Nucleic acid probe for capturing target sequence, design method and application thereof

Abstract

The invention provides a nucleic acid probe for capturing a target sequence, a design method and application thereof. According to the design method disclosed by the invention, two ends of a probe respectively cover flanking areas of a target sequence. The two ends are connected through a linker, and in addition, a probe with over-high homology with a whole genome is filtered out. According to the method, non-specific binding of homologous sequences can be greatly reduced, data waste caused by repeated sequences is greatly reduced, the capture efficiency of the probe and the detection effect of the depth coefficient are effectively improved, and the designed probe has the detection performance of high specificity, high sensitivity and high positive detection rate.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology detection, and in particular to a nucleic acid probe for capturing a target sequence, a design method and an application thereof. Background Art

[0002] Microsatellite instability (MSI) is a genetic phenomenon that refers specifically to the abnormal change in the length of microsatellite sequences (also called simple repeat sequences) in an individual's DNA due to insertion or deletion mutations. Microsatellites are short DNA sequences consisting of 1 to 6 base pairs that are repeated many times in the genome to form tandem repeat regions. Under normal circumstances, the DNA replication mechanism can accurately replicate these repeat sequences, but in the case of microsatellite instability, the mismatch repair (MMR) system may not be able to correctly repair these replication errors due to functional defects, resulting in changes in the number of repeat units in the microsatellite sequence.

[0003] Microsatellite instability (MSI) testing has a wide range of uses, mainly in the following aspects: 1. Cancer diagnosis and prognosis: MSI is associated with many types of cancer, especially colorectal cancer, but can also appear in tumors in other parts of the body, such as endometrial cancer, gastric cancer, and pancreatic cancer. MSI testing helps to identify and classify cancer early, and for some types of tumors, MSI status can be used as a prognostic indicator. For example, patients with stage II colon cancer with MSI-H (high microsatellite instability) may have a good survival rate even without adjuvant chemotherapy. 2. Genetic risk assessment: Some MSI-positive tumors are associated with hereditary cancer syndromes, such as Lynch Syndrome, a condition with increased susceptibility to hereditary cancer due to mutations in the MMR gene. By testing MSI, it can help identify family members at risk of hereditary cancer and conduct early monitoring and intervention. 3. Guide treatment decisions: MSI status has an important impact on the treatment response of tumors. In particular, MSI-H tumors typically have a higher tumor mutation burden (TMB) and are more likely to express PD-L1, which makes them more sensitive to immunotherapies such as immune checkpoint inhibitors. Therefore, MSI has become an important biomarker for predicting response to immunotherapy. 4. Biomarker research: With the development of precision medicine, MSI, as a biomarker, plays a key role in the molecular typing of cancer and the selection of personalized treatment options. It can help doctors choose the most suitable treatment strategy for patients, improve treatment efficacy and reduce unnecessary side effects.

[0004] At present, the main technical routes for detecting microsatellite instability (MSI) include the following: 1. Immunohistochemistry (IHC): Indirectly judge the MSI status by detecting the expression of mismatch repair (MMR) proteins (such as MLH1, MSH2, MSH6, PMS2) in tumor tissues. 2. Polymerase chain reaction (PCR) + capillary electrophoresis: This is one of the currently recognized "gold standard" methods for MSI detection. A series of microsatellite loci in tumor tissues and normal tissues (usually surrounding non-tumor tissues or blood) are amplified by PCR, and then capillary electrophoresis is used to separate the amplified products and compare the fragment lengths of tumor and normal samples to determine whether there is a length difference, thereby judging the stability of the microsatellite loci. 3. Next-generation sequencing (NGS): With the development of high-throughput sequencing technology, NGS has also been used for MSI detection, especially when there is no suitable control sample or a large number of gene mutations need to be detected at the same time. By sequencing and analyzing the microsatellite loci in tumor samples, the changes in microsatellite sequences can be accurately evaluated.

[0005] The probe hybridization capture + NGS strategy is increasingly becoming one of the mainstream methods for studying MSI. The key to the success of this strategy lies in the design of MSI probes. Since MSI is often located in the repetitive regions of the genome and microsatellites themselves are composed of highly repetitive short sequences, it is extremely challenging to design probes that can span these repetitive sequences without causing self-hybridization or forming secondary structures. The depth coefficient of MSI sites captured by conventional probe designs is often low, which affects the interpretation of clinical results. In order to enhance the capture of MSI sequences, some probe designs will design probes at far positions on both sides of the site to capture MSI sequences through the probe flank capture ability, but this will increase the size of the capture interval and waste effective data; some designs will design multiple probes on the MSI site to enhance the capture of the MSI region by increasing the probability of molecular collisions, but the increase in this non-specific probe will also greatly increase the capture of non-specific sequences. In addition, some designs may require special chemical modifications or probe structure designs to solve this problem. The above probe design strategies will lead to different degrees of increase in probe costs, while improving capture performance, it also brings negative effects such as non-specific capture and decreased uniformity. Therefore, an efficient MSI probe design strategy that does not bring negative effects as much as possible is urgently needed. Summary of the invention

[0006] In order to solve at least one of the above problems, the present disclosure provides a nucleic acid probe for capturing a target sequence, a design method and its application. The nucleic acid probe designed using the method provided by the present disclosure can reduce the nonspecific binding of homologous sequences and the data waste caused by repeated sequences, effectively improve the capture efficiency and depth coefficient of the probe, and thus improve the detection specificity and sensitivity.

