CN113416769B - Method, composition and use for detecting microsatellite instability of non-control sample based on next generation sequencing technology - Google Patents

Abstract

The invention discloses a method, a composition and application for detecting microsatellite instability of a sample without a control sample based on a next-generation sequencing technology. The method of the invention comprises the steps of utilizing the amplification primer composition to perform library construction and sequencing on a tissue sample; extracting sample data from the sequencing data, and further extracting a required sequencing sequence; calculating the length of the sequencing sequence, counting the number of sequencing sequences distributed by each length, and selecting the length with the most distributed sequencing sequences as a calculation value of the biomarker; calculating the average value and the standard deviation of the sequence lengths corresponding to the amplification primers in each biomarker in a human blood database according to the amplification primer composition; and calculating the Z value, judging the stability of the biomarker based on the absolute value of the Z value, and further judging the stability of the tissue sample based on the stability of the biomarker. The method of the invention does not need a reference sample, thereby reducing the cost and the complexity of analysis.

Description

Method, composition and use for detecting microsatellite instability of non-control sample based on next generation sequencing technology

The application is a divisional application of Chinese patent application 201811050997.6, the application date of the original application is 09 and 10 months in 2018, and the invention is named as a method, a composition and an application for detecting the instability of a microsatellite without a reference sample based on a second-generation sequencing technology.

Technical Field

The invention belongs to the field of gene detection, and particularly relates to a method for detecting microsatellite instability of a non-control sample based on a next-generation sequencing technology.

Background

In prokaryotic and eukaryotic genomes, a number of short, tandemly repeated DNA sequences (1-6 bases), i.e.microsatellite sequences (MS), are widely distributed. In the process of DNA replication, these sequences are frequently subjected to a small range of base deletion, insertion or substitution, and exhibit instability. Namely MicroSatellite Instability (MSI). The MSI phenomenon was first discovered in 1993 by Jacobs et al in colorectal cancer, is associated with carcinogenesis and can be used for cancer detection (William R. Jacobs, et al, science,1993, 260. 5930 genomes of 18 cancers were analyzed by Ronald J Hause et al in 2016 using genome-wide exon sequencing technology, and MSI was found to occur in cancer cells of 14 cancer patients, with a maximum incidence of endometrial MSI as 30%, and 19% of gastric and colon cancers (Classification and characterization of microsatellite instability 18cancer types. Hause RJ, pritcard CC, shendive J, salipant SJ. Nat Med.2016Nov;22 (11): 1342-1350. Doi. According to the frequency with which MSI is detected in colorectal cancer, it can be classified into three categories, microcutellite stability (MSS), microcutellite stability-low (MSI-L) and Microcutellite stability-high (MSI-H) (free actuation of PTEN by promoter methylation in Microsatellite activity-high specific biological markers. Goel A, arnold CN, niedzwieecki D, hydrocarbons JM, dowlell JM, wasserman L, compton C, mayer RJ, bertagolli MM, boland CR. Cancer Res.2004May 1 (9): 3014-21). The NCCN colorectal cancer guide mentions that MSI detection should be performed in patients with a history of colorectal cancer, and can be used as a good marker for colorectal cancer prognosis; and the MSI-H colorectal cancer stage II patients have better prognosis (https:// www.nccn.org/professional/physioian _ gls/pdf/color. Pdf). The FDA approved the immunochemotherapeutic drug nivolumab for patients with metastatic colorectal cancer in 7 months of 2017 (https:// www.cancer.gov/news-events/cancer-currentblog/2017/nivolumab-FDA-colotectal), while approved the combination of ipilimumab and nivolumab in 7 months of 2018 for the treatment of MSI-H/mismatch repier patients with metastatic and rectal cancer (dMMR) above 12 years of age (https:// www.fda.gov/drugs/informationdrugs/aproproved stems/ucm613227. Htm). It can be seen that MSI is detected in colorectal cancer and its importance in clinical applications.

