CN113293204B - Primer composition, kit and method for detecting microsatellite instability based on second-generation sequencing platform - Google Patents

Abstract

The invention discloses a primer composition and a method for detecting microsatellite instability based on a second-generation sequencing platform. The primer composition comprises an upstream primer set and a downstream primer set, wherein each primer in the upstream primer set comprises a UID sequence at the 5' end and a binding region complementary to one primer in a PCR universal primer pair, and each primer in the downstream primer set comprises a binding region complementary to the other primer in the PCR universal primer pair; wherein each primer in the upstream primer set specifically binds upstream of a mutation site of at least one gene in the specific gene group, and each primer in the downstream primer set specifically binds downstream of the mutation site, respectively. The method of the invention simplifies the detection process to a great extent and reduces the detection cost, and has the characteristics of high flux, high sensitivity and high specificity.

Description

Primer composition, kit and method for detecting microsatellite instability based on second-generation sequencing platform

The application relates to a sub-filed application of Chinese patent application 201810957432.X, the application date of the original application is 2018, 08 and 21, and the application is a primer composition, a kit and a method for detecting microsatellite instability based on a second-generation sequencing platform.

Technical Field

The invention belongs to the field of gene detection, and particularly relates to a primer composition and a method for detecting microsatellite instability based on a second-generation sequencing platform.

Background

Colorectal cancer is a common malignant tumor of the digestive tract, and in the common malignant tumor death of China, colorectal cancer patients occupy the fifth place in men and the sixth place in women. The new occurrence rate of colorectal cancer in China exceeds 25 ten thousand per year, the death rate is about 14 ten thousand, and the new occurrence rate and the death rate account for 20% of colorectal cancer cases in the same period worldwide. Therefore, reducing the incidence and mortality of colon cancer in China is an unprecedented major clinical science problem.

Survival rates of 24% -58% 5 years after radical resection of colorectal cancer, on average only 40%, postoperative recurrence and metastasis are important causes of death. Colorectal cancer pathogenesis and genomic instability are closely related, mainly including chromosomal instability (chromosomal instability, CI) and microsatellite instability (microsatellite instability, MSI).

Microsatellite instability refers to the appearance of new microsatellite alleles due to the hypermutated state of the cell caused by DNA mismatch repair (MISMATCH REPAIR, MMR). It includes insertion and deletion mutations in short tandem DNA repeats (microsatellites), as well as nucleotide substitutions throughout the genome. MSI is a diagnostic marker for gastrointestinal, endometrial and colorectal tumors. About 15% of colorectal cancer patients have MSI phenomena, with more than 90% of typical hereditary non-polyposis colorectal cancer (HEREDITARY NONPOLYPOSIS COLERECTAL CANCER, HNPCC) patients being MSI-type, indicating MSI can be an important marker for judging HNPCC patients; compared with MSS (microsatellite stabilized) colon cancer, the prognosis of colorectal cancer patients carrying MSI is better, and the drug response of the colorectal cancer patients carrying MSI is different, so that MSI can be used as an independent predictor of colorectal cancer prognosis, and MSI detection is significant for colorectal cancer patients. Recent studies indicate that MSI may be a marker for immune checkpoint blocking therapy. Mismatch repair refers to the repair process by which a nucleotide sequence is restored to normal in a DNA molecule containing mismatched bases. The MMR gene family contains 9 genes, mainly used to correct mismatched base pairs on DNA duplex, and also to repair small fragment nucleotide insertions or deletions due to replication slippage. MMR gene mutation or promoter methylation can lead to MMR gene function deletion, thereby causing DNA molecules containing mismatched bases, nucleotide insertions or deletions to be unable to repair normally, and finally causing a wide MSI phenomenon.

In detecting MSI in cancer cells, MSI sequence changes can be detected directly, or the occurrence of MSI can be determined by detecting MMR gene deletion. The MSI status of patients is clinically detected mainly by Immunohistochemical (IHC) staining or Polymerase Chain Reaction (PCR) methods. MMR gene defect detection often relies on immunohistochemistry (protein level), whereas MSI detection generally relies on molecular means, PCR detection (DNA level). IHC is mainly used for detecting MMR protein (MLH 1, MSH2, MSH6 and PMS 2) expression. Immunohistochemical detection can directly identify MMR-deficient genes that lead to MSI occurrence, but about 5% -11% of MSI occurrence does not appear to be deficient in MMR protein. Some missense mutations in MMR proteins lose MMR function but can be recognized by antibody detection, and this is therefore an advantage of molecular detection. PCR is mainly to use specific primers to amplify microsatellite loci in cancer tissue samples one by one or multiplex fluorescence PCR, the amplified products are subjected to fragment size analysis by gel electrophoresis and compared with normal control samples, the sequence change condition of the amplified products is checked, and 5 loci (NR-27, NR-24, NR-21, BAT-25 and BAT-26) are usually detected.