[0007] According to one aspect of the present disclosure, a nucleic acid probe for capturing a target sequence is provided, wherein the nucleic acid probe does not cover the target sequence, and comprises: a first fragment covering an upstream flanking sequence of the target sequence, a second fragment covering a downstream flanking sequence of the target sequence, and a linker connecting the first fragment and the second fragment.

[0008] In some embodiments, the target sequence includes a target sequence containing a non-conservative sequence such as a point mutation, a repeated sequence, a copy number variation, an insertion or deletion sequence and / or a fusion sequence.

[0009] In some embodiments, the target sequence comprises a microsatellite locus.

[0010] In some embodiments, the target sequence includes a microsatellite instability site.

[0011] In some embodiments, the first fragment and the second fragment are the same or different lengths.

[0012] In some embodiments, the nucleotide sequence of the linker is randomly generated.

[0013] In some embodiments, the nucleotide sequences of the first fragment and the second fragment may be consistent with the positive strand of the chromosome or the negative strand of the chromosome.

[0014] In some embodiments, the length of the probe is not particularly limited, for example, it can be between 10 bases and 300 bases. In some embodiments, the length of the probe can be between 30 and 150 bases. In some embodiments, the length of the probe can be between 40 bases and 80 bases. In some embodiments, the nucleotide sequence of the first fragment and the second fragment is usually consistent with the positive strand or negative strand of the chromosome. In some embodiments, the nucleotide sequence of the first fragment and the second fragment is usually more than 15 consecutive pairs with the positive strand or negative strand of the chromosome to have capture ability.

[0015] In some embodiments, the length of the linker is not particularly limited, for example, it can be between 3 bases and 20 bases. For example, it can be 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, 10 bases, 11 bases, 12 bases, 13 bases, 14 bases, 15 bases, 16 bases, 17 bases, 18 bases, 19 bases or 20 bases.

[0016] In some embodiments, a linker generally has blocking ability as long as it is not contiguously paired with a target sequence.

[0017] In some embodiments, the nucleic acid probe may include one or more of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and nucleic acid derivatives such as peptide nucleic acid (PNA), morpholino (MNA), bridged nucleic acid (BNA), locked nucleic acid (LNA), glycol nucleic acid (GNA), and threose nucleic acid (TNA).

[0018] In some embodiments, any one or more nucleotides in the probe have a functional group, and the functional group includes biotin, an amino acid or polypeptide tag (such as poly-histidine), glutathione, heparin, and a sugar.

[0019] In some embodiments, the number of nucleic acid probes includes a combination of one or more.

[0020] In some embodiments, the linkers of the nucleic acid probes are the same or different in length.

[0021] In some embodiments, the nucleotide sequences of the linkers of the nucleic acid probes are different.

[0022] In some embodiments, the nucleic acid probe has a nucleotide sequence as shown in any one or more of SEQ ID NOs: 1 to 15, or a nucleotide sequence having 85% sequence identity thereto.

[0023] According to another aspect of the present disclosure, a method for designing the nucleic acid probe of the present disclosure is provided, the method comprising the following steps:

[0024] A1) taking the target region as a target, obtaining a first fragment covering the upstream flanking sequence of the target sequence, and a second fragment covering the downstream flanking sequence of the target sequence;

[0025] A2) using a linker to connect the first fragment and the second fragment to form a nucleic acid probe that captures the target sequence.

[0026] In some embodiments, the design method further comprises the following steps:

[0027] A3) performing homology filtering on the nucleic acid probes obtained in step A2) to screen out nucleic acid probes with low homology.

[0028] In some embodiments, the screening in step A4) comprises the following steps:

[0029] B1) counting the sequence identity of the homologous sequences of the nucleic acid probes obtained in step A2) on the genome;

[0030] B2) counting the number P of homologous sequences of the nucleic acid probe on the genome whose sequence identity is higher than a threshold value M;

[0031] B3) Screening out nucleic acid probes whose number P is lower than a threshold value N.

[0032] In some embodiments, the threshold value M = 60% to 99.9%.

[0033] In some embodiments, the threshold value M=60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%.

[0034] In some embodiments, the threshold N=3-100.

[0035] In some embodiments, the threshold N=3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100.

[0036] In some embodiments, the screening in step A4) is achieved using a blast tool.

[0037] According to another aspect of the present disclosure, a design system is provided, the system is used to execute the design method, and the system includes the following modules:

[0038] A fragment screening module is used to screen out the first fragment and the second fragment covering the upstream and downstream flanking regions of the target sequence respectively;

[0039] The connection module connects the two fragments through the linker to form a complete probe.