There are many methods for detecting MSI based on NGS, and the main methods can be classified as by comparing the length size distribution difference of tissue and control microsatellite sequences (application of next generation sequencing technology in micro satellite association testing. Genes (base). 2015Feb 12 (1): 46-59. Doi.

Disclosure of Invention

In order to solve at least part of the technical problems in the prior art, the invention provides a method for detecting microsatellite instability (MSI) without a control sample based on a next-generation sequencing technology. The method of the invention makes up the vacancy of the prior art, thereby reducing the cost of experiment, sequencing and analysis. Specifically, the present invention includes the following.

In a first aspect of the present invention, there is provided a method for detecting microsatellite instability without a control sample based on a second generation sequencing technology, which comprises the following steps:

(1) Establishing a library of the tissue sample by using the amplification primer composition to obtain a sample library, and sequencing the sample library to obtain sequencing data, wherein a control sample is not included in the step;

(2) Extracting sample data from the sequencing data by using the specific tag sequence of the sample, and extracting a sequencing sequence corresponding to the biomarker of each MSI from the sample data according to the amplification primer composition;

(3) Calculating the length of the sequencing sequence of each biomarker, counting the number of sequencing sequences distributed in each length, and selecting the length with the most distributed sequencing sequences in each biomarker as the calculated value of the biomarker;

(4) Calculating the average value and the standard deviation of the sequence length with the maximum sequencing sequence corresponding to the amplification primer in each biomarker in a human blood database according to the amplification primer composition;

(5) Calculating a Z value using formula (I), considering the biomarker to be unstable if the | Z value | > =3, considering the biomarker to be stable if the | Z value | <3, considering the tissue sample to be high frequency unstable, i.e., MSI-H type, if there are more than 2 biomarkers to be unstable, and considering the tissue sample to be stable, i.e., MSS type, if there are less than 1 biomarkers to be unstable, wherein formula (I): z value = (calculated-mean)/standard deviation.

According to the method for detecting microsatellite instability in a non-control sample based on the secondary sequencing technology of the present invention, preferably each primer in the amplification primer composition specifically binds to at least a partial sequence of a biomarker of MSI, wherein the biomarker is selected from at least one gene selected from the group consisting of KIT, MSH2, BIRC3, SLC7A8, ZNF2, MAP4K3, REEP5, DEFB105A, DEFB B, ACVR2A, RNF, DOCK3, GTF2IP1, LOC100093631, ARHGEF12, NOMO1, PIP5K1A, KIF (dist. =4,175bp) and DDX59 (dist. =19,111bp).

According to the method for detecting microsatellite instability in a sample without a control based on the second generation sequencing technology, the amplification primer composition preferably comprises a first amplification primer pair, a second amplification primer pair, a third amplification primer pair, a fourth amplification primer pair and a fifth amplification primer pair, wherein each amplification primer pair is specifically bound to at least partial sequences of 5 different biomarkers of MSI.

According to the method for detecting microsatellite instability of a sample without a control based on the next generation sequencing technology, the amplification primer composition preferably comprises a sequence shown in SEQ ID NO. 1-10.

According to the method for detecting microsatellite instability in a control-free sample based on the second-generation sequencing technology, preferably, the step (1) is performed by a process comprising a first amplification cycle and a second amplification cycle, wherein the first amplification cycle comprises 20 cycles of amplification procedures using 50 ng/. Mu.l or less of DNA from the tissue sample as a template, 98 ℃ 30s,98 ℃ 10s,58 ℃ 15s,72 ℃ 20s,72 ℃ 2min, and 4 ∞ in total, and the second amplification cycle comprises 20 cycles of amplification procedures using 98 ℃ 30s,98 ℃ 10s,58 ℃ 15s,72 ℃ 20s,72 ℃ 2min, and 4 ∞, as a template, and the first amplification cycle comprises 30s,98 ℃ 10s,58 ℃ 15s,72 ℃ 20s, and 72 ℃ 2min, and 4 ∞.

