Probe and method for detecting tiny residual focus

Abstract

The invention discloses a probe and a method for detecting tiny residual focuses. The probe for detecting the tiny residual focus is a four-layer encrypted imbricated hybrid capture probe, two layers of probes are wild probes, two layers of probes are mutant probes, the nucleic acid sequence of the wild probes comprises the sequence as shown in SEQ ID NO.1-SEQ ID NO.74, and the nucleic acid sequence of the mutant probes comprises the sequence as shown in SEQ ID NO.75-SEQ ID NO.149. The invention further discloses a kit for detecting the tiny residual focus. According to the invention, the four-layer encrypted imbricated hybrid capture probe for detecting the tiny residual focus is designed, a tiny residual focus molecule detection method with high sensitivity and good specificity is provided, the low-frequency mutation capture efficiency is high, and the mutation frequency detection limit of ct DNA containing the tiny residual focus can be as low as 0.005%.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a probe and a method for detecting micro residual lesions. Background Art

[0002] Minimal residual disease (MRD) in solid tumors refers to the small amount of tumor cells or DNA fragments that remain in the body of patients with solid tumors after radical treatment (such as surgery, chemoradiotherapy, etc.). These remnants can be detected through circulating tumor DNA (ctDNA) in the blood, but are difficult to detect with traditional imaging or pathology methods. The presence of MRD is significantly associated with the risk of tumor recurrence. Its detection can help assess treatment efficacy, predict recurrence, and guide subsequent treatment decisions.

[0003] MRD detection based on next-generation sequencing (NGS) is currently the mainstream technology in the field of solid tumors. Its core is to identify extremely low-abundance ct DNA mutation signals in the blood through highly sensitive methods. NGS technology routes are mainly divided into two categories:

[0004] Tumor-informed (tissue prior strategy): Tumor tissue samples need to be obtained in advance, and patient-specific mutation sites are screened through whole exome sequencing (WES) or large panel testing, and then a personalized panel is customized for subsequent ct DNA tracking.

[0005] Tumor-naive (tissue-free strategy): It does not rely on tumor tissue and directly detects high-frequency mutations in the blood through a fixed panel. However, its sensitivity is low (for example, the sensitivity for early lung cancer in the Lung-CLiP study was only 41%-67%). Moreover, due to the fixed panel, it can only cover hotspot mutation areas and cannot comprehensively monitor the mutation sites of patients with solid tumors, which may lead to missed detection.

[0006] Both analytical methods have their advantages and disadvantages. Tumor-informed approaches offer relatively higher accuracy, but they require tissue sample results and require sequencing depths exceeding 200,000 for subsequent monitoring samples. Furthermore, the need to synthesize personalized panels each time poses significant challenges to synthesis capacity, batch stability, and timeliness. Tumor-naive approaches, due to their use of fixed panels, are more convenient to perform and facilitate quality control. However, they cannot meet the needs of personalized tumor heterogeneity. Panel sizing is crucial: too large a panel wastes sequencing data, while too small a panel may underrepresent mutation sites. Furthermore, due to the inherent sensitivity limitations of next-generation sequencing platforms, a significant amount of background noise mutations around 0.1% are generated, leading to high false-positive rates due to interference from wild-type sequences. Without tissue mutations as a reference, setting filtering thresholds is difficult, and detection thresholds generally require higher thresholds, resulting in inefficient capture of low-frequency mutations and poor sensitivity.

[0007] Therefore, there is an urgent need to provide a tumor-informed MRD molecular detection method / kit with higher sensitivity and better specificity to help clinicians accurately identify MRD-positive patients, predict recurrence and guide subsequent treatment. Summary of the Invention

[0008] In response to the shortcomings of existing technologies and practical needs, the present invention provides a probe and method for detecting minimal residual disease (MRD). The purpose is to provide a highly sensitive and specific tumor-informed MRD molecular detection method. By optimizing the probe design for the mutation sites detected by initial WES, more accurate subsequent monitoring can be performed, reducing the detection limit of ctDNA mutation frequency to 0.005%.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In the first aspect, the present invention provides a probe for detecting minimal residual lesions, wherein the probe for detecting minimal residual lesions is a four-layer encrypted shingled hybridization capture probe, wherein two layers of probes are wild-type probes and two layers are mutant probes, the nucleic acid sequence of the wild-type probe includes the sequence described in SEQ ID NO.1-SEQ ID NO.74, and the nucleic acid sequence of the mutant probe includes the sequence described in SEQ ID NO.75-SEQ ID NO.149.

[0011] The present invention provides a highly sensitive and specific molecular detection probe for minimal residual lesions. The probe is a four-layer encrypted shingled hybridization capture probe, which adds two layers of mutant probes to two layers of wild-type probes. It has high efficiency in capturing low-frequency mutations, and the detection limit of ct DNA mutation frequency containing minimal residual lesions can be as low as 0.005%.

[0012] (1) Point mutation

[0013] AKT1 gene p.E17K

[0014] >chr14:105246515-105246615(Wild 1)

[0015] SEQ ID NO.1:

[0016] GAGGAAGTAGCGTGGCCGCCAGGTCTTGATGTACTCCCCTACAGACGTGCGGGTGGTGAGAGC CACGCACACTCTACCCGTCAGACCCTCGCCAGGCAGC.

[0017] >chr14:105246485-105246585(Wild 2)

[0018] GTAGCCAATGAAGGTGCCATCATTCTTGAGGAGGAAGTAGCGTGGCCGCCAGGTCTTGATGTAC TCCCCTACAGACGTGCGGGTGGTGAGAGCCACGCA (SEQ ID NO. 2).

[0019] >chr14:105246500-105246600 (mutation 1)

[0020] GTAGCCAATGAAGGTGCCATCATTCTTGAGGAGGAAGTAGCGTGGCCGCCAGGTCTTGATGTAC TTCCCTACAGACGTGCGGGTGGTGAGAGCCACGCAC (SEQ ID NO. 75).

[0021] >chr14:105246500-105246600 (mutation 2)

[0022] GAGGAAGTAGCGTGGCCGCCAGGTCTTGATGTACTTCCCTACAGACGTGCGGGTGGTGAGAGCC ACGCACACTCTACCCGTCAGACCCTCGCCAGGCAGC (SEQ ID NO. 76).

[0023] ALK gene p.F1174L

[0024] >chr2:29443659-29443759 (wild type 1)

[0025] CTCACCCCAATGCAGCGAACAATGTTCTGGTGGTTGAATTTGCTGCAGAGCAGAGAGGGATGTA ACCAAAATTAACTGAGCTGAGTCTGGGCAAATCTTA (SEQ ID NO.3).

[0026] >chr2:29443629-29443729 (wild type 2)

[0027] AGCAGGATGAACCGGGGCAGGGATTGCAGGCTCACCCCAATGCAGCGAACAATGTTCTGGTGG TTGAATTTGCTGCAGAGCAGAGAGGGATGTAACCAAA (SEQ ID NO.4).

[0028] >chr2:29443644-29443744 (mutation 1)

[0029] AGCAGGATGAACCGGGGCAGGGATTGCAGGCTCACCCCAATGCAGCGAACAATGTTCTGGTGG TTTAATTTGCTGCAGAGCAGAGAGGGATGTAACCAAA (SEQ ID NO.77).

[0030] >chr2:29443644-29443744 (mutation 2)

[0031] CTCACCCCAATGCAGCGAACAATGTTCTGGTGGTTTAATTTGCTGCAGAGCAGAGAGGGATGTA ACCAAAATTAACTGAGCTGAGTCTGGGCAAATCTTA (SEQ ID NO.78).

[0032] BRAF gene V600K

[0033] >chr7:140453101-140453201 (wild type 1)

[0034] TTCAAACTGATGGGACCCACTCCATCGAGATTTCACTGTAGCTAGACCAAAATCACCTATTTTTA CTGTGAGGTCTTCATGAAGAAATATATCTGAGGTG(SEQ ID NO.5).

[0035] >chr7:140453071-140453171(wild type 2)

[0036] TACCATCCACAAAATGGATCCAGACAACTGTTCAAACTGATGGGACCCACTCCATCGAGATTTCA CTGTAGCTAGACCAAAATCACCTATTTTTACTGTG(SEQ ID NO.6).

[0037] >chr7:140453086-140453186(mutation 1)

[0038] TACCATCCACAAAATGGATCCAGACAACTGTTCAAACTGATGGGACCCACTCCATCGAGATTTCT TTGTAGCTAGACCAAAATCACCTATTTTTACTGTG(SEQ ID NO.79).

[0039] >chr7:140453086-140453186(mutation 2)

[0040] TTCAAACTGATGGGACCCACTCCATCGAGATTTCTTTGTAGCTAGACCAAAATCACCTATTTTTAC TGTGAGGTCTTCATGAAGAAATATATCTGAGGTG(SEQ ID NO.80).<000008​​​​​​​​​​​

[0045] TTACCATCCACAAAATGGATCCAGACAACTGTTCAAACTGATGGGACCCACTCCATCGAGATTTC ACTGTAGCTAGACCAAAATCACCTATTTTTACTGT(SEQ ID NO.8).

[0046] >chr7:140453085 - 140453185(Mutation 1)

[0047] TTACCATCCACAAAATGGATCCAGACAACTGTTCAAACTGATGGGACCCACTCCATCGAGATTTC TCTGTAGCTAGACCAAAATCACCTATTTTTACTGT(SEQ ID NO.81).

[0048] >chr7:140453085 - 140453185(Mutation 2)

[0049] GTTCAAACTGATGGGACCCACTCCATCGAGATTTCTCTGTAGCTAGACCAAAATCACCTATTTTT ACTGTGAGGTCTTCATGAAGAAATATATCTGAGGT(SEQ ID NO.82).

[0050] BRCA1 gene p.R1443*

[0051] >chr17:41234415 - 41234515(Wild type 1)

[0052] CACACCTTTTTCTGATGTGCTTTGTTCTGGATTTCGCAGGTCCTCAAGGGCAGAAGAGTCACTTA TGATGGAAGGGTAGCTGTTAGAAGGCTGGCTCCCA(SEQ ID NO.9).

[0053] >chr17:41234385 - 41234485(Wild type 2)

[0054] GCTTAAGATATCAGTGTTTGGCCAACAATACACACCTTTTTCTGATGTGCTTTGTTCTGGATTTCG CAGGTCCTCAAGGGCAGAAGAGTCACTTATGATG(SEQ ID NO.10).

[0055] >chr17:41234400-41234500 (mutation 1)

[0056] GCTTAAGATATCAGTGTTTGGCCAACAATACACACCTTTTTCTGATGTGCTTTGTTCTGGATTTCA CAGGTCCTCAAGGGCAGAAGAGTCACTTATGATG (SEQ ID NO. 83).

[0057] >chr17:41234400-41234500 (mutation 2)

[0058] CACACCTTTTTCTGATGTGCTTTGTTCTGGATTTCACAGGTCCTCAAGGGCAGAAGAGTCACTTA TGATGGAAGGGTAGCTGTTAGAAGGCTGGCTCCCA (SEQ ID NO. 84).

[0059] BRCA2 gene p.N1784Tfs*7 (mutation type deletion)

[0060] >chr13:32913801-32913901(Wild 1)

[0061] TGGTATTGAGCCAGTATTGAAGAATGTTGAAGATCAAAAAAACACTAGTTTTTCCAAAGTAATAT CCAATGTAAAAGATGCAAATGCATACCCACAAACT (SEQ ID NO. 11).

[0062] >chr13:32913771-32913871(Wild 2)

[0063] AGGATATCTCTCAAAAAATAAACTTGATTCTGGTATTGAGCCAGTATTGAAGAATGTTGAAGATC AAAAAAACACTAGTTTTTCCAAAGTAATATCCAAT (SEQ ID NO. 12).

[0064] >chr13:32913786-32913887 (mutation 1)

[0065] AGGATATCTCTCAAAAAATAAACTTGATTCTGGTATTGAGCCAGTATTGAAGAATGTTGAAGATC AAAAAACACTAGTTTTTCCAAAGTAATATCCAATG(SEQ ID NO.85).

[0066] >chr13:32913786-32913887(Mutation 2)

[0067] TGGTATTGAGCCAGTATTGAAGAATGTTGAAGATCAAAAAACACTAGTTTTTCCAAAGTAATATC CAATGTAAAAGATGCAAATGCATACCCACAAACTG(SEQ ID NO.86).

[0068] BRCA2 gene p.K1691Nfs*15(Mutant has a deletion)[[ID=])

[0069] >chr13:32913523-32913623(Wild type 1)

[0070] AAAACTTCTGTGAGTCAGACTTCATTACTTGAAGCAAAAAAATGGCTTAGAGAAGGAATATTTG ATGGTCAACCAGAAAGAATAAATACTGCAGATTATG(SEQ ID NO.13).

[0071] >chr13:32913493-32913593(Wild type 2)

[0072] GCCTTAGCTTTTTACACAAGTTGTAGTAGAAAAACTTCTGTGAGTCAGACTTCATTACTTGAAGC AAAAAAATGGCTTAGAGAAGGAATATTTGATGGTC(SEQ ID NO.14).

[0073] >chr13:32913508-32913609(Mutation 1)

[0074] GCCTTAGCTTTTTACACAAGTTGTAGTAGAAAAACTTCTGTGAGTCAGACTTCATTACTTGAAGC AAAAAATGGCTTAGAGAAGGAATATTTGATGGTCA(SEQ ID NO.87).

[0075] >chr13:32913508-32913609(Mutation 2)

[0076] AAAACTTCTGTGAGTCAGACTTCATTACTTGAAGCAAAAAATGGCTTAGAGAAGGAATATTTGAT GGTCAACCAGAAAGAATAAATACTGCAGATTATGT(SEQ ID NO.88).

