A library of standards, kits and methods of use for correcting igh amplification bias

Abstract

The application discloses a standard sample library for correcting IGH amplification bias, a kit and a use method, realizes reduction of more than 100 times of amplification bias of multiplex PCR to 2-10 times through a PCR product connection library process and a long molecular tag with a fixed base structure, effectively reduces interference of different IGH rearrangement primary structure differences in combination with a specific combined standard sample library, and can reduce more than 50 times of fluctuation of one ten thousandth of tumor cell proportion to 2-3 times. Therefore, the application not only simplifies an experimental process, but also has technical advantages of excellent detection performance, low cost and high throughput, and can be applied to detection of lymphoid tumor immune group libraries, and provides precise technical support for detection of IGH rearrangement mutations in multiple myeloma, lymphoma and lymphoid leukemia.

Description

Technical Field

[0001] This invention relates to the field of immune repertoire sequencing technology, and more particularly to a standard library, kit, and method of use for correcting IGH amplification bias. Background Technology

[0002] Minimal residual disease (MRD) refers to the condition in which a small number of cancer cells remain in the body of a patient with leukemia / lymphoma / myeloma / solid tumor after achieving complete remission (CR) through treatment (10). 8 -10 9 This may lead to recurrence. Because conventional imaging cannot detect it, more accurate and sensitive methods are needed to detect residual cancer cells. Current NCCN guidelines and expert consensus recommend using IGH rearrangement detection for MRD in hematologic malignancies.

[0003] Currently, the detection methods for IGH rearrangements include multicolor flow cytometry, PCR, ddPCR, and NGS. Internationally, high-throughput sequencing (NGS) is recommended as the detection method for MRD. Among them, the detection sensitivity of NGS is much higher than that of flow cytometry, especially when the proportion of tumor cells is less than one in ten thousand, NGS can benefit more positive patients.

[0004] However, the drawbacks of existing NGS-based quantification technologies are that they all rely on two rounds of PCR for library construction. This approach amplifies amplification bias, leading to inaccurate tumor cell quantification. This is the primary cause of bias and inaccurate quantification. Secondly, quantification using a single cell reference internal control suffers from significant batch effects, often exhibiting large fluctuations in low-abundance tumor cells, resulting in inaccurate quantification. Furthermore, different rearrangement primary structures and lengths also affect tumor cell quantification. Finally, the reaction of multiple primers in the system results in inconsistent amplification efficiencies for each IGH rearrangement, leading to inaccurate tumor cell quantification. This is particularly severe in the detection of target-dominant clones. Summary of the Invention

[0005] In view of the aforementioned shortcomings and deficiencies of existing technologies, this invention provides a one-round PCR and ligation method combined with a specific combination of standard libraries, thereby providing a simple, efficient, and low-cost high-throughput detection process. This reduces amplification bias caused by two rounds of PCR, and further reduces quantification errors by using a specific combination of standard libraries that are compatible with different rearrangements in actual samples. Finally, by using long molecular tags with fixed base structures, it solves the problem of quantification distortion of tumor cells caused by amplification bias, expands the accuracy of quantitative detection of lymphoid leukemia immune repertoires, and significantly reduces reagent and labor costs.

[0006] On one hand, the present invention provides a standard library for correcting IGH amplification bias. The standard library includes five standards with the standard names VJ118, VJ311, VDJ428, VDJ510 and VJ741, respectively. Among them, VJ118 includes the sequence SEQ ID No: 093, VJ311 includes the sequence SEQ ID No: 094, VDJ428 includes the sequence SEQ ID No: 095, VDJ510 includes the sequence SEQ ID No: 096 and VJ741 includes the sequence SEQ ID No: 097.

[0007] In a further technical solution, the standard library covers the rearrangement regions of V1, V3, V4, and V5, the amplification length is 149bp-338bp, and the VDJ and VJ rearrangements.

[0008] On the other hand, the present invention also provides a kit for correcting IGH amplification bias, the kit comprising a primer set of sequences SEQ ID No: 001-SEQ ID No: 090, a long molecular tag primer with a fixed base structure of sequence SEQ ID No: 092, and a standard library mixture of sequences SEQ ID No: 093-SEQ ID No: 097.

[0009] In a further technical solution, the kit also includes 10×PCR Buffer, dNTPs, and AmpliTaq Gold. ™ DNA polymerase, MgCl2, FEA Buffer V2, FEA Enzyme Mix V2, T4 ligation buffer, T4 ligase and universal adapters for next-generation sequencing.

[0010] In this invention, long molecular tags with a specific combination of fixed base structures cover 12 or more random bases. Simultaneously, a fixed base is designed for every 4 bases in the molecular tag to correct GC bias during primer synthesis. This fixed base design must follow the order A>T>G>C. Because synthesis errors and biases during random base synthesis can lead to an increased proportion of identical base tandem sequences such as AAAAA, GGGGG, and TTTTT, resulting in a significantly higher sequencing error rate and classification errors, the introduction of fixed bases can reduce the number of identical base tandem sequences. Furthermore, the random base sequence synthesis process can result in a biased proportion of G and C bases, disrupting the balance. Therefore, A and T are preferentially designed as fixed bases.

[0011] The present invention also provides a method of using the reagent kit based on the present invention, which includes the following steps: S1. Extract total DNA from the leukemia sample to be tested and mix it with a standard library mixture containing sequences SEQ ID No: 093-SEQ ID No: 097; S2. Multiplex PCR amplification of the mixed DNA product was performed using primers containing the primer set with the sequence SEQ ID No: 001-SEQ ID No: 090 and the long molecular tag primer with the fixed base structure of the sequence SEQ ID No: 092, to obtain the PCR-amplified DNA; S3. The PCR-amplified DNA is ligated with adapters, constructed into a library, and then sequenced to obtain the total amount of DNA library and sequencing data. S4. Perform bioinformatics filtering analysis, calculate reads caused by filtering amplification bias, and simultaneously determine the proportion of tumor cells based on a standard library of specific combinations with known copy numbers.