[0040] In some embodiments, the design system further comprises a homologous sequence screening module for screening out probes with low homology to genomic sequences as nucleic acid probes.

[0041] According to another aspect of the present disclosure, a nucleic acid probe is provided, wherein the probe is designed according to the design method or design system of the present disclosure.

[0042] According to another aspect of the present disclosure, a kit is provided, wherein the kit comprises the nucleic acid probe provided by the present disclosure.

[0043] According to another aspect of the present disclosure, a detection method is provided, which comprises: using the nucleic acid probe or the kit provided by the present disclosure to detect a target sequence in a sample to be tested of a subject.

[0044] In some embodiments, the target sequence includes a target sequence containing a non-conservative sequence such as a point mutation, a repeated sequence, a copy number variation, an insertion or deletion sequence and / or a fusion sequence.

[0045] In some embodiments, the target sequence comprises a microsatellite locus.

[0046] In some embodiments, the target sequence includes a microsatellite instability site.

[0047] In some embodiments, the detection method further comprises determining whether the sample to be tested is microsatellite instability positive based on the detection result.

[0048] In some embodiments, the detection method further comprises: determining that the subject has cancer by determining that the sample to be tested is microsatellite instability positive.

[0049] In some embodiments, the sample to be tested includes a tissue or body fluid sample.

[0050] In some embodiments, the body fluid sample includes: saliva or saliva supernatant, whole blood, serum, plasma, milk, urine, lumbar or ventricular CSF, lymph fluid, prostatic fluid, semen, sputum, stool diluted supernatant, tears, tumor cells, alveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab, cerebrospinal fluid, pleural effusion, bile, amniotic fluid, peritoneal fluid, aqueous humor, vitreous humor, vaginal discharge diluted supernatant and their processed products.

[0051] In some embodiments, the tissue sample includes tissue, paraffin sections, and processed materials thereof.

[0052] According to another aspect of the present disclosure, a storage medium is provided, and when the computer program is executed by a processor, the steps of the design method, design system and / or detection method described in the present disclosure are implemented.

[0053] According to another aspect of the present disclosure, provided is use of the nucleic acid probe, the design method and / or the design system provided by the present disclosure in detecting a target sequence.

[0054] According to another aspect of the present disclosure, provided is use of the nucleic acid probe, the design method and / or the design system provided by the present disclosure in preparing a product for target sequence detection or cancer detection.

[0055] In some embodiments, the use includes early screening, diagnosis, auxiliary diagnosis or prediction of prognosis of cancer.

[0056] In some embodiments, the target sequence includes a target sequence containing a non-conservative sequence such as a point mutation, a repeated sequence, a copy number variation, an insertion or deletion sequence and / or a fusion sequence.

[0057] In some embodiments, the target sequence comprises a repetitive sequence.

[0058] In some embodiments, the target sequence comprises a microsatellite locus.

[0059] In some embodiments, the target sequence includes a microsatellite instability site.

[0060] Beneficial effects:

[0061] The design method disclosed in the present invention is to make the two ends of the probe cover the flanking region of the target sequence respectively. The two ends are connected by a linker, and such a design can play a role in shielding repetitive sequences. On the other hand, probes with too high homology to the whole genome are filtered out to enhance specificity.

[0062] This design method can greatly reduce the nonspecific binding of homologous sequences through sequence design and a limited number of probes without special chemical modification or probe structure design, greatly reduce the data waste caused by repeated sequences, and effectively improve the capture efficiency of the probe and the detection effect of the depth coefficient. The designed probe has the detection performance of high specificity, high sensitivity and high positive detection rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The schematic diagram of the design of the nucleic acid probe for capturing the target sequence is shown, wherein red represents linker6, red represents the upstream and downstream of the probe, gray represents the target DNA, and "AAAAAA" represents the repetitive sequence of the MSI site.

[0064] Figure 2 A bar graph of the capture efficiency of different probe sets is shown.

[0065] Figure 3 The histogram of the MSI depth coefficient when adding a conventional MSI probe and adding 15 MSI probes shown in Table 2 is shown. DETAILED DESCRIPTION

[0066] The present disclosure provides a method for designing a nucleic acid probe, comprising the following steps: (1) obtaining genomic sequences on both sides of the MSI site; (2) generating a linker sequence; (3) assembling a complete probe sequence; and (4) evaluating probe homology. Through the above steps, a probe can be designed for a given MSI site of any length, and the target region can be captured without introducing nonspecific MSI sequences.

[0067] A nucleic acid probe for capturing a target sequence obtained by the above design method does not cover the target sequence, and the nucleic acid probe comprises: a first segment covering an upstream flanking sequence of the MSI, a second segment covering a downstream flanking sequence of the MSI, and a linker connecting the first segment and the second segment, such as Figure 1 shown.

[0068] definition

[0069] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0070] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0071] The expression "about" as used herein is understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is not aware of the use of the term based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0072] The term "microsatellite" refers to a string of repeated bases in the human genome, with the repeat unit length ranging from 1-6 bp.

