A probe and method for detecting microresidual lesions

Abstract

The application discloses a probe and a method for detecting micro residual lesions. The probe for detecting micro residual lesions is a four-layer encrypted imbricated hybrid 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 comprises the sequence described in SEQ ID NO. 1-SEQ ID NO. 74, and the nucleic acid sequence of the mutant probe comprises the sequence described in SEQ ID NO. 75-SEQ ID NO. 149. The four-layer encrypted imbricated hybrid capture probe for detecting micro residual lesions is designed, a micro residual lesion molecular detection method with high sensitivity and good specificity is provided, the low-frequency mutation capture efficiency is high, and the detection limit of the ct DNA mutation frequency containing micro residual lesions can be as low as 0.005%.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a probe and method for detecting minute residual lesions. Background Technology

[0002] Minimal residual disease (MRD) in solid tumors refers to a small number of tumor cells or DNA fragments remaining in the body of patients with solid tumors after radical treatment (such as surgery, radiotherapy, and chemotherapy). These residues may be detected through circulating tumor DNA (ctDNA) in the bloodstream, but are difficult to detect using traditional imaging or pathological methods. The presence of MRD is significantly associated with the risk of tumor recurrence, and its detection can help assess treatment effectiveness, predict recurrence, and guide subsequent treatment decisions.

[0003] Next-generation sequencing (NGS)-based MRD detection is currently the mainstream technology in the field of solid tumors. Its core principle is to identify extremely low-abundance ctDNA mutation signals in the blood using highly sensitive methods. NGS technology mainly falls into two categories:

[0004] Tumor-informed strategy: This requires obtaining tumor tissue samples in advance, screening for patient-specific mutation sites through whole exome sequencing (WES) or large panel detection, and then customizing a personalized panel for subsequent ct DNA tracking.

[0005] Tumor-naive strategy: It does not rely on tumor tissue and directly detects high-frequency mutations in the blood through a fixed panel. However, it has low sensitivity (e.g., the sensitivity for early lung cancer in the Lung-CLiP study was only 41%-67%). Furthermore, due to panel fixation, it can only cover hotspot mutation regions and cannot comprehensively monitor mutation sites in patients with solid tumors, which may result in missed detections.

[0006] Both of the above analytical methods have their advantages and disadvantages. The Tumor-informed approach has relatively higher accuracy, but it requires tissue sample testing results and a sequencing depth of over 200,000X for subsequent monitoring samples. Furthermore, since personalized panels need to be synthesized each time, it presents significant challenges in terms of synthesis capacity, batch stability, and timeliness. The Tumor-naive approach, using fixed panels, is more convenient for testing and facilitates quality control; however, it cannot meet the needs of individualized tumor heterogeneity. Panel size is also a critical issue; too large a panel wastes sequencing data, while too small a panel fails to cover mutation sites. Moreover, due to the inherent limitations of the second-generation sequencing platform's detection sensitivity, a large number of background noise mutations occur around 0.1%, and wild-type sequence interference leads to a high false positive rate. Without tissue mutations as a reference, it is difficult to set a filtering threshold, generally requiring a higher detection threshold, resulting in insufficient low-frequency mutation capture efficiency and thus poor detection sensitivity.

[0007] Therefore, there is an urgent need to provide a more sensitive and specific tumor-informed MRD molecular detection method / kit to help clinicians accurately identify MRD-positive patients, predict relapse, and guide subsequent treatment. Summary of the Invention

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

[0009] To achieve this objective, the present invention adopts the following technical solution:

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

[0011] This invention provides a highly sensitive and specific molecular detection probe for minimal residual disease (ctDNA). The probe is a four-layer encrypted shingled hybridization capture probe, which adds two mutant probes to two wild-type probes. It has high low-frequency mutation capture efficiency, so that the detection limit of ctDNA mutation frequency containing ctDNA can be as low as 0.005%.

[0012] (I) 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. seventh eight).

[0032] BRAF gene V600K

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

[0034] It should be noted that in the translation of the sequence part in item , "seventh eight" is a literal translation of "78" in Chinese. It is recommended to check and correct it according to the actual situation to ensure the accuracy of the sequence number.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).

[0041] BRAF gene V600E

[0042] >chr7:140453100-140453200(wild type 1)

[0043] GTTCAAACTGATGGGACCCACTCCATCGAGATTTCACTGTAGCTAGACCAAAATCACCTATTTTT ACTGTGAGGTCTTCATGAAGAAATATATCTGAGGT(SEQ ID NO.7).

[0044] >chr7:140453070-140453170(wild type 2)

[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 (mutant with 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 (Deletion in the mutant)

[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] >chr13:3291268�-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] The EGFR gene p.L747_P753delinsS (mutant form) has a 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(Mutant has duplication)

[0177] >chr17:3788095-37881054(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).

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

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

[0208] >chr4:55592134-55592228(Mutation 1)

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

[0210] >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.​​​​​​​​​​​​

[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] >chr12:25398234-25398334(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)

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

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

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

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

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

[0248] KRAS gene G13D

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

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

[0251] >chr12:25398215-...(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] >chr1:115258711-115258811(Wild type 1)

[0259] TTAGCTGGATTGTCAGTGCGCTTTTCCCAACACCACCTGCTCCAACCACCACCAGTTTGTACTCA GTCATTTCACACCAGCAAGAACCTGTTGGAAACCA(SEQ ID NO.55).

