished, and the detection sensitivity and specificity are high (by carrying out genome stability detection on 64 colorectal cancer samples verified by MSI-PCR (MSI-PCR), 14 MSI-H samples and 50 MSS samples, the microsatellite loci screened by the method are detected correctly, and the sensitivity and specificity are both 100 percent, and the method is concretely shown in the following table 2).
Table 2:
in a preferred embodiment, the microsatellite sites are colorectal cancer tissue marker microsatellite sites, preferably the colorectal cancer tissue marker microsatellite sites include one or more of the sites shown in table 1.
Compared with the existing NGS method, the 9 sites are fewer, so that the problem of low accuracy caused by introducing excessive sites is solved, the relevance to an unstable state is high, and the accuracy, sensitivity and specificity of sample judgment are improved.
Example 4
The embodiment provides a kit for detecting the genome stability state, which comprises microsatellite loci, wherein the microsatellite loci are one or more than one microsatellite loci. When the kit is used for detecting the genome stability state of a sample, the accuracy, sensitivity and specificity of sample judgment are improved due to the high correlation between the microsatellite loci in the kit and the unstable state.
Example 5
Using the 9 microsatellite loci obtained from the screening in example 2, the stability status of tumor tissues and genomes of leukocytes of 43 cases (10 cases of them were verified as MSI-H samples by MSI-PCR gold criteria and 33 cases were verified as MSS samples by MSI-PCR gold criteria) of colorectal cancer samples were tested, and fig. 2 was plotted according to the test results (the score of MSI on ordinate is the average of the length differences at all microsatellite loci of each sample), and the results of fig. 2 show: among them, 10 MSI-H samples are correctly divided into MSI-H, and 33 MSS samples are correctly divided into MSS. The specificity sensitivity is 100%. Therefore, the microsatellite loci screened by the method can be used for correctly distinguishing the microsatellite high instability sample and the microsatellite stability sample in the colorectal cancer sample, so that all samples can be correctly classified.
Through the above description of the embodiments, those skilled in the art can clearly understand that the method according to the above embodiments can be implemented by software plus a necessary general hardware platform, and certainly can also be implemented by hardware, but the former is a better implementation mode in many cases. Based on such understanding, the technical solutions of the present invention may be embodied in the form of a software product, which is stored in a storage medium (e.g., ROM/RAM, magnetic disk, optical disk) and includes instructions for enabling a terminal device (e.g., a mobile phone, a computer, a server, or a network device) to execute the method according to the embodiments of the present invention.
Corresponding to the above manner, the present application further provides a device for screening microsatellite loci related to genome stability and a device for detecting genome stability, which are used to implement the above embodiments and preferred embodiments, and are not described again after having been described. As used below, the term "module" may be a combination of software and/or hardware that implements a predetermined function. Although the means described in the embodiments below are preferably implemented in software, an implementation in hardware, or a combination of software and hardware is also possible and contemplated.
This is further illustrated below in connection with alternative embodiments.
Example 6
In this embodiment, there is provided an apparatus for screening tumor tissue marker microsatellite loci, as shown in fig. 3, the apparatus comprising: the system comprises a determination module 10, a verification module 20, a selection classification module 30, a site screening module 40 and an intersection acquisition module 50, wherein the determination module 10 is used for determining the states of the microsatellite sites of tumor tissues and blood cells of a plurality of samples and the states of the samples by adopting a first method; a verification module 20 for verifying the sample states of the plurality of samples using a second method different from the first method; a selecting and classifying module 30, configured to select multiple samples with consistent sample states determined by the first method and the second method, and divide the multiple samples with consistent sample states into a first type sample of an MSS and a second type sample of an MSI-H according to the sample states of the samples; the site screening module 40 is used for respectively screening a plurality of microsatellite sites with the highest correlation with the first type of sample to form a first site set and a plurality of microsatellite sites with the highest correlation with the second type of sample to form a second site set; and an intersection acquiring module 50, configured to acquire an intersection of the first set of sites and the second set of sites, so as to obtain a microsatellite locus related to genome stability.