[0012] In a further technical solution, the PCR reaction conditions in step S2 are as follows: Step 1: 95℃, 10min; Step 2: 95℃, 45sec; Step 3: 60℃, 45sec; Step 4: 72℃, 90sec; Steps 2 to 4 are cycled 35 times; Step 5: 72℃, 7min; Step 6: Hold at 4℃.

[0013] In a further technical solution, in step S3, the adapter ligation involves ligating the PCR-amplified DNA with a second-generation universal adapter to obtain a ligation product that can be directly sequenced.

[0014] In a further technical solution, step S4, the calculation of reads due to amplification bias includes the following process: Calculate the same rearranged reads before and after deduplication using long molecular tag primer sequences with fixed base structures. 1 minus Mi equals the amplification Dup, calculated using the following formula: ; Here, the reads after deduplication are denoted as Xi, the reads before deduplication are denoted as Yi, the proportion of amplification preference after different rearrangement types is denoted as Mi, and i represents the number of tens of thousands of rearrangement types.

[0015] In a further technical solution, step S4, the tumor cell percentage includes the following calculation steps: The tumor cell percentage is calculated using a long molecular tag primer sequence with a fixed base structure and a standard library. The nanograms of sample input per case are known to be Ck, and the total number of cells per sample is E. The sample contained 30 ng of human genomic DNA, totaling 10,000 copies. The total copy number was calculated, and then divided by 2 to determine the total cell count. The copy number of a specific combination of standard library samples was denoted as Aj, the deduplicated reads of that standard library were denoted as Bj, and the deduplicated reads of tens of thousands of rearrangements were denoted as Xi. The number of tumor cells D in this sample was: Finally, the percentage of tumor cells in this sample (Mj) is calculated by dividing the number of tumor cells in this sample by the total number of cells in this sample and multiplying by 100%. Finally, the average tumor cell percentage Mj of each standard in the standard library is calculated to obtain the final tumor cell percentage of the sample, where k represents the number of samples, j represents the number of standard libraries for this specific combination, and i represents the number of tens of thousands of rearrangements detected.

[0016] The beneficial effects of this invention are: Given the three major shortcomings of existing immunohistochemistry library detection for lymphoid leukemia: firstly, the overall process is complex, with two rounds of amplification further amplifying bias and leading to high costs for patients; secondly, using a single cell reference internal control for quantification results in significant batch effects, failing to consider different rearrangement types and amplification lengths, leading to 50-fold fluctuations in quantification of tumor cells below 0.01%; and thirdly, inconsistent multiplex amplification efficiency leads to inaccurate tumor cell quantification, as existing molecular tags do not consider rearrangement types, synthesis biases, and errors, thus only correcting less than 40% of amplification duplications (Dup), with amplification bias still exceeding 100-fold. This invention first eliminates the second round of amplification in the library construction process to reduce amplification bias, then optimizes a long molecular tag with a fixed base structure to remove amplification bias, combined with an optimized standard library combination for accurate tumor cell quantification, ultimately ensuring the accuracy of tumor cell quantification. The data model after deduplication in this invention reduces the Dup bias by at least 75% compared to the data model before deduplication, with an amplification bias of only 2-10 times. Compared to existing technologies that can only correct less than 40% of Dup, with amplification bias still exceeding 100-fold, this represents a significant performance improvement. The deduplication standard library data model of this invention shows that even when tumor cells account for only one ten-thousandth, without considering highly complex rearrangements, there will be large quantitative fluctuations, with a fluctuation range of about 50 times. However, the standard library data model of this invention comprehensively considers complex subtyping and can stabilize the quantitative analysis of tumor cells at 2-3 times. Combined with the long molecular tag with fixed base structure mentioned above, the quantitative analysis of tumor cells can be stabilized at about 0.7-1 times.

[0017] The method of this invention uses DNA as a detection sample, which is first mixed with a specific standard library. Then, it uses a set of long molecular tag primers with a dedicated fixed base structure for landing PCR targeted amplification. The data is then obtained through a standardized purification, library construction, and next-generation sequencing process. Finally, the data is processed using preference correction and tumor cell quantification methods to simultaneously determine the proportion of tumor cells in the sample. Furthermore, this invention clarifies the application scenarios of this technology in the detection of rearrangements and recurrence monitoring in lymphoid hematologic malignancies.

[0018] This invention not only simplifies the experimental procedure, but also has the technical advantages of excellent detection performance, low cost and high throughput. It can be applied to the detection of lymphoid tumor immune repertoires and provides precise technical support for the detection of IGH rearrangement mutations in multiple myeloma, lymphoma and lymphoid leukemia. Attached Figure Description

[0019] Figure 1 The graph shows the correlation between NGS and the proportion of tumor cells detected by flow cytometry.

[0020] Figure 2 A graph showing the amplification preference results for different rearrangement types.

[0021] Figure 3 This is a graph showing the results of genotyping types and amplification preferences.

[0022] Figure 4 The graph shows the number of reads and the classification results before and after molecular tag correction.

[0023] Figure 5 This is a graph showing the amplification preference levels before and after molecular tag correction.

[0024] Figure 6 This is a graph showing the results of amplification preference levels for specific samples. Detailed Implementation

[0025] To better explain and facilitate understanding of this invention, a detailed description of the invention is provided below with reference to the accompanying drawings and specific embodiments. All raw materials used in this invention are commercially available, and experimental methods not specifically described are based on conventional operating methods known to those skilled in the art.

[0026] This invention provides a process from experimental DNA targeted enrichment and ligation library construction to analytical amplification bias correction and specific standard library for tumor cell quantification. By using ligation library construction and a set of long molecular tag primers with a dedicated fixed base structure to correct amplification bias, the performance of this technology can be improved by more than 25% compared to existing technologies. Finally, the proportion of tumor cells in the sample is determined using a specific standard library, and the quantitative fluctuation can be reduced from 50 times to 2-3 times. Furthermore, this invention clarifies the application scenarios of this technology in the detection of rearrangements and recurrence monitoring in lymphoid hematologic malignancies.