According to the method for detecting microsatellite instability of a sample without a control sample based on the second generation sequencing technology, the sequencing in the step (1) is preferably second generation sequencing.

The method for detecting microsatellite instability in a sample without control based on the next generation sequencing technology is preferably used, wherein the tissue sample is potential cancer tissue and does not comprise blood or components thereof.

According to the method for detecting microsatellite instability of a control-free sample based on the next generation sequencing technology, preferably, a step of adding a specific tag sequence to the obtained sample library after the library is built in the step (1).

In a second aspect of the present invention, there is provided a composition for detecting microsatellite instability in a control-free sample based on a second generation sequencing technique, which comprises an amplification primer composition for pooling a tissue sample to obtain a sample library, a tag sequence specific to the sample, and reagents for primer extension and amplification reactions.

In a third aspect of the invention, there is provided the use of a composition according to the second aspect of the invention in the preparation of a detection agent for detecting microsatellite instability in a sample without a control based on a secondary sequencing technique.

The method of the invention not only does not need a blood control sample which is necessary for the conventional detection of microsatellite instability (MSI), but also saves the steps of experiment, sequencing and analysis of the control sample, thereby reducing the cost and the complexity of analysis.

Drawings

FIG. 1 shows the result of detection of sample1 tissue by a 3730 read micro kit.

FIG. 2 shows the result of the test of sample1 blood with a 3730 read micro kit. As can be seen from a comparison of FIGS. 1 and 2, BAT25 was unstable, BAT26 was unstable, MONO27 was stable, NR21 was unstable, and NR24 was stable.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.

"microsatellite instability" as defined herein, sometimes also referred to as "MSI", refers to a mutation in a gene characterized by an alteration in the length of the DNA microsatellite repeat sequence, and is a type of genomic instability that can lead to a variety of tumors, such as colorectal cancer.

The term "no control sample" as used herein means that no control, reference or standard is required in the process of detecting microsatellite instability, including controls, references or standards from the same individual, and also including controls, references or standards from different individuals. This is in marked contrast to the prior art methods. In the prior art, DNA extracted from leukocytes in blood is usually used as a control. The method of the invention avoids obtaining normal tissues as contrast by operation or biopsy material taking, and reduces the pain of patients.

[ method for detecting microsatellite instability without control sample based on second-generation sequencing technology ]

In a first aspect of the invention, there is provided a method, sometimes also referred to as "the method of the invention", for detecting microsatellite instability in a sample without a control based on a second generation sequencing technique, comprising at least the following steps (1) to (5).

Step (1)

The step (1) of the invention comprises a library building step and a sequencing step. Specifically, the step (1) comprises the steps of utilizing the amplification primer composition to build a library of the tissue sample to obtain a sample library, and sequencing the sample library to obtain sequencing data. It should be noted that no control sample is required in this step.

The library construction of the present invention includes a first round amplification and a second round amplification process. The invention finds that the sample library suitable for the back-end sequencing is very favorable to be obtained through two rounds of amplification respectively, and the detection accuracy is improved. Preferably, the first round of amplification according to the present invention comprises amplification using DNA from a tissue sample as a template, using a program of 20 cycles of 98 ℃ 30s,98 ℃ 10s,58 ℃ 15s,72 ℃ 20s,72 ℃ 2min, and 4 ℃ infinity. The concentration of the template in the first round of amplification is 50 ng/. Mu.l or less, preferably 40 ng/. Mu.l or less, and more preferably 20 ng/. Mu.l or less. If the concentration is too high, the two rounds of amplification are not favored. The second round of amplification of the present invention includes a procedure of amplification using the first round of amplification product as a template, and using a program of 98 ℃ 30s,98 ℃ 10s,58 ℃ 15s,72 ℃ 20s,72 ℃ 2min, and 4 ℃ infinity for 20 cycles. Wherein the amount of template (i.e., first round amplification product) for the second round amplification can be the entire first round amplification product, or portions thereof can be used.