[0260] >chr1:115258681-115258781(Wild type 2)

[0261] GATCATATTCATCTACAAAGTGGTTCTGGATTAGCTGGATTGTCAGTGCGCTTTTCCCAACACCAC CTGCTCCAACCACCACCAGTTTGTACTCAGTCAT(SEQ ID NO.56).

[0262] >chr1:115258696-115258796(Mutation 1)

[0263] 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] [[ID=​​​​​​​​​​​​​​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)

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

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

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

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

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

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

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

[0314] FGFR3::TACC3

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

[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 type)

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

[0335] >chr10:43609947-43610047(RET wild type)

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

[0337] >chr10_chr10:35-135(Fusion 1)

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

[0339] >chr10_chr10:65-165(Fusion 2)

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

[0341] CD74::ROS1

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

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

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

[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 (Merge 2)

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

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

[0351] Secondly, the present invention provides the application of the probe described in the first aspect for detecting minimal residual lesions in the preparation of products for detecting minimal residual lesions.

[0352] Thirdly, the present invention provides a kit for detecting minimal residual lesions, the kit comprising the probe for detecting minimal residual lesions described in the first aspect.

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

[0354] (1) Sample preparation: gDNA was extracted from the FFPE and peripheral blood samples submitted by the patient for the first test; ctDNA was extracted from the non-invasive blood samples submitted by the patient for subsequent tests.

[0355] (2) Whole exome sequencing: The nucleic acid of FFPE sample and the nucleic acid gDNA of peripheral blood sample were used to construct libraries, hybridize whole exome probes and capture them for sequencing 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. The personalized capture probes include the probes for detecting minimal residual lesions described in the first aspect.

[0357] (4) ct DNA sequencing detection: construct a library of ct DNA, select a ct DNA sample with known negative sequencing results to construct a library, and hybridize the two libraries obtained simultaneously with step (3) a customized personalized capture probe panel to capture ct DNA;

[0358] (5) Sequencing of minimal residual lesions: The captured ct DNA was sequenced using a sequencer;

[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 genes.

[0360] Preferably, the screening criteria for selecting 30-40 somatic mutation sites from the whole exome sequencing results in step (3) are: somatic mutation sites with gene mutation frequencies similar to or half that of tumor cells.

[0361] Preferably, the somatic mutation site is a somatic cell type I mutation site, a somatic cell type II mutation site, or a site in somatic cell type III mutation where the base mutation type is transversion.

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

[0363] Type II variants: These are variants that have potential clinical significance in treatment, prognosis, and diagnosis. They generally refer to variants in other types of cancer for which there are targeted therapies.

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

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

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

[0367] Preferably, the step (6) of comparing the ct DNA 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 cell 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. It can effectively distinguish them by detecting the content and mutation frequency of microresidual lesions in the samples, thereby improving the sensitivity and accuracy of detection.

[0369] Fifthly, the present invention provides an apparatus for detecting minimal residual lesions, the apparatus 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 gDNA from the FFPE sample and peripheral blood sample submitted by the patient for the first test; and extracting ctDNA from the cell-free nucleic acid from the non-invasive blood sample submitted by the patient for subsequent tests.

[0371] The detection module is used to perform the following: constructing libraries from FFPE sample nucleic acid and peripheral blood sample nucleic acid gDNA, hybridizing whole exome probes and capturing them onto the instrument 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, the personalized capture probes including the probes for detecting minimal residual lesions as described in the first aspect, constructing libraries from sample ct DNA, and simultaneously selecting ct DNA samples with known negative sequencing results for library construction, and hybridizing the two libraries obtained with the customized personalized capture probe panel to capture ct DNA;

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

[0373] Preferably, the screening criteria for selecting 30-40 somatic mutation sites are: somatic mutation sites with gene mutation frequencies similar to or half that of tumor cells.

[0374] Preferably, the somatic mutation site is a somatic cell type I mutation site, a somatic cell type II mutation site, or a site in somatic cell type III mutation where the base mutation type is transversion.

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

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

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

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

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

[0380] (1) This invention discloses a molecular detection method for solid tumor MRD with good specificity, high sensitivity and lower detection limit. This method uses a tumor-informed strategy to first perform WES detection on tumor tissue and paired peripheral blood, and then selects specific sites for personalized probe customization based on the WES results. The main reason for the high sensitivity and lower detection limit of this invention is that a four-layer probe encryption design is used in the personalized probe design and synthesis, of which two layers are mutant probes and two layers are wild-type probes. The probes in each layer are staggered by 10-20 bp. 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 detection limit of this method can be as low as 0.005%.

[0381] (2) In the bioinformatics analysis process, this invention uses TNscope software and Vardict software to detect point mutations simultaneously and merges the two result files to reduce the false positive rate. The four-color fluorescence imaging sequencing instrument (GeneMind) is used for detection, which can greatly reduce the background noise of sequencing and further improve the reliability of the detection results. In addition, this invention has obvious price advantages and aims to reduce the economic burden on patients through cost-effective gene testing services, while also helping to assist in the precise diagnosis and treatment in clinical practice. Attached Figure Description

[0382] Figure 1 This is a flowchart of the detection process of the present invention;

[0383] Figure 2 This is a schematic diagram of the probe used to detect minute residual lesions according to the present invention. Detailed Implementation

[0384] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0385] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

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

[0387] Table 1

[0388]

[0389]

[0390] Reagent kits 1-8 are from the manufacturer: Nanoda (Nanjing) Biotechnology Co., Ltd.; model numbers are as follows:

[0391] ① Enzyme digestion and library preparation module: EZ DNALibrary Preparation Module v2, 96 doses (Reagent kits 1, 2, 4, and 5);

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

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

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

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

[0396] Reagent kits 9-15 are from the manufacturer: Aijitaikang Biotechnology (Beijing) Co., Ltd.; model number as follows:

[0397] ①Library building module: Fast Library Prep Kit v2.0, 96 doses (kit 9);

[0398] ② Universal adapter kit: UMIAdapter & UDI Pirimer 1-96 (Reagent kits 10, 11, 12);

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

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

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

[0402] Example 1

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

[0404] Step 1, Sample Preparation: Nucleic acid (gDNA) is extracted from the FFPE and peripheral blood samples submitted by the patient for the first time; cell-free nucleic acid (ctDNA) is extracted from the non-invasive blood samples submitted by the patient for subsequent tests.