[0027] The specific verification examples are as follows:

[0028] Example 1 This embodiment provides a PCR amplification system based on DNA clinical samples, first optimizing the IGH rearrangement to ensure sufficient yield for subsequent ligation reactions.

[0029] (1) The sequences of the primers for multiplex PCR based on the detection of IGH rearrangement are shown in Table 1. The primers were synthesized by Shanghai Qingke Biotechnology Co., Ltd. The primers SEQ ID No: 001-SEQ ID No: 091 were mixed in equal proportions and then diluted to 10 μM.

[0030] Table 1: Primer Design Sequences SEQ ID No: 001 TCGCAGACCCTCTCACTCACCTGT SEQ ID No: 002 GATCTCCTGTAAGGGTTCTGGATACAGC SEQ ID No: 003 AAACCCACACAGACCCTCACGCTGA SEQ ID No: 004 AAACCCACAGAGACCCTCACGCTGA SEQ ID No: 005 AAACCCACACAGACCCTCACACTGA SEQ ID No: 006 CTCAGTGAAGGTCTCCTGCAAGG SEQ ID No: 007 CTCGGTGAAGGTCTCCTGCAAGG SEQ ID No: 008 CTCAGTGAAGGTTTCCTGCAAGG SEQ ID No: 009 GGCTACAGTGAAAATCTCCTGCAAGG SEQ ID No: 010 TTCGGAGACCCTGTCCCTCACCTG SEQ ID No: 011 TTCGGACACCCTGTCCCTCACCTG SEQ ID No: 012 TTCACAGACCCTGTCCCTCACCTG SEQ ID No: 013 TTCGGAGACCCCGTCCCTCACCTG SEQ ID No: 014 GGGGACCCTGTCCCTCACCTG SEQ ID No: 015 TGGGGGGTCCCTGAGACTCTCCTG SEQ ID No: 016 TGGGGGGTCCCTTAGACTCTCCTG SEQ ID No: 017 AGGGCGGTCCCTGAGACTCTCCTG SEQ ID No: 018 AGGGCCGTCCCTGAGACTCTCCTG SEQ ID No: 019 TGGGGGGTCCCTGAAACTCTCCTG SEQ ID No: 020 TGGCAGGTCCCTGAGACTCTCCTG SEQ ID No: 021 TGGAGGGTCCCTGAGACTCTCCTG SEQ ID No: 022 TGGGAGGTCCCTGAGACTCTCCTG SEQ ID No: 023 TGGGGGGGCCCTGAGACTCTCC SEQ ID No: 024 TGGATCAGGCAGTCCCCATCGAGA SEQ ID No: 025 CTGGGTGCGCCAGATGCCC SEQ ID No: 026 TGTGAGCTGGATCCGTCAGCC SEQ ID No: 027 TGTGGGCTGGATCCGTCAGCC SEQ ID No:028 GTGCGACAGGCCCCTGGACA SEQ ID No:029 GGGTGCGACAGGCCACTGGACA SEQ ID No:030 GTGCGCCAGGCCCCCGGACA SEQ ID No:031 GGGTGCGACAGGCTCGTGGACA SEQ ID No:032 GGGTGCAACAGGCCCCTGGAAA SEQ ID No:033 GGGTGCGACAGGCTCCTGGAAA SEQ ID No:034 GTGCGACAGGCCCCCGGACA SEQ ID No:035 GTGCGACAGGCCCCCAGACA SEQ ID No:036 TCCGCCAGCCCCCAGGGAAG SEQ ID No:037 TCCGGCAGCCCCCAGGGAAG SEQ ID No:038 TCCGGCAGCCCACCAGGGAAG SEQ ID No:039 TCCGCCAGCACCCAGGGAAG SEQ ID No:040 TCCGGCAGCCCGCCGGGA SEQ ID No:041 TCCGGCAGCCGCCGGGGA SEQ ID No:042 TCCGGCAGCCCGCTGGGAAG SEQ ID No:043 TCCGCCAGCCCCTAGGGAAG SEQ ID No:044 GTCCGCCAGGCTCCAGGGAA SEQ ID No:045 TTCCGCCAGGCTCCAGGGAA SEQ ID No:046 GTCCGCCAGGCTTCCGGGAA SEQ ID No:047 GTCCGTCAAGCTCCGGGGAA SEQ ID No:048 ATCCGCCAGGCTCCAGGGAA SEQ ID No:049 GTCCGCCAAGCTCCAGGGAA SEQ ID No:050 GTCCGCCAGGCTCCAGGCAA SEQ ID No:051 GTCCGCCAGGCCCCAGGCAA SEQ ID No:052 GTCCGCCAGGCTCCGGGCAA SEQ ID No:053 GGGTCCGTCAAGCTCCAGGGAAG SEQ ID No:054 TGGGTCCGCCAAGCTACAGGAAA SEQ ID No:055 GTCCGCCAGCCTCCAGGGAA SEQ ID No:056 GTCCGGCAAGCTCCAGGGAA SEQ ID No: 057 GTGCGAGCTGGATCCGTCAGC SEQ ID No: 058 GCAGCCTAAAGGCTGAGGACACT SEQ ID No: 059 TCTGTGACTCCCGAGGACACGGCT SEQ ID No: 060 GTGGAGCAGCCTGAAGGCCT SEQ ID No: 061 TGACCAACATGGACCCTGTGGACA SEQ ID No: 062 ACATGGAGCTGAGCAGCCTGAGAT SEQ ID No: 063 ACATGGAGCTGAGCAGGCTGAGAT SEQ ID No: 064 ACATGGAGCTGAGGAGCCTGAGAT SEQ ID No: 065 CATGGAGCTGAGGAGCCTAAGATCTGA SEQ ID No: 066 AGCTCTGTGACCGCCGCGGAC SEQ ID No: 067 AGCTCTGTGACCGCCGTGGACA SEQ ID No: 068 AGCTCTGTGACCGCTGCAGACACG SEQ ID No: 069 AGCTCTGTGACCGCTGCGGACA SEQ ID No: 070 AGCTCTGTGACTGCCGCAGACACG SEQ ID No: 071 AGCTCTGTGACTGCAGCAGACACG SEQ ID No: 072 AGCTCTGTGACTGCCGCGGACA SEQ ID No: 073 AGCTCTGTGACCGCGGACGCG SEQ ID No: 074 GCTCTGTGACCGCCGCGGAC SEQ ID No: 075 AGCTCTGTGACCGCCGCAGACA SEQ ID No: 076 AGCTCTGTGACCGCTGACACGG SEQ ID No: 077 CAAATGAACAGCCTGAGAGCCGAGGAC SEQ ID No: 078 CAAATGAACAGCCTGAAAACCGAGGAC SEQ ID No: 079 CAAATGAACAGTCTGAAAACCGAGGAC SEQ ID No: 080 CAAATGATCAGCCTGAAAACCGAGGAC SEQ ID No: 081 AAATGAACAGTCTGAGAACTGAGGACACC SEQ ID No: 082 CAAATGAACAGTCTGAGAGCCGAGGAC SEQ ID No: 083 CAAATGAACAGCCTGAGAGCTGAGGAC SEQ ID No: 084 CAAATGAGCAGCCTGAGAGCTGAGGAC SEQ ID No: 085 CAAATGAACAGCCTGAGAGACGAGGAC SEQ ID No: 086 CAAATGGGCAGCCTGAGAGCTGAGGAC SEQ ID No: 087 CAAATGAACAGCCTGAGAGCCGGGG SEQ ID No: 088 CAAATGAACAGTCTGAGAGCTGAGGAC SEQ ID No: 089 CAAATGAGCAGTCTGAGAGCTGAGGAC SEQ ID No: 090 CACGCTAAAGGCTGAGGACACTG SEQ ID No: 091 CTYACCTGARGAGACRGTGAC SEQ ID No: 092 NNNANNNTNNNNCTYACCTGARGAGACRGTGAC (2) One DNA clinical sample 04-62005 was selected. The proportion of tumor cells in the sample was confirmed to be 9.95% by flow cytometry. The sample was diluted with negative leukocyte DNA to a tumor cell proportion of 1%. 1200 ng of the diluted DNA was taken, and the final concentration was 60 ng / µL.