In a preferred embodiment, the assay module comprises: the first acquisition unit is used for acquiring sequencing data of tumor tissues and blood cells of a plurality of samples and all microsatellite loci in an area covered by the sequencing data; the statistical unit is used for counting the difference value delta d of the length d of the repeated sequences of the sequencing data of the tumor tissues and the blood cells of each sample at each microsatellite locus; and the judging unit is used for judging the state of the microsatellite loci according to the relation between the difference value delta d and the first threshold value, and judging the sample state of each sample according to the relation between the mean value or the median of the difference values delta d of all the microsatellite loci of each sample and the second threshold value.
In a preferred embodiment, the judging unit includes: the first judgment submodule is used for judging the state of the microsatellite locus to be unstable when the difference value delta d is larger than or equal to a first threshold value; and the second judgment submodule is used for judging the state of the microsatellite locus to be stable when the difference value delta d is less than the first threshold value.
In a preferred embodiment, the determining unit further includes: the third judgment submodule is used for judging the state of the sample to be MSI-H when the mean value or the median of the difference values delta d of the sample at all the microsatellite loci is larger than or equal to a second threshold value; and the fourth judgment submodule is used for judging the state of the sample to be MSS when the mean value or the median of the difference values delta d of the sample at all the microsatellite loci is less than the second threshold value.
In a preferred embodiment, the site screening module comprises: the first screening submodule is used for taking a union set of the unstable-state microsatellite loci of each sample in the first class of samples, performing descending order arrangement on the unstable-state microsatellite loci according to the difference value delta d, and selecting the upper m-position microsatellite loci as a first locus set; the second screening submodule is used for taking a union set of the stable-state microsatellite loci of each sample in the second type of sample, performing ascending arrangement on the microsatellite loci according to the difference value delta d, and selecting the microsatellite loci arranged at the front n positions as a second locus set; wherein m and n are each independently a natural number of 2 or more.
In a preferred embodiment, the verification module is a PCR module, the PCR module comprising: the detection submodule is used for detecting the stability of each sample at five microsatellite loci of R-27, NR-24, NR-21, BAT-25 and BAT-26 through PCR; the sixth judgment submodule is used for judging the sample state of the sample to be MSI-H when the states of more than or equal to 2 microsatellite loci are unstable; and the seventh judging submodule is used for judging the sample state of the sample to be MSS when the state of the microsatellite locus is not detected to be unstable.
In a preferred embodiment, the sequencing data is sequencing data of a capture library.
In a preferred embodiment, the tumor tissue is colorectal cancer tissue.
Example 7
In this embodiment, a device for detecting genome stability is provided, where the device detects the genome stability of a sample to be detected by using a microsatellite locus related to genome stability, and the microsatellite locus related to genome stability is one or more microsatellite loci selected from the above-mentioned microsatellite loci or any one of the above-mentioned screening methods.
The device judges the stability state of each site according to the method for judging the stability state of the microsatellite sites (namely, the stability state is judged according to the relation between the difference value delta d and the first threshold), and then synthesizes the stability state of each site to evaluate the genome stability state of the sample to be detected (namely, the stability state is judged according to the relation between the mean value or the median of the difference values delta d of all the microsatellite sites and the second threshold).
From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the stability state of the sample is detected by adopting two different methods, the two samples with the same verification state are taken as objects, the intersection of the most relevant multiple microsatellite loci in the sample in the highly unstable state and the most relevant multiple microsatellite loci in the sample in the stable state is further obtained, so that the microsatellite loci with obvious differences in the two types of samples in the highly unstable state and the highly unstable state are obtained, and the microsatellite loci can be used as the microsatellite loci with the specificity or the sign of tumor tissues. The microsatellite loci screened by the screening method are highly related to the unstable state of the genome, so that the microsatellite loci screened by the screening method are high in accuracy, high in detection sensitivity and specificity and more beneficial to accurately judging the stable state of the genome microsatellite of the sample. For the genome stability detection device and the detection kit of the colon cancer sample, the accuracy of sample judgment is improved by a relatively small site number. In addition, the sample determination speed is very fast due to the small number of sites.