[0405] Step 2, Whole Exome Sequencing (WES): Library construction was performed on the nucleic acids of FFPE samples and peripheral blood samples, WES probes were hybridized and captured and loaded onto the sequencing machine to obtain WES sequencing results.

[0406] Step 3: Personalized Probe Design: Select 30-40 somatic mutation sites from the WES results for personalized probe design and customization. The probes are four-layer encrypted shingled hybridization capture probes, with two layers being wild-type probes and two layers being mutant probes. The nucleic acid sequences of the wild-type probes include the sequences described in SEQ ID NO.1-SEQ ID NO.74, and the nucleic acid sequences of the mutant probes include the sequences described in SEQ ID NO.75-SEQ ID NO.149. Site selection needs to meet the following conditions and priorities: the gene mutation frequency is similar to or half of the tumor cell content, i.e., the master cloning gene site (homozygous or heterozygous); priority is given to type I and type II mutations, and for type III mutations, priority is given to sites with transversions; priority is given to somatic mutation sites with a mutation frequency >10%.

[0407] Step 4, ctDNA sequencing detection: Construct a library of ctDNA, hybridize it with the negative ctDNA sample library using the customized probe panel from step 3, and capture it.

[0408] Step 5, MRD sequencing: Sequencing was performed using a Genemind SURFSeq 5000 sequencer (PE100).

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

[0410] Example 2

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

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

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

[0414] 1.1 Take the components from kit 1 for the experiment. Take samples according to the amount of DNA input 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 shown in Table 2 below and prepare the reaction system on an ice box.

[0416] Table 2

[0417] DNA samples (FFPE and peripheral blood) 10 (500 ng / 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 an ice plate.

[0419] 1.3 Shake the mixture in the PCR tube to mix well and centrifuge. Place it on a PCR instrument to perform the reaction as shown in Table 3 below.

[0420] Table 3

[0421] 25℃ 15 / 23 min (FFPE / peripheral blood) 1 65℃ 30min 1 4℃ Keep 1

[0422] 2. Connector connection:

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

[0424] Table 4

[0425] Reagent 5 2 Reagent 6 26 Total volume 28

[0426] 2.2 Adapter ligation: First, add 2 μL of reagent 4 to the reaction system of step 1, up to the bottom of the PCR tube, then add 28 μL of the prepared reagent, vortex to mix, centrifuge, and place on a PCR instrument to perform the reaction as shown in Table 5 below:

[0427] Table 5

[0428] 20℃ Keep 1 20℃ 15min 1 4℃ Keep 1

[0429] 3. Purification of ligation products:

[0430] 3.1 Mixing magnetic beads: Shake reagent 7 until it is mixed evenly (Important note: Reagent 7 must be mixed evenly before purification, otherwise it will affect the purification effect).

[0431] 3.2 Add magnetic beads: Add 40 μL (0.5 times the volume) of reagent 7 to each well. Use a pipette to pipette 10 times. If air bubbles are generated 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 min to allow the PCR products to bind to the magnetic beads.

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

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

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

[0436] Important Note: Avoid contact between 80% ethanol and air. Otherwise, the ethanol concentration will change, affecting the cleaning effect on the magnetic beads. Prepare only enough 80% ethanol for each use.

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

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

[0439] 3.9 Dry the magnetic beads: Keep the PCR reaction plate on the magnetic rack and leave it at room temperature for 2-5 minutes.

[0440] Important Note: Do not allow the magnetic beads to become overly dry. Small cracks appearing in the middle of the bead cluster indicate that the beads are sufficiently dry. If large cracks appear running through the entire cluster, or if the beads break into small, thin pieces, they are overly dry. Overly dry magnetic beads will be difficult to resuspend.

[0441] 3.10 Resuspending the magnetic beads: Remove the PCR plate / tube from the magnetic rack and immediately add 20 μL of nuclease-free water to each tube to resuspend the magnetic beads. Gently pipette the suspension 10 times. If air bubbles appear 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 kit 5 and thaw them at room temperature.

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

[0445] Table 6

[0446] Reagent 8 25 Reagent 9 5 The magnetic bead suspension obtained in step 3 20 Total volume 50

[0447] Note that you should first add 25 μL of reagent 8 to the suspension obtained in step 3, shake to mix and centrifuge; then add 5 μL of reagent 9 below the surface of the above suspension.

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

[0449] Table 7

[0450]

[0451] The above reaction process yields the library.

[0452] 5. Library purification:

[0453] 5.1 Mixing magnetic beads: Shake reagent 7 until it is mixed evenly (Important note: Reagent 7 must be mixed evenly before purification, otherwise it will affect the purification effect).