MSI can be classified into high microsatellite instability (MSI-H), low satellite instability (MSI-L) or microsatellite stability (MSS) according to the degree. In general, MSI-H is the case when more than 2 sites are unstable during detection; if 1 locus is unstable during detection, the MSI-L is obtained; if no detection site is unstable, it is MSS.

At present, most of the diagnosis methods for hereditary tumor in the market stay at the detection level of single tumor or a plurality of sites, and the methods have different defects, such as complex operation, complex primer or probe design, difficult interpretation of results, low flux and the like; in the multiplex PCR detection, different primers have interference, and the selection and concentration of the primers have higher requirements; the immunohistochemical method has low specificity and repeatability, high sample quality requirement and complex operation. Therefore, the existing detection method for the instability of the microsatellite is difficult to meet the detection requirements of large sample quantity, multiple detection sites, wide distribution, high detection accuracy and the like. Therefore, a novel detection means is urgently needed, which is simple and rapid to operate in experiments, and has high sensitivity and repeatability so as to meet the demands of markets and medical treatment.

Disclosure of Invention

In order to solve at least part of the technical problems in the prior art, the invention provides a primer composition, a kit and a method for detecting microsatellite instability based on a second generation sequencing platform. The invention improves the sensitivity of diagnosis, can sequence multiple samples simultaneously, and greatly reduces the cost while screening a large sample amount. Specifically, the present invention includes the following.

In a first aspect of the present invention, there is provided a primer composition comprising an upstream primer set and a downstream primer set, each primer in the upstream primer set comprising a UID sequence at the 5' end and a binding region complementary to one primer in a PCR universal primer pair, each primer in the downstream primer set comprising a binding region complementary to the other primer in the PCR universal primer pair, respectively; wherein each primer in the upstream primer set specifically binds upstream of a mutation site of at least one gene in the following gene group, and each primer in the downstream primer set specifically binds downstream of the mutation site, respectively; the gene group included KIT、MSH2、BIRC3、SLC7A8、ZNF2、MAP4K3、REEP5、DEFB105A、DEFB105B、ACVR2A、RNF43、DOCK3、GTF2IP1、LOC100093631、ARHGEF12、NOMO1、PIP5K1A、KIF14(dist.＝4,175bp) and DDX59 (dist.=19, 111 bp).

In certain embodiments, the mutation site comprises: at least one of 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, monoo-27 mutation of MAP4K3 gene, D5S346 mutation of REEP5 gene, a (a 9 mutation of DEFB105A or DEFB105B gene, a (A8 mutation of ACVR2A gene, a (C7 mutation of RNF43 gene, a (C7 mutation of DOCK3 gene, a (T13 mutation of GTF2IP1 or LOC100093631 gene, a (T8 (C) 5 mutation of ARHGEF gene, a (a 9 mutation of NOMO gene, a (T) 9 (C) mutation of PIP5K1A gene, a (T8 mutation of KIF14 (dist=4,175 bp) gene, and a (T8 mutation of DDX 59=19,111 bp).

In certain embodiments, the upstream primer set is selected from the group consisting of the sequences set forth in SEQ ID nos: 1-18; the downstream primer group is selected from the group consisting of a primer sequence of SEQ ID No: 19-36.

In a second aspect of the invention, there is provided a kit for detecting microsatellite instability based on a second generation sequencing platform comprising a primer composition according to the first aspect of the invention.

In a third aspect of the invention, there is provided a method of detecting microsatellite instability based on a second generation sequencing platform comprising the steps of:

(1) Hybridizing a tissue sample with a primer composition, wherein the primer composition is according to any one of claims 1-3;

(2) Filling up gap gaps under the condition suitable for DNA polymerase or DNA ligase reaction to obtain target DNA;

(3) And amplifying by using the target DNA as a template and utilizing a PCR universal primer pair to obtain a sequencing library.

In certain embodiments, the method of detecting microsatellite instability based on a second generation sequencing platform further comprises (4) double-ended sequencing the sequencing file using second generation sequencing to obtain double-ended sequencing data for a sample.

In certain embodiments, the method for detecting microsatellite instability based on a second generation sequencing platform further comprises (5) recording the double-ended sequencing data as R1 and R2 respectively, extracting UIDs of R1 sequences, comparing the files after UIDs are extracted to a reference genome, filtering the comparison results, extracting the double-ended sequencing sequences of the reserved comparison results, and splicing.