[0031] (3) Amplification: The sample was amplified using two different PCR programs. 4 µL of the primer set mixed in step (1) was added to an eight-tube PCR array, along with 5 µL of 10×PCR Buffer (Thermo Fisher Scientific, 4311806), 5 µL of dNTP (Toyobo, NTP-201), and 0.6 µL of AmpliTaq Gold. ™ DNA polymerase (Thermo Fisher Scientific, 4311806), 3 µL of MgCl2 (Thermo Fisher Scientific, 4311806), 20 µL of the DNA diluted in step (2), and water to make up to 50 µL. Repeat 3 times.

[0032] The reaction conditions 1 include: Step 1: 95℃, 10 min; Step 2: 95℃, 45 sec; Step 3: 60℃, 45 sec; Step 4: 72℃, 90 sec; Steps 2 to 4 are repeated 25 times; Step 5: 72℃, 7 min; Step 6: Hold at 4℃.

[0033] Reaction conditions 2 include: Step 1: 95℃, 10 min; Step 2: 95℃, 45 sec; Step 3: 60℃, 45 sec; Step 4: 72℃, 90 sec; Steps 2 to 4 are repeated 35 times; Step 5: 72℃, 7 min; Step 6: Hold at 4℃.

[0034] Once amplification is complete and the temperature has cooled to 4°C, the amplified DNA can be removed. Add 100 μL of DNA purification magnetic beads to each well, incubate at room temperature for 5 minutes, then place on a magnetic rack. After clarification, discard the supernatant. Add 180 μL of freshly prepared 80% ethanol to each well, then pipette in the opposite direction of the magnetic beads 8 times. Incubate for 30 seconds, then aspirate all supernatant. Repeat this process, adding 180 μL of freshly prepared 80% ethanol to each well, then pipette in the opposite direction of the magnetic beads 8 times. Incubate for 30 seconds, then aspirate all supernatant. Air dry for 5 minutes, then add 20 μL of pure water. Incubate at room temperature for 5 minutes, then place on a magnetic rack. After clarification, retain the supernatant.

[0035] (4) Quality control: The total amount of DNA under different amplification systems was detected. The quality control results are shown in Table 2.

[0036] Table 2: List of DNA yields under different PCR amplification conditions

[0037] (5) Results and conclusions: Amplification reaction condition 2 can significantly increase the yield of the product to meet the input of subsequent library construction and ligation reactions, thereby reducing the amplification bias of the present invention. Therefore, amplification reaction condition 2 is more conducive to the subsequent experimental application of the product of the present invention.

[0038] Example 2: This embodiment is based on the optimized amplification system of Example 1. One clinical DNA sample was selected and tested using a single standard and a standard library to confirm that there was sufficient yield for the subsequent ligation reaction.

[0039] (1) The sequences of the primers for multiplex PCR based on the detection of IGH rearrangement are shown in Table 1. The primers were synthesized by Shanghai Qingke Biotechnology Co., Ltd. The primers SEQ ID No: 001-SEQ ID No: 091 were mixed in equal proportions and then diluted to 10 μM.

[0040] (2) One DNA clinical sample 04-62005 was selected. The proportion of tumor cells in the sample was confirmed by flow cytometry to be 9.95%. The sample was diluted with negative leukocyte DNA to a tumor cell proportion of 1%, 0.1%, and 0.01%, with a final concentration of 60 ng / µL. 1% and 0.01% were mixed with single standard VJ311, and 1%, 0.1%, and 0.01% were mixed with a standard library. The standard was designed by the company and synthesized by Shanghai Qingke Biotechnology Co., Ltd. (see Table 3).