[0454] 5.2 Add magnetic beads: Add 50 μL (1 volume) of reagent 7 to each well. Use a pipette to pipette 10 times. If air bubbles are generated 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 min to allow the PCR products to bind to the magnetic beads.

[0456] 5.4 Separating magnetic beads: Place the PCR plate / tube on a magnetic rack for 5 minutes, until the solution becomes clear.

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

[0458] 5.6 Cleaning the magnetic beads: Keep the PCR plate / tube on the magnetic rack. Add 150 μL of freshly prepared 80% ethanol to each tube. Incubate for 30 seconds, then aspirate all supernatant. Be careful not to touch the magnetic beads.

[0459] Important Note: Avoid contact between 80% ethanol and air. Otherwise, the ethanol concentration will change, affecting the cleaning effect on the magnetic beads. Prepare only enough 80% ethanol for each use.

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

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

[0462] 5.9 Dry the magnetic beads: Keep the PCR reaction plate on the magnetic rack and let it sit at room temperature for 2-5 minutes.

[0463] Important Note: Do not allow the magnetic beads to become overly dry. Small cracks appearing in the middle of the bead cluster indicate that the beads are sufficiently dry. If large cracks appear running through the entire cluster, or if the beads break into small, thin pieces, they are overly dry. Overly dry magnetic beads will be difficult to resuspend.

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

[0465] 5.11 Separate the supernatant: Place the PCR plate / tube back into the magnetic rack until the solution becomes clear. This process may take 5 minutes. Transfer 30 μL of the supernatant to a new PCR tube to obtain the purified library.

[0466] 6.1 Qubit quantification: Take 1 μL of the 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 was used to detect the library length, and the main peak was between 250-550bp.

[0468] 7. Library hybridization:

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

[0470] Table 8

[0471] Reagent 10 5 Reagent 11 2 Total volume 50

[0472] Take 1000 ng of the obtained library according to the concentration and place it in a PCR tube. Add the prepared 7 μL mixture and place it in a vacuum filter (V-AQ mode, 60℃) to dry into a dry powder for about 40 min.

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

[0474] Table 9

[0475] 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 dried library powder, vortex to mix, centrifuge, and then place on a PCR instrument to perform the reaction as shown in Table 10 below:

[0477] Table 10

[0478] 95℃ 30s 1 65℃ 16h 1

[0479] 8. Document capture

[0480] 8.1 Remove reagents 15 and 16 from the refrigerator in kit 7 and allow them to equilibrate at room temperature for 30 minutes before use.

[0481] 8.2 Take 50 μL of reagent 16 (capture magnetic beads) for each sample, place it on a magnetic rack, discard the supernatant, add 100 μL of reagent 15 to resuspend the magnetic beads, mix well, centrifuge briefly, place on a magnetic rack, and discard the supernatant after the liquid has completely clarified; repeat the above steps once.

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

[0483] Table 11

[0484] Reagent 12 8.5 Reagent 13 2.7 Reagent 1 5.8 Total volume 17

[0485] Transfer the resuspended magnetic beads to a new eight-cell array (17 μL per well) and incubate at 65°C for 5 min in a constant temperature mixer.

[0486] 8.4 Capture:

[0487] 8.4.1 Transfer the preheated resuspended magnetic beads to the capture product, pipette the resuspended magnetic beads and incubate in a PCR instrument (65℃) for 45 min. Gently pipette and mix thoroughly every 10-12 min for a total of 4 times to keep the magnetic beads in suspension.

[0488] 8.4.2 After incubation, remove the PCR tube from the PCR instrument 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 it to a new 1.5 mL EP tube preheated to 65°C.

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

[0490] 8.4.4 Quickly add 150 μL of 65℃ ES Wash Buffer to the EP tube again, gently blow and aspirate 15 times to mix thoroughly, and incubate at 65℃ for 5 min.

[0491] 8.4.5 After brief centrifugation, place on a magnetic rack for 30 seconds. Once the liquid is completely clear, aspirate and discard the supernatant, discarding as much residual buffer as possible. Add 150 μL of room temperature ES Wash Buffer and gently pipette 15 times to mix thoroughly. Transfer the entire reaction solution with the magnetic beads to a new 1.5 mL low-adsorption EP tube and incubate at room temperature for 2 minutes. During this time, vortex for 30 seconds and let stand for 30 seconds, alternating between the two to ensure thorough mixing.

[0492] 8.4.6 After the above EP tubes are briefly centrifuged, place them on a magnetic rack for 30s. After the liquid is completely clear, discard the residual buffer and add 150μL of room temperature ES Wash Buffer. Gently blow and aspirate 15 times to mix thoroughly. Incubate at room temperature for 2min, vortexing for 30s and letting stand for 30s during the incubation period. Repeat this process to ensure thorough mixing.

[0493] 8.4.7 After briefly centrifuging the above centrifuge tubes, place them on a magnetic rack for 30 seconds. Once the liquid is completely clear, discard any remaining buffer. After brief centrifugation, use a small pipette tip to remove any remaining liquid.

[0494] 8.4.8 Remove the EP tube from the magnetic rack, add 22.5 μL of Nuclease Free Water, gently pipette 10 times to ensure thorough mixing, and 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, allow them to equilibrate to room temperature, and then prepare the reaction solution according to the following system (Table 12):

[0497] Table 12

[0498] Reagent 17 25 Reagent 18 2.5 The magnetic bead mixture obtained in step 8 22.5 Total volume 50

[0499] 9.2 Vortex and centrifuge the mixture in the PCR tube, then place it on a PCR instrument for the following reaction (Table 13):

[0500] Table 13

[0501]

[0502] 10. Library purification after capture:

[0503] 10.1 Library Purification:

[0504] 10.1.1 After briefly centrifuging the amplification product from step 9, add 50 μL of the product to the amplification product. Mix SP Beads thoroughly and incubate at 25°C for 10 minutes.