In certain embodiments, the method for detecting microsatellite instability based on a second generation sequencing platform further comprises (6) for a tissue sample and a control sample, separately counting the length of the spliced sequences for each microsatellite locus, and if the tissue sample and the control sample are off-peak and the number of intermediate bases is greater than or equal to 2, then the locus is considered unstable. Preferably, the condition for retaining the alignment is that the alignment is unique to the reference genome and that the alignment and random bases are de-duplicated and the best quality sequence is retained.

In certain embodiments, the method of detecting microsatellite instability based on a second generation sequencing platform further comprises (7) interpreting the criteria as: the number of unstable sites is equal to 0, and the tissue sample is MSS type; the number of unstable sites is equal to 1, and the tissue sample is MSI-L type; the number of unstable positioning points is more than or equal to 2, and the tissue sample is MSI-H type.

The method of the invention is not only efficient, systematic and economical, but also has the greatest advantage of improving the sensitivity of diagnosis. The invention has the advantages of small target area, obviously reduced sequencing cost, high sequencing depth of 1000× (30×ofconventional whole genome sequencing), and low-frequency mutation information discovery. In the post genome era, the demand for resequencing of candidate segments of genome is increasing, the attention of people to sequences exceeds a few SNP, the range of candidate segments can be between 5Kb and 100Kb, the conventional PCR method or target region hybridization capture sequencing is expensive, and the method well solves the problem. The method can be used for sequencing multiple samples simultaneously, and greatly reduces the cost while screening a large amount of samples.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions 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. In addition, for numerical ranges in the present invention, it is understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, 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 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.

The term "microsatellite instability", sometimes also referred to as "MSI", as used herein, refers to a mutation in a gene characterized by a change in the length of a DNA microsatellite repeat sequence, a type of genomic instability that can lead to a variety of tumors, such as colorectal cancer.

[ Primer composition ]

In a first aspect of the invention, a primer composition is provided for detecting microsatellite instability based on a second generation sequencing platform.

The primer composition of the invention comprises an upstream primer set and a downstream primer set, wherein each primer in the upstream primer set respectively comprises a 5' -end UID sequence and a binding region complementary to one primer in the PCR universal primer pair, and each primer in the downstream primer set respectively comprises a binding region complementary to the other primer in the PCR universal primer pair. Wherein each primer in the upstream primer set specifically binds upstream of a mutation site of at least one gene in the gene group, and each primer in the downstream primer set specifically binds downstream of the mutation site, respectively. That is, one primer in the upstream primer set can constitute a primer pair with one primer in the downstream primer set, and when bound to a specific sequence of a corresponding gene, a specific mutation site in the gene can be amplified. Preferably, the distance between the upstream primer and its corresponding downstream primer is 100bp-1000kb, preferably 110bp-500kb, more preferably 120bp-300kb, still more preferably 150bp-250kb. In a preferred embodiment, the 5' UID sequence overlaps at least partially or completely with the binding region in the primers of the upstream primer set. In a preferred embodiment, the binding region is located at the 3' end of each primer in the downstream primer set.

The UID refers to a molecular tag, after the original sample is subjected to specific hybridization, a specific tag sequence is added to each fragment to distinguish different fragments in the same sample, and in the subsequent data analysis, errors caused by DNA polymerase or amplification and sequencing processes can be eliminated through the tag sequences. The molecular tags of the invention generally consist of random sequences (e.g., NNNNNNN) of 50nt or less, more preferably 25nt or less, still more preferably 20nt or less, still more preferably 15nt or less, or degenerate bases (NNNRNYN). On the other hand, the random sequence has a length of preferably 3nt or more, more preferably 5nt or more, and still more preferably 6nt or more. Tumor heterogeneity can limit detection of drive mutations, particularly low frequency drive mutations, and the design of the primers of the invention is advantageous for increasing the sequencing depth of the target region, particularly for low frequency mutation discovery and in tumor research and application. In addition, the primer composition provided by the invention can identify the error replication introduced in the experimental process, and reduce false positive.

The gene group in the present invention includes at least one of KIT, MSH2, BIRC3, SLC7A8, ZNF2, MAP4K3, REEP5, DEFB105A (also referred to as DEFB 105B), ACVR2A, RNF, DOCK3, GTF2IP1 (also referred to as LOC 100093631), ARHGEF12, NOMO1, PIP5K1A, KIF (dist.=4,175 bp) and DDX59 (dist.=19,111 bp). Preferably, the gene group of the present invention consists of all the genes described above, and such gene group is more comprehensive and highly accurate for the detection of microsatellite instability. Table 1 shows information on each gene in the gene group of the present invention.