[0073] The term "microsatellite instability" refers to a state characterized by having genomic instability in a specific repetitive DNA sequence (e.g., short tandem repeats or simple sequence repeats). The method for detecting microsatellite instability includes any method known in the art. MSI can be classified as MSI-high (MSI-H), MSI-low (MSI-L) and MS-stable (MSS), wherein instability in two or more markers is classified as MSI-H, instability in one marker is only classified as MSI-L, and instability not observed in five markers is classified as MSS. If more than 30% of the tested markers show instability (MSI-H), MSI can also be classified as MSI-H, if 10-30% of the tested markers show instability, it is classified as MSI-L, and if less than 10% of the tested markers show instability, it is classified as MSS. 5, 6, 7, 8, 9, 10 or more than 10 markers can be used to test the MSI state. In some embodiments, a cancer is classified as MSS. A cancer may also have an unknown MSI status, i.e., the tumor is not known to have an MSI-H status.

[0074] The term "DNA" used in this disclosure, the full name of which is "deoxyribonucleic acid" (abbreviated as DNA), is one of the four biological macromolecules contained in biological cells, one of the nucleic acids. DNA carries the genetic information necessary for the synthesis of RNA and proteins, and is an indispensable biological macromolecule for the development and normal functioning of organisms. DNA is a macromolecular polymer composed of deoxynucleotides. Deoxynucleotides are composed of bases, deoxyribose and phosphate. There are four types of bases: adenine (A), guanine (G), thymine (T) and cytosine (C).

[0075] The term "probe" used in the present disclosure refers to an oligonucleotide that can bind to a target nucleic acid of a complementary sequence through one or more types of chemical bonds (usually through complementary base pairing, usually through hydrogen bond formation). Depending on the stringency of the hybridization conditions, the probe can bind to a target sequence that lacks complete complementarity with the probe sequence. There may be any number of base pairs that will interfere with the hybridization between the target sequence described in the text and the single-stranded sequence. However, if the number of mutations is so large that hybridization does not occur even under the least stringent hybridization conditions, the sequence is not a complementary target sequence. The probe may be single-stranded, or partially single-stranded and partially double-stranded. The strand type (strandedness) of the probe is described by the properties of the structure, composition, and target sequence. The probe may be directly labeled or indirectly labeled by biotin, which is later bound by, for example, a streptavidin complex. In some embodiments, the term "probe" comprises an oligonucleotide chain, or an oligonucleotide chain complementary thereto.

[0076] The term "probe set" as used in the present disclosure generally refers to a collection of more than one probe, which achieves the positioning and / or quantification of the target nucleic acid by recognizing and binding (by hybridization pairing) to the target sequence. Each probe in the probe combination is generally an oligonucleotide, such as a single-stranded DNA molecule or RNA. In some embodiments, the term "probe" comprises an oligonucleotide chain, or an oligonucleotide chain complementary to the oligonucleotide chain.

[0077] The term "sequence identity" as used in the present disclosure refers to the "percentage of sequence identity" or "percentage of sequence identity" between two polynucleotides, i.e., the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide, a gap present in the target sequence is not counted. Similarly, since the target sequence nucleotides are counted and the nucleotides from the reference sequence are not counted, the gaps present in the reference sequence are not counted. At least 85% sequence identity includes a continuous segment having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity over the entire length of the sequence.

[0078] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The comparison of the sequence and the determination of the sequence identity percentage between the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite that can be obtained from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. Government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used for comparing nucleic acid sequences, and BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0079] The term "tumor" as used herein refers to a mass or neoplasm, which itself is defined as an abnormal new growth of cells that usually grow faster than normal cells and will continue to grow if not treated, sometimes causing damage to adjacent structures. Tumors can vary greatly in size. Tumors can be solid or fluid-filled. Tumors can refer to benign (non-malignant, usually harmless) or malignant (capable of metastasizing) growths. Some tumors may contain benign neoplastic cells (e.g., carcinoma in situ) while also containing malignant cancer cells (e.g., adenocarcinoma). It should be understood to include neoplasms located in multiple locations throughout the body. Therefore, for the purposes of this disclosure, tumors include primary tumors, lymph nodes, lymphatic tissue, and metastatic tumors. Non-limiting examples of such cancers include biliary tract cancer, bladder cancer, transitional cell carcinoma, urothelial carcinoma, breast cancer, cervical cancer, cervical squamous cell carcinoma, rectal cancer, colorectal cancer, colon cancer, hereditary nonpolyposis colorectal cancer, colorectal adenocarcinoma, gastrointestinal stromal tumor (GIST), endometrial cancer, endometrial stromal sarcoma, esophageal cancer, esophageal squamous cell carcinoma, esophageal adenocarcinoma, ocular melanoma, uveal melanoma, gallbladder cancer, gallbladder adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma , transitional cell carcinoma, urothelial carcinoma, Wilms tumor, liver cancer, hepatoepithelial carcinoma, hepatocellular carcinoma, bile duct carcinoma, hepatoblastoma, lung cancer, non-small cell lung cancer (NSCLC), nasopharyngeal carcinoma (NPC), neuroblastoma, oral cancer, oral squamous cell carcinoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, pseudopapillary neoplasm, acinar cell carcinoma, prostate cancer, skin cancer, melanoma, malignant melanoma, cutaneous melanoma, small intestine cancer, stomach cancer, gastric epithelial carcinoma, uterine cancer, or uterine sarcoma.