[0505] 10.1.2 After briefly centrifuging the PCR tube, 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 wall of the PCR tube, being careful not to disturb the magnetic beads. Let it stand for 30 seconds, then use a pipette to remove and discard the supernatant.

[0507] 10.1.4 Repeat step 10.3 once; after briefly centrifuging the PCR tube, place it on a magnetic rack and use a 10μL pipette tip to remove a small amount of residual ethanol, being careful not to aspirate 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 has completely evaporated (Note: Do not dry it too much, otherwise it will affect the magnetic bead recovery efficiency).

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

[0510] 10.1.7 After briefly centrifuging the PCR tube, place it on a magnetic rack for 2 minutes until the liquid is completely clear. Carefully transfer the supernatant to a new 1.5 mL EP tube for storage using a pipette, being careful not to aspirate the magnetic beads.

[0511] 10.2. Document Quality Inspection:

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

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

[0514] 11. Sequencing:

[0515] Sequencing was performed using the Genemind sequencing platform and the SURFSeq 5000 sequencer. It is recommended to use the PE150 or PE100 sequencing chip for sequencing, with 15G of data per sample.

[0516] Example 3

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

[0518] 1. Use the bioinformatics software shown in Table 14 below for data analysis:

[0519] Table 14

[0520]

[0521] 2. The quality control requirements for sequencing data are as follows: Taking into account the actual sample sequencing quality and the requirements for the mutation detection software to accurately detect mutations, the quality control threshold requirements for sequencing data are as follows (Table 15).

[0522] Table 15

[0523] Average sequencing depth (X) (tissue) On average, each base was sequenced an average of [number] times. ≥200X (tissue) Average sequencing depth (X) (peripheral blood) On average, each base was sequenced an average of [number] times. ≥100X(peripheral blood) Base ratio ≥100x (tissue) Percentage of bases sequenced more than 100 times ≥85％ ≥50x base ratio (peripheral blood) Percentage of bases sequenced more than 50 times ≥85％ Sequence alignment rate (OnTarget) The degree of matching between sequencing results and the reference genome ≥95％ Q30 The reliability of this base is 99.9%. ≥85％

[0524] Example 4

[0525] A tumor-informed molecular detection kit and method for MRD (subsequent monitoring of MRD).

[0526] 1. Customized subsequent site design:

[0527] From the initial WES results, select 35 loci that meet the criteria in the overview. Upload the chromosomal coordinates of the loci to 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. Construction of ct DNA library:

[0529] 2.1 Take reagents 19 and 20 from kit 9 ​​for the experiment. Take samples according to the amount of ct DNA added (66 ng). Take 0.2 mL PCR tubes according to the number of samples and label the tube caps with the sample numbers.

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

[0531] Table 16

[0532] ctDNA sample 10 (66 ng / 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 an ice plate.

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

[0535] Table 17

[0536] 30℃ 30min 1 65℃ 30min 1 4℃ Keep 1

[0537] 3. Connector connection

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

[0539] Table 18

[0540] Reagent 1 10 Reagent 21 5 Reagent 22 30 Reagent 23 5 The mixture obtained in step 2 60 Total volume 110

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

[0542] Table 19

[0543] 22℃ Keep 1 22℃ 15min 1 4℃ Keep 1

[0544] 4. Purification of ligation products:

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

[0546] 4.2 Add magnetic beads: Add 88 μL (0.8 times the volume) of reagent 24 to each well. Use a pipette to pipette 10 times. If air bubbles are generated 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 min to allow the PCR products to bind to the magnetic beads.

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

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

[0550] 4.6 Cleaning the magnetic beads: Keep the PCR plate / tube on the magnetic rack. Add 200 μL of freshly prepared 80% ethanol to each tube. Incubate for 30 seconds, then aspirate all supernatant. Be careful not to touch the magnetic beads.

[0551] Important Note: Avoid contact between 80% ethanol and air. Otherwise, the ethanol concentration will change, affecting the cleaning effect on the magnetic beads. Prepare only enough 80% ethanol for each use.

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

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

[0554] 4.9 Dry the magnetic beads: Keep the PCR reaction plate on the magnetic rack and let it sit at room temperature for 5 minutes.

[0555] Important Note: Do not allow the magnetic beads to become overly dry. Small cracks appearing in the middle of the bead cluster indicate that the beads are sufficiently dry. If large cracks appear running through the entire cluster, or if the beads break into small, thin pieces, they are overly dry. Overly dry magnetic beads will be difficult to resuspend.

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

[0557] 4.11 Absorb the supernatant: After briefly centrifuging the PCR tube, place it on a magnetic rack for 2 minutes until the solution becomes clear. Use a pipette to aspirate 20 μL of the supernatant and transfer it to a new PCR tube. Label the tube and prepare for the index-PCR reaction.

[0558] 5. Index PCR amplification:

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

[0560] Prepare the reaction solution according to the system in Table 20 below:

[0561] Table 20

[0562] Reagent 25 25 Reagent 26 5 The supernatant obtained in step 3 20 Total volume 50

[0563] Note that you should first add 25 μL of reagent 25 to the supernatant obtained in step 3, shake to mix and centrifuge; then add 5 μL of reagent 26 below the surface of the above suspension.