TABLE 1.17 genes and microsatellite locus information thereof

The mutation sites in the above genes of the present invention may include: at least one of 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, monoo-27 mutation of MAP4K3 gene, D5S346 mutation of REEP5 gene, a (a 9 mutation of DEFB105A or DEFB105B gene, a (A8 mutation of ACVR2A gene, a (C7 mutation of RNF43 gene, a (C7 mutation of DOCK3 gene, a (T13 mutation of GTF2IP1 or LOC100093631 gene, a (T8 (C) 5 mutation of ARHGEF gene, a (a 9 mutation of NOMO gene, a (T) 9 (C) mutation of PIP5K1A gene, a (T8 mutation of KIF14 (dist=4,175 bp) gene, and a (T8 mutation of DDX 59=19,111 bp). Preferably, the primer composition of the present invention corresponds to all the above mutation sites. In certain embodiments, the upstream primer set of the primer composition of the invention is selected from the group consisting of the sequences set forth in SEQ ID nos: 1-18. The downstream primer set is selected from the group consisting of the sequences of SEQ ID No: 19-36.

TABLE 2-1 information on exemplary primer compositions

TABLE 2-2 information on exemplary primer compositions

Note that: in the upstream primer or the downstream primer, the sequence indicated by the lower case letter corresponds to the intron region, and the sequence indicated by the upper case letter corresponds to the exon region.

Each primer in the primer composition of the present invention is capable of specifically binding to a part of the sequence of a specific gene, but unlike the sequence or region between the amplification primer pairs of conventional PCR primers, each primer in the upstream primer of the primer composition of the present invention and its corresponding downstream primer are bound to the same DNA or single-stranded both sides of the gene. For example, for BAT25 mutation of KIT gene, the upstream primer in the upstream primer set binds to the upstream side of a single strand containing the BAT25 mutation site, while the downstream primer corresponding to the upstream primer binds to the downstream side of the same single strand (i.e., single strand containing the BAT25 mutation site).

The form of the primer composition of the present invention is not particularly limited, and may be 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 the primer compositions, may exist as each primer alone, or may exist as a mixture of two or more partial primer compositions in various mixtures.

[ Kit for detecting microsatellite instability based on second-generation sequencing platform ]

In a second aspect of the invention, there is provided a kit for detecting microsatellite instability (sometimes also referred to as a "kit of the invention") based on a second generation sequencing platform, comprising a primer composition according to the first aspect of the invention.

In addition to comprising the primer composition according to the first aspect of the invention, the kit of the invention may preferably comprise reagents which provide primer extension and amplification reactions. For example, in some embodiments, the kit may further comprise 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). Alternatively, the kit may comprise reagents for performing a hybridization assay. The detection reagent may comprise nucleotide analogs and/or labeling moieties, such as directly detectable moieties, e.g., fluorophores (fluorescent dyes) or radioisotopes, or indirectly detectable moieties, e.g., members of binding pairs, e.g., biotin, or enzymes capable of catalyzing non-soluble colorimetric or luminescent reactions (luminometric reaction). In addition, the kit may further comprise at least one container containing a reagent for electrophoresis detection of nucleic acids. Such reagents include those for direct detection of nucleic acids, such as fluorescent intercalators or silver staining reagents, or those for detection of labeled nucleic acids. Kits may also include precautions related to regulating manufacturing, use, or marketing of the diagnostic kit in a form prescribed by a government agency. The kit may also be provided with detailed instructions for use, storage and troubleshooting. The kit may also optionally be provided in a suitable device, preferably for robotic operation in a high throughput setting.

The components of the kit may be provided as a dry powder. When the reagents and/or components are provided as dry powders, the powders may be restored by the addition of a suitable solvent. It is contemplated that the solvent may also be disposed in another container. The container will typically include at least one vial, test tube, flask, bottle, syringe, and/or other container means, with the solvent optionally being placed in aliquots. The kit may further comprise a 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 in which additional components may be placed separately. However, various combinations of components may be included in the container.

Kits of the invention may also include components that retain or maintain DNA or RNA, such as agents that are resistant to degradation by nucleic acids. Such components may be, for example, either RNase-free or nuclease with protection against RNase. Any of the compositions or reagents described herein may be a component in a kit.