[0077] BRCA2 gene p.D1420Y

[0078] >chr13:32912714-32912814(Wild type 1)

[0079] AGTTAACTGCTACTAAAACGGAGCAAAATATAAAAGATTTTGAGACTTCTGATACATTTTTTCAG ACTGCAAGTGGGAAAAATATTAGTGTCGCCAAAGA(SEQ ID NO.15).

[0080] >chr13:32912684-32912784(Wild type 2)

[0081] CATGTCATGGTAATACTTCAAATAAAGAACAGTTAACTGCTACTAAAACGGAGCAAAATATAAAA GATTTTGAGACTTCTGATACATTTTTTCAGACTGC(SEQ ID NO.16).

[0082] >chr13:32912699-32912799(Mutation 1)

[0083] CATGTCATGGTAATACTTCAAATAAAGAACAGTTAACTGCTACTAAAACGGAGCAAAATATAAAA TATTTTGAGACTTCTGATACATTTTTTCAGACTGC(SEQ ID NO.89).

[0084] >chr13:32912699-32912799(Mutation 2)

[0085] AGTTAACTGCTACTAAAACGGAGCAAAATATAAAATATTTTGAGACTTCTGATACATTTTTTCAGA CTGCAAGTGGGAAAAATATTAGTGTCGCCAAAGA(SEQ ID NO.90).

[0086] CDK12 gene p.P250H

[0087] >chr17:37619037-37619137 (wild 1)

[0088] GGGAGCTTCTTATGGCCAAGATTATGACCTTAGTCCCTCACGATCTCATACCTCGAGCAATTATGA CTCCTACAAGAAAAGTCCTGGAAGTACCTCGAGA (SEQ ID NO. 17).

[0089] >chr17:37619007-37619107(Wild 2)

[0090] TGACAGTCCCAAACAAGATGATAGCCCCTCGGGAGCTTCTTATGGCCAAGATTATGACCTTAGTC CCTCACGATCTCATACCTCGAGCAATTATGACTCC (SEQ ID NO. 18).

[0091] >chr17:37619022-37619122 (mutation 1)

[0092] TGACAGTCCCAAACAAGATGATAGCCCCTCGGGAGCTTCTTATGGCCAAGATTATGACCTTAGTC ACTCACGATCTCATACCTCGAGCAATTATGACTCC (SEQ ID NO. 91).

[0093] >chr17:37619022-37619122 (mutation 2)

[0094] GGGAGCTTCTTATGGCCAAGATTATGACCTTAGTCACTCACGATCTCATACCTCGAGCAATTATGA CTCCTACAAGAAAAGTCCTGGAAGTACCTCGAGA (SEQ ID NO. 92).

[0095] EGFR gene p.L858R

[0096] >chr7:55259479-55259579(Wild 1)

[0097] AACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAA AGAATACCATGCAGAAGGAGGCAAAGTAAGGAGGTGG(SEQ ID NO.19).

[0098] >chr7:55259449-55259549(wild type 2)

[0099] CGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGG GCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATAC(SEQ ID NO.20).

[0100] >chr7:55259464-55259564(mutation 1)

[0101] CGACCTGGCAGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGG GCGGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATAC(SEQ ID NO.93).

[0102] >chr7:55259464-55259564(mutation 2)

[0103] AACACCGCAGCATGTCAAGATCACAGATTTTGGGCGGGCCAAACTGCTGGGTGCGGAAGAGAA AGAATACCATGCAGAAGGAGGCAAAGTAAGGAGGTGG(SEQ ID NO.94).

[0104] EGFR gene p.E746_A750del(deletion occurred in the mutant)

[0105] >chr7:55242436-55242536(wild type 1)

[0106] AGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAA GGAAATCCTCGATGTGAGTTTCTGCTTTGCTGTGTG(SEQ ID NO.21).

[0107] >chr7:55242406-55242506(Wild 2)

[0108] TGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTA AGAGAAGCAACATCTCCGAAAGCCAACAAGGAAAT (SEQ ID NO. 22).

[0109] >chr7:55242414-55242529 (mutation 1)

[0110] CTCTCTCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAA AACATCTCCGAAAGCCAACAAGGAAATCCTCGATG (SEQ ID NO. 95).

[0111] >chr7:55242414-55242529 (mutation 2)

[0112] GAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAAACATCTCCGAAAGCCAACAAGGAAATCC TCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCCA (SEQ ID NO. 96).

[0113] EGFR gene p.L747_P753delinsS (mutation type deletion)

[0114] >chr7:55242443-55242543 (wild 1)

[0115] TAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATC CTCGATGTGAGTTTCTGCTTTGCTGTGTGGGGGTCC (SEQ ID NO. 23).

[0116] >chr7:55242413-55242513(Wild 2)

[0117] GGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAA GCAACATCTCCGAAAGCCAACAAGGAAATCCTCGAT(SEQ ID NO.24).

[0118] >chr7:55242419-55242537(Mutation 1)

[0119] TCTGTCATAGGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAA TCGAAAGCCAACAAGGAAATCCTCGATGTGAGTTTC(SEQ ID NO.97).

[0120] >chr7:55242419-55242537(Mutation 2)

[0121] TGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATCGAAAGCCAACAAGGAAATCCTCGATGTG AGTTTCTGCTTTGCTGTGTGGGGGTCCATGGCTCTG(SEQ ID NO.98).

[0122] EGFR gene p.T790M

[0123] >chr7:55249035-55249135(Wild type 1)

[0124] GGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCCTGG ACTATGTCCGGGAACACAAAGACAATATTGGCTCC(SEQ ID NO.25).

[0125] >chr7:55249005-55249105(Wild type 2)

[0126] CGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATC ACGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTAT(SEQ ID NO.26).

[0127] >chr7:55249020-55249120(Mutation 1)

[0128] CGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATC ATGCAGCTCATGCCCTTCGGCTGCCTCCTGGACTAT(SEQ ID NO.99).

[0129] >chr7:55249020-55249120(Mutation 2)

[0130] GGGCATCTGCCTCACCTCCACCGTGCAGCTCATCATGCAGCTCATGCCCTTCGGCTGCCTCCTGG ACTATGTCCGGGAACACAAAGACAATATTGGCTCC(SEQ ID NO.100).

[0131] EGFR gene p.D770_N771insG

[0132] >chr7:55248976-55249076(Wild type 1)

[0133] TCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCA TCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGC(SEQ ID NO.27).

[0134] >chr7:55248946-55249046(Wild type 2)

[0135] CATGCGAAGCCACACTGACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGG ACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCC(SEQ ID NO.28).

[0136] >chr7:55248963-55249060(Mutation 1)

[0137] TGCGAAGCCACACTGACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGAC GGTAACCCCCACGTGTGCCGCCTGCTGGGCATCTGC(SEQ ID NO.101).

[0138] >chr7:55248963-55249060(Mutation 2)

[0139] CCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACGGTAACCCCCACGTGTGCCGCCTGCTGGGC ATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAG(SEQ ID NO.102).

[0140] EGFR gene p.S768I

[0141] >chr7:55248969-55249069(Wild type 1)

[0142] CTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTG CTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATC(SEQ ID NO.29).

[0143] >chr7:55248939-55249039(Wild type 2)

[0144] TGGCCACCATGCGAAGCCACACTGACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCC AGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGC(SEQ ID NO.30).

[0145] >chr7:55248954-55249054(Mutation 1)

[0146] TGGCCACCATGCGAAGCCACACTGACGTGCCTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCC ATCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGC(SEQ ID NO.103).

[0147] >chr7:55248954-55249054(Mutation 2)

[0148] CTCTCCCTCCCTCCAGGAAGCCTACGTGATGGCCATCGTGGACAACCCCCACGTGTGCCGCCTG CTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATC(SEQ ID NO.104).

[0149] EGFR gene p.L861Q

[0150] >chr7:55259488-55259588 (wild type 1)

[0151] GCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGCTGGGTGCGGAAGAGAAAGAATACCAT GCAGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTC (SEQ ID NO.31).

[0152] >chr7:55259458-55259558 (wild type 2)

[0153] AGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAA ACTGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAA (SEQ ID NO.32).

[0154] >chr7:55259473-55259573 (mutation 1)

[0155] AGCCAGGAACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAA ACAGCTGGGTGCGGAAGAGAAAGAATACCATGCAGAA (SEQ ID NO.105).

[0156] >chr7:55259473-55259573 (mutation 2)

[0157] GCATGTCAAGATCACAGATTTTGGGCTGGCCAAACAGCTGGGTGCGGAAGAGAAAGAATACCAT GCAGAAGGAGGCAAAGTAAGGAGGTGGCTTTAGGTC (SEQ ID NO.106).

[0158] EGFR gene p.G719C

[0159] >chr7:55241671-55241771 (wild type 1)

[0160] TGAAGGAAACTGAATTCAAAAAGATCAAAGTGCTGGGCTCCGGTGCGTTCGGCACGGTGTATAA GGTAAGGTCCCTGGCACAGGCCTCTGGGCTGGGCCG(SEQ ID NO.33).

[0161] >chr7:55241641-55241741(wild type 2)

[0162] AAGCTCCCAACCAAGCTCTCTTGAGGATCTTGAAGGAAACTGAATTCAAAAAGATCAAAGTGCT GGGCTCCGGTGCGTTCGGCACGGTGTATAAGGTAAG(SEQ ID NO.34).

[0163] >chr7:55241656-55241756(mutation 1)

[0164] AAGCTCCCAACCAAGCTCTCTTGAGGATCTTGAAGGAAACTGAATTCAAAAAGATCAAAGTGCT GTGCTCCGGTGCGTTCGGCACGGTGTATAAGGTAAG(SEQ ID NO.107).

[0165] >chr7:55241656-55241756(mutation 2)

[0166] TGAAGGAAACTGAATTCAAAAAGATCAAAGTGCTGTGCTCCGGTGCGTTCGGCACGGTGTATAA GGTAAGGTCCCTGGCACAGGCCTCTGGGCTGGGCCG(SEQ ID NO.108).

[0167] ERBB2 gene p.Y772_A775dup (duplication occurs in the mutant)

[0168] >chr17:37880955-37881055(wild type 1)

[0169] TCTCAGCGTACCCTTGTCCCCAGGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCC CGCCTTCTGGGCATCTGCCTGACATCCACGGTGCA(SEQ ID NO.35).

[0170] >chr17:37880925-37881025 (Wild type 2)

[0171] GTGTTTGGGGGTGTGTGGTCTCCCATACCCTCTCAGCGTACCCTTGTCCCCAGGAAGCATACGTG ATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCT (SEQ ID NO.36).

[0172] >chr17:37880946-37881034 (Mutation 1)

[0173] GGGGGTGTGTGGTCTCCCATACCCTCTCAGCGTACCCTTGTCCCCAGGAAGCATACGTGATGGCT TACGTGATGGCTGGTGTGGGCTCCCCATATGTCTC (SEQ ID NO.109).

[0174] >chr17:37880946-37881034 (Mutation 2)

[0175] CGTACCCTTGTCCCCAGGAAGCATACGTGATGGCTTACGTGATGGCTGGTGTGGGCTCCCCATAT GTCTCCCGCCTTCTGGGCATCTGCCTGACATCCAC (SEQ ID NO.110).

[0176] ERBB2 gene p.Y772_A775dup (duplication in the mutant)

[0177] >chr17:37880954-37881054 (Wild type 1)

[0178] CTCTCAGCGTACCCTTGTCCCCAGGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTC CCGCCTTCTGGGCATCTGCCTGACATCCACGGTGC (SEQ ID NO.37).

[0179] >chr17:37880924-37881024 (Wild type 2)

[0180] GGTGTTTGGGGGTGTGTGGTCTCCCATACCCTCTCAGCGTACCCTTGTCCCCAGGAAGCATACGT GATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCC(SEQ ID NO.38).

[0181] >chr17:37880945-37881033(Mutation 1)

[0182] TGGGGGTGTGTGGTCTCCCATACCCTCTCAGCGTACCCTTGTCCCCAGGAAGCATACGTGATGGC ATACGTGATGGCTGGTGTGGGCTCCCCATATGTCT(SEQ ID NO.111).

[0183] >chr17:37880945-37881033(Mutation 2)

[0184] GCGTACCCTTGTCCCCAGGAAGCATACGTGATGGCATACGTGATGGCTGGTGTGGGCTCCCCATA TGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCA(SEQ ID NO.112).

[0185] IDH1 gene p.R132G

[0186] >chr2:209113077-209113177(Wild type 1)

[0187] GCCAACATGACTTACTTGATCCCCATAAGCATGACGACCTATGATGATAGGTTTTACCCATCCACT CACAAGCCGGGGGATATTTTTGCAGATAATGGCT(SEQ ID NO.39).

[0188] >chr2:209113047-209113147(Wild type 2)

[0189] GAAAAAAAAAACATGCAAAATCACATTATTGCCAACATGACTTACTTGATCCCCATAAGCATGAC GACCTATGATGATAGGTTTTACCCATCCACTCACA(SEQ ID NO.40).

[0190] >chr2:209113062-209113162(Mutation 1)

[0191] GAAAAAAAAAACATGCAAAATCACATTATTGCCAACATGACTTACTTGATCCCCATAAGCATGAC CACCTATGATGATAGGTTTTACCCATCCACTCACA(SEQ ID NO.113).

[0192] >chr2:209113062-209113162(Mutation 2)

[0193] GCCAACATGACTTACTTGATCCCCATAAGCATGACCACCTATGATGATAGGTTTTACCCATCCACT CACAAGCCGGGGGATATTTTTGCAGATAATGGCT(SEQ ID NO.114).