[0041] Table 3: Standard Product Library Combinations and Sequences

[0042] (3) Amplification: Take 4 µL of the primer set synthesized in step (1) and add it to an eight-tube PCR array. Add 5 µL of 10×PCR Buffer (Thermo Fisher Scientific, 4311806), 5 µL of dNTP (Toyobo, NTP-201), and 0.6 µL of AmpliTaq Gold. ™ DNA polymerase (Thermo Fisher Scientific, 4311806), 3µL of MgCl2 (Thermo Fisher Scientific, 4311806), 20µL of the DNA mixture from step (2), and water to make up to 50µL. The amplification reaction conditions were: Step 1: 95℃, 10min; Step 2: 95℃, 45sec; Step 3: 60℃, 45sec; Step 4: 72℃, 90sec; Steps 2 to 4 were repeated 35 times; Step 5: 72℃, 7min; Step 6: 4℃. The amplified DNA was removed when the temperature dropped to 4℃. 100µL of DNA purification magnetic beads were added to each well of the amplified DNA. After incubation at room temperature for 5min, the DNA was placed on a magnetic rack and the supernatant was discarded after clarification. 180µL of freshly prepared 80% ethanol was added to each well, and then pipetted 8 times in the opposite direction of the magnetic beads. After incubation for 30s, all supernatant was aspirated. Repeatedly add 180 μL of freshly prepared 80% ethanol to each well, then pipette along the opposite direction of the magnetic bead 8 times. After incubation for 30 seconds, remove all supernatant, air dry for 5 minutes, add 20 μL of pure water, let stand at room temperature for 5 minutes, then place on a magnetic rack and retain the supernatant after clarification.

[0043] (4) Adapter ligation: Take 1000 ng of amplified DNA, add 5 μL of FEA Buffer V2 (Novizan, ND627), add water to make up to 40 μL, mix well, add 10 μL of FEA Enzyme Mix V2 for enzyme digestion and fragmentation (Novizan, ND627), react at 37℃ for 5 min, react at 65℃ for 30 min, and hold at 4℃. Add 10 μL of T4 ligation buffer (NEB, E7445L), 4 μL of T4 ligase (NEB, E7445L), 5 μL of next-generation sequencing universal adapter (Novizan, N34201) to the above reaction system, add water to make up to 100 μL, react at 20℃ for 15 min, and hold at 4℃.

[0044] (5) After purification, add 60 μL of DNA purification magnetic beads to each well of the ligation product from step (4). After incubating at room temperature for 5 min, place the well on a magnetic rack and discard the supernatant after clarification. Then add 180 μL of freshly prepared 80% ethanol to each well and pipette in the opposite direction of the magnetic beads 8 times. After incubation for 30 s, aspirate all the supernatant. Repeat the process of adding 180 μL of freshly prepared 80% ethanol to each well and pipette in the opposite direction of the magnetic beads 8 times. After incubation for 30 s, aspirate all the supernatant, air dry for 5 min, add 10 μL of pure water, incubate at room temperature for 5 min, place the well on a magnetic rack, and retain the supernatant after clarification. Measure 1 G of data per sample using a high-throughput sequencer.

[0045] (6) Bioinformatics analysis: The proportion of tumor cells was calculated using a standard library. Given that the nanograms of each sample input are Ck, and the total number of cells E per sample is: The copy number of a specific combination of standard libraries is denoted as Aj, the reads of a specific combination of standard libraries before deduplication is denoted as Yj, the reads of tens of thousands of rearrangements before deduplication are denoted as Yi, and the number of tumor cells D1 in this sample is: Finally, the percentage of tumor cells in this sample (Mj) is calculated by dividing the number of tumor cells in this sample by the total number of cells in this sample and multiplying by 100%. Finally, the average tumor cell percentage Mj of each standard in the standard library is calculated to obtain the final tumor cell percentage of the sample, where k represents the number of samples, j represents the number of standard libraries for this specific combination, and i represents the number of tens of thousands of rearrangements detected.

[0046] (7) Results Analysis and Conclusions: Based on the standard library combination designed in this invention, this standard library fully considers the regions with high complexity (V1, V3, V4, V5), fully considers amplification lengths of 149bp-338bp, fully considers VDJ rearrangement and DJ rearrangement, and fully considers primer detection rearrangement types. When the proportion of tumor cells in the sample is 1%, the fluctuation of the quantitative value of a single standard is 3-4 times, while when the proportion of tumor cells in the sample is 0.01%, the fluctuation of the quantitative value of a single standard is about 50 times. At this time, the standard library combination designed in this invention can reduce the fluctuation of the value of 0.1% tumor cell proportion and 0.01% tumor cell proportion to 2-3 times, improve the accuracy of the value determination (see Table 4), and the correlation between the value determination results and the flow cytometry results reaches 0.969. Moreover, the NGS detection results are more sensitive than flow cytometry results. Figure 1 As shown.

[0047] Table 4: Standard Library Combinations and Proportion of Tumor Cells

[0048] Example 3: This embodiment is based on the optimized quantitative standard library of Example 2. One commercial DNA standard Romas cell line (CBP60263D) was selected. Long molecular tag primers with and without fixed base structure were used to confirm the effect of long molecular tags with fixed base structure on multiple response preference correction.

[0049] (1) The sequences of primers for multiplex PCR based on the detection of IGH rearrangement are shown in Table 1. The primers were synthesized by Shanghai Qingke Biotechnology Co., Ltd. Primers SEQ ID No: 001-SEQ ID No: 091 were mixed in equal proportions and diluted to 10 μM. Primers SEQ ID No: 001-SEQ ID No: 090 and primer SEQ ID No: 092 were mixed in equal proportions and diluted to 10 μM. Primer SEQ ID No: 092 is a long molecular tag primer with a fixed base structure.

[0050] (2) One commercially available DNA standard, Romas cell line (CBP60263D), with a known percentage of tumor cells was selected. The sample was diluted with negative leukocyte DNA to a tumor cell percentage of 1% and a final concentration of 60 ng / µL. The sample was then mixed with a standard library, which was designed by the company and synthesized by Shanghai Qingke Biotechnology Co., Ltd. (see Table 3).