After the step of creating a library according to the present invention is completed, it is preferable to include a step of adding a specific tag sequence to the obtained sample library. Any sequence known in the art may be used for the specific tag sequence. Preferably, a base sequence of 6 or 8bp of barcode (index) used for sequencing is used. The addition of specific tag sequences is used to distinguish between different sample libraries.

The sequencing of the invention is carried out by a second generation sequencing technique. Paired-end sequencing or single-end sequencing can be performed using next-generation sequencing techniques. Second generation sequencing can be performed using instruments or platforms known in the art, such as Illumina MiniSeq, nextSeq, and the like. The length of the sequencing data obtained by the second-generation sequencing is generally 50 to 500bp, preferably 80 to 450bp, more preferably 100 to 400bp, and further preferably 150 to 300bp.

The tissue sample of step (1) of the present invention is any tissue from an individual. It can be from any part of the body. Preferably a potentially cancerous tissue, and also preferably a tissue sample of the invention does not comprise blood or components derived from blood, such as plasma or cells in its blood.

The amplification primer composition of step (1) of the present invention comprises a combination of a plurality of different primers for obtaining a sample library, which is different from a sequencing primer at the time of sequencing. The amplification primer composition of the present invention comprises a plurality of pairs of primer pairs for amplifying a specific biomarker or a partial sequence thereof. That is, each primer of the amplification primer composition of the present invention specifically binds to at least a partial sequence of the biomarker of MSI. Preferably, the biomarker is from at least one gene selected from the group consisting of KIT, MSH2, BIRC3, SLC7A8, ZNF2, MAP4K3, REEP5, DEFB105A, DEFB B, ACVR2A, RNF, DOCK3, GTF2IP1, LOC100093631, ARHGEF12, NOMO1, PIP5K1A, KIF14 (dist. =4,175bp), and DDX59 (dist. =19,111bp). More preferably, the biomarker is selected from the group consisting of at least one BAT25 mutation of KIT gene, BAT26 mutation of MSH2 gene, NR27 mutation of BIRC3 gene, NR21 mutation of SLC7A8 gene, NR24 mutation of ZNF2 gene, mon-27 mutation of MAP4K3 gene, D5S346 mutation of REEP5 gene, (a) 9 mutation of DEFB105A or DEFB105B gene, (a) 8 mutation of ACVR2A gene, (C) 7 mutation of RNF43 gene, (C) 7 mutation of DOCK3 gene, T13 mutation of GTF2IP1 or LOC100093631 gene, (T) 8 (C) 5 mutation of ARHGEF12 gene, (a) 9 mutation of NOMO1 gene, (T) 9 (C) 6 mutation of PIP5K1A gene, KIF14 (dist =4,175bp) mutation and DDX 19 bp (19,1118) mutation of gene.

In certain embodiments, the amplification primer compositions of the invention comprise a first amplification primer pair, a second amplification primer pair, a third amplification primer pair, a fourth amplification primer pair, and a fifth amplification primer pair, wherein each amplification primer pair specifically binds to at least a partial sequence of 5 different biomarkers BAT25, BAT26, MONO27, NR21, and NR24, respectively. More preferably, the amplification primer composition of the present invention comprises primers having the sequences shown in SEQ ID NOS: 1 to 10.

BAT25 primer:

primer 1: TCTGCATTTTAACTATGGCTC (SEQ ID NO: 1)

Primer 2: CTCGCCTCCAAGAATGTAAGT (SEQ ID NO: 2)

BAT26 primer:

primer 1: CTGCGGTAATCAAGTTTTTAG (SEQ ID NO: 3)

Primer 2: AACCATTCAACATTTTTAACCC (SEQ ID NO: 4)

MONO27 primer:

primer 1: GAAATGGTGGGAACCCAG (SEQ ID NO: 5)

Primer 2: GGTGGATCAAATTTCACTTGG (SEQ ID NO: 6)

NR21 primer:

primer 1: GAGTCGCTGGCACAGTTCTA (SEQ ID NO: 7)