[0194] IDH2 gene p.R172M

[0195] >chr15:90631802-90631902(Wild type 1)

[0196] CTCCACCCTGGCCTACCTGGTCGCCATGGGCGTGCCTGCCAATGGTGATGGGCTTGGTCCAGCC AGGGACTAGGCGTGGGATGTTTTTGCAGATGATGGG(SEQ ID NO.41).

[0197] >chr15:90631772-90631872(Wild type 2)

[0198] TCGGGGGGTGCCCAGGTCAGTGGATCCCCTCTCCACCCTGGCCTACCTGGTCGCCATGGGCGTG CCTGCCAATGGTGATGGGCTTGGTCCAGCCAGGGAC(SEQ ID NO.42).

[0199] >chr15:90631787-90631887(Mutation 1)

[0200] TCGGGGGGTGCCCAGGTCAGTGGATCCCCTCTCCACCCTGGCCTACCTGGTCGCCATGGGCGTG CATGCCAATGGTGATGGGCTTGGTCCAGCCAGGGAC(SEQ ID NO.115).

[0201] >chr15:90631787-90631887(Mutation 2)

[0202] CTCCACCCTGGCCTACCTGGTCGCCATGGGCGTGCATGCCAATGGTGATGGGCTTGGTCCAGCC AGGGACTAGGCGTGGGATGTTTTTGCAGATGATGGG(SEQ ID NO.116).

[0203] KIT gene p.Y503_F504insAY / p.Y503_F504insAY(Mutant has an insertion)

[0204] >chr4:55592146-55592246(Wild type 1)

[0205] TGTAAGGCTTACAACGATGTGGGCAAGACTTCTGCCTATTTTAACTTTGCATTTAAAGGTAACAA CAAAGGTATATTTCTTTTTAATCCAATTTAAGGGG(SEQ ID NO.43). [[ID=...]]

[0206] >chr4:55592116-55592216(Wild type 2) ...

[0207] AGTGCATTCAAGCACAATGGCACGGTTGAATGTAAGGCTTACAACGATGTGGGCAAGACTTCTG CCTATTTTAACTTTGCATTTAAAGGTAACAACAAAG(SEQ ID NO.44).

[0208] >chr4:5559|2134-55592228(Mutation 1) [[ID=...]]

[0209] GCATTCAAGCACAATGGCACGGTTGAATGTAAGGCTTACAACGATGTGGGCAAGACTTCTGCCT ATGCCTATTTTAACTTTGCATTTAAAGGTAACAACA(SEQ ID NO.117).

[0210] (注意:原文中

[0206] 、 、 等标签后的省略号部分,由于原内容未给出完整信息,所以翻译时保留原样。如果这是格式排版问题,请提供完整准确的原文以便更准确翻译。)>chr4:55592134-55592228(Mutation 2)

[0211] AAGGCTTACAACGATGTGGGCAAGACTTCTGCCTATGCCTATTTTAACTTTGCATTTAAAGGTAA CAACAAAGGTATATTTCTTTTTAATCCAATTTAAG(SEQ ID NO.118).

[0212] KIT gene p.D816V

[0213] >chr4:55599285-55599385(Wild type 1)

[0214] GATCACAAAGATTTGTGATTTTGGTCTAGCCAGAGACATCAAGAATGATTCTAATTATGTGGTTAA AGGAAACGTGAGTACCCATTCTCTGCTTGACAGT(SEQ ID NO.45).

[0215] >chr4:55599255-55599355(Wild type 2)

[0216] AGCCAGAAATATCCTCCTTACTCATGGTCGGATCACAAAGATTTGTGATTTTGGTCTAGCCAGAG ACATCAAGAATGATTCTAATTATGTGGTTAAAGGA(SEQ ID NO.46).

[0217] >chr4:55599270-55599370(Mutation 1)

[0218] AGCCAGAAATATCCTCCTTACTCATGGTCGGATCACAAAGATTTGTGATTTTGGTCTAGCCAGAG TCATCAAGAATGATTCTAATTATGTGGTTAAAGGA(SEQ ID NO.119).

[0219] >chr4:55599270-55599370(Mutation 2)

[0220] GATCACAAAGATTTGTGATTTTGGTCTAGCCAGAGTCATCAAGAATGATTCTAATTATGTGGTTAA AGGAAACGTGAGTACCCATTCTCTGCTTGACAGT(SEQ ID NO.120).

[0221] KRAS gene p.G12D

[0222] >chr12:25398248-25398348 (wild type 1)

[0223] TTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCACCAGCTCCAACTACCACAAGTTTATATTCA GTCATTTTCAGCAGGCCTTATAATAAAAATAATGA (SEQ ID NO.47).

[0224] >chr12:25398218-25398318 (wild type 2)

[0225] GATCATATTCGTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCAC CAGCTCCAACTACCACAAGTTTATATTCAGTCAT (SEQ ID NO.48).

[0226] >chr12:25398233-25398333 (mutation 1)

[0227] GATCATATTCGTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCAT CAGCTCCAACTACCACAAGTTTATATTCAGTCAT (SEQ ID NO.121).

[0228] >chr12:25398233-25398333 (mutation 2)

[0229] TTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCATCAGCTCCAACTACCACAAGTTTATATTCAG TCATTTTCAGCAGGCCTTATAATAAAAATAATGA (SEQ ID NO.122).

[0230] KRAS gene p.G12C

[0231] >chr12:25398249-25398349 (wild type 1)

[0232] TAGCTGTATCGTCAAGGCACTCTTGCCTACGCCACCAGCTCCAACTACCACAAGTTTATATTCAG TCATTTTCAGCAGGCCTTATAATAAAAATAATGAA(SEQ ID NO.49).

[0233] >chr12:25398219-25398319(wild type 2)

[0234] ATCATATTCGTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCACC AGCTCCAACTACCACAAGTTTATATTCAGTCATT(SEQ ID NO.50).

[0235] >chr12:2xxxxx-25398334(mutation 1)

[0236] ATCATATTCGTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCACA AGCTCCAACTACCACAAGTTTATATTCAGTCATT(SEQ ID NO.123).

[0237] >chr12:25398234-25398334(mutation 2)

[0238] TAGCTGTATCGTCAAGGCACTCTTGCCTACGCCACAAGCTCCAACTACCACAAGTTTATATTCAG TCATTTTCAGCAGGCCTTATAATAAAAATAATGAA(SEQ ID NO.124).

[0239] KRAS gene p.Q61H

[0240] >chr12:25380239-25380339(wild type 1)

[0241] CTCATGTACTGGTCCCTCATTGCACTGTACTCCTCTTGACCTGCTGTGTCGAGAATATCCAAGAG ACAGGTTTCTCCATCAATTACTACTTGCTTCCTGT(SEQ ID NO.51).

[0242] >chr12:25380209-25380309(wild type 2) Note: In the original text, there seems to be an error in the "2xxxxx" part in the "ID=9" line. It should be a correct genomic location information. The translation is done based on the provided text as accurately as possible.

[0243] GCAAATACACAAAGAAAGCCCTCCCCAGTCCTCATGTACTGGTCCCTCATTGCACTGTACTCCTC TTGACCTGCTGTGTCGAGAATATCCAAGAGACAGG(SEQ ID NO.52).

[0244] >chr12:25380224-25380324(Mutation 1)

[0245] GCAAATACACAAAGAAAGCCCTCCCCAGTCCTCATGTACTGGTCCCTCATTGCACTGTACTCCTC GTGACCTGCTGTGTCGAGAATATCCAAGAGACAGG(SEQ ID NO.125).

[0246] >chr12:25380224-25380324(Mutation 2)

[0247] CTCATGTACTGGTCCCTCATTGCACTGTACTCCTCGTGACCTGCTGTGTCGAGAATATCCAAGAG ACAGGTTTCTCCATCAATTACTACTTGCTTCCTGT(SEQ ID NO.126).

[0248] KRAS gene G13D

[0249] >chr12:25398245-25398345(Wild type 1)

[0250] GAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCACCAGCTCCAACTACCACAAGTTTATAT TCAGTCATTTTCAGCAGGCCTTATAATAAAAATAA(SEQ ID NO.53).

[0251] >chr12:25398215-25398315(Wild type 2)

[0252] TTGGATCATATTCGTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGC CACCAGCTCCAACTACCACAAGTTTATATTCAGT(SEQ ID NO.54).

[0253] >chr12:25398230-25398330(Mutation 1)

[0254] TTGGATCATATTCGTCCACAAAATGATTCTGAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGT CACCAGCTCCAACTACCACAAGTTTATATTCAGT(SEQ ID NO.127).

[0255] >chr12:25398230-25398330(Mutation 2)

[0256] GAATTAGCTGTATCGTCAAGGCACTCTTGCCTACGTCACCAGCTCCAACTACCACAAGTTTATATT CAGTCATTTTCAGCAGGCCTTATAATAAAAATAA(SEQ ID NO.128).

[0257] NRAS gene p.G12A

[0258] ​​​​​​​​​​​​​​​GATCATATTCATCTACAAAGTGGTTCTGGATTAGCTGGATTGTCAGTGCGCTTTTCCCAACACCAG CTGCTCCAACCACCACCAGTTTGTACTCAGTCAT(SEQ ID NO.129).

[0264] >chr1:115258696-115258796(Mutation 2)

[0265] TTAGCTGGATTGTCAGTGCGCTTTTCCCAACACCAGCTGCTCCAACCACCACCAGTTTGTACTCA GTCATTTCACACCAGCAAGAACCTGTTGGAAACCA(SEQ ID NO.130).

[0266] NRAS gene p.Q61R

[0267] >chr1:115256493-115256593(Wild type 1)

[0268] TCATGTATTGGTCTCTCATGGCACTGTACTCTTCTTGTCCAGCTGTATCCAGTATGTCCAACAAAC AGGTTTCACCATCTATAACCACTTGTTTTCTGTA(SEQ ID NO.57).

[0269] >chr1:115256463-115256563(Wild type 2)

[0270] CAAATACACAGAGGAAGCCTTCGCCTGTCCTCATGTATTGGTCTCTCATGGCACTGTACTCTTCTT GTCCAGCTGTATCCAGTATGTCCAACAAACAGGT(SEQ ID NO.58).

[0271] >chr1:115256478-115256578(Mutation 1)

[0272] CAAATACACAGAGGAAGCCTTCGCCTGTCCTCATGTATTGGTCTCTCATGGCACTGTACTCTTCT CGTCCAGCTGTATCCAGTATGTCCAACAAACAGGT(SEQ ID NO.131).

[0273] >chr1:115256478-115256578(Mutation 2)

[0274] TCATGTATTGGTCTCTCATGGCACTGTACTCTTCTCGTCCAGCTGTATCCAGTATGTCCAACAAAC AGGTTTCACCATCTATAACCACTTGTTTTCTGTA(SEQ ID NO.132).

[0275] PDGFRA gene p.D842V

[0276] >chr4:55152057-55152157(Wild type 1)

[0277] AATTGTGAAGATCTGTGACTTTGGCCTGGCCAGAGACATCATGCATGATTCGAACTATGTGTCGA AAGGCAGTGTACGTCCTCACTTCCCTCACTGGTCA(SEQ ID NO.59).

[0278] >chr4:55152027-55152127(Wild type 2)

[0279] TGCTCGCAACGTCCTCCTGGCACAAGGAAAAATTGTGAAGATCTGTGACTTTGGCCTGGCCAGA GACATCATGCATGATTCGAACTATGTGTCGAAAGGC(SEQ ID NO.60).

[0280] >chr4:55152042-55152142(Mutation 1)

[0281] TGCTCGCAACGTCCTCCTGGCACAAGGAAAAATTGTGAAGATCTGTGACTTTGGCCTGGCCAGA GTCATCATGCATGATTCGAACTATGTGTCGAAAGGC(SEQ ID NO.133).

[0282] >chr4:55152042-55152142(Mutation 2)

[0283] AATTGTGAAGATCTGTGACTTTGGCCTGGCCAGAGTCATCATGCATGATTCGAACTATGTGTCGA AAGGCAGTGTACGTCCTCACTTCCCTCACTGGTCA(SEQ ID NO.134).

[0284] PIK3CA gene p.H1047R

[0285] >chr3:178952049-178952149 (wild type 1)

[0286] TTTGGAGTATTTCATGAAACAAATGAATGATGCACATCATGGTGGCTGGACAACAAAAATGGATT GGATCTTCCACACAATTAAACAGCATGCATTGAAC (SEQ ID NO.61).

[0287] >chr3:178952019-178952119 (wild type 2)

[0288] CCTAGCCTTAGATAAAACTGAGCAAGAGGCTTTGGAGTATTTCATGAAACAAATGAATGATGCAC ATCATGGTGGCTGGACAACAAAAATGGATTGGATC (SEQ ID NO.62).

[0289] >chr3:178952034-178952134 (mutation 1)

[0290] CCTAGCCTTAGATAAAACTGAGCAAGAGGCTTTGGAGTATTTCATGAAACAAATGAATGATGCAC GTCATGGTGGCTGGACAACAAAAATGGATTGGATC (SEQ ID NO.135).

[0291] >chr3:178952034-178952134 (mutation 2)

[0292] TTTGGAGTATTTCATGAAACAAATGAATGATGCACGTCATGGTGGCTGGACAACAAAAATGGATT GGATCTTCCACACAATTAAACAGCATGCATTGAAC (SEQ ID NO.136).