[0051] (3) Amplification: Take 4 µL of the primer set synthesized in step (1) and add it to an eight-tube PCR array. Add 5 µL of 10×PCR Buffer (Thermo Fisher Scientific, 4311806), 5 µL of dNTP (Toyobo, NTP-201), and 0.6 µL of AmpliTaq Gold. ™DNA polymerase (Thermo Fisher Scientific, 4311806), 3 µL of MgCl2 (Thermo Fisher Scientific, 4311806), 20 µL of the DNA diluted in step (2), and water to make up to 50 µL. The amplification reaction conditions were: Step 1: 95℃, 10 min; Step 2: 95℃, 45 sec; Step 3: 60℃, 45 sec; Step 4: 72℃, 90 sec; Steps 2 to 4 were repeated 35 times; Step 5: 72℃, 7 min; Step 6: 4℃. The amplified DNA was removed when the temperature dropped to 4℃. 100 µL of DNA purification magnetic beads were added to each well of the amplified DNA. After incubation at room temperature for 5 min, the DNA was placed on a magnetic rack and the supernatant was discarded after clarification. 180 µL of freshly prepared 80% ethanol was added to each well, and then pipetted 8 times in the opposite direction of the magnetic beads. After incubation for 30 s, all supernatant was aspirated. Repeatedly add 180 μL of freshly prepared 80% ethanol to each well, then pipette along the opposite direction of the magnetic bead 8 times. After incubation for 30 seconds, remove all supernatant, air dry for 5 minutes, add 20 μL of pure water, place at room temperature for 5 minutes, place on a magnetic rack, and retain the supernatant after clarification. The product concentration is around 100 ng, which fully meets the requirements for library construction. The amplification product yield information is shown in Table 5.

[0052] Table 5: Yield Results of Amplified Products

[0053] 4) Adapter ligation: Take 1000 ng of amplified DNA, add 5 μL of FEA Buffer V2 (Novizan, ND627), add water to make up to 40 μL, mix well, then add 10 μL of FEA Enzyme Mix V2 for enzyme digestion and fragmentation (Novizan, ND627), incubate at 37℃ for 5 min, incubate at 65℃ for 30 min, and hold at 4℃. Add 10 μL of T4 ligation buffer (NEB, E7445L), 4 μL of T4 ligase (NEB, E7445L), and 5 μL of universal next-generation sequencing adapter (Novizan, N34201) to the above reaction system, add water to make up to 100 μL, incubate at 20℃ for 15 min, and hold at 4℃.

[0054] (5) After purification, add 60 μL of DNA purification magnetic beads to the ligation product in step (4), place at room temperature for 5 min, then place on a magnetic rack and discard the supernatant after clarification. Then add 180 μL of freshly prepared 80% ethanol to each well, then pipette in the opposite direction of the magnetic beads 8 times, incubate for 30 s, and then aspirate all the supernatant. Repeat the process of adding 180 μL of freshly prepared 80% ethanol to each well, then pipette in the opposite direction of the magnetic beads 8 times, incubate for 30 s, then aspirate all the supernatant, air dry for 5 min, add 10 μL of pure water, place at room temperature for 5 min, then place on a magnetic rack and retain the supernatant after clarification. A single sample was sequenced using a high-throughput sequencer to obtain 1G of data. The data output met the requirements. Data volume information is shown in Table 6.

[0055] Table 6: Library sequencing data volume and data quality control results

[0056] (6) Bioinformatics analysis: The proportion of tumor cells was calculated using long molecular tag primer sequences with fixed base structures and a standard library. The reads after deduplication were denoted as Xi, and the reads before deduplication were denoted as Yi. The proportion of amplification preference after different rearrangement types was calculated and denoted as Mi. 1 minus Mi is the amplification of Dup, where i represents the number of tens of thousands of rearrangements. The tumor cell percentage was calculated using long molecular tag primer sequences with fixed base structures and a standard library. The nanograms of data input per sample were known as Ck, and the total number of cells per sample, E, was: E = Ck / 30 * 10000 / 2. The copy number of the standard library for a specific combination was denoted as Aj, the deduplicated reads of the standard library for a specific combination were denoted as Bj, and the deduplicated reads of tens of thousands of rearrangements were denoted as Xi. The number of tumor cells in this sample, D, was: D = Xi / Bj * Aj / 2. Finally, the tumor cell percentage Mj in this sample was obtained by dividing the number of tumor cells in this sample by the total number of cells in this sample and multiplying by 100%: Mj = D / E * 100%. The average tumor cell percentage Mj of each standard in the standard library was then calculated as the final tumor cell percentage of the sample, where k represents the number of samples, j represents the number of standard libraries for this specific combination, and i represents the number of tens of thousands of rearrangements detected.

[0057] (7) Results and Conclusions: The long molecular tag primer set with fixed base structure designed based on the present invention can be compatible with the deduplication of tens of millions of template molecules. The long molecular tag with fixed base structure must be ≥12 bases. At the same time, a fixed base is designed for every 4 bases of the molecular tag to correct GC bias and synthesis errors during primer synthesis. The design of the fixed base must refer to the fixed sequence A>T>G>C. The fixed bases are random bases. After comparing and analyzing the data before and after molecular tag correction, the more reads of the dominant rearrangement, the greater its corresponding amplification bias. Therefore, correction in positive samples is very necessary. The amplification Dup of most rearrangements fluctuates between 0% and 80%, which indicates that the primary structure and length of the IGH rearrangement are also the core reasons affecting the bias. Figure 2 and Figure 3 As shown, by analyzing all rearrangement types before and after deduplication, the Dup fluctuation caused by amplification bias was reduced by 70% after correction by molecular tags with fixed base structures, such as... Figure 4 As shown, further analysis of the deduplication effect of the top 50 rearrangements with the highest read distribution reveals that the amplification bias after correction by this molecular tag was reduced by 75%-80% compared to the data before deduplication, with an amplification bias of less than 10 times. This is significantly better than existing technologies, which can only correct less than 40% of the Dup while still maintaining an amplification bias of over 100 times, demonstrating a significant performance improvement. Figure 5 As shown in Table 7, after correction using a long molecular tag primer set with a fixed base structure combined with a standard library, the error between the tumor cell percentage and the theoretical value was about 1, which is better than that of long molecular tags without a fixed base structure.