[0080] The terms "hybridization" or "binding" used in the present disclosure refer to the pairing (including partial complementarity) of complementary (including partially complementary) polynucleotide chains. Hybridization and the strength of hybridization (for example, the strength of binding between polynucleotide chains) are affected by many factors well known in the art, including the degree of complementarity between polynucleotides, the stringency of related conditions, which are affected by conditions such as salt concentration, the melting temperature (Tm) of the formed hybrid, the presence of other components, the molar concentration of the hybridized chain, and the G:C content of the polynucleotide chain. When a polynucleotide chain is said to be "hybridized" with another polynucleotide chain, it means that there is a certain complementarity between the two polynucleotide chains, or the two polynucleotide chains form a hybrid under high stringency conditions. When a polynucleotide chain is said not to hybridize with another polynucleotide chain, it means that there is no sequence complementarity between the two polynucleotide chains, or the two polynucleotide chains do not form a hybrid under high stringency conditions.

[0081] The term "complementary" as used in the present disclosure refers to the concept of sequence complementarity between regions of two polynucleotide chains or between two regions of the same polynucleotide chain. It is known that the adenine base of the first region of the polynucleotide chain can form a specific hydrogen bond ("base pair") with the base of the second region of the polynucleotide chain that is antiparallel and complementary to the first region (if the base is thymine or uracil). Similarly, it is known that the cytosine base of the first polynucleotide chain can base pair with the base of the second polynucleotide chain that is antiparallel and complementary to the first region (if the base is guanine). If at least one nucleotide in the first region can base pair with the base of the second region when the first region of the polynucleotide is arranged in an antiparallel and complementary manner with the second region of the same or another different polynucleotide, the two regions are complementary. Therefore, the two complementary polynucleotide chains do not need to base pair at every nucleotide position. "Complementary" means that the first polynucleotide chain is 100% or "completely" complementary to the second polynucleotide chain, and therefore base pairing is formed at every nucleotide site. "Complementary" also means that the first polynucleotide strand is not 100% complementary (eg, 90%, or 80% or 70% complementary) and contains mismatched nucleotides at one or more nucleotide positions. In one embodiment, two complementary polynucleotide strands are capable of hybridizing to each other under high stringency hybridization conditions.

[0082] The term "stringent hybridization conditions" as used in the present disclosure is defined as conditions that can be determined empirically under which a probe or primer specifically hybridizes to a target sequence but not to non-target sequences. The term "stringent conditions" is functionally defined in terms of the hybridization of a nucleic acid probe to a target nucleic acid (i.e., to a specific nucleic acid sequence of interest) by the specific hybridization method discussed in Sambrook et al., 1989, 9.52-9.55. See also Sambrook et al., 1989 9.47-9.52, 9.56-9.58; Kanehisa 1984 Nucl. Acids Res. 12: 203-213; and Wetmur et al. 1968 J. Mol. Biol. 31: 349-370. Appropriate stringent conditions that promote DNA hybridization are known to those skilled in the art.

[0083] As used in this disclosure, the term "sequencing" refers to the process of determining the sequence (eg, the identity and order of monomeric units) of a biological molecule, eg, a nucleic acid such as DNA or RNA. Exemplary sequencing methods include, but are not limited to, targeted sequencing, single molecule real-time sequencing, exon or exome sequencing, intron sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy terminator sequencing, whole genome sequencing, hybridization sequencing, pyrophosphate sequencing, capillary electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high throughput sequencing, massively parallel signature sequencing, emulsion PCR, low denaturation temperature co-amplification PCR (COLD-PCR), multiplex PCR, reversible dye terminator sequencing, paired end sequencing, near-term sequencing, exonuclease sequencing, ligation sequencing, short read sequencing, single molecule sequencing, sequencing by synthesis, real-time sequencing, reverse terminator sequencing, nanopore sequencing, 454 sequencing, Solexa Genome Analyzer sequencing, SOLiD™ sequencing, MS-PET sequencing, DNA nanoball sequencing (DNBSEQ), composite probe anchor polymerase sequencing (cPAS), and combinations thereof. In some embodiments, sequencing can be performed by a genetic analyzer, such as a genetic analyzer commercially available from Illumina, Inc., Pacific Biosciences, Inc., Applied Biosystems / Thermo Fisher Scientific, or Shenzhen MGI Intelligent Manufacturing Technology Co., Ltd., etc. For example, BGI DNBseq sequencing platforms such as BGISEQ-500, BGISEQ-50, MGISEQ-2000, MGISEQ-200, DNBSEQ-T7, DNBSEQ-G99, DNBSEQ-T20X2, or Illumina's HiSeq2000, HiSeq2500, HiSeq4000, HiSeqX10, NovaSeq6000, etc.