[0564] 5.2 Vortex and centrifuge the mixture in the PCR tube, then place it on a PCR instrument to perform the reactions shown in Table 21 below:

[0565] Table 21

[0566]

[0567] The above reaction yields the ct DNA library.

[0568] 6. Library purification:

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

[0570] 6.2 Add magnetic beads: Add 50 μL (1 volume) of reagent 24 to each well. Use a pipette to pipette 10 times. If air bubbles are generated 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 min to allow the PCR products to bind to the magnetic beads.

[0572] 6.4 Separating magnetic beads: Place the PCR plate / tube on a magnetic rack for 5 minutes, until the solution becomes clear.

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

[0574] 6.6 Cleaning the magnetic beads: Keep the PCR plate / tube on the magnetic rack. Add 150 μL of freshly prepared 80% ethanol to each tube. Incubate for 30 seconds, then aspirate all supernatant. Be careful not to touch the magnetic beads.

[0575] Important Note: Avoid contact between 80% ethanol and air. Otherwise, the ethanol concentration will change, affecting the cleaning effect on the magnetic beads. Prepare only enough 80% ethanol for each use.

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

[0577] 6.8 Remove residual ethanol: Completely remove any remaining trace amounts of ethanol from each tube. Centrifuge briefly for 15 seconds, return the PCR plate / tube to the magnetic rack, and use a 10-20 μL pipette tip to remove any remaining ethanol solution from the bottom of the tube.

[0578] 6.9 Dry the magnetic beads: Keep the PCR reaction plate on the magnetic rack and let it sit at room temperature for 5 minutes.

[0579] Important Note: Do not allow the magnetic beads to become overly dry. Small cracks appearing in the middle of the bead cluster indicate that the beads are sufficiently dry. If large cracks appear running through the entire cluster, or if the beads break into small, thin pieces, they are overly dry. Overly dry magnetic beads will be difficult to resuspend.

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

[0581] 6.11 Separate the supernatant: Place the PCR plate / tube back into the magnetic rack until the solution becomes clear. This process may take 5 minutes. Transfer 30 μL of the supernatant to a new PCR tube to obtain the purified library.

[0582] 7. Document Quality Inspection:

[0583] 7.1 Qubit quantification: Take 1 μL of the 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] 7.2 Qseq monitoring of library length showed that the main peak was between 250-550 bp.

[0585] 8. Library hybridization:

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

[0587] Table 22

[0588] Reagent 27 13 Reagent 28 5 Reagent 28 2 Reagent 29 5 Reagent 30 2 Reagent 1 3 Total volume 30

[0589] Take 1000 ng of the obtained library (the ctDNA library to be tested and the negative library) according to the concentration and place it in a PCR tube. Place it in a vacuum filter (V-AQ mode, 60℃) to dry into a dry powder for about 25 min.

[0590] 8.2 Add 30 μL of hybridization mixture to the dried library powder, vortex to mix, centrifuge, and then place on a PCR instrument to perform the reaction as shown in Table 23 below:

[0591] Table 23

[0592] 85℃ 5min 1 60℃ 16 1

[0593] 9. Document capture

[0594] 9.1 Preparations before capture

[0595] 9.1.1 Remove reagents 32 and 33 from the refrigerator in kit 14 and allow them to equilibrate to room temperature.

[0596] 9.1.2 Take 30 μL of Reagent 32 (capture magnetic beads) for each hybridization library, place it on a magnetic rack, discard the supernatant, add 180 μL of Reagent 32 to resuspend the magnetic beads, mix well, centrifuge briefly, place on a magnetic rack, and discard the supernatant after the liquid is completely clear; repeat the above steps 2 times.

[0597] 9.1.3 Add 180 μL of reagent 32 resuspension magnetic beads to each hybridization library: Transfer the hybridization product to the resuspension magnetic beads, place them on a vertical rotary mixer, rotate at 5 r, and bind at room temperature for 20 min.

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

[0599] 9.1.5 Remove the PCR tube from the magnetic rack and add 150 μL of TargetSeq solution preheated to 60°C. WashBuffer 2v2, gently aspirate and mix, centrifuge briefly, place on a constant temperature shaker or metal bath, and incubate at 60℃ for 5 min.

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

[0601] 9.1.7 Repeat steps 9.5-9.6 four times.

[0602] 9.1.8 Keep the PCR tube on the magnetic rack, add 200 μL of 80% ethanol to the PCR tube, let it stand for 30 seconds, and then completely discard the ethanol solution (you can use a 10 μL pipette to discard the residual ethanol). Let the magnetic beads air dry at room temperature to allow the residual ethanol to evaporate completely.

[0603] 9.1.9 Add 24 μL of Nuclease-Free Water to the PCR tube, remove the PCR tube from the magnetic rack, briefly vortex to resuspend and mix 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℃, allow them to equilibrate to room temperature, and then prepare the reaction solution according to the system shown in Table 24 below:

[0606] Table 24

[0607] Reagent 34 1 Reagent 35 25 The 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, then place it on a PCR instrument to perform the reactions shown in Table 25 below:

[0609] Table 25

[0610]

[0611] 9.3 Purification after capture:

[0612] 9.3.1 After briefly centrifuging the amplification product from 9.2, add 55 μL of IGT Pure Beads and mix well. 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 until the solution becomes 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, 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 briefly to remove the liquid adhering to the tube wall to the bottom, and then use a 10 μL pipette to discard the remaining ethanol solution at the bottom).