[ Method for detecting microsatellite instability based on second-generation sequencing platform ]

In a third aspect of the invention, there is provided a method for detecting microsatellite instability based on a second generation sequencing platform (sometimes also referred to as the "method of the invention") comprising at least the following steps (1) - (3), optionally further comprising steps (4) - (7).

Step (1) of the present invention is a hybridization step. Specifically, a tissue sample is hybridized with the primer composition according to the first aspect of the present invention. The hybridization process involves denaturing the DNA in the tissue sample, then gradient cooling and constant temperature hybridization. Wherein the DNA denaturation temperature is usually from 90℃to 100℃or lower, for example, 92℃94℃96℃98℃and the like. The denaturation time (i.e., denaturation temperature retention time) is generally 0.5 to 15 minutes, preferably 1 to 10 minutes, more preferably 2 to 5 minutes. Gradient cooling refers to the process of gradually decreasing the denaturation temperature to the hybridization temperature. The preferred gradient is 0.5-3deg.C/min, more preferably 1deg.C/min. Constant temperature hybridization refers to a reaction process that is carried out at a constant hybridization temperature for 5 to 24 hours, preferably 8 to 15 hours.

In a preferred embodiment, the distance between the upstream primer and the corresponding downstream primer in the primer composition is 100bp to 1000kb, preferably 110bp to 500kb, more preferably 120bp to 300kb, still more preferably 150bp to 250kb after binding to DNA in the tissue sample, respectively. That is, the size of the gap to be filled is 100bp to 1000kb, preferably 110bp to 500kb, more preferably 120bp to 300kb, still more preferably 150bp to 250kb. The tissue sample of the present invention is a test sample from a subject, comprising a DNA component. In certain embodiments, the hybridization process is performed in a hybridization mixture solution comprising a primer composition. In particular the composition of the hybridization mixture is known in the art. Reference may be made to the specifications of, for example, molecular cloning.

The step (2) of the invention is a gap filling step. Specifically, gaps between the upstream primer and its corresponding downstream primer are filled under conditions suitable for DNA polymerase or DNA ligase reaction, thereby forming a complete amplified strand complementary to the original single-stranded sequence and fully embodying the original sequence information, i.e., the target DNA. As described above, the size of the gap of the present invention is 100bp to 1000kb, preferably 110bp to 500kb, more preferably 120bp to 300kb, still more preferably 150bp to 250kb. The gap filling process in the step (2) is a one-way amplification process. The DNA polymerase and DNA ligase in step (2) may use enzymes known in the art or commercially available enzymes, and the conditions suitable for the DNA polymerase or DNA ligase reaction are conditions which can be readily determined by those skilled in the art as required. Which comprises subjecting the hybridized sample to single base replication in a buffer containing DNA polymerase and DNA ligase at 50-65℃for 30-120 minutes, preferably 40-100 minutes, and then preserving at low temperature (e.g.4℃). Followed by extension at 35-39℃for 20-60 minutes. Finally, the enzyme is deactivated at high temperature (e.g., 90-100 ℃). The buffer in step (2) may further include substances required for single-stranded extension such as dNTPs and NAD.

Step (3) of the present invention is a PCR amplification step. Specifically, the target DNA is used as a template to be amplified by using a PCR universal primer pair to obtain a sequencing library. PCR amplification conditions are known in the art and can be adjusted as desired. In certain embodiments, the amplification conditions are as follows: pre-denaturing for 30s at 98 ℃ and then entering a polymerase chain reaction amplification stage: denaturation at 98℃for 10s, annealing at 62℃for 30s, extension at 72℃for 20s, and 21 cycles; finally, the mixture is stretched for 20s at 72 ℃ and is kept stand at 4 ℃.

Step (4) of the present invention is a second generation sequencing step. Specifically, the double-end sequencing is carried out on the sequencing file by utilizing a second-generation sequencing technology, so that double-end sequencing data of a sample are obtained. Second generation sequencing can be performed using instruments or platforms known in the art. For example Illumina MiniSeq, nextSeq, etc.

It should be noted that, the negative control experiment is preferably performed simultaneously during steps (1) to (3) of the present invention. Wherein the control sample is the subject's blood leukocyte DNA.

Steps (5) - (7) of the present invention are data analysis and result judgment steps. In an exemplary embodiment, the data analysis and result determination process is as follows:

1. after sequencing, double-ended sequencing data were obtained for each sample, designated R1 and R2, respectively.

2. Extracting the molecular tag of the R1 sequence, and comparing the file after random base extraction with a reference genome.

3. The filter comparison results require that the following conditions be met: 1) Unique comparison to the reference genome; 2) The one with the best quality is removed by aligning the positions and random bases and retained.