[0293] TP53 gene p.R273H

[0294] >chr17:7577084-7577184 (wild type 1)

[0295] CTGTGCGCCGGTCTCTCCCAGGACAGGCACAAACACGCACCTCAAAGCTGTTCCGTCCCAGTAG ATTACCACTACTCAGGATAGGAAAAGAGAAGCAAGA(SEQ ID NO.63).

[0296] >chr17:7577054-7577154(wild type 2)

[0297] GCTCCCCTTTCTTGCGGAGATTCTCTTCCTCTGTGCGCCGGTCTCTCCCAGGACAGGCACAAACA CGCACCTCAAAGCTGTTCCGTCCCAGTAGATTACC(SEQ ID NO.64).

[0298] >chr17:7577069-7577169(mutation 1)

[0299] GCTCCCCTTTCTTGCGGAGATTCTCTTCCTCTGTGCGCCGGTCTCTCCCAGGACAGGCACAAACA TGCACCTCAAAGCTGTTCCGTCCCAGTAGATTACC(SEQ ID NO.137).

[0300] >chr17:7577069-7577169(mutation 2)

[0301] CTGTGCGCCGGTCTCTCCCAGGACAGGCACAAACATGCACCTCAAAGCTGTTCCGTCCCAGTAG ATTACCACTACTCAGGATAGGAAAAGAGAAGCAAGA(SEQ ID NO.138).

[0302] (II) Fusion mutation

[0303] EML4::ALK

[0304] >chr2:42524041-42524141(EML4 wild type)

[0305] AATAAATTGAACCAACATAAGTTAGGCACTATCTGTACGAAATTAATGTTTTAATTAAATGTTTTT CAAATCCATTCACCTGAATGTCTAAGCTTGGCAG(SEQ ID NO.65).

[0306] >chr2:29447548-29447648 (ALK wild type)

[0307] ATATGGTGCCATCCCTCAAAGGGACAGGATAATAGGAGCTAACACTTGTTGCATGGTTACTACGT GCTCGGCAATTTACACATTTCAATTCATTCGATCC (SEQ ID NO. 66).

[0308] >chr2_chr2:35-135(fusion 1)

[0309] TCAAATCCATTCACCTGAATGTCTAAGCTTGGCAGATATGGTGCCATCCCTCAAAGGGACAGGAT AATAGGAGCTAACACTTGTTGCATGGTTACTACGT (SEQ ID NO. 139).

[0310] >chr2_chr2:65-165 (fusion 2)

[0311] TACGAAATTAATGTTTTAATTAAATGTTTTTCAAATCCATTCACCTGAATGTCTAAGCTTGGCAGATATGGTGCCATCCCTCAAAGGGACAGGATAATAG (SEQ ID NO. 140).

[0312] >chr2_insert_chr2:74-174 (fusion 3) new sequence inserted after breakpoint fusion (insertion sequence in the standard)

[0313] TTCACCTGAATGTCTAAGCTTGGCAGGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAAT AGGAACTTCATATGGTGCCATCCCTCAAAGGGACA (SEQ ID NO. 141).

[0314] FGFR3::TACC3

[0315] >chr4:1808665-1808765 (FGFR3 wild-type)

[0316] GTGCTGGCTCTGGCCTGGTGCCACCCGCCTATGCCCCTCCCCCTGCCGTCCCCGGCCATCCTGCC CCCCAGAGTGCTGAGGTGTGGGGCGGGCCTTCTGG(SEQ ID NO.67).

[0317] >chr4:1729563-1729663(TACC3 wild)

[0318] CCCAGGTGCCCTGGCTGACCTGGACTGCTCAAGCTCTTCCCAGAGCCCAGGAAGTTCTGAGAA CCAAATGGTGTCTCCAGGAAAAGTGTCTGGCAGCCCT(SEQ ID NO.68).

[0319] >chr4_chr4:35-135(Fusion 1)

[0320] CCCCAGAGTGCTGAGGTGTGGGGCGGGCCTTCTGGCCCAGGTGCCCTGGCTGACCTGGACTGC TCAAGCTCTTCCCAGAGCCCAGGAAGTTCTGAGAACC(SEQ ID NO.142).

[0321] >chr4_chr4:65-165(Fusion 2)

[0322] CCTCCCCCTGCCGTCCCCGGCCATCCTGCCCCCCAGAGTGCTGAGGTGTGGGGCGGGCCTTCTG GCCCAGGTGCCCTGGCTGACCTGGACTGCTCAAGCT(SEQ ID NO.143).

[0323] TPM3::NTRK1

[0324] >chr1:154137491-154137591(TPM3 wild)

[0325] GCCACTGCACCTGGCCTGAGCATTCCATTTAAAGAGAAACAAAATAATAAAGGCATTGATAGAG ATGAGAAGGCCACAATAGCTCTAAAGGTAGATTTAG(SEQ ID NO.69).

[0326] >chr1:156843542-156843642(NTRK1 wild)

[0327] TGAGACCAGCTTCATCTTCACTGAGTTCCTGGAGCCGGCAGCCAATGAGACCGTGCGGCACGGG TGTCTGCGCCTCAACCAGCCCACCCACGTCAACAAC(SEQ ID NO.70).

[0328] >chr1_chr1:35-135(Fusion 1)

[0329] TGAGAAGGCCACAATAGCTCTAAAGGTAGATTTAGTGAGACCAGCTTCATCTTCACTGAGTTCCT GGAGCCGGCAGCCAATGAGACCGTGCGGCACGGGT(SEQ ID NO.144).

[0330] >chr1_chr1:65-165(Fusion 2)

[0331] GAAACAAAATAATAAAGGCATTGATAGAGATGAGAAGGCCACAATAGCTCTAAAGGTAGATTTA GTGAGACCAGCTTCATCTTCACTGAGTTCCTGGAGC(SEQ ID NO.145).

[0332] CCDC6::RET

[0333] >chr10:61638614-61638714(CCDC6 wild)

[0334] GAAGATATCCCTTCCCTCCAAGCACACAAACTGCATCTTTTCCTTTCCTTTCCCTTATGGCTTGGC CTAAATGAATAGTCATAATTGTTAAATAATTCCT(SEQ ID NO.71). [[ID=##]]

[0335] >chr10:43609947-4361\0047(RET wild)

[0336] TGCCGCACGGTGATCGCAGCCGCTGTCCTCTTCTCCTTCATCGTCTCGGTGCTGCTGTCTGCCTT CTGCATCCACTGCTACCACAAGTTTGCCCACAAGC(SEQ ID NO.72).

[0337] It should be noted that there seems to be an unclear or incorrect "chr10:43609947-4361\0047" in the original text. I've translated it as is, but it might need to be corrected in the original source for a more accurate translation.>chr10_chr10:35-135(Fusion 1)

[0338] CCTAAATGAATAGTCATAATTGTTAAATAATTCCTTGCCGCACGGTGATCGCAGCCGCTGTCCTCT TCTCCTTCATCGTCTCGGTGCTGCTGTCTGCCTT(SEQ ID NO.146).

[0339] >chr10_chr10:65-16(5(Fusion 2)

[0340] TCTTTTCCTTTCCTTTCCCTTATGGCTTGGCCTAAATGAATAGTCATAATTGTTAAATAATTCCTTGCCGCACGGTGATCGCAGCCGCTGTCCTCTTCTC(SEQ ID NO.147).

[0341] CD74::ROS1

[0342] >chr5:149783665-149783765(CD74 wild)

[0343] TTCTATCCCGGTGCTGTCTCTAATTTGCTATGTAACTTTGGACAAGTCCCCTTCCCTCTAGGATTC AGGGTCCTGAAGTAGAAGGTCAAAGGGCCACCCT(SEQ ID NO.73).

[0344] >chr6:117646629-117646729(ROS1 wild)

[0345] CTGGATTACTTAATCCCTCTCTGAAATACCCACAATGGCTCTCCATTTACTGCTTTCAGAATCAGC TACAAACTTCTTTGTGGCATGTGAGGTCTTCTGT(SEQ ID NO.74).

[0346] >chr5_chr6:35-135(Fusion 1)

[0347] CAGGGTCCTGAAGTAGAAGGTCAAAGGGCCACCCTCTGGATTACTTAATCCCTCTCTGAAATAC CCACAATGGCTCTCCATTTACTGCTTTCAGAATCAG(SEQ ID NO.148).

[0348] >chr5_chr6:65-165(fusion 2)

[0349] CTTTGGACAAGTCCCCTTCCCTCTAGGATTCAGGGTCCTGAAGTAGAAGGTCAAAGGGCCACCC TCTGGATTACTTAATCCCTCTCTGAAATACCCACAA (SEQ ID NO. 149).

[0350] Preferably, the length of the wild-type probe and the mutant probe is 90-110bp (e.g., 91bp, 92bp, 95bp, 100bp, 105bp or 110bp), and the length of the bases staggered between probes is 10-20bp (e.g., 10bp, 11bp, 12bp, 13bp, 15bp or 20bp).

[0351] In a second aspect, the present invention provides use of the probe for detecting minimal residual lesions described in the first aspect in preparing a product for detecting minimal residual lesions.

[0352] In a third aspect, the present invention provides a kit for detecting minimal residual lesions, wherein the kit comprises the probe for detecting minimal residual lesions according to the first aspect.

[0353] In a fourth aspect, the present invention provides a method for detecting minimal residual disease for purposes other than disease diagnosis and / or treatment, the method comprising the following steps:

[0354] (1) Sample preparation: Nucleic acid extraction is performed on the FFPE samples and peripheral blood samples submitted by the patient for the initial examination to obtain gDNA; free nucleic acid extraction is performed on the non-invasive blood samples submitted by the patient for subsequent examination to obtain ctDNA;

[0355] (2) Whole exome detection: FFPE sample nucleic acid and peripheral blood sample nucleic acid gDNA are used to construct libraries, hybridize with whole exome probes and capture on the machine to obtain whole exome sequencing results;

[0356] (3) Personalized capture probe design: 30-40 (e.g., 30, 31, 32, 35, 38, or 40) somatic mutation sites are screened from the whole exome sequencing results, and personalized capture probes are designed and customized for the screened mutation sites, wherein the personalized capture probes include the probe for detecting minimal residual lesions described in the first aspect;

[0357] (4) ct DNA sequencing detection: construct a ct DNA library, select a ct DNA sample with a known negative sequencing result for library construction, and simultaneously hybridize the two constructed libraries with the customized capture probe panel in step (3) to capture ct DNA;

[0358] (5) Minimal residual disease sequencing: Use a sequencer to sequence the captured ct DNA;

[0359] (6) Bioinformatics analysis: The ct DNA sequencing results are compared with the human reference genome to obtain the point mutation results of the sample gene.

[0360] Preferably, the screening criteria for selecting 30-40 somatic mutation sites from the whole exome sequencing results in step (3) is: somatic mutation sites whose gene mutation frequency is similar to or half of the tumor cell content.

[0361] Preferably, the somatic mutation site is a somatic class I variation mutation site, a somatic class II variation mutation site, and a site in which the base mutation type in somatic class III variation is a transversion.

[0362] Class I variants have important clinical significance for treatment, prognosis, and diagnosis, mainly because such variants have targeted medications, including those approved by the NMPA, FDA, or national drug regulatory authorities; or there are professional clinical guidelines, such as the CSCO diagnosis and treatment guidelines, the NCCN clinical practice guidelines, etc., that recommend medications.

[0363] Class II mutations have potential clinical significance for treatment, prognosis, and diagnosis, generally referring to mutations in other cancer types that can be targeted with medication.

[0364] Preferably, the somatic mutation site is a somatic variation site with a mutation frequency >10%.

[0365] Preferably, the somatic mutation site is a somatic mutation site that avoids simple repeat sequences and large insertion / deletion mutations.

[0366] Preferably, the somatic mutation site is a somatic mutation site whose GC content of the probe for detecting minimal residual lesions described in the first aspect of the claim is between 30% and 70% (for example, 30%, 32%, 40%, 50%, 60% or 70%).

[0367] Preferably, the step (6) of comparing the ctDNA sequencing results with the human reference genome to obtain the point mutation results of the sample gene includes: comparing the sequencing results with the human reference genome, performing sequence alignment, marking repetitive sequences, somatic mutation detection and germline mutation detection to obtain the point mutation results of the sample gene.

[0368] The method of the present invention can be used to distinguish nucleic acid samples from different sources. By detecting the content and mutation frequency of microresidual lesions in the samples, effective differentiation can be made, thereby improving the sensitivity and accuracy of detection.

[0369] In a fifth aspect, the present invention provides a device for detecting minimal residual lesions, the device comprising a nucleic acid extraction module, a detection module, and a sequencing analysis module;

[0370] The nucleic acid extraction module is used to perform the following operations: extracting nucleic acids from FFPE samples and peripheral blood samples submitted by patients for initial testing to obtain gDNA; extracting free nucleic acids from non-invasive blood samples submitted by patients for subsequent testing to obtain ctDNA;

[0371] The detection module is configured to perform the following steps: constructing a library of FFPE sample nucleic acid and peripheral blood sample nucleic acid gDNA, hybridizing the whole exome probe and capturing the whole exome probe on a machine to obtain whole exome sequencing results, screening 30-40 somatic cell mutation sites from the whole exome sequencing results, designing and customizing personalized capture probes for the screened mutation sites, wherein the personalized capture probes include the probe for detecting minimal residual lesions described in the first aspect, constructing a library of sample ct DNA, and simultaneously selecting a ct DNA sample with a known negative sequencing result for library construction, and simultaneously hybridizing the two constructed libraries with a customized personalized capture probe panel to capture ct DNA;

[0372] The sequencing analysis module is used to perform the following operations: using a sequencer to sequence the captured ct DNA, comparing the ct DNA sequencing results with the human reference genome, performing sequence alignment, marking repeated sequences, somatic cell variation detection, and germline mutation detection to obtain point mutation results of the sample gene.