[0084] The term "targeted sequencing" as used in the present disclosure refers to a technique for capturing and sequencing target fragments in a DNA sample using a biotin-labeled DNA or RNA probe. The probe may be labeled with biotin. Each nucleotide in the probe of the present invention may be synthesized chemically using, for example, a general-purpose DNA synthesizer (e.g., Model 394 manufactured by Applied Biosystems). Any other method well known in the art may also be used to synthesize oligonucleotides, such as probes.

[0085] The term "subject" as used in this disclosure refers to any animal, mammal or human. The subject has, may have or is suspected of having one or more diseases. The subject may have cancer, the subject may show symptoms associated with cancer, the subject may not show symptoms associated with cancer, or the subject may not have been diagnosed with cancer. In some embodiments, the subject is a human.

[0086] As used herein, the term "computer-readable medium" (e.g., data storage, data storage, etc.) or "computer-readable storage medium" refers to any medium that participates in providing instructions to a processor for execution. Such media can take many forms, including but not limited to non-volatile media, volatile media, and transmission media. Examples of non-volatile media may include but are not limited to optical disks, solid-state disks, magnetic disks, such as storage devices. Examples of volatile media may include but are not limited to dynamic memory, such as memory.

[0087] Common forms of computer readable media include, for example, floppy disks, diskettes, hard disks, magnetic tape or any other magnetic medium, CD-ROMs, any other optical medium, punch cards, paper tape, any other physical medium with patterns of holes, RAM, PROM and EPROM, FLASH-EPROM, any other memory chip or cassette, or any other tangible medium from which a computer can read.

[0088] In addition to computer-readable media, data can be provided as a signal on a transmission medium included in a communication device or system to provide one or more instruction sequences to a processor of a computer system for execution. For example, a communication device may include a transceiver with a signal indicating instructions and data. The instructions and data are configured to cause one or more processors to implement the functions outlined in this disclosure. Representative examples of data communication transmission connections may include, for example, telephone modem connections, wide area networks (WANs), local area networks (LANs), infrared data connections, NFC connections, etc.

[0089] The term "diagnosis" as used herein refers not only to "judgment", but also includes "determination", "differentiation", "identification", "inspection", "detection" and "prediction". It includes distinguishing / differentiating or assisting in distinguishing / differentiating cancer and benign lesions, judging / determining cancer and the degree of development of cancer, predicting the risk of cancer, treatment effect, prognosis and risk of recurrence. In some embodiments, the diagnosis includes distinguishing or assisting in distinguishing cancer and benign lesions, judging the degree of development of cancer, or predicting the risk of cancer, treatment effect, prognosis and risk of recurrence.

[0090] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be noted that for those skilled in the art, several variations and improvements made on the basis of this invention and creation concept all belong to the protection scope of the present invention. In the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications. The equipment, instruments, reagents and / or kits used in the following examples are not mentioned in terms of their sources, and are all commercially available on the market, or obtained by conventional methods known to those skilled in the art.

[0091] Example

[0092] The instruments, equipment and consumables are shown in Table 1.

[0093] Table 1 Recommended instruments and consumables

[0094]

[0095]

[0096] Embodiment 1:

[0097] Taking the probe length of 120 nt as an example, the probe is designed according to the following steps:

[0098] (1) Obtain the genomic sequences on both sides of the MSI site. If the given MSI site is chr pos1 pos2 (standard bed file format, where pos1 refers to the first base of the MSI site and pos2 refers to the last base of the MSI site), then obtain the sequence 1 sequence through position chr pos1-57 pos1 and obtain the sequence 2 sequence through chr pos2 to pos2+57;

[0099] (2) Generate a linker sequence. Use the python random.choice() random function to generate a 6nt DNA sequence containing four bases, AGCT, and name it linker6.

[0100] (3) Assemble the complete probe sequence, and use the linker6 sequence of step (2) to connect the sequence 1 and sequence 2 generated in step (1) to assemble a complete 120nt probe sequence. The generated sequence structure is sequence1-linker6-sequence2. Use linker6 to connect the relatively specific sequences on both sides of the MSI to shield the repetitive sequences. Refer to the cot-1 component in the hybridization capture experiment;

[0101] (4) Probe homology assessment: For the designed MSI probes, the blast tool is used to calculate the number of times each probe appears in the whole genome according to the homology of 75% (-perc_identify 0.75), and then highly homologous probes are filtered out according to the homology threshold (for example, probes with more than 3 homologous times are eliminated. If the site is very important, the value can be increased from 3 to 100. Too large a value will cause nonspecific capture) to form the final probe panel for subsequent hybridization experiments.