4. And extracting the double-end sequencing sequences of the reserved comparison results, and splicing the double-end sequencing sequences.

5. And respectively counting the length of the spliced sequence for each microsatellite locus for the tissue sample and the control sample. If the tissue sample and the control sample are off-peak and the number of bases in the middle is greater than or equal to 2, the site is considered unstable.

6. Interpretation criteria: the number of unstable sites is equal to 0, and the sample is MSS type; the number of unstable sites is equal to 1, and the sample is MSI-L type; the number of unstable positioning points is more than or equal to 2, and the sample is MSI-H type.

The method can effectively identify the mutation, and is used for analyzing mutation conditions, copy number mutation, microsatellite instability and the like in a specific genome region. Compared with whole genome sequencing and other target region capture sequencing, the method has high efficiency, and can ensure high-depth sequencing while reducing the sequencing cost.

Examples

1. Reagent and sample preparation

1.1 Reagent: AMPure XP loads, qubit,80% ethanol (freshly prepared) and Low TE or enzyme-free Water

1.2 Preparation work

1.2.1 Dissolving and dispensing 20mM NAD+

The NAD+ powder has stable property, and is dissolved and packaged for initial use according to the following requirements

1) 1000 Μl of enzyme-free water was added to the NAD+ powder and the solubilization was complete to obtain 20mM NAD+ liquid.

2) Vortex for 10 seconds, and repeat the vibration for 10 seconds if there is still unmelted.

3) Mu.l of 20mM NAD+ liquid per tube was dispensed and placed on ice.

The daily remaining 20mM NAD+ was discarded and not frozen for secondary use. 20mM NAD+ can be stored at-20℃for 3 months.

1.2.2 Dissolving Oligo Pool (containing SEQ ID NO: 1-38)

1) 28. Mu.l of enzyme-free water was added to the Oligo Pool powder, and the mixture was vortexed and shaken for 10 seconds to dissolve the mixture completely to obtain a liquid.

2) Incubate at room temperature for 15 min until complete reconstitution.

3) 200 Μl of pipettor was added to 97 μ lHybInhancer to the dissolved Oligo Pool liquid, and the mixture was stored at-15℃to-25 ℃. The single-tube Oligo Pool/HybInhancer mixture can be subjected to 24 reactions.

The Oligo Pool/HybInhancer mixture can be stored at-15deg.C to-25deg.C for 3 months.

1.2.3 Based on the Qubit amount, the starting amount was 100ng.

1.2.4 Enzymes were removed from-20℃at least 10 minutes in advance, placed on ice or in a refrigerator at 4℃to reach 4℃before use.

The other reagents except enzyme are thawed, and then centrifuged after short-time shaking, and stored on ice.

2. Specific flow

2.1 Hybridization

Hybridization was performed as follows:

2.1.1 the following reagents were mixed. The mixture of Buffer H and Oligo Pool/HybInhancer may be premixed.

The mixing ratio is as follows:

the Oligo Pool is a hybridization primer Pool capable of specifically identifying a target region, and in the hybridization process, each pair of upstream primers has 10bp UMI, so that the subsequent analysis is facilitated.

2.1.2 Mixing the above systems uniformly and centrifuging at a low speed. And (3) placing the sample on a PCR instrument, opening a hot cover, and running a preset program.

The samples were incubated on the PCR apparatus at 56℃for 18 hours, and maintained at 56℃until the next step.

2.2 Gap Filling

2.2.1 DNTPs were diluted five times from 10mM to 2mM using enzyme-free water.

2.2.2 The following premix was added directly to 16. Mu.l of hybridization product.

The day of the discard was left over NAD+ and 2mM dNTPs.

2.2.3 Shaking and mixing evenly. The following procedure was run on a PCR instrument:

the heat cover was opened and incubated at 56℃for 60 minutes and stored at 4 ℃.

2.2.4 The following mixtures were prepared and added directly to the respective reaction tubes.

2.2.5 Shaking and mixing evenly. Under the condition of opening the hot cover, the following procedure was run on the PCR instrument:

2.3 PCR amplification

2.3.1 The PCR reaction solution, all enzymes, freeze thawing reagent and reaction mixture are needed to be used on ice. PCR premix reaction solutions were prepared according to the following table. Shaking, mixing, centrifuging for a short time, and placing on ice for standby.

2.3.2 PCR premix and primers were added to 0.2ml hybridization tubes (step 1.2 reactant).

Ensuring that the primer is finally added into the reaction system.

The Primer suggests PrimerMIX or homemade Index Primer matched to NEB library kit.