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 (22)
1. A method for screening for microsatellite loci associated with genomic stability, said method comprising:
determining the state of the microsatellite loci of the tumor tissues and the blood cells of the plurality of samples and the state of the samples by adopting a first method;
verifying a sample state of a plurality of the samples using a second method different from the first method;
selecting a plurality of samples with consistent sample states determined by the first method and the second method, and dividing the plurality of samples with consistent sample states into a first type sample of an MSS and a second type sample of an MSI-H according to the sample states of the samples;
respectively screening a plurality of microsatellite loci with highest correlation with the first type of samples to form a first locus set and a plurality of microsatellite loci with highest correlation with the second type of samples to form a second locus set;
and taking the intersection of the first site set and the second site set to obtain the microsatellite sites related to the genome stability.
2. The screening method according to claim 1, wherein the determining the state of the microsatellite loci of tumor tissues and blood cells in each of the plurality of samples and the state of the sample by the first method comprises:
obtaining sequencing data for tumor tissue and blood cells of each of a plurality of said samples and all microsatellite loci within a region encompassed by said sequencing data;
counting the difference value deltad of the length d of the repeated sequences of the tumor tissue and the blood cells of each sample at each microsatellite locus;
and judging the state of the microsatellite loci according to the relation between the difference value delta d and a first threshold, and judging the sample state of each sample according to the relation between the mean value or the median of the difference value delta d of all the microsatellite loci of each sample and a second threshold.
3. The screening method of claim 2, wherein determining the status of the microsatellite locus based on the relationship between the difference Δ d and the first threshold comprises:
when the difference value delta d is larger than or equal to a first threshold value, judging that the state of the microsatellite locus is unstable;
and when the difference value delta d is smaller than the first threshold value, judging that the state of the microsatellite locus is stable.
4. The screening method of claim 3, wherein determining the sample status of each sample according to the relationship between the mean or median of the differences Δ d between all the microsatellite loci of each sample and the second threshold comprises:
when the mean value or the median of the difference values delta d of the sample at all the microsatellite loci is larger than or equal to a second threshold value, judging that the state of the sample is MSI-H;
and when the mean value or the median of the differences delta d of the sample at all the microsatellite loci is less than a second threshold value, judging that the state of the sample is MSS.
5. The screening method of claim 2, wherein screening the plurality of microsatellite loci most closely related to the first type of sample to form a first set of loci and the plurality of microsatellite loci most closely related to the second type of sample to form a second set of loci respectively comprises:
merging the microsatellite loci in the unstable state of each sample in the first class of samples, performing descending order arrangement on the microsatellite loci in the unstable state according to the difference value delta d, and selecting the microsatellite loci in the first m positions as the first locus set;
merging the stable-state microsatellite loci of each sample in the second type of sample, arranging the microsatellite loci in an ascending order according to the difference value delta d, and selecting the microsatellite loci arranged at the top n positions as the second locus set;
wherein m and n are each independently a natural number of 2 or more.
6. The screening method according to any one of claims 1 to 5, wherein the second method is a method of PCR comprising:
detecting the stability of each sample at five microsatellite loci of R-27, NR-24, NR-21, BAT-25 and BAT-26 by PCR,
when the states of more than or equal to 2 microsatellite loci are unstable, judging that the sample state of the sample is MSI-H;
and when the state of the micro-satellite position is not detected to be unstable, determining the sample state of the sample to be MSS.
7. The screening method of any one of claims 2 to 5, wherein the sequencing data is sequencing data of a capture library; preferably, the length d of the repetitive sequence of the sequencing data of the tumor tissue and the blood cells at each microsatellite locus is calculated according to the Euclidean distance.
8. The screening method according to claim 1, wherein the tumor tissue is colorectal cancer tissue.
9. A microsatellite locus which is a marker of tumour tissue, wherein said microsatellite locus has been selected by the screening method according to any one of claims 1 to 8.
10. Microsatellite locus according to claim 9, wherein said microsatellite locus is a microsatellite locus for detecting the genomic stability status of a colorectal cancer sample, preferably said microsatellite locus comprises one or more of the loci shown in the following table:
。
11. A kit for detecting the stability status of a genome, the kit comprising a microsatellite locus, wherein said microsatellite locus is a microsatellite locus according to claim 9 or 10.