[0616] 9.3.5 Ensure the PCR tubes are on a magnetic rack and allowed to stand at room temperature for 5 minutes to allow the magnetic beads to dry completely and any residual ethanol to evaporate.

[0617] 9.3.6 Add 25 μL of Nuclease-Free Water, remove the PCR tube from the magnetic rack, mix by pipetting or vortexing, and let stand at room temperature for 2 minutes.

[0618] 9.3.7 Brief centrifugation: Place the PCR tube on a magnetic rack for 2 minutes until the solution becomes clear.

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

[0620] 9.4 Document Quality Inspection:

[0621] 9.4.1 Take 1 μL of the library and use the Qubit dsDNAHS Assay Kit reagents to determine the library concentration on a Qubit 4.0 Fluorometer, and record the library concentration.

[0622] 9.4.2 Take 1 μL of the library and use a fragment analyzer to perform fragment quality control. The fragment size should be basically the same as the pre-library size.

[0623] 10. Sequencing:

[0624] Sequencing was performed using the Genemind sequencing platform and the SURFSeq 5000 sequencer. It is recommended to use the PE150 or PE100 sequencing chip for sequencing, with 3GB of data per sample.

[0625] Example 5

[0626] Bioinformatics analysis workflow and data quality control for MRD molecular detection results of solid tumors.

[0627] 1. Use the bioinformatics software shown in Table 26 below for data analysis:

[0628] Table 26

[0629] Fastp v0.23.2 To the connector Vardict v202112.04 Sequence alignment, consensus sequence generation based on UMI information, and variant detection. annovar v2020 Annotation SNV / Indel variants delly v0.9.1 Detecting gene fusion variants cnvkit v0.9.10 Detection of SNV / Indel variants bcftools v1.8 Merge VCF files

[0630] 2. The quality control requirements for sequencing data are as follows: Taking into account the actual sample sequencing quality and the requirements for the mutation detection software to accurately detect mutations, the quality control threshold requirements for sequencing data are shown in Table 27 below.

[0631] Table 27

[0632] Average sequencing depth (X) On average, each base was sequenced an average of [number] times. ≥200,000X ≥2000x base ratio Percentage of bases sequenced more than 2000 times ≥90％ ≥500x base ratio Percentage of bases sequenced more than 500 times ≥85％ Sequence alignment rate (OnTarget) The degree of matching between sequencing results and the reference genome ≥95％ Q30 The reliability of this base is 99.9%. ≥85％

[0633] Example 6

[0634] The accuracy and repeatability of the MRD molecular detection kit for solid tumors.

[0635] 1. Verification of the accuracy and repeatability of ctDNA standard SNV / Indel: The ctDNA standard with a mutation frequency (VAF) of 0.5% was diluted to VAFs of 0.1%, 0.05%, 0.02%, 0.01%, and 0.005%. 66 ng of each frequency was added for molecular detection of MRD in solid tumors, and each group was run in parallel to obtain accuracy and repeatability results. (Quantitative analysis using digital PCR by the standard manufacturer showed that the actual frequencies of the four loci in the BRCA1, BRCA2, and CDK12 genes in this standard were higher than the labeled frequencies, by 4-5 times.)

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

[0637] Table 28

[0638]

[0639]

[0640] Sequencing tests were performed on ct DNA standards with VAF = 0.1%. Both parallel tests 1 and 2 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 met the expected results.

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

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

[0643] Table 29

[0644]

[0645]

[0646] Sequencing was performed on ct DNA standards with VAF = 0.05%. Parallel 1 detected 31 SNV mutation sites within the detection range, with a detection rate of 100%; parallel 2 detected 30 SNV mutation sites within the detection range, with a detection rate of 96.7%. The measured mutation frequency was basically consistent with the theoretical mutation frequency, which met the expected results.

[0647] Intra-batch consistency: One ct DNA standard (GW-OCTM802) with VAF=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 is 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 was performed on the ct DNA standard with VAF=0.02%. Parallel 1 detected a total of 28 SNV mutation sites within the detection range, with a detection rate of 90.3%; parallel 2 detected a total of 26 SNV mutation sites within the detection range, with a detection rate of 83.8%. The measured mutation frequency was basically consistent with the theoretical mutation frequency, which met the expected results.

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

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

[0655] Table 31

[0656]

[0657]

[0658] Sequencing was performed on the ct DNA standard with VAF=0.01%. Parallel 1 detected a total of 20 SNV mutation sites within the detection range, with a detection rate of 64.5%; parallel 2 detected a total of 21 SNV mutation sites within the detection range, with a detection rate of 67.7%. The measured mutation frequency was basically consistent with the theoretical mutation frequency, which met the expected results.

[0659] Intra-batch consistency: One ct DNA standard (GW-OCTM802) with VAF=0.01% was replicated twice within the same batch, and the intra-batch consistency was 77.4%, which met the expected results.

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

[0661] Table 32

[0662]

[0663]

[0664] Sequencing was performed on ctDNA standards with VAF = 0.01%. Parallel 1 detected 12 SNV mutation sites within the detection range, with a detection rate of 38.7%; Parallel 2 detected 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 met the expected results.

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

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

[0667] 2. Fusion accuracy and repeatability verification of ct DNA standard: The ct DNA standard with a mutation frequency (VAF) of 0.5% was diluted to VAF of 0.1% and 0.05%, and 66ng was added for each frequency for solid tumor MRD molecular detection. Each group was also performed in parallel to obtain accuracy and repeatability results.