2.3.3 Blowing up and down for 20 times so as to mix the reaction solution uniformly.

2.3.4 After short centrifugation, the mixture was placed on ice.

2.3.5 The following procedure was set up in the PCR apparatus. When the hot cap is preheated to 105 ℃, the hot cap is quickly transferred into a PCR reaction tube and a program is run.

2.4 Library purification

2.4.1 Taking out the AMPure XP magnetic beads 30 minutes in advance, vibrating and mixing uniformly, and placing the mixture at room temperature for standby.

2.4.2 Add 65. Mu.l of resuspended AMPure XP beads to 100. Mu.l of PCR reaction (if the PCR reaction was less than 100. Mu.l, add water to make up). Blowing up and down for 10 times until the materials are evenly mixed.

2.4.3 Incubation for 10 min at room temperature.

2.4.4 After transient centrifugation, the beads were separated from the supernatant by placing on a magnetic rack for 3 minutes. When the supernatant clear, carefully transfer supernatant to a new tube, discard the beads. (note: non-disposable supernatant)

2.4.5 Add 30. Mu.l of post-resuspension AMPure XP beads to the supernatant. Mix well and incubate at room temperature for 10 minutes.

2.4.6, And then placing the mixture on a magnetic rack for 3 minutes to separate the magnetic beads from the supernatant. After the supernatant was cleared, the supernatant was carefully removed, and the beads were retained. (note: non-disposable magnetic beads)

2.4.7 200 Μl of freshly prepared 80% ethanol was added to the sample tube, which was placed on a magnetic rack. The tube wall was rotated 720 degrees at room temperature, allowed to stand until the supernatant was clear, and the supernatant was discarded.

2.4.8 Repeating step 7 once.

2.4.9 Uncapping, and drying at room temperature until the surface of the magnetic bead is matte, so as to avoid excessive drying. (note: not overdrying, would result in low recovery)

2.4.10 Remove the reaction tube from the magnetic rack and add 25. Mu.l low TE resuspended beads.

2.4.11 Shaking and mixing uniformly, and incubating for 3 to 5 minutes at room temperature.

2.4.12 The reaction tube was placed on a magnetic rack to separate the magnetic beads and incubated until the liquid was clear.

2.4.13 Transfer supernatant to new tube, indicating information on sample name, library type, date of library construction, index/barcode, etc.

The library was stored at-20 ℃.

3. Sequencing on machine

After library construction, QPCR quantification was performed for each library, and mixing was performed based on the quantitative results and the amount of data required for each library. The well-mixed sample cell is denatured and diluted to a proper on-machine concentration. The sequence was determined using a MiniSeq sequencer from illuminea, MINISEQ HIGH Output REAGENT KIT kit, 310cycles on-machine, PE.times.151, index 8bp. Data for analysis may be obtained.

4. Information analysis

Taking a sample test as an example, marking a cancer tissue sample of the sample as test_cancer, marking a normal control sample of the sample as test_normal, and respectively performing on-machine sequencing to obtain double-end sequencing results of test_cancer_1.Fq.gz, test_cancer_2.Fq.gz, test_normal_1.Fq.gz and test_normal_2.Fq.gz. The format of the sequencing result file is FASTQ, and each 4 rows of the FASTQ format file represent a sequencing sequence, wherein the first row of the file is a sequence header (read ID), and information such as a sequencer, a sequencing position, index of a marked sample and the like is recorded; the second row is a DNA template sequence comprising four bases of ATGC; the third row "+" symbol is either the same as the first row; and the fourth behavior sequencing quality is in one-to-one correspondence with the bases of the second row.

The following steps are performed for test_cancer and test_normal, respectively, and are taken here as an example of test_cancer: 1) The molecular tag of each sequence in the test_cancer_1.Fq file, i.e., UMI, is extracted and recorded, and the result file is denoted as test_cancer_1_UMI. Fq. And (3) processing the sequence of the R2 file, only recording the corresponding UMI in the R1 file, and recording the result file as test_cancer_2_UMI.

2) The test_cancer_1_umi.fq and the test_cancer_2_umi.fq were aligned in a double-ended alignment onto the human reference genome hg19.fa using alignment software, the result of the alignment being test_cancer.

3) Converting test_cancer.sam into a test_cancer.bam format, sorting results, selecting comparison results of R1 and R2 which are respectively and only compared with a reference genome, performing comparison result de-duplication according to the comparison position and UMI recorded in the step 1), and recording the de-duplication result as test_cancer.sort.de-duplication. If the UMI of the alignment position and sequence are identical, only the best quality result is retained, which represents a DNA copy.