[0373] Preferably, the screening criteria for selecting 30-40 somatic mutation sites is: somatic mutation sites whose gene mutation frequency is close to or half of the tumor cell content.

[0374] Preferably, the somatic mutation site is a somatic class I variation mutation site, a somatic class II variation mutation site, and a site in which the base mutation type in somatic class III variation is a transversion.

[0375] Preferably, the somatic mutation site is a somatic variation site with a mutation frequency >10%.

[0376] Preferably, the somatic mutation site is a somatic mutation site that avoids simple repeat sequences and large insertion / deletion mutations.

[0377] Preferably, the somatic mutation site is a somatic mutation site whose GC content of the probe for detecting minimal residual lesions described in the first aspect is between 30% and 70%.

[0378] In a sixth aspect, the present invention provides the use of the probe for detecting minimal residual lesions described in the first aspect, the kit for detecting minimal residual lesions described in the third aspect, the method for detecting minimal residual lesions described in the fourth aspect, or the device for detecting minimal residual lesions described in the fifth aspect in the preparation of a product for diagnosing and / or treating cancer.

[0379] Compared with the prior art, the present invention has the following beneficial effects:

[0380] (1) The present invention discloses a molecular detection method for solid tumor MRD with good specificity, high sensitivity and lower detection limit. The method adopts a tumor-informed strategy to first perform WES detection on tumor tissue and paired peripheral blood, and then selects specific sites based on the WES results to customize personalized probes. The main reason for the high sensitivity and lower detection limit of the method of the present invention is that a four-layer probe encryption design is adopted during the design and synthesis of personalized probes, wherein two layers are mutant probes and two layers are wild-type probes, and the distance between each layer of probes is staggered by 10-20bp bases. This design can improve the detection efficiency of low-frequency mutation sites. The detection limit of conventional solid tumor MRD (tumor-informed strategy) on the market is 0.01%, while the minimum detection limit of this method can be as low as 0.005%;

[0381] (2) The present invention uses TNscope software and Vardict software to simultaneously detect point mutations in the bioinformatics analysis process, and merges the two result files to reduce the false positive rate; the use of a sequencer (GeneMind) with four-color fluorescence camera sequencing for detection can greatly reduce the background noise of sequencing and further improve the reliability of the detection results. In addition, the present invention has obvious price advantages and aims to reduce the financial pressure of patients through cost-effective genetic testing services, while facilitating clinical precision diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0382] Figure 1 It is a detection flow chart of the present invention;

[0383] Figure 2 Schematic diagram of the probe for detecting minimal residual lesions of the present invention. DETAILED DESCRIPTION

[0384] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0385] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0386] The specific components of the kit of the present invention are shown in Table 1 below.

[0387] Table 1

[0388]

[0389]

[0390] Kits 1-8 are from the manufacturer: Nanoda (Nanjing) Biotechnology Co., Ltd.; the models are as follows:

[0391] ① Enzyme digestion library construction module: EZ DNA Library Preparation Module v2, 96 servings (Kits 1, 2, 4, and 5);

[0392] ②Universal truncated double-ended unique connector module: Universal Stubby Adapter (UDI) Module Set E2, 96 servings (Kit 3);

[0393] ③ Hybridization capture reagent: ES Hybrid Capture Reagents, 96 units (Kits 6, 7, and 8);

[0394] ④General blocking sequence: NanoBlockers(for NXT), 96 people (reagents 10 and 11);

[0395] *Components 10 and 11 of kit 6 are blocking sequences, and the other components are hybridization capture reagents;

[0396] Kits 9-15 are from the manufacturer: Aigitaikang Biotechnology (Beijing) Co., Ltd.; the models are as follows:

[0397] ①Library building modules: Fast Library Prep Kit v2.0, 96 servings (Kit 9);

[0398] ②Universal adapter kit: UMIAdapter & UDIPrimer 1-96 (Kits 10, 11, and 12);

[0399] ③TargetSeq Hybridization Elution Kit: TargetSeq Hyb&Wash Kit v2.0 (Kits 13, 14, and 15);

[0400] ④General blocking sequence: Eco Universal Blocking Oligo (reagents 28, 29);

[0401] *Components 28 and 29 of kit 13 are blocking sequences, and the other components are hybridization reagents.

[0402] Example 1

[0403] This embodiment provides a method for molecular detection of minimal residual disease (MRD) based on a tumor-informed strategy (tissue-a priori strategy), which includes at least the following steps:

[0404] Step 1. Sample preparation: Extract nucleic acid (gDNA) from the patient's initial FFPE samples and peripheral blood samples; extract free nucleic acid (ctDNA) from the patient's subsequent non-invasive blood samples.

[0405] Step 2: Whole Exome Sequencing (WES) detection: FFPE sample nucleic acid and peripheral blood sample nucleic acid are used to construct libraries, hybridize with WES probes, and capture on the machine to obtain WES sequencing results.

[0406] Step 3. Personalized probe design: 30-40 somatic mutation sites are screened from the WES results for personalized probe design and customization. The probe is a four-layer encrypted shingled hybridization capture probe, of which two layers are wild-type probes and two layers are mutant probes. The nucleic acid sequence of the wild-type probe includes the sequence described in SEQ ID NO.1-SEQ ID NO.74, and the nucleic acid sequence of the mutant probe includes the sequence described in SEQ ID NO.75-SEQ ID NO.149. Site selection must meet the following conditions and priorities: the gene mutation frequency is similar to or half of the tumor cell content, that is, the main clone gene site (homozygous or heterozygous); Class I and Class II variants are preferred, and for Class III variants, sites with base mutation types of transversion are preferred; somatic variant sites with a mutation frequency >10% are preferred.

[0407] Step 4. ctDNA sequencing detection: Construct a ctDNA library and simultaneously hybridize and capture the personalized probe panel customized in step 3 with the negative ctDNA sample library.

[0408] Step 5. MRD sequencing: Use Genemind SURFSeq 5000 sequencer (PE100) for sequencing.

[0409] Step 6. Bioinformatics analysis: The sequencing results are compared with the human reference genome to perform sequence alignment, mark repetitive sequences, detect somatic mutations, and detect germline mutations to obtain point mutation results of the sample gene.

[0410] Example 2

[0411] Based on tumor-informed MRD molecular detection kits and detection methods (WES for initial diagnosis).

[0412] The detection method of this embodiment includes the following steps:

[0413] 1. WES detection: Library construction of FFPE samples and peripheral blood gDNA.

[0414] 1.1 Take the components in kit 1 for the experiment, sample according to the DNA input amount of 500ng, take 0.2mL PCR tubes according to the number of samples, and mark the sample number on the tube cap.

[0415] 1.2 Add the components in the order listed in Table 2 below and set up the reaction system on an ice box.

[0416] Table 2

[0417] Components Volume (μL) DNA samples (FFPE and peripheral blood) 10 (500ng / nucleic acid concentration) Reagent 1 30 Reagent 2 5 Reagent 3 5 Total volume 50

[0418] Reagent 2 and Reagent 3 can be premixed on ice.

[0419] 1.3 Vortex and centrifuge the mixture in the PCR tube, then place it in a PCR instrument to perform the reaction as shown in Table 3.

[0420] Table 3

[0421] temperature Reaction time Number of cycles 25℃ 15 / 23 min (FFPE / peripheral blood) 1 65℃ 30min 1 4℃ Keep 1

[0422] 2. Connector connection:

[0423] 2.1 Preparation before ligation: Take out reagent 5 and reagent 6 from -20℃, thaw them at room temperature, and then prepare the reaction system on an ice box (Table 4):

[0424] Table 4

[0425] Components Volume (μL) Reagent 5 2 Reagent 6 26 Total volume 28

[0426] 2.2 Adapter ligation: First, take 2 μL of reagent 4 and add it to the reaction system in step 1, add it to the bottom of the PCR tube, then add 28 μL of the above-prepared reagent, shake and mix, centrifuge, and place it on the PCR instrument to carry out the reaction as shown in Table 5 below:

[0427] Table 5

[0428] temperature Reaction time Number of cycles 20℃ Keep 1 20℃ 15min 1 4℃ Keep 1

[0429] 3. Purification of ligation product:

[0430] 3.1 Mixing magnetic beads: Oscillate reagent 7 until it is evenly mixed (Important note: Be sure to mix reagent 7 evenly before purification, otherwise it will affect the purification effect).

[0431] 3.2 Adding Magnetic Beads: Add 40 μL (0.5 volume) of Reagent 7 to each well. Pipet up and down 10 times. If bubbles form at the bottom of the tube during pipetting, briefly centrifuge and remix.

[0432] 3.3 Product Binding: Incubate the reaction plate / tube at room temperature for 5 minutes to allow the PCR products to bind to the magnetic beads.

[0433] 3.4 Separation of magnetic beads: Place the PCR plate / tube on a magnetic stand for 5 minutes until the solution becomes clear.

[0434] 3.5 Remove the supernatant: Carefully remove the supernatant from each tube, while being careful not to touch or blow away the magnetic beads.

[0435] 3.6 Wash the magnetic beads: Place the PCR plate / tubes on the magnetic rack. Add 150 μL of freshly prepared 80% ethanol to each tube. Incubate for 30 seconds and then aspirate the supernatant. Be careful not to touch the magnetic beads.

[0436] Important: Avoid contact between 80% ethanol and air. This can cause the ethanol concentration to change, affecting the effectiveness of cleaning the magnetic beads. Prepare only enough 80% ethanol for each use.

[0437] 3.7 Secondary Cleaning: Repeat the previous step. The remaining 80% ethanol can be used for purification of the second round of PCR products.

[0438] 3.8 Remove remaining ethanol: Completely remove any traces of ethanol from each tube. Briefly centrifuge for 10-15 seconds, place the PCR plate / tubes back on the magnetic stand, and use a 10-20 μL pipette tip to remove any remaining ethanol from the bottom of the tube.

[0439] 3.9 Dry the magnetic beads: Place the PCR reaction plate on the magnetic stand at room temperature for 2-5 minutes.

[0440] Important: Do not overdry the beads. They are sufficiently dry when small cracks appear in the center of the bead mass. If large cracks appear throughout the bead mass, or if the beads break into small flakes, they are overdried. Overdried beads will be difficult to resuspend.

[0441] 3.10 Resuspend the beads: Remove the PCR plate / tubes from the magnetic stand and immediately add 20 μL of nuclease-free water to each tube to resuspend the beads. Gently pipette the suspension 10 times. If bubbles form at the bottom of the tube, briefly centrifuge and remix.

[0442] 4. Index PCR amplification:

[0443] 4.1 Reagent preparation: Take out reagents 8 and 9 from reagent kit 5 and thaw them at room temperature.

[0444] Prepare the reaction solution according to the system in Table 6:

[0445] Table 6

[0446] Components Volume (μL) Reagent 8 25 Reagent 9 5 Magnetic bead suspension obtained in step 3 20 Total volume 50

[0447] Note: First, take 25 μL of reagent 8 and add it to the suspension obtained in step 3, shake to mix and centrifuge; then take 5 μL of reagent 9 and add it below the liquid surface of the above suspension.

[0448] 4.2 Vortex and centrifuge the mixture in the PCR tube, then place it in a PCR instrument and perform the following reaction (Table 7):

[0449] Table 7

[0450]

[0451] After the above reaction, a library is obtained.

[0452] 5. Library purification:

[0453] 5.1 Mixing magnetic beads: Oscillate reagent 7 until it is evenly mixed (Important note: Be sure to mix reagent 7 evenly before purification, otherwise it will affect the purification effect).

[0454] 5.2 Adding magnetic beads: Add 50 μL (1 volume) of Reagent 7 to each well. Pipet up and down 10 times. If bubbles form at the bottom of the tube during pipetting, briefly centrifuge and remix.

[0455] 5.3 Product Binding: Incubate the reaction plate / tube at room temperature for 5 minutes to allow the PCR products to bind to the magnetic beads.

[0456] 5.4 Separation of magnetic beads: Place the PCR plate / tube on a magnetic stand for 5 minutes until the solution becomes clear.

[0457] 5.5 Remove the supernatant: Carefully remove the supernatant from each tube, while being careful not to touch or blow away the magnetic beads.

[0458] 5.6 Wash the magnetic beads: Place the PCR plate / tubes on the magnetic stand. Add 150 μL of freshly prepared 80% ethanol to each tube. Incubate for 30 seconds and then aspirate the supernatant. Be careful not to touch the magnetic beads.

[0459] Important: Avoid contact between 80% ethanol and air. This can cause the ethanol concentration to change, affecting the effectiveness of cleaning the magnetic beads. Prepare only enough 80% ethanol for each use.

[0460] 5.7 Secondary Cleaning: Repeat the previous step. The remaining 80% ethanol can be used for purification of the second round of PCR products.

[0461] 5.8 Remove remaining ethanol: Completely remove any traces of ethanol from each tube. Briefly centrifuge for 10-15 seconds, place the PCR plate / tubes back on the magnetic stand, and use a 10-20 μL pipette tip to remove any remaining ethanol from the bottom of the tube.

[0462] 5.9 Dry the magnetic beads: Place the PCR reaction plate on the magnetic stand at room temperature for 2-5 minutes.

[0463] Important: Do not overdry the beads. They are sufficiently dry when small cracks appear in the center of the bead mass. If large cracks appear throughout the bead mass, or if the beads break into small flakes, they are overdried. Overdried beads will be difficult to resuspend.

[0464] 5.10 Resuspend the magnetic beads: Remove the PCR plate / tubes from the magnetic stand and immediately add 32 μL of nuclease-free water to each tube to resuspend the magnetic beads. Gently pipette the suspension 10 times. If bubbles form at the bottom of the tube, briefly centrifuge and remix.