[0058] Table 7: Standard Library Combinations and Proportion of Tumor Cells

[0059] Example 4: To further illustrate the specificity of this kit, this example uses 6 clinical samples with negative IGH rearrangements for specificity evaluation to confirm the specificity of the IGH detection kit.

[0060] (1) The sequences of primers for multiplex PCR based on the detection of IGH rearrangement are shown in Table 1. The primers were synthesized by Shanghai Qingke Biotechnology Co., Ltd. Primers SEQ ID No: 001-SEQ ID No: 091 were mixed in equal proportions and diluted to 10 μM. Primers SEQ ID No: 001-SEQ ID No: 090 and primer SEQ ID No: 092 were mixed in equal proportions and diluted to 10 μM. Primer SEQ ID No: 092 is a long molecular tag primer with a fixed base structure.

[0061] (2) Six clinical samples with negative IGH rearrangement were selected, with a final concentration of 100 ng / µL.

[0062] (3) Amplification: Take 4 µL of the primer set synthesized in step (1) and add it to an eight-tube PCR array. Add 5 µL of 10×PCR Buffer (Thermo Fisher Scientific, 4311806), 5 µL of dNTP (Toyobo, NTP-201), and 0.6 µL of AmpliTaq Gold. ™ DNA polymerase (Thermo Fisher Scientific, 4311806), 3 µL of MgCl2 (Thermo Fisher Scientific, 4311806), 15 µL of the DNA diluted in step (2), and water to make up to 50 µL. The amplification reaction conditions were as follows: Step 1: 95 °C, 10 min; Step 2: 95 °C, 45 sec; Step 3: 60 °C, 45 sec; Step 4: 72 °C, 90 sec; Steps 2 to 4 were repeated 35 times; Step 5: 72 °C, 7 min; Step 6: 4 °C. The amplified DNA was removed when the temperature dropped to 4 °C. 100 µL of DNA purification magnetic beads were added to each well of the amplified DNA. After incubation at room temperature for 5 min, the DNA was placed on a magnetic rack and the supernatant was discarded after clarification. 180 µL of freshly prepared 80% ethanol was added to each well, and then pipetted 8 times in the opposite direction of the magnetic beads. After incubation for 30 s, all supernatant was aspirated. Repeatedly add 180 μL of freshly prepared 80% ethanol to each well, then pipette along the opposite direction of the magnetic bead 8 times. After incubation for 30 seconds, remove all supernatant, air dry for 5 minutes, add 20 μL of pure water, place at room temperature for 5 minutes, place on a magnetic rack, and retain the supernatant after clarification. The product concentration is approximately 100 ng.

[0063] (4) Adapter ligation: Take 500 ng of amplified DNA, add 5 μL of FEA Buffer V2 (Novizan, ND627), add water to make up to 40 μL, mix well, and then add 10 μL of FEA Enzyme Mix V2 for enzyme digestion and fragmentation (Novizan, ND627). Incubate at 37℃ for 5 min, then at 65℃ for 30 min, and maintain at 4℃. Add 10 μL of T4 ligation buffer (NEB, E7445L), 4 μL of T4 ligase (NEB, E7445L), and 5 μL of universal next-generation sequencing adapter (Novizan, N34201) to the above reaction system, add water to make up to 100 μL, incubate at 20℃ for 15 min, and maintain at 4℃.

[0064] (5) After purification, add 60 μL of DNA purification magnetic beads to each well of the ligation product from step (4). After incubating at room temperature for 5 min, place the well on a magnetic rack and discard the supernatant after clarification. Then add 180 μL of freshly prepared 80% ethanol to each well and pipette in the opposite direction of the magnetic beads 8 times. After incubation for 30 s, aspirate all the supernatant. Repeat the process of adding 180 μL of freshly prepared 80% ethanol to each well and pipette in the opposite direction of the magnetic beads 8 times. After incubation for 30 s, aspirate all the supernatant, air dry for 5 min, add 10 μL of pure water, incubate at room temperature for 5 min, place the well on a magnetic rack, and retain the supernatant after clarification. A single sample was sequenced using a high-throughput sequencer to obtain 2G of data, and the data output met the requirements.

[0065] (6) Bioinformatics analysis: The amplification bias correction effect is judged by analyzing the continuity of reads of each IGH rearrangement in the negative sample, and the specificity of the kit is judged by analyzing whether a dominant clone appears in reads of each IGH rearrangement in the negative sample.

[0066] (7) Results Analysis and Conclusions: Based on the specificity performance verification of 6 negative samples, it was found that the first 41 rearrangement types were uniform across different samples, with amplification fluctuations of less than 2-fold, no obvious amplification bias, and no dominant clones. This indicates that the specificity of the kit is 100%. (See [link to kit]) Figure 6 As shown.

[0067] Example 5: To further illustrate the sensitivity of this kit, this example uses two MRD clinical samples, each repeated 10 times, to evaluate the sensitivity and confirm the sensitivity of the IGH detection kit. Since the total amount of DNA that can be extracted from 90% of clinical samples is around 5ug, the sensitivity at 1.5ug is verified using MRD clinical samples here.

[0068] (1) The sequences of primers for multiplex PCR based on the detection of IGH rearrangement are shown in Table 1. The primers were synthesized by Shanghai Qingke Biotechnology Co., Ltd. Primers SEQ ID No: 001-SEQ ID No: 091 were mixed in equal proportions and diluted to 10 μM. Primers SEQ ID No: 001-SEQ ID No: 090 and primer SEQ ID No: 092 were mixed in equal proportions and diluted to 10 μM. Primer SEQ ID No: 092 is a long molecular tag primer with a fixed base structure.

[0069] (2) Two MRD clinical samples were selected, each repeated 10 times, with a final concentration of 100 ng / µL.