Primer 2: CTGGTCACTCGCGTTTACAA (SEQ ID NO: 8)

The NR24 primer:

primer 1: ATTGTGCCATTGCATTCCAA (SEQ ID NO: 9)

Primer 2: GTGTCTTGCTGAATTTTACCTCCTGAC (SEQ ID NO: 10)

The sequences of the different primers are shown in SEQ ID NO 1-10 above, while the present invention provides the sequences of SEQ ID NO 1-10 above in a computer readable form. In the event that the sequence set forth herein differs from the sequence set forth in computer readable form, the content of the sequence set forth herein controls.

Step (2)

Step (2) of the present invention is a step of extracting a desired sequencing sequence from sequencing data. Specifically, the method comprises the steps of extracting sample data from sequencing data by using a specific label sequence of a sample, and extracting a sequencing sequence corresponding to a biomarker of each MSI from the sample data according to an amplification primer composition.

As described above, the specific tag sequence is a sequence for labeling sample data. General sequencing data can be distinguished from sample data by specific tag sequences. After sample data is extracted by using the specific label sequence, a sequencing sequence corresponding to each biomarker is further extracted according to each amplification primer in the amplification primer composition. That is, the sequencing sequence amplified by the amplification primer pair as at least a portion of the sequence of each biomarker is generally between 100-300bp, preferably between 150-250 bp.

For example, in the case where the amplification primers include a first amplification primer pair, a second amplification primer pair, a third amplification primer pair, a fourth amplification primer pair, and a fifth amplification primer pair, and the first amplification primer pair specifically binds to at least a partial sequence of BAT25, a sequencing sequence amplified by the first amplification primer pair can be extracted by the first amplification primer pair. Similarly, the sequencing sequence amplified by the second amplification primer can be extracted by the second amplification primer pair.

Step (3)

Step (3) of the present invention is a step of calculating a calculated value of each biomarker. Specifically, the method comprises the steps of calculating the length of a sequencing sequence corresponding to each biomarker, counting the number of sequencing sequences distributed in each length, and selecting the length with the most distributed sequencing sequences in each biomarker as the calculated value of the biomarker.

Step (4)

The step (4) of the present invention comprises calculating the average value and standard deviation of the sequence lengths corresponding to the amplification primers in each biomarker in a human blood database based on the amplification primer composition. Wherein the human blood database is a library composed of known data. It may use a combination of currently known data or it may be a new database by collecting and combining currently published data. In addition, each company or unit can collect and form a human blood database according to needs.

Step (5)

Step (5) of the present invention is a step of calculating a Z value and evaluating whether the biomarker is stable based on the absolute value of the Z value. Specifically, the method comprises the following steps of using a formula (I): z value = (calculated value-average)/standard deviation calculated Z value. The biomarker is considered to be unstable if the | Z value | > =3, and stable if the | Z value | < 3.

Step (5) of the present invention further comprises the step of assessing whether the tissue sample is stable based on the instability of the biomarker. In particular, the stability of at least 5 biomarkers from the same tissue sample was evaluated according to the above procedure. If more than 2 biomarkers are unstable, the tissue sample is considered to be high frequency unstable, i.e., MSI-H. If less than 1 biomarker is unstable, the tissue sample is considered to be stable, i.e., MSS-type.

[ composition for detecting microsatellite instability in a sample without control based on a second generation sequencing technique ]

In a second aspect of the invention, compositions for detecting microsatellite instability in a sample without a control based on next generation sequencing techniques are provided, which are sometimes abbreviated herein as "compositions of the invention". The composition at least comprises an amplification primer composition for performing library building on a tissue sample to obtain a sample library, a specific tag sequence of the sample and reagents for primer extension and amplification reaction.

The specific tag sequences of the amplification primer composition and the sample of the present invention have been described in the first aspect of the present invention, and are not described in detail herein for the repeated parts. The following is a supplementary explanation made on the basis of the disclosure of the first aspect.