[0465] 5.11 Separate the supernatant: Place the PCR plate / tube back on the magnetic stand until the solution becomes clear, which may take 5 minutes. Pipette 30 μL of the supernatant into a new PCR tube to obtain the purified library.

[0466] 6.1 Qubit quantification: Take 1 μL of library and use Qubit to detect the library concentration. The library quality control standard is: concentration greater than 2 ng / μL and total amount not less than 200 ng.

[0467] 6.2Qseq detected the library length, and the main peak was between 250-550bp.

[0468] 7. Library hybridization:

[0469] 7.1 Remove reagents 10 and 11 from -20°C in kit 6, thaw at room temperature, and prepare the reaction solution according to the following system (Table 8):

[0470] Table 8

[0471] Components Volume (μL) Reagent 10 5 Reagent 11 2 Total volume 50

[0472] 1000 ng of the obtained library was drawn into a PCR tube according to the concentration, and after adding 7 μL of the prepared mixed system, it was placed in a vacuum filter (V-AQ mode, 60°C) to dry into a dry powder for about 40 minutes.

[0473] 7.2 Remove reagents 12, 13, and 14 from -20°C in kit 6, thaw at room temperature, and prepare the reaction solution according to the system in Table 9 below:

[0474] Table 9

[0475] Components Volume (μL) Reagent 12 8.5 Reagent 13 2.7 Reagent 14 4 Reagent 1 1.8 Total volume 17

[0476] 7.3 Add 17 μL of hybridization mixture to the drained library powder, shake and mix thoroughly, centrifuge, and place in a PCR instrument to perform the following reactions as shown in Table 10:

[0477] Table 10

[0478] temperature Reaction time Number of cycles 95℃ 30s 1 65℃ 16h 1

[0479] 8. Library Capture

[0480] 8.1 Take out reagents 15 and 16 from the refrigerator and equilibrate them at room temperature for 30 minutes before use.

[0481] 8.2 Take 50 μL of Reagent 16 (capture beads) from each sample, place it on a magnetic rack, discard the supernatant, add 100 μL of Reagent 15 to resuspend the beads, mix thoroughly, centrifuge briefly, place it on a magnetic rack, wait until the liquid is completely clear, and discard the supernatant. Repeat the above steps once more.

[0482] 8.3 Prepare magnetic bead resuspension solution according to the table below to resuspend the magnetic beads (Table 11):

[0483] Table 11

[0484] Components Volume (μL) Reagent 12 8.5 Reagent 13 2.7 Reagent 1 5.8 Total volume 17

[0485] The resuspended magnetic beads were transferred to a new eight-well plate (17 μL per well), and placed in a thermomixer at 65°C for 5 min.

[0486] 8.4 Capture:

[0487] 8.4.1 Transfer the preheated, resuspended capture beads to the capture product. After pipetting to resuspend the beads, place in a PCR instrument (65°C) and incubate for 45 minutes. Gently pipette and mix thoroughly every 10-12 minutes for a total of 4 times to keep the beads suspended.

[0488] 8.4.2 After incubation, remove the PCR tube from the thermal cycler and add 150 μL of ES Wash Buffer at 65°C. Gently pipette 15-20 times to thoroughly mix the hybridization system containing the magnetic beads, and transfer the tube to a new 1.5 mL EP tube preheated at 65°C.

[0489] 8.4.3 Place the EP tube on the magnetic rack for 30 seconds. Once the liquid is completely clear, use a pipette to remove and discard the supernatant.

[0490] 8.4.4 Quickly add 150 μL of 65°C ES Wash Buffer to the EP tube again. Gently pipette 15 times to mix thoroughly. Incubate at 65°C for 5 minutes.

[0491] 8.4.5 After instant centrifugation, place the tube on a magnetic rack for 30 seconds. After the liquid is completely clarified, remove the supernatant and discard any residual buffer. Add 150 μL of room temperature ES Wash Buffer and mix thoroughly by gently pipetting up and down 15 times. Transfer the entire reaction solution containing the magnetic beads to a new 1.5 mL low-adsorption EP tube and incubate at room temperature for 2 minutes. Vortex and mix thoroughly for 30 seconds and then let it stand for 30 seconds, alternating between these steps to ensure thorough mixing.

[0492] 8.4.6 Centrifuge the EP tube briefly and place it on a magnetic rack for 30 seconds. After the liquid is completely clear, discard any remaining buffer and add 150 μL of room temperature ES Wash Buffer. Mix thoroughly by gently pipetting up and down 15 times. Incubate at room temperature for 2 minutes, vortexing for 30 seconds and then letting it stand for 30 seconds, alternating between these steps to ensure thorough mixing.

[0493] 8.4.7 Centrifuge the above-mentioned centrifuge tube briefly and place it on a magnetic rack for 30 seconds. After the liquid is completely clear, discard the remaining buffer. After the brief centrifugation, use a small pipette tip to aspirate and discard the remaining liquid.

[0494] 8.4.8 Remove the EP tube from the magnetic stand, add 22.5 μL of Nuclease Free Water, and gently pipette 10 times to ensure even mixing. Transfer the entire magnetic bead mixture to a new 0.2 mL PCR tube.

[0495] 9. Capture PCR amplification:

[0496] 9.1 Remove reagents 17 and 18 from kit 8 from -20°C, equilibrate to room temperature, and prepare the reaction solution according to the following system (Table 12):

[0497] Table 12

[0498] Components Volume (μL) Reagent 17 25 Reagent 18 2.5 Magnetic bead mixture obtained in step 8 22.5 Total volume 50

[0499] 9.2 Vortex and centrifuge the mixture in the PCR tube. Place the mixture in a PCR instrument and perform the following reaction (Table 13):

[0500] Table 13

[0501]

[0502] 10. Post-capture library purification:

[0503] 10.1 Library purification:

[0504] 10.1.1 After centrifugation of the amplified product in step 9, add 50 μL SP Beads, mix well, and incubate at 25°C for 10 min.

[0505] 10.1.2 Centrifuge the PCR tube briefly and place it on a magnetic rack for 5 minutes until the liquid is completely clear. Use a pipette to remove and discard the supernatant.

[0506] 10.1.3 Slowly add 150 μL of 80% ethanol along the side of the PCR tube, taking care not to disturb the magnetic beads. Let it stand for 30 seconds, and use a pipette to remove and discard the supernatant.

[0507] 10.1.4 Repeat step 10.3 once; centrifuge the PCR tube briefly and place it on a magnetic rack. Use a 10 μL pipette tip to remove any remaining ethanol, being careful not to absorb the magnetic beads.

[0508] 10.1.5 Open the PCR tube cap and let it stand at room temperature for about 5 minutes until the ethanol evaporates completely (Note: Do not over-dry, otherwise it will affect the efficiency of magnetic bead recovery).

[0509] 10.1.6 Remove the PCR tube from the magnetic stand, add 22 μL / 40 μL Nuclease Free Water to the tube, vortex to mix, and incubate at room temperature for 10 minutes.

[0510] 10.1.7 Centrifuge the PCR tube briefly and place it on a magnetic rack for 2 minutes until the liquid is completely clear. Use a pipette to carefully transfer the supernatant to a new 1.5 mL EP tube for storage, taking care not to absorb the magnetic beads.

[0511] 10.2. Library Quality Control:

[0512] 10.2.1 Qubit quantification: Take 1 μL of library and use Qubit to detect the library concentration.

[0513] 10.2.2Qseq detected the library length, and the main peak was between 250-550bp.

[0514] 11. Sequencing:

[0515] Sequencing was performed using the Genemind sequencing platform and the SURFSeq 5000 sequencer. PE150 or PE100 sequencing chips were recommended, with 15G of data per sample.

[0516] Example 3

[0517] Bioinformatics analysis process and data quality control of WES molecular detection results for solid tumors.

[0518] 1. Data analysis was performed using the bioinformatics software shown in Table 14 below:

[0519] Table 14

[0520]

[0521] 2. Data quality control requirements are as follows: Taking into account the actual sample sequencing quality and the requirement for accurate variant detection by variant detection software, the sequencing data quality control threshold requirements are as shown in Table 15.

[0522] Table 15

[0523] index illustrate Require Average sequencing depth (X) (tissue) The average number of times each base is sequenced ≥200X (tissue) Average sequencing depth (X) (peripheral blood) The average number of times each base is sequenced ≥100X(peripheral blood) ≥100x base ratio (tissue) The percentage of bases sequenced more than 100 times ≥85％ ≥50x base ratio (peripheral blood) The percentage of bases sequenced more than 50 times ≥85％ Sequence alignment rate (OnTarget) How well the sequencing results match the reference genome ≥95％ Q30 The reliability of this base is 99.9% ≥85％

[0524] Example 4

[0525] Based on tumor-informed MRD molecular detection kits and detection methods (follow-up monitoring of MRD).

[0526] 1. Subsequent site design and customization:

[0527] Screen 35 loci that meet the outlined conditions from the results of the initial WES, upload the chromosome coordinates of the loci to the website http: / / 111.203.192.126:8888 / home / , select the genome type as hg19 and the density as 4X, and submit the task. After the probe is designed, it can be synthesized and mailed to the department.

[0528] 2.ct DNA library construction:

[0529] 2.1 Take reagents 19 and 20 from kit 9 for the experiment. Sample according to the input amount of ct DNA 66ng. Take 0.2mL PCR tubes according to the number of samples and mark the sample number on the tube cap.

[0530] 2.2 Add the components in the following order and set up the reaction system on an ice box (Table 16):

[0531] Table 16

[0532] Components Volume (μL) ctDNA samples 10(66ng / nucleic acid concentration) Reagent 1 40 Reagent 19 3 Reagent 20 7 Total volume 60

[0533] Reagent 19 and reagent 20 can be premixed on ice.

[0534] 2.3 Vortex and centrifuge the mixture in the PCR tube, then place it in a PCR instrument and perform the following reaction (Table 17):

[0535] Table 17

[0536] temperature Reaction time Number of cycles 30℃ 30min 1 65℃ 30min 1 4℃ Keep 1

[0537] 3. Connector connection

[0538] 3.1 Preparation before ligation: Remove reagents 21, 22, and 23 from the reagent kit 10 from -20°C, equilibrate to room temperature, and then prepare the reaction system on an ice box (Table 18):

[0539] Table 18

[0540] Components Volume (μL) Reagent 1 10 Reagent 21 5 Reagent 22 30 Reagent 23 5 The mixed solution obtained in step 2 60 Total volume 110

[0541] 3.2 Adapter ligation: First, take 5 μL of reagent 21 and add it to the reaction system in step 2, add it to the bottom of the PCR tube, then add 45 μL of the other reagents prepared above, shake and mix, centrifuge, and place it on the PCR instrument to perform the following reaction (Table 19):

[0542] Table 19

[0543] temperature Reaction time Number of cycles 22℃ Keep 1 22℃ 15min 1 4℃ Keep 1

[0544] 4. Purification of ligation product:

[0545] 4.1 Mixing magnetic beads: Oscillate reagent 24 until it is evenly mixed (Important note: Be sure to mix reagent 24 evenly before purification, otherwise it will affect the purification effect).

[0546] 4.2 Adding Magnetic Beads: Add 88 μL (0.8 volume) of Reagent 24 to each well. Pipet up and down 10 times. If bubbles form at the bottom of the tube during pipetting, briefly centrifuge and remix.

[0547] 4.3 Product Binding: Incubate the reaction plate / tube at room temperature for 5 minutes to allow the PCR products to bind to the magnetic beads.

[0548] 4.4 Separation of magnetic beads: Place the PCR plate / tube on a magnetic stand for 5 minutes until the solution becomes clear.

[0549] 4.5 Remove the supernatant: Carefully remove the supernatant from each tube, while being careful not to touch or blow away the magnetic beads.

[0550] 4.6 Wash the magnetic beads: Place the PCR plate / tubes on the magnetic stand. Add 200 μL of freshly prepared 80% ethanol to each tube. Incubate for 30 seconds and then aspirate the supernatant. Be careful not to touch the magnetic beads.

[0551] Important: Avoid contact between 80% ethanol and air. This can cause the ethanol concentration to change, affecting the effectiveness of cleaning the magnetic beads. Prepare only enough 80% ethanol for each use.

[0552] 4.7 Secondary Cleaning: Repeat the previous step. The remaining 80% ethanol can be used for purification of the second round of PCR products.

[0553] 4.8 Remove remaining ethanol: Completely remove any traces of ethanol from each tube. Briefly centrifuge for 10-15 seconds, place the PCR plate / tubes back on the magnetic stand, and use a 10-20 μL pipette tip to remove any remaining ethanol from the bottom of the tube.

[0554] 4.9 Dry the magnetic beads: Place the PCR reaction plate on the magnetic stand at room temperature for 5 minutes.

[0555] Important: Do not overdry the beads. They are sufficiently dry when small cracks appear in the center of the bead mass. If large cracks appear throughout the bead mass, or if the beads break into small flakes, they are overdried. Overdried beads will be difficult to resuspend.

[0556] 4.10 Resuspend the magnetic beads: Remove the PCR plate / tubes from the magnetic stand and immediately add 22 μL of nuclease-free water to each tube to resuspend the magnetic beads. Gently pipette the suspension 10 times. If bubbles form at the bottom of the tube, briefly centrifuge and remix.

[0557] 4.11 Aspirate the supernatant: Centrifuge the PCR tube briefly and place it on a magnetic rack for 2 minutes. After the solution is clarified, use a pipette to aspirate 20 μL of the supernatant and transfer it to a new PCR tube. Label it and prepare for the index-PCR reaction.