4) For each microsatellite locus, a sequence satisfying the following conditions is retained according to the position of the designed primer on the genome: the alignment start position of one end sequence is the same as the upstream primer of the locus at the start position of the genome, and the alignment end position of the other end sequence is the same as the downstream primer of the locus at the end position of the genome. Sequences meeting the conditions are considered as amplification products of the pair of amplification primers, i.e. the target region of interest. If the alignment start position of one end sequence and the upstream primer of the site are the same at the start position of the genome and the alignment end position of the other end sequence and the downstream primer of the site are different at the end position of the genome, then the sequence meeting this condition is not considered to be the target amplification product required by us. Conversely, if the alignment start position of one end sequence and the upstream primer of the site are different at the start position of the genome and the alignment end position of the other end sequence and the downstream primer of the site are the same at the end position of the genome, then the sequence meeting the situation is not considered to be the target amplification product required by us.

5) For each microsatellite locus, the FLAG of the sequence reserved in 4) is divided into two files according to sequence comparison and respectively marked as R1_UMI_seq and R2_UMI_seq, the sequence seq1 and the sequence seq2 corresponding to the sequence ID in the two files are combined into one sequence, and the combined sequence is stored in the Merged _sequence file.

For the tissue sample and the control sample, the sequence length in the Merged _sequence file of the corresponding microsatellite loci is counted respectively. If the lengths of the tissue sample and the control sample are staggered, and the difference of the peak values is more than or equal to 2, the locus is considered to be unstable.

If the number of unstable sites is equal to 0, the sample is MSS type; the number of unstable sites is equal to 1, and the sample is MSI-L type; the number of unstable positioning points is more than or equal to 2, and the sample is MSI-H type.

5. Summary of results

For 84 patients, the traditional PCR gold standard detection is carried out simultaneously to verify the detection result of the invention, and the experimental stability and the feasibility of the method are verified. The traditional PCR gold standard carries out multiplex amplification on six single nucleotide repeated sites, and the PCR method finally determines 46 MSI-H samples, 1 MSI-L sample and 37 MSS samples. Compared with the PCR analysis result, the MSI detection result is completely consistent with the PCR result, the sensitivity is 100%, and the specificity is 100%.

TABLE 3 results of 84 patients using PCR gold standards and the present method to determine microsatellite instability

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

Sequence listing

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Claims (1)

1. A method for detecting microsatellite instability based on a second generation sequencing platform for non-disease diagnostic purposes, comprising the steps of:

(1) Hybridizing a tissue sample to a primer composition, wherein the primer composition comprises an upstream primer set and a downstream primer set, and each primer in the upstream primer set and its corresponding downstream primer bind to both single strands of the same DNA or gene, each primer in the upstream primer set respectively comprising a UID sequence at the 5 'end and a binding region complementary to one primer of a PCR universal primer pair, each primer in the downstream primer set respectively comprising a binding region complementary to the other primer of the PCR universal primer pair and located at the 3' end of each primer, wherein each primer in the upstream primer set respectively specifically binds to a single strand upstream of a mutation site of at least one gene in the following gene set, and each primer in the downstream primer set respectively specifically binds to a single strand downstream of the mutation site, the upstream primer set comprising SEQ ID No:1-18, said downstream primer set consisting of SEQ ID No: 19-36;

(2) Filling up gap gaps under the condition suitable for DNA polymerase or DNA ligase reaction to obtain target DNA;

(3) Amplifying the target DNA serving as a template by using a PCR universal primer pair to obtain a sequencing library;

(4) Performing double-end sequencing on the sequencing library by using second-generation sequencing to obtain double-end sequencing data of a sample;

(5) The double-end sequencing data are respectively marked as R1 and R2, UIDs of the R1 sequences are extracted, files after UIDs are extracted are compared to a reference genome, comparison results are filtered, the reserved double-end sequencing sequences of the comparison results are extracted and spliced, wherein the condition of the reserved comparison results is that only comparison is carried out on the reference genome, and a sequence with the best quality is reserved according to the comparison position and random base de-duplication;

(6) For a tissue sample and a control sample, respectively counting the length of a spliced sequence for each microsatellite locus, and if the tissue sample and the control sample are in peak staggering and the middle difference of the base number is more than or equal to 2, considering the locus to be unstable;

(7) The interpretation criteria are: the number of unstable sites is equal to 0, and the tissue sample is MSS type; the number of unstable sites is equal to 1, and the tissue sample is MSI-L type; the number of unstable positioning points is more than or equal to 2, and the tissue sample is MSI-H type.