The form in which the amplification primer combination of the present invention is present is not particularly limited, and it may be present in the form of a dry powder or a solution. The primer composition of the present invention may be in the form of a mixture of all primers, or may be in the form of a mixture of each primer alone, or may be in the form of a mixture of two or more kinds of partial primers, or may be in the form of a plurality of different mixtures.

The reagents for primer extension and amplification reactions of the present invention may employ reagents or components known in the art. For example, in some embodiments, reagents for primer extension and amplification reactions may include one or more of the following components: DNA polymerase (such as thermostable DNA polymerase, etc.), polymerase chain reaction buffer, reverse transcription buffer, and deoxynucleoside triphosphates (dntps). Optionally, reagents for performing the hybridization assay are included. Nucleotide analogues and/or labelling moieties may also be included, such as directly detectable moieties, e.g. fluorophores (fluorescent dyes) or radioisotopes, or indirectly detectable moieties, e.g. members of binding pairs such as biotin, or enzymes capable of catalysing non-soluble colorimetric or luminescent reactions (luminescence reactions). In addition, the composition of the present invention may further comprise at least one container for a reagent for electrophoretic detection of nucleic acids. Such reagents include those that directly detect nucleic acids, such as fluorescent chimeric or silver stained reagents, or those used to detect labeled nucleic acids.

[ use ]

In a third aspect of the invention, there is provided the use of a composition according to the second aspect of the invention, sometimes referred to as "use of the invention", in the preparation of a detector for detecting microsatellite instability in a sample without control based on a second generation sequencing technique.

The detection agent of the present invention may be provided in the form of a kit. Where provided in kit form, the kits of the invention may further comprise precautions relating to the regulated manufacture, use or sale of the diagnostic kit in a form prescribed by a governmental agency. The kit may also be provided with detailed instructions for use, storage, and troubleshooting. The kit may optionally also be provided in a suitable robotic operated device, preferably for high throughput settings.

The components of the kit of the invention may be provided as dry powders. When the reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is contemplated that the solvent may also be disposed in another container. The container will typically comprise at least one vial, test tube, flask, bottle, syringe, and/or other container means, optionally in which the solvent is placed in equal portions. The kit may further comprise second container means for containing a sterile, pharmaceutically acceptable buffer and/or other solvent.

Where more than one component is present in a kit, the kit will also typically comprise a second, third or other additional container into which additional components may be separately placed. In addition, combinations of various components may be included in the container.

Kits of the invention may also include components that retain or maintain DNA, such as agents that are resistant to nucleic acid degradation. Such components may be, for example, nucleases either without RNase or with protection against RNase. Any of the compositions or reagents described herein can be a component of a kit.

Example 1

A single tissue sample of 10 samples was taken, which was known to verify MSI results by a 3730 read microtest kit (patent No.: ZL 2011 1 0152226. X). In total 3 MSI-H stable high frequency forms and 7 MSS stable forms.

MSI is detected by the analytical method of the present invention. The method comprises the following specific steps:

this example is illustrated by sample1 (3730 read micro kit verification results are shown in FIGS. 1 and 2).

1. Selecting 5 biomarkers for detecting microsatellite instability (MSI), namely BAT25, BAT26, MONO27, NR21 and NR24, and designing amplification primers for each marker as follows:

BAT25 primer:

primer 1: TCTGCATTTTAACTATGGCTC

Primer 2: CTCGCCTCCAAGAATGTAAGT

BAT26 primer:

primer 1: CTGCGGTAATCAAGTTTTTAG

Primer 2: AACCATTCAACATTTTTAACCC

MONO27 primer:

primer 1: GAAATGGTGGGAACCCAG

Primer 2: GGTGGATCAAATTTCACTTGG

NR21 primer:

primer 1: GAGTCGCTGGCACAGTTCTA

Primer 2: CTGGTCACTCGCGTTTACAA

The NR24 primer:

primer 1: ATTGTGCCATTGCATTCCAA

Primer 2: GTGTCTTGCTGAATTTTACCTCCTGAC

2. Designing a primer according to the step 1, and performing conventional amplicon library building on the single tissue sample 1; the extracted gDNA is diluted to the corresponding nano drop concentration according to the requirements of a kit, the sample DNA of sample1 is 20ng/ul, and a 20ul system is prepared according to the following conditions: phoenix buffer 5ul, taq0.1ul, dNTP 2ul and sample1 sample DNA 40ng, different amounts of forward and reverse primers are added according to different sites, the final concentration of MONO27 and BAT26 is 900nM, the final concentration of NR24 is 600nM, and the rest sites are 250nM; performing PCR amplification by using a life amplification instrument, wherein the amplification procedure comprises the following steps: 20 cycles of 30s at 98 ℃, 10s at 98 ℃, 15s at 58 ℃, 20s at 72 ℃, 2min at 72 ℃, and infinity at 4 ℃; after the product purified by 1.6 times of magnetic beads is eluted by 20ul of water, 19ul of the product is taken as a template for the next round, and a second round of 30ul of amplification system is prepared as follows: phoenix buffer 6ul, taq 0.15ul, dNTP 3ul, template 19ul, forward and reverse primers 1ul respectively; performing PCR amplification by using a life amplification instrument, wherein the amplification procedure comprises the following steps: 20 cycles of 30s at 98 ℃, 10s at 98 ℃, 15s at 58 ℃, 20s at 72 ℃, 2min at 72 ℃, and infinity at 4 ℃; after purifying the product by 1.6 times of magnetic beads, eluting with 20ul of water, and carrying out Qubit and 2100 quality inspection.

3. Posing each biomarker library, adding 1 specific tag sequence to the posing library of 5 biomarkers in the sample of sample1, and performing double-end reading length 300bp sequencing by using an Illumina Miseq sequencer.

4. And (3) splitting the data of the sample1 sample from the total data obtained by sequencing according to the specific tag sequence.

5. According to the primer sequences of the 5 biomarkers in the step 1, extracting the sequencing data of the 5 biomarkers from the sequencing data of sample 1.

6. The length of the sequencing sequence of 5 biomarkers is calculated respectively, and the number of the sequencing sequence distributed in each length is counted.

7. Selecting the length with the most distributed sequencing sequence number in the 5 biomarkers to represent the 'calculated value' of the biomarker, so as to obtain the 'calculated value' of BAT25 as 122; BAT26 "Calculations" 177; "calculated" for MONO27 is 169; "calculated value" of NR21 is 107; the "calculated value" of NR24 was 133.

8. Calculating the average value and standard deviation of the length with the maximum number of distributed sequencing sequences of the 5 biomarkers in a human blood sample database (self-accumulation) according to the forward and reverse amplification primers of the 5 biomarkers designed in the step 1, wherein the average value of BAT25 is 123.9038462, and the standard deviation is 0.533564241; BAT26 has an average value of 179.0666667 and a standard deviation of 0.393122697; the average value of MONO27 is 171.5357143 and the standard deviation is 2.088620995; the average value of NR21 is 110.9642857 and the standard deviation is 0.631427984; the average value of NR24 is 133.8653846 with a standard deviation of 0.595039829.

9. The Z-score values (Z values) were calculated using the "calculated values" of 5 biomarkers obtained in step 7 and the mean and standard deviation of blood for the corresponding biomarkers obtained in step 8, giving a Z-score value of-3.568166693 for BAT25, of-5.25705 for BAT26, of-1.21406 for MONO27, of-6.278286386 for NR21, and of-1.454330573 for NR24, respectively.

10. Biomarker stability judgment standard: the biomarker is considered to be unstable if | Z-score | > =3, and stable if | Z-score | < 3. Based on this criterion, BAT25, BAT26, NR21 were judged to be unstable, MONO27, NR24 were judged to be stable. The result is consistent with the verification result of the 3730 micro-reading kit.