[0558] 5. Index PCR amplification:

[0559] 5.1 Reagent Preparation: Take out reagents 25 and 26 from reagent kit 12 and equilibrate them to room temperature.

[0560] Prepare the reaction solution according to the following table 20:

[0561] Table 20

[0562] Components Volume (μL) Reagent 25 25 Reagent 26 5 The supernatant obtained in step 3 20 Total volume 50

[0563] Note that 25 μL of reagent 25 was first added to the supernatant obtained in step 3, shaken to mix, and centrifuged; then 5 μL of reagent 26 was added below the liquid surface of the above suspension.

[0564] 5.2 The mixed system in the PCR tube was shaken and centrifuged, and then placed on a PCR instrument to carry out the reaction shown in Table 21 below:

[0565] Table 21

[0566]

[0567] After the above reaction, a ct DNA library is obtained.

[0568] 6. Library purification:

[0569] 6.1 Mixing magnetic beads: Oscillate reagent 24 until it is evenly mixed (Important note: Be sure to mix reagent 24 evenly before purification, otherwise it will affect the purification effect).

[0570] 6.2 Adding Magnetic Beads: Add 50 μL (1 volume) of Reagent 24 to each well. Pipet up and down 10 times. If bubbles form at the bottom of the tube during pipetting, briefly centrifuge and remix.

[0571] 6.3 Product Binding: Incubate the reaction plate / tube at room temperature for 5 minutes to allow the PCR products to bind to the magnetic beads.

[0572] 6.4 Separation of magnetic beads: Place the PCR plate / tube on a magnetic stand for 5 minutes until the solution becomes clear.

[0573] 6.5 Remove the supernatant: Carefully remove the supernatant from each tube while being careful not to touch or blow away the magnetic beads.

[0574] 6.6 Wash the magnetic beads: Place the PCR plate / tubes on the magnetic stand. Add 150 μL of freshly prepared 80% ethanol to each tube. Incubate for 30 seconds and then aspirate the supernatant. Be careful not to touch the magnetic beads.

[0575] Important: Avoid contact between 80% ethanol and air. This can cause the ethanol concentration to change, affecting the effectiveness of cleaning the magnetic beads. Prepare only enough 80% ethanol for each use.

[0576] 6.7 Secondary Cleaning: Repeat the previous step. The remaining 80% ethanol can be used for purification of the second round of PCR products.

[0577] 6.8 Remove remaining ethanol: Completely remove any traces of ethanol from each tube. Briefly centrifuge for 15 seconds, place the PCR plate / tubes back on the magnetic stand, and use a 10-20 μL pipette tip to remove any remaining ethanol from the bottom of the tube.

[0578] 6.9 Dry the magnetic beads: Place the PCR reaction plate on the magnetic stand at room temperature for 5 minutes.

[0579] Important: Do not overdry the beads. They are sufficiently dry when small cracks appear in the center of the bead mass. If large cracks appear throughout the bead mass, or if the beads break into small flakes, they are overdried. Overdried beads will be difficult to resuspend.

[0580] 6.10 Resuspend the magnetic beads: Remove the PCR plate / tubes from the magnetic stand and immediately add 32 μL of nuclease-free water to each tube to resuspend the magnetic beads. Gently pipette the suspension 10 times. If bubbles form at the bottom of the tube, briefly centrifuge and remix.

[0581] 6.11 Separate the supernatant: Place the PCR plate / tube back on the magnetic stand until the solution becomes clear, which may take 5 minutes. Pipette 30 μL of the supernatant into a new PCR tube to obtain the purified library.

[0582] 7. Library quality control:

[0583] 7.1 Qubit quantification: Take 1 μL of library and use Qubit to detect the library concentration. The library quality control standard is: concentration greater than 2 ng / μL and total amount not less than 200 ng.

[0584] 7.2Qseq monitored the library length, with the main peak between 250-550 bp.

[0585] 8. Library hybridization:

[0586] 8.1 Remove reagents 27, 28, 29, 30, and 31 from -20°C in kit 13, thaw at room temperature, and prepare the reaction solution according to the system shown in Table 22 below:

[0587] Table 22

[0588] Components Volume (μL) Reagent 27 13 Reagent 28 5 Reagent 28 2 Reagent 29 5 Reagent 30 2 Reagent 1 3 Total volume 30

[0589] 1000 ng of the obtained library (ctDNA library to be tested and negative library) was aspirated according to the concentration and placed in a PCR tube, which was then placed in a vacuum filter (V-AQ mode, 60°C) and dried into dry powder for about 25 minutes.

[0590] 8.2 Add 30 μL of hybridization mixture to the drained library powder, shake and mix thoroughly, centrifuge, and place in a PCR instrument to perform the following reactions as shown in Table 23:

[0591] Table 23

[0592] temperature Reaction time Number of cycles 85℃ 5min 1 60℃ 16 1

[0593] 9. Library Capture

[0594] 9.1 Preparation before capture

[0595] 9.1.1 Remove reagents 32 and 33 from the refrigerator and equilibrate to room temperature.

[0596] 9.1.2 Take 30 μL of Reagent 32 (capture beads) from each hybridization library, place it on a magnetic rack, discard the supernatant, add 180 μL of Reagent 32 to resuspend the beads, mix thoroughly, centrifuge briefly, place it on a magnetic rack, wait until the liquid is completely clear, and discard the supernatant. Repeat the above steps twice.

[0597] 9.1.3 Add 180 μL of Reagent 32 to each hybridization library and resuspend the magnetic beads. Transfer the hybridization product to the resuspended magnetic beads and place on a vertical rotating mixer at 5 rpm for 20 min at room temperature.

[0598] 9.1.4 Remove the PCR tube, centrifuge briefly, and place on a magnetic rack for 2 minutes. After the solution is clear, discard the supernatant.

[0599] 9.1.5 Remove the PCR tube from the magnetic stand and add 150 μL of TargetSeq preheated at 60°C. Add 2x2 WashBuffer, pipette gently to mix, centrifuge briefly, place on a constant temperature shaker or metal bath, and incubate at 60°C for 5 min.

[0600] 9.1.6 Remove the PCR tube, centrifuge briefly, and place on a magnetic rack for 2 minutes. After the solution is clear, discard the supernatant.

[0601] 9.1.7 Repeat 9.5-9.6 four times.

[0602] 9.1.8 Keep the PCR tube on the magnetic rack and add 200 μL of 80% ethanol to the PCR tube. After standing for 30 seconds, completely discard the ethanol solution (you can use a 10 μL pipette to discard residual ethanol). Dry the magnetic beads at room temperature to completely evaporate the residual ethanol.

[0603] 9.1.9 Add 24 μL of Nuclease-Free Water to the PCR tube, remove the PCR tube from the magnetic stand, vortex briefly to resuspend the magnetic beads, and perform the capture PCR amplification reaction.

[0604] 9.2 Post-capture amplification:

[0605] 9.2.1 Remove reagents 34 and 35 from kit 15 from -20°C, equilibrate to room temperature, and prepare the reaction mixture according to the system shown in Table 24 below:

[0606] Table 24

[0607] Components Volume (μL) Reagent 34 1 Reagent 35 25 Magnetic bead suspension obtained in step 9.1 24 Total volume 50

[0608] 9.2.3 Vortex and centrifuge the mixture in the PCR tube. Place the mixture in a PCR instrument and perform the reaction as shown in Table 25 below:

[0609] Table 25

[0610]

[0611] 9.3 Post-capture purification:

[0612] 9.3.1 Centrifuge the amplified product from 9.2 briefly, then add 55 μL of IGT Pure Beads, mix thoroughly, and incubate at 25°C for 5 min.

[0613] 9.3.2 Centrifuge briefly and place the PCR tube on a magnetic rack for 3 minutes to allow the solution to clear.

[0614] 9.3.3 Keep the PCR tube on the magnetic rack, discard the supernatant, add 200 μL of 80% ethanol solution to the PCR tube, and let it stand for 30 seconds.

[0615] 9.3.4 Keep the PCR tube on the magnetic rack, discard the supernatant, and add 200 μL of 80% ethanol solution to the PCR tube again. Let it stand for 30 seconds and then completely discard the supernatant (you can centrifuge it briefly to centrifuge the liquid on the wall to the bottom of the tube, and then use a 10 μL pipette to discard the ethanol solution remaining at the bottom).

[0616] 9.3.5 Ensure that the PCR tube is on the magnetic rack and stand at room temperature for 5 minutes to dry the magnetic beads and allow the residual ethanol to evaporate completely.

[0617] 9.3.6 Add 25 μL of Nuclease-Free Water, remove the PCR tube from the magnetic stand, pipette or vortex to mix, and let it stand at room temperature for 2 minutes.

[0618] 9.3.7 Centrifuge briefly and place the PCR tube on a magnetic rack for 2 minutes to allow the solution to clear.

[0619] 9.3.8 Use a pipette to aspirate 23 μL of supernatant and transfer it to a new PCR tube. Store the captured library in a -20°C refrigerator. The captured library can be stored in a -20°C refrigerator for one month.

[0620] 9.4 Library Quality Control:

[0621] 9.4.1 Take 1 μL of library and measure the library concentration using the Qubit dsDNA HS Assay Kit reagent on a Qubit 4.0 Fluorometer. Record the library concentration.

[0622] 9.4.2 Take 1 μL of the library and perform fragment quality check using a fragment analyzer. The fragment size should be basically consistent with the pre-library size.

[0623] 10. Sequencing:

[0624] Sequencing was performed using the Genemind sequencing platform and the SURFSeq 5000 sequencer. PE150 or PE100 sequencing chips were recommended, with 3G of data per sample.

[0625] Example 5

[0626] Bioinformatics analysis process and data quality control of solid tumor MRD molecular detection results.

[0627] 1. Data analysis was performed using the bioinformatics software shown in Table 26 below:

[0628] Table 26

[0629] software Software version Function Fastp v0.23.2 Remove the connector Vardict v202112.04 Sequence alignment, consensus sequence generation based on UMI information, and mutation detection annovar v2020 Annotation of SNV / Indel variants delly v0.9.1 Detection of gene fusion variants cnvkit v0.9.10 Detect SNV / Indel variants bcftools v1.8 Merge vcf files

[0630] 2. Data quality control requirements are as follows: Taking into account the actual sample sequencing quality and the requirement for accurate variant detection by the variant detection software, the sequencing data quality control threshold requirements are shown in Table 27 below.

[0631] Table 27

[0632] index illustrate Require Average sequencing depth (X) The average number of times each base is sequenced ≥200,000X ≥2000x base ratio The percentage of bases sequenced more than 2000 times ≥90％ ≥500x base ratio The percentage of bases sequenced more than 500 times ≥85％ Sequence alignment rate (OnTarget) How well the sequencing results match the reference genome ≥95％ Q30 The reliability of this base is 99.9% ≥85％

[0633] Example 6

[0634] Accuracy and reproducibility of solid tumor MRD molecular detection kit.

[0635] 1. Verification of SNV / Indel Accuracy and Reproducibility of ctDNA Standards: ctDNA standards with a mutation frequency (VAF) of 0.5% were diluted to VAFs of 0.1%, 0.05%, 0.02%, 0.01%, and 0.005%. 66 ng of each VAF was used for molecular MRD testing of solid tumors, with one replicate run per group to verify accuracy and reproducibility. (Digital PCR quantification by the standard manufacturer revealed that the actual frequencies of four loci in the BRCA1, BRCA2, and CDK12 genes in this standard were slightly higher, 4-5 times the labeled frequencies.)

[0636] 1.1VAF(SNV)=0.1%The accuracy and repeatability results are shown in Table 28 below:

[0637] Table 28

[0638]

[0639]

[0640] Sequencing of ct DNA standards with a VAF of 0.1% was performed. Parallel 1 and parallel 2 both detected 31 SNV mutation sites within the detection range, with a detection rate of 100%. The measured mutation frequency was basically consistent with the theoretical mutation frequency, which was in line with the expected results.

[0641] Intra-batch consistency: One ct DNA standard (GW-OCTM802) with a VAF of 0.1% was repeated twice within the same batch, and SNVs / Indels were consistently detected, with an intra-batch consistency of 100%.

[0642] 1.2VAF(SNV)=0.05% The accuracy and repeatability results are shown in Table 29 below:

[0643] Table 29

[0644]

[0645]

[0646] Sequencing of ct DNA standards with a VAF of 0.05% revealed 31 SNV mutations within the detection range in parallel 1, with a detection rate of 100%. Parallel 2 detected 30 SNV mutations within the detection range, with a detection rate of 96.7%. The observed mutation frequencies were generally consistent with the theoretical frequencies, meeting expectations.

[0647] Intra-batch consistency: One ct DNA standard (GW-OCTM802) with a VAF of 0.05% was repeated twice within the same batch, and 30 SNV / Indel sites were consistently detected, with an intra-batch consistency of 96.7%, which was in line with the expected results.

[0648] 1.3VAF(SNV)=0.02% The accuracy and repeatability results are shown in Table 30 below:

[0649] Table 30

[0650]

[0651]

[0652] Sequencing of ct DNA standards with a VAF of 0.02% revealed 28 SNV mutations within the detection range in parallel 1, with a detection rate of 90.3%. Parallel 2 detected 26 SNV mutations within the detection range, with a detection rate of 83.8%. The observed mutation frequencies were generally consistent with the theoretical frequencies, meeting expectations.

[0653] Intra-batch consistency: One ct DNA standard (GW-OCTM802) with a VAF of 0.02% was repeated twice within the same batch, and the intra-batch consistency was 87%, which was in line with the expected results.