[0102] Using the above method, 15 probes were designed for 15 MSI sites that are strongly correlated with cancer occurrence and have clinical detection value (as shown in Table 2, with a total size of 1.8K). They were mixed with the FGFR probe group (see patent document with application number 202110533156.6) for hybridization capture experiments, and the performance of this design and the conventional MSI probe (sequence as shown in SEQ ID NO: 16 to 30) in terms of capture efficiency and depth coefficient were compared.

[0103] Table 2. 15 MSI probe sequences designed in Example 1

[0104]

[0105]

[0106]

[0107] Embodiment 2:

[0108] 1. Sample preparation

[0109] 1. For plasma samples, use Streck or Kangwei blood collection tubes to collect 8-10mL of peripheral blood. Immediately after collection, gently invert and mix 8-10 times to avoid coagulation and hemolysis of the sample. Separate the plasma from the whole blood by centrifugation, and dispense the supernatant (plasma) into 5mL centrifuge tubes prepared in advance. The separated plasma is stored at -20±5℃.

[0110] 2. The storage time of FFPE samples should not exceed 3 years. The samples should be confirmed to contain tumor cells (tumor cell content should be ≥10%), and the number of slice samples should be 10-15 (3-4μm thick, tissue area>25mm2).

[0111] 2. Inspection Method

[0112] 1. cfDNA extraction

[0113] The whole blood samples were centrifuged at 1,600 g and 16,000 g to separate plasma and remove cell debris in the plasma. Then, the magnetic bead method was used to extract plasma cfDNA using the Maelstrom 2400 free DNA extractor.

[0114] After adding proteinase K and incubation buffer to paraffin-embedded tissue (FFPE) for incubation outside the machine, magnetic bead method whole genome DNA extraction was performed using Promega Maxwell 16 instrument. Whole genome DNA was fragmented using VAFA ultrasonic crusher, and the main peak size of fragmented DNA was 200-250bp. The fragmented samples were purified by magnetic beads to recover fragmented DNA.

[0115] 2. Library Construction

[0116] The cfDNA is end-repaired and "A" is added, and then the pre-capture intermediate library is obtained through the process of adapter connection, purification, pre-capture PCR (Non-C-PCR) and purification. The qualified samples of the intermediate library concentration quality control are then subjected to subsequent hybridization elution.

[0117] 3. Hybridization capture

[0118] The library that has passed the concentration quality control is subjected to the process of pooling, evaporation, hybridization of mixed probes, elution, PCR of eluted products and purification to obtain the common library after hybridization. The common library is sequenced after passing the concentration and fragment distribution quality control.

[0119] 4. Sequencing and Data Analysis

[0120] The sequencing was performed using a DNBSEQ-T7 sequencer. The sequencing experiment was performed according to the instructions provided by the manufacturer.

[0121] Embodiment 3:

[0122] According to the method described in Example 2, plasma samples of tumor patients who have undergone tumor tissue sequencing were selected for testing. Sample composition: plasma samples of 193 patients with pan-cancer tumor tissues with MSS and 13 plasma samples of patients with MSI-H tumor tissues and MSAF (maximum somatic variation frequency)>0.4%. Use the 15 probes (size 1.8K) shown in Table 2 and mix them with the FGFR probe set in use (see patent document with application number 202110533156.6) to perform hybridization capture experiments, and compare the performance of this design with the conventional MSI probe design in terms of capture efficiency and depth coefficient. The results are as follows. Figures 2-3 The "Maximum System Allele Frequency" (MSAF) refers to the maximum frequency of an allele in a specific population or database.

[0123] from Figure 2 It can be seen that when no MSI probe is added, the capture efficiency of the FGFR probe group (see patent document with application number 202110533156.6) is about 48%. When 15 conventional MSI probes are added, the capture efficiency drops to 42%. However, when 15 probes shown in Table 2 are added, the capture efficiency is the same as the capture efficiency without adding any MSI probe, both of which are 48%.

[0124] from Figure 3 It can be seen that the 15 MSI probes designed by the method described in Example 1 have an average depth factor that is 32% higher than that of conventional MSI probes, and have good uniformity. The depth factor = the number of bases in the MSI probe region / the average depth of the probe panel.

[0125] Embodiment 4:

[0126] The capture probes shown in Table 2 were used to detect the plasma samples of the tumor patients as described in Example 3, and the MSI status of the samples was determined according to the sequencing results. The test results are shown in Table 3. The test results of the tumor tissue samples were used as reference results to evaluate the detection performance of the 15 probes shown in Table 2, wherein the results of plasma samples with microsatellite instability (MSI) were positive, and the results of plasma samples with microsatellite stability (MSS) were negative, and the statistical results of the detection performance were shown in Table 4.

[0127] As shown in Tables 3 and 4, by comparing the MSI status of tumor tissue and the MSI detection status of plasma, under the condition of MSAF>0.4%, the specificity was 100.0%, the sensitivity was 92.3%, and the positive detection rate was 100.0%.

[0128] Table 3 Sample test results

[0129]

[0130]

[0131] Table 4 Performance results

[0132]

[0133] Sensitivity is the proportion of positive results from the plasma test method among positive results from tumor tissue samples. Specificity is the proportion of negative results from the test method among negative results from the reference method. Positive Predictive Value (PPV) is the probability that a subject with a positive test result actually has the disease.