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

[0669] Table 33

[0670]

[0671] Sequencing was performed on ct DNA standards with VAF (Fusion) = 0.1%. 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 5 Fusion 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 met the expected results.

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

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

[0674] Table 34

[0675]

[0676] Sequencing was performed on ctDNA standards with VAF (Fusion) = 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 met the expected results.

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

[0678] Example 6

[0679] Minimum input and repeatability validation of the MRD molecular detection kit for solid tumors.

[0680] 1. Validation of the dosage and repeatability of ctDNA standard SNV / Indel: The ctDNA standard with a mutation frequency (VAF) of 0.5% was diluted to a VAF of 0.01%, and 66ng, 44ng, 22ng, and 10ng were added respectively for the molecular detection of solid tumor MRD. Each group was also performed in parallel to obtain the minimum dosage and repeatability results.

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

[0682] Table 35

[0683]

[0684] 66 ng of ct DNA standard with VAF(SNV) = 0.01% was added for sequencing detection. Parallel 1 detected 16 SNV / Indel mutation sites within the detection range, with a detection rate of 51.6%; Parallel 2 detected 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 met the expected results.

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

[0686] The results of adding 44 ng of 1.2VAF(SNV) = 0.01% ctDNA standard and the repeatability verification are shown in Table 36 below:

[0687] Table 36

[0688]

[0689]

[0690] 44 ng of ct DNA standard with VAF(SNV) = 0.01% was added for sequencing detection. 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 met the expected results.

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

[0692] The results of adding 22 ng of 1.3VAF(SNV) = 0.01% ct DNA standard and the repeatability verification are shown in Table 37 below:

[0693] Table 37

[0694]

[0695]

[0696] 22 ng of ctDNA standard with VAF(SNV) = 0.01% was added 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 met the expected results.

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

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

[0699] Table 38

[0700]

[0701]

[0702] 10 ng of ct DNA standard with VAF(SNV) = 0.01% was added for sequencing detection. 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 met the expected results.

[0703] The expert consensus states that for MRD molecular detection of solid tumors, a positive result is achieved with the detection of two out of 16 sites. In this experiment, when VAF = 0.01%, a detection rate of over 29% was achieved with an input of 22 ng. Therefore, the minimum nucleic acid input in this experiment can be as low as 22 ng. (After removing four high-frequency sites from the standard, the detection rate in parallel experiment 2 with an input of 10 ng was low, with only one site detected; therefore, the minimum input was set at 22 ng.)

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

[0705] In summary, this invention designs a four-layer encrypted shingled hybridization capture probe for detecting minimal residual disease (MRD), providing a highly sensitive and specific molecular detection method for MRD, with high low-frequency mutation capture efficiency, enabling the detection limit of ctDNA mutation frequency containing MRD to be as low as 0.005%.

[0706] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A probe for detecting minute residual lesions, characterized in that, The probe used to detect minimal residual lesions is a four-layer encrypted shingled hybridization capture probe, wherein two layers are wild-type probes and two layers are mutant probes. The wild-type probes include probes with nucleic acid sequences as shown in SEQ ID NO.1-SEQ ID NO.74, and the mutant probes include probes with nucleic acid sequences as shown in SEQ ID NO.75-SEQ ID NO.

149.

2. The use of the probe for detecting minimal residual lesions as described in claim 1 in the preparation of products for detecting minimal residual lesions.

3. A reagent kit for detecting minimal residual lesions, characterized in that, The kit includes the probe for detecting minute residual lesions as described in claim 1.

4. A device for detecting minute 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 gDNA from the FFPE sample and peripheral blood sample submitted by the patient for the first test; and extracting ctDNA from the cell-free nucleic acid from the non-invasive blood sample submitted by the patient for subsequent tests. The detection module is used to perform the following: constructing libraries from FFPE sample nucleic acid and peripheral blood sample nucleic acid gDNA, hybridizing whole exome probes and capturing them for sequencing 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 probes for detecting minimal residual lesions as described in claim 1, constructing libraries from sample ct DNA, and simultaneously selecting ct DNA samples with known negative sequencing results for library construction, and hybridizing the two libraries obtained simultaneously with the customized personalized capture probe panel to capture ct DNA; The sequencing analysis module is used to perform the following: sequencing the captured ctDNA using a sequencer, comparing the ctDNA sequencing results with the human reference genome, performing sequence alignment, marking repetitive sequences, detecting somatic variations, and detecting germline mutations to obtain point mutation results of the sample genes.

5. The device for detecting minute residual lesions according to claim 4, characterized in that, The screening criteria for selecting 30-40 somatic mutation sites are: somatic mutation sites with gene mutation frequencies similar to or half that of tumor cells.

6. The device for detecting minute residual lesions according to claim 4, characterized in that, The somatic mutation sites are somatic cell type I mutation sites, somatic cell type II mutation sites, and somatic cell type III mutation sites where the base mutation type is transversion.

7. The device for detecting minute residual lesions according to claim 4, characterized in that, The somatic mutation sites are somatic mutation sites with a mutation frequency >10%.

8. The device for detecting minute residual lesions according to claim 4, characterized in that, The somatic mutation sites are somatic mutation sites that avoid simple repetitive sequences and large fragment insertion / deletion mutations.

9. The use of the probe for detecting minimal residual disease as described in claim 1, the kit for detecting minimal residual disease as described in claim 3, or the device for detecting minimal residual disease as described in any one of claims 4-8 in the preparation of products for diagnosing solid tumors.

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