11. Tissue stability judgment standard: if more than 2 biomarkers are unstable, the tissue sample is considered to be of the high frequency unstable (MSI-H) type, and if 0 or 1 biomarker is unstable, the tissue sample is considered to be of the stable (MSS) type. Based on this criterion, 4 markers of sample1 were unstable, and thus sample1 was judged to be unstable at high frequency (MSI). This is consistent with the 3730 read micro kit validation results.

Examples 2 to 10

Verification was performed in the same manner as in example 1, except that the tissue samples were changed to sample2 to 10, respectively. The results of the verification of examples 2-10 are shown in Table 1.

Table 1 table of verification results of the method of the present invention

Note: "+" indicates instability, "-" indicates stability, and the grey portion in the table indicates an unstable marker.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

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<210> 10

<211> 27

<212> DNA

<213> Artificial sequence ()

<400> 10

gtgtcttgct gaattttacc tcctgac 27

Claims (4)

1. A method for detecting microsatellite instability of a sample without a control based on a next generation sequencing technology is characterized by comprising the following steps:

(1) Utilizing an amplification primer composition with a sequence shown in SEQ ID NO. 1-10 to library gDNA (deoxyribonucleic acid) from a tissue sample to obtain a sample library, sequencing the sample library to obtain sequencing data, wherein a control sample is not included in the step, the tissue sample is a potential cancer tissue and does not include blood or components thereof, and adding a specific tag sequence into the obtained sample library after the library is built in the step (1);

(2) Extracting sample data from the sequencing data by using the specific tag sequence of the sample, and extracting a sequencing sequence corresponding to each MSI biomarker from the sample data according to the amplification primer composition, wherein the biomarkers are BAT25, BAT26, MONO27, NR21 and NR24, and each primer in the amplification primer composition specifically binds to at least a partial sequence of the MSI biomarker;

(3) Calculating the length of the sequencing sequence of each biomarker, counting the number of sequencing sequences distributed in each length, and selecting the length with the most distributed sequencing sequences in each biomarker as the calculated value of the biomarker;

(4) Calculating the average value and standard deviation of the sequence length of the maximum sequencing sequence corresponding to the amplification primers in each biomarker in a human blood database according to the amplification primer composition of the sequences shown in SEQ ID NO. 1-10, wherein the average value of BAT25 is 123.9038462, the standard deviation is 0.533564241, the average value of BAT26 is 179.0666667, the standard deviation is 0.393122697, the average value of MONO27 is 171.5357143, the standard deviation is 2.8620995, the average value of NR21 is 110.9642857, the standard deviation is 0.631427984, the average value of NR24 is 133.8653846, and the standard deviation is 0.595039829;

(5) Calculating a Z value using formula (I), wherein the biomarker is considered unstable if the | Z value | > =3, and stable if the | Z value | <3, wherein formula (I): z value = (calculated-mean)/standard deviation;

considering the tissue sample as being high frequency unstable, i.e., MSI-H type, if more than 2 biomarkers are unstable, and considering the tissue sample as being stable, i.e., MSS type, if less than 1 biomarker is unstable;

wherein the sequencing in step (1) is second-generation sequencing.

2. The method for detecting microsatellite instability in a non-control sample based on secondary sequencing technology according to claim 1 wherein the amplification primer composition comprises a first amplification primer pair, a second amplification primer pair, a third amplification primer pair, a fourth amplification primer pair and a fifth amplification primer pair, wherein each of the amplification primer pairs specifically binds to at least a partial sequence of 5 different biomarkers of MSI.

3. A composition for detecting microsatellite instability of a non-control sample based on a next-generation sequencing technology is characterized by comprising an amplification primer composition for performing library building on a tissue sample to obtain a sample library, a specific tag sequence of the sample and reagents for primer extension and amplification reaction, wherein the sequence of the amplification primer composition is shown as SEQ ID NO. 1-10.

4. Use of a composition according to claim 3 in the preparation of a detector for detecting microsatellite instability in a sample without a control based on a second generation sequencing technique.

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