12. An apparatus for detecting the genome stability state, wherein the apparatus detects the genome stability state of a test sample using the stability state of a microsatellite locus, said microsatellite locus being according to claim 9 or 10.
13. A screening device for microsatellite loci associated with genomic stability, said screening device comprising:
a measuring module for measuring the state of the microsatellite loci of the tumor tissue and the blood cells of each of the plurality of samples and the state of the sample by using a first method;
a verification module for verifying a sample state of a plurality of the samples using a second method different from the first method;
a selecting and classifying module, configured to select a plurality of samples with the consistent sample states determined by the first method and the second method, and classify the plurality of samples with the consistent sample states into a first type sample of an MSS and a second type sample of an MSI-H according to the sample states of the samples;
the site screening module is used for respectively screening a plurality of microsatellite sites with the highest correlation with the first type of sample to form a first site set and a plurality of microsatellite sites with the highest correlation with the second type of sample to form a second site set;
and the intersection acquisition module is used for acquiring the intersection of the first site set and the second site set to obtain the microsatellite sites related to the genome stability.
14. The screening device of claim 13, wherein the assay module comprises:
a first obtaining unit, configured to obtain sequencing data of tumor tissue and blood cells of each of the plurality of samples and all microsatellite loci in an area covered by the sequencing data;
a counting unit, configured to count a difference Δ d between the sequencing data of the tumor tissue and the blood cells of each sample at the length d of the repeat sequence of each microsatellite locus;
and the judging unit is used for judging the state of the microsatellite loci according to the relation between the difference value delta d and a first threshold value, and judging the sample state of each sample according to the relation between the mean value or the median of the difference values delta d of all the microsatellite loci of each sample and a second threshold value.
15. The screening apparatus according to claim 14, wherein the judging unit includes:
the first judgment submodule is used for judging that the state of the microsatellite locus is unstable when the difference value delta d is larger than or equal to a first threshold value;
and the second judgment submodule is used for judging that the state of the microsatellite locus is stable when the difference value delta d is less than the first threshold value.
16. The screening apparatus according to claim 15, wherein the judging unit further includes:
the third judgment submodule is used for judging the state of the sample to be MSI-H when the mean value or the median of the difference values delta d of the sample at all the microsatellite loci is larger than or equal to a second threshold value;
and the fourth judgment submodule is used for judging that the state of the sample is MSS when the mean value or the median of the difference values delta d of the sample at all the microsatellite loci is less than a second threshold value.
17. The screening apparatus of claim 14, wherein the site screening module comprises:
the first screening submodule is used for merging the microsatellite loci in the unstable state of each sample in the first class of samples, performing descending order arrangement on the microsatellite loci in the unstable state according to the difference value delta d, and selecting the microsatellite loci in the previous m positions as the first locus set;
the second screening submodule is used for taking a union set of the stable-state microsatellite loci of each sample in the second type of sample, arranging the microsatellite loci in an ascending order according to the difference value delta d, and selecting the microsatellite loci arranged at the front n positions as the second locus set;
wherein m and n are each independently a natural number of 2 or more.
18. The screening apparatus of any one of claims 13 to 17, wherein the validation module is a PCR module comprising:
a detection submodule for detecting the stability of each sample at five microsatellite loci of R-27, NR-24, NR-21, BAT-25 and BAT-26 by PCR,
a sixth judging submodule, configured to judge that the sample state of the sample is MSI-H when the states of not less than 2 microsatellite loci are unstable;
and the seventh judging submodule is used for judging that the sample state of the sample is MSS when the state of the microsatellite locus is not detected to be unstable.
19. The screening apparatus of any one of claims 14 to 17, wherein the sequencing data is sequencing data of a capture library;
preferably, the length d of the repetitive sequence of the sequencing data of the tumor tissue and the blood cells at each microsatellite locus is calculated according to the Euclidean distance.
20. The screening apparatus of claim 13, wherein the tumor tissue is colorectal cancer tissue.
21. A storage medium, in which a computer program is stored, wherein the computer program is arranged to perform the method of any of claims 1 to 8 when executed.
22. An electronic device comprising a memory and a processor, wherein the memory has stored therein a computer program, and wherein the processor is arranged to execute the computer program to perform the method of any of claims 1 to 8.
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