[0654] 1.4VAF(SNV)=0.01%Accuracy and repeatability results are shown in Table 31 below:

[0655] Table 31

[0656]

[0657]

[0658] Sequencing of ct DNA standards with a VAF of 0.01% revealed 20 SNV mutations within the detection range in parallel 1, with a detection rate of 64.5%. Parallel 2 detected 21 SNV mutations within the detection range, with a detection rate of 67.7%. The observed mutation frequencies were generally consistent with the theoretical frequencies, meeting expectations.

[0659] Intra-batch consistency: One ct DNA standard (GW-OCTM802) with a VAF of 0.01% was repeated twice within the same batch, and the intra-batch consistency was 77.4%, which was in line with the expected result.

[0660] 1.5VAF(SNV)=0.005% Accuracy and repeatability results are shown in Table 32 below:

[0661] Table 32

[0662]

[0663]

[0664] Sequencing was performed on ctDNA standards with a VAF of 0.01%. Parallel 1 detected a total of 12 SNV mutation sites within the detection range, with a detection rate of 38.7%; parallel 2 detected a total of 10 SNV mutation sites within the detection range, with a detection rate of 32.2%. The measured mutation frequency was basically consistent with the theoretical mutation frequency, which was in line with the expected results.

[0665] Intra-batch consistency: One ctDNA standard (GW-OCTM802) with a VAF of 0.005% was repeated twice within the same batch, with an intra-batch consistency of 67.7%, which was in line with the expected results.

[0666] Expert consensus indicates that MRD molecular detection of solid tumors is considered positive if two out of 16 sites are detected. In this experiment, when VAF = 0.005%, the site detection rate can reach more than 30%, so the minimum detection limit of the mutation frequency in this experiment can be as low as 0.005%.

[0667] 2. Verification of the accuracy and repeatability of ct DNA standard Fusion: ct DNA standards with a mutation frequency (VAF) of 0.5% were diluted to VAFs of 0.1% and 0.05%, and 66 ng was used at each frequency for solid tumor MRD molecular detection. An additional parallel run was performed for each group to obtain accuracy and repeatability results.

[0668] 2.1 The accuracy and repeatability of the ct DNA standard test with VAF (Fusion) = 0.1% are shown in Table 33 below:

[0669] Table 33

[0670]

[0671] Sequencing of ct DNA standards with a VAF(Fusion) of 0.1% revealed four fusion mutation sites within the detection range in parallel 1, with a detection rate of 80%. Parallel 2 detected five fusion mutation sites within the detection range, with a detection rate of 100%. The observed mutation frequencies were essentially consistent with the theoretical frequencies, meeting expectations.

[0672] Intra-batch consistency: One ct DNA standard (GW-OCTM802) with a VAF of 0.1% was repeated twice within the same batch, and the intra-batch consistency was 80%, which was in line with the expected results.

[0673] 2.2 The accuracy and repeatability of the ct DNA standard test with VAF (Fusion) = 0.05% are shown in Table 34 below:

[0674] Table 34

[0675]

[0676] Sequencing was performed on ctDNA standards with a VAF(Fusion) of 0.05%. Parallel 1 detected a total of 4 fusion mutation sites within the detection range, with a detection rate of 80%; parallel 2 detected a total of 2 fusion mutation sites within the detection range, with a detection rate of 40%. The measured mutation frequency was basically consistent with the theoretical mutation frequency, which was consistent with the expected results.

[0677] Intra-batch consistency: One ctDNA standard (GW-OCTM802) with a VAF of 0.05% was repeated twice within the same batch, with an intra-batch consistency of 60%, which was in line with the expected results.

[0678] Example 6

[0679] Minimum input and repeatability verification of solid tumor MRD molecular detection kit.

[0680] 1. Verification of SNV / Indel Input and Repeatability of ctDNA Standards: Dilute the ctDNA standard with a mutation frequency (VAF) of 0.5% to a VAF of 0.01%. 66ng, 44ng, 22ng, and 10ng of the standard were injected for solid tumor MRD molecular detection, and each group was tested once in parallel to obtain the minimum input and repeatability results.

[0681] The results of the repeatability verification of ct DNA standard with VAF(SNV)=0.01% (66 ng) are shown in Table 35 below:

[0682] Table 35

[0683]

[0684] 66 ng of ct DNA standard with VAF(SNV)=0.01% was sequenced. Parallel 1 detected a total of 16 SNV / Indel mutation sites within the detection range, with a detection rate of 51.6%. Parallel 2 detected a total of 18 SNV / Indel mutation sites within the detection range, with a detection rate of 40%. The measured mutation frequency was basically consistent with the theoretical mutation frequency, which was consistent with the expected results.

[0685] Intra-batch consistency: 66 ng of ctDNA standard (GW-OCTM802) with VAF = 0.01% was added and repeated twice within the same batch, and the intra-batch consistency was 62%, which was in line with the expected results.

[0686] 1.2 ctDNA standard with VAF (SNV) = 0.01% was added in 44 ng and the repeatability verification results are shown in Table 36:

[0687] Table 36

[0688]

[0689]

[0690] 44 ng of ct DNA standard with VAF(SNV)=0.01% was sequenced. Parallel 1 detected a total of 17 SNV / Indel mutation sites within the detection range, with a detection rate of 54.8%; parallel 2 detected a total of 12 SNV / Indel mutation sites within the detection range, with a detection rate of 38.7%. The measured mutation frequency was basically consistent with the theoretical mutation frequency, which was consistent with the expected results.

[0691] Intra-batch consistency: 44 ng of a ct DNA standard (GW-OCTM802) with a VAF of 0.01% was tested twice within the same batch, and the intra-batch consistency was 70.9%, which was in line with the expected results.

[0692] 1.3 ct DNA standard with VAF (SNV) = 0.01% was added in 22 ng and the repeatability verification results are shown in Table 37:

[0693] Table 37

[0694]

[0695]

[0696] 22 ng of ctDNA standard with VAF(SNV)=0.01% was injected for sequencing detection. Parallel 1 detected a total of 9 SNV / Indel mutation sites within the detection range, with a detection rate of 29%; parallel 2 detected a total of 13 SNV / Indel mutation sites within the detection range, with a detection rate of 41.9%. The measured mutation frequency was basically consistent with the theoretical mutation frequency, which was in line with the expected results.

[0697] Intra-batch consistency: 22 ng of ctDNA standard (GW-OCTM802) with VAF = 0.01% was added and repeated twice within the same batch, with an intra-batch consistency of 67.7%, which was in line with the expected results.

[0698] 1.4 ctDNA standard with VAF (SNV) = 0.01% was added with 10 ng and the repeatability verification results are shown in Table 38:

[0699] Table 38

[0700]

[0701]

[0702] 10 ng of ct DNA standard with VAF(SNV)=0.01% was sequenced. Parallel 1 detected a total of 10 SNV / Indel mutation sites within the detection range, with a detection rate of 32.2%; parallel 2 detected a total of 8 SNV / Indel mutation sites within the detection range, with a detection rate of 25.8%. The measured mutation frequency was basically consistent with the theoretical mutation frequency, which was consistent with the expected results.

[0703] Expert consensus states that solid tumor MRD molecular testing is considered positive if two of 16 sites are detected. In this experiment, when VAF = 0.01%, the detection rate of the test sites reached over 29% with an input of 22 ng, so the minimum input of nucleic acid for this experiment can be as low as 22 ng. (Excluding the four high-frequency sites in the standard, the detection rate was low with a 10 ng input in two parallel runs, with only one site detected, so the minimum input was set at 22 ng.)

[0704] Intra-batch consistency: 10 ng of ctDNA standard (GW-OCTM802) with VAF = 0.01% was injected twice within the same batch, and the intra-batch consistency was 77.4%, which was in line with the expected results.

[0705] In summary, the present invention designs a four-layer encrypted shingled hybridization capture probe for detecting minimal residual lesions, providing a highly sensitive and specific molecular detection method for minimal residual lesions. It has a high efficiency in capturing low-frequency mutations, and the detection limit of ctDNA mutation frequency containing minimal residual lesions can be as low as 0.005%.

[0706] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A probe for detecting minimal residual lesions, characterized in that: The probe for detecting minimal residual lesions is a four-layer encrypted shingled hybridization capture probe, wherein two layers of probes are wild-type probes and two layers are mutant probes. The nucleic acid sequence of the wild-type probe includes the sequence described in SEQ ID NO.1-SEQ ID NO.74, and the nucleic acid sequence of the mutant probe includes the sequence described in SEQ ID NO.75-SEQ ID NO.

149.

2. The probe for detecting minimal residual lesions according to claim 1, wherein The length of the wild-type probe and the mutant probe is 90-110 bp, and the length of the bases staggered between the probes is 10-20 bp.

3. Use of the probe for detecting minimal residual lesions according to claim 1 or 2 in the preparation of a product for detecting minimal residual lesions.

4. A kit for detecting minimal residual lesions, characterized in that: The kit comprises the probe for detecting minimal residual lesions according to claim 1 or 2.

5. A method for detecting minimal residual disease for purposes other than disease diagnosis and / or treatment, characterized in that: The method comprises the following steps: (1) Sample preparation: Nucleic acid extraction is performed on the FFPE samples and peripheral blood samples submitted by the patient for the initial examination to obtain gDNA; free nucleic acid extraction is performed on the non-invasive blood samples submitted by the patient for subsequent examination to obtain ctDNA; (2) Whole exome detection: FFPE sample nucleic acid and peripheral blood sample nucleic acid gDNA are used to construct libraries, hybridize with whole exome probes and capture on the machine to obtain whole exome sequencing results; (3) Personalized capture probe design: 30-40 somatic mutation sites are screened from the whole exome sequencing results, and personalized capture probes are designed and customized for the screened mutation sites, wherein the personalized capture probes include the probe for detecting minimal residual lesions according to claim 1 or 2; (4) ct DNA sequencing detection: construct a ct DNA library, select ct DNA samples with known negative sequencing results for library construction, and simultaneously hybridize the two constructed libraries with the customized capture probe panel in step (3) to capture ct DNA; (5) Minimal residual disease sequencing: Use a sequencer to sequence the captured ct DNA; (6) Bioinformatics analysis: The ct DNA sequencing results are compared with the human reference genome to obtain the point mutation results of the sample gene.

6. The method for detecting minimal residual lesions for purposes other than disease diagnosis and / or treatment according to claim 5, characterized in that: The screening criteria for selecting 30-40 somatic mutation sites from the whole exome sequencing results in step (3) are: somatic mutation sites with a gene mutation frequency similar to or half of the tumor cell content; Preferably, the somatic mutation site is a somatic class I mutation site, a somatic class II mutation site, and a somatic class III mutation site in which the base mutation type is a transversion; Preferably, the somatic mutation site is a somatic variation site with a mutation frequency >10%; Preferably, the somatic mutation site is a somatic mutation site that avoids simple repeat sequences and large insertion / deletion mutations; Preferably, the somatic mutation site is a somatic mutation site whose GC content of the probe for detecting minimal residual lesions according to claim 1 or 2 is between 30% and 70%.

7. The method according to claim 5 or 6, characterized in that The step (6) of comparing the ctDNA sequencing results with the human reference genome to obtain the point mutation results of the sample gene includes: comparing the sequencing results with the human reference genome, performing sequence alignment, marking repeated sequences, somatic mutation detection and germline mutation detection to obtain the point mutation results of the sample gene.

8. A device for detecting minimal residual lesions, characterized in that: The device includes a nucleic acid extraction module, a detection module and a sequencing analysis module; The nucleic acid extraction module is used to perform the following operations: extracting nucleic acids from FFPE samples and peripheral blood samples submitted by patients for initial testing to obtain gDNA; extracting free nucleic acids from non-invasive blood samples submitted by patients for subsequent testing to obtain ctDNA; The detection module is configured to perform the following steps: constructing a library of FFPE sample nucleic acid and peripheral blood sample nucleic acid gDNA, hybridizing the whole exome probe and capturing the whole exome probe on a machine to obtain whole exome sequencing results, screening 30-40 somatic mutation sites from the whole exome sequencing results, designing and customizing personalized capture probes for the screened mutation sites, wherein the personalized capture probes include the probe for detecting minimal residual lesions according to claim 1 or 2, constructing a library of sample ct DNA, and simultaneously selecting a ct DNA sample with a known negative sequencing result for library construction, and simultaneously hybridizing the two constructed libraries with the customized personalized capture probe panel to capture ct DNA; The sequencing analysis module is used to perform the following operations: using a sequencer to sequence the captured ctDNA, comparing the ctDNA sequencing results with the human reference genome, performing sequence alignment, marking repeated sequences, somatic cell variation detection, and germline mutation detection to obtain point mutation results of the sample gene.

9. The device for detecting minimal residual lesions according to claim 8, characterized in that: The screening criteria for selecting 30-40 somatic mutation sites are: somatic mutation sites with a gene mutation frequency close to or half of that in tumor cells; Preferably, the somatic mutation site is a somatic class I mutation site, a somatic class II mutation site, and a somatic class III mutation site in which the base mutation type is a transversion; Preferably, the somatic mutation site is a somatic variation site with a mutation frequency >10%; Preferably, the somatic mutation site is a somatic mutation site that avoids simple repeat sequences and large insertion / deletion mutations; Preferably, the somatic mutation site is a somatic mutation site whose GC content of the probe for detecting minimal residual lesions according to claim 1 or 2 is between 30% and 70%.

10. Use of the probe for detecting minimal residual lesions according to claim 1 or 2, the kit for detecting minimal residual lesions according to claim 4, the method for detecting minimal residual lesions according to any one of claims 5 to 7, or the device for detecting minimal residual lesions according to claim 8 or 9 in the preparation of a product for diagnosing and / or treating cancer.

Images