[0070] (3) Amplification: Take 4 µL of the primer set synthesized in step (1) and add it to an eight-tube PCR array. Add 5 µL of 10×PCR Buffer (Thermo Fisher Scientific, 4311806), 5 µL of dNTP (Toyobo, NTP-201), and 0.6 µL of AmpliTaq Gold. ™ DNA polymerase (Thermo Fisher Scientific, 4311806), 3 µL of MgCl2 (Thermo Fisher Scientific, 4311806), 15 µL of the DNA diluted in step (2), and water to make up to 50 µL. The amplification reaction conditions were as follows: Step 1: 95 °C, 10 min; Step 2: 95 °C, 45 sec; Step 3: 60 °C, 45 sec; Step 4: 72 °C, 90 sec; Steps 2 to 4 were repeated 35 times; Step 5: 72 °C, 7 min; Step 6: 4 °C. The amplified DNA was removed when the temperature dropped to 4 °C. 100 µL of DNA purification magnetic beads were added to each well of the amplified DNA. After incubation at room temperature for 5 min, the DNA was placed on a magnetic rack and the supernatant was discarded after clarification. 180 µL of freshly prepared 80% ethanol was added to each well, and then pipetted 8 times in the opposite direction of the magnetic beads. After incubation for 30 s, all supernatant was aspirated. Repeatedly add 180 μL of freshly prepared 80% ethanol to each well, then pipette along the opposite direction of the magnetic bead 8 times. After incubation for 30 seconds, remove all supernatant, air dry for 5 minutes, add 20 μL of pure water, place at room temperature for 5 minutes, place on a magnetic rack, and retain the supernatant after clarification. The product concentration is approximately 100 ng.

[0071] (4) Adapter ligation: Take 500 ng of amplified DNA, add 5 μL of FEA Buffer V2 (Novizan, ND627), add water to make up to 40 μL, mix well, and then add 10 μL of FEA Enzyme Mix V2 for enzyme digestion and fragmentation (Novizan, ND627). Incubate at 37℃ for 5 min, then at 65℃ for 30 min, and maintain at 4℃. Add 10 μL of T4 ligation buffer (NEB, E7445L), 4 μL of T4 ligase (NEB, E7445L), and 5 μL of universal next-generation sequencing adapter (Novizan, N34201) to the above reaction system, add water to make up to 100 μL, incubate at 20℃ for 15 min, and maintain at 4℃.

[0072] (5) After purification, add 60 μL of DNA purification magnetic beads to each well of the ligation product from step (4). After incubating at room temperature for 5 min, place the well on a magnetic rack and discard the supernatant after clarification. Then add 180 μL of freshly prepared 80% ethanol to each well and pipette in the opposite direction of the magnetic beads 8 times. After incubation for 30 s, aspirate all the supernatant. Repeat the process of adding 180 μL of freshly prepared 80% ethanol to each well and pipette in the opposite direction of the magnetic beads 8 times. After incubation for 30 s, aspirate all the supernatant, air dry for 5 min, add 10 μL of pure water, incubate at room temperature for 5 min, place the well on a magnetic rack, and retain the supernatant after clarification. The MRD samples were sequenced using a high-throughput sequencer to obtain 2G of data, and the data output met the requirements.

[0073] (6) Bioinformatics analysis: The limit of detection of the kit is determined by analyzing the detection of IGH rearrangements in sensitive samples.

[0074] (7) Results Analysis and Conclusions: Based on the existing experimental system, 0.0005% of MRD samples were successfully detected. IGH Rearrangement. The above results indicate that with a DNA input of 1.5 μg, the limit of detection (LOD) of the product is 0.0005%. Furthermore, by analyzing the quantitative results of tumor cells, it was found that the quantitative results of the internal control in 20 MRD samples with a tumor cell content of 0.0005% were around 0.0016%, with the tumor cell content fluctuating by 2-3 times. The quantitative results were accurate, as shown in Table 8.

[0075] Table 8: Sensitivity Samples and Proportion of Tumor Cells

[0076] Example 6: To further illustrate the accuracy of this kit, this example uses 10 MRD clinical samples with competing product test results for accuracy evaluation. Since the total amount of DNA that can be extracted from 90% of clinical samples is around 5ug, the accuracy of MRD clinical samples here is verified at 1.5ug.

[0077] (1) The sequences of primers for multiplex PCR based on the detection of IGH rearrangement are shown in Table 1. The primers were synthesized by Shanghai Qingke Biotechnology Co., Ltd. Primers SEQ ID No: 001-SEQ ID No: 091 were mixed in equal proportions and diluted to 10 μM. Primers SEQ ID No: 001-SEQ ID No: 090 and primer SEQ ID No: 092 were mixed in equal proportions and diluted to 10 μM. Primer SEQ ID No: 092 is a long molecular tag primer with a fixed base structure.

[0078] (2) Ten MRD clinical samples were selected, with a final concentration of 100 ng / µL.

[0079] (3) Amplification: Take 4 µL of the primer set synthesized in step (1) and add it to an eight-tube PCR array. Add 5 µL of 10×PCR Buffer (Thermo Fisher Scientific, 4311806), 5 µL of dNTP (Toyobo, NTP-201), and 0.6 µL of AmpliTaq Gold. ™ DNA polymerase (Thermo Fisher Scientific, 4311806), 3 µL of MgCl2 (Thermo Fisher Scientific, 4311806), 15 µL of the DNA diluted in step (2), and water to make up to 50 µL. The amplification reaction conditions were as follows: Step 1: 95 °C, 10 min; Step 2: 95 °C, 45 sec; Step 3: 60 °C, 45 sec; Step 4: 72 °C, 90 sec; Steps 2 to 4 were repeated 35 times; Step 5: 72 °C, 7 min; Step 6: 4 °C. The amplified DNA was removed when the temperature dropped to 4 °C. 100 µL of DNA purification magnetic beads were added to each well of the amplified DNA. After incubation at room temperature for 5 min, the DNA was placed on a magnetic rack and the supernatant was discarded after clarification. 180 µL of freshly prepared 80% ethanol was added to each well, and then pipetted 8 times in the opposite direction of the magnetic beads. After incubation for 30 s, all supernatant was aspirated. Repeatedly add 180 μL of freshly prepared 80% ethanol to each well, then pipette along the opposite direction of the magnetic bead 8 times. After incubation for 30 seconds, remove all supernatant, air dry for 5 minutes, add 20 