[0134] In summary, the feasibility of this design method and the improvement of detection performance have been verified from the aspects of capture efficiency and MSI probe depth coefficient performance.

[0135] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A nucleic acid probe for capturing a target sequence, characterized in that: The nucleic acid probe does not cover the target sequence, and the nucleic acid probe comprises: Covering the first fragment of the upstream flanking sequence of the target sequence, A second segment covering the downstream flanking sequence of the target sequence, and A linker connects the first fragment and the second fragment.

2. The nucleic acid probe according to claim 1, characterized in that The target sequence includes point mutations, repeated sequences, copy number variations, insertion or deletion sequences and / or fusion sequences; Preferably, the target sequence includes a microsatellite locus; Preferably, the target sequence includes a microsatellite instability site; The length of the first fragment or the second fragment ranges from 10 to 300 nt; Preferably, the length of the first fragment or the second fragment ranges from 30 to 150 nt; Preferably, the length of the first fragment or the second fragment ranges from 40 to 80 nt. Preferably, the first segment and the second segment have the same or different lengths; Preferably, the linker length is 3 to 20 nt.

3. The nucleic acid probe according to claim 1, characterized in that The nucleic acid probe has a nucleotide sequence as shown in any one or more of SEQ ID NOs: 1 to 15, or a nucleotide sequence having 85% sequence identity therewith.

4. A method for designing a nucleic acid probe according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: A1) taking the target sequence as the target, obtaining a first fragment covering the upstream flanking sequence of the target sequence, and a second fragment covering the downstream flanking sequence of the target sequence; A2) connecting the first fragment and the second fragment using a linker to form a nucleic acid probe that captures the target sequence; and Optionally, A3) performing homology filtering on the nucleic acid probes obtained in step A2) to screen out nucleic acid probes with low homology.

5. The design method according to claim 4, characterized in that: The screening in step A3) comprises the following steps: B1) counting the sequence identity of the homologous sequences of the nucleic acid probes obtained in step A2) on the genome; B2) counting the number P of homologous sequences of the nucleic acid probe on the genome whose sequence identity is higher than a threshold value M; B3) screening out nucleic acid probes whose number P is lower than a threshold value N; Preferably, the threshold value M=60% to 99.9%; Preferably, the threshold N=3-100.

6. A nucleic acid probe design system, characterized in that: The system is used to execute the design method according to claim 4 or 5, and the system includes the following modules: A fragment screening module is used to screen out the first fragment and the second fragment covering the upstream and downstream flanking regions of the target sequence respectively; The connection module connects the two fragments through the linker to form a complete probe; and Optionally, a homologous sequence screening module screens out probes with low homology to the genomic sequence as nucleic acid probes.

7. A kit comprising the nucleic acid probe according to any one of claims 1 to 3.

8. A detection method, comprising: The target sequence in the test sample of the subject is detected using the nucleic acid probe described in any one of claims 1 to 3 or the kit described in claim 7.

9. The detection method according to claim 8, characterized in that: The target sequence includes point mutations, repeated sequences, copy number variations, insertion or deletion sequences and / or fusion sequences; Preferably, the target sequence includes a microsatellite locus; Preferably, the target sequence includes a microsatellite instability site; Preferably, the detection method further comprises judging the sample to be tested as microsatellite instability positive according to the detection result; Preferably, the detection method further comprises: judging that the subject has cancer by judging that the sample to be tested is microsatellite instability positive; Preferably, the sample to be tested includes a tissue or body fluid sample; Preferably, the body fluid sample includes: saliva or saliva supernatant, whole blood, serum, plasma, milk, urine, lumbar or ventricular CSF, lymph, prostatic fluid, semen, sputum, stool diluted supernatant, tears, tumor cells, alveolar lavage fluid, sputum, pus, nasopharyngeal swab, oral swab, cerebrospinal fluid, pleural effusion, bile, amniotic fluid, peritoneal fluid, aqueous humor, vitreous humor, vaginal discharge diluted supernatant and their processed products; Preferably, the tissue sample includes tissues, paraffin sections and processed materials thereof.

10. A storage medium, wherein the computer program, when executed by a processor, implements the steps of the design method of claim 4 or 5, the design system of claim 6 and / or the detection method of claim 8 or 9.

11. Use of the nucleic acid probe according to any one of claims 1 to 3, the design method according to claim 4 or 5, the design system according to claim 6, the kit according to claim 7, and / or the detection method according to claim 8 or 9 in target sequence detection.

12. The use according to claim 11, characterized in that The use includes early screening, diagnosis, auxiliary diagnosis or prediction of prognosis of cancer, Preferably, the target sequence includes point mutations, repeated sequences, copy number variations, insertion or deletion sequences and / or fusion sequences; Preferably, the target sequence includes a microsatellite locus; Preferably, the target sequence includes a microsatellite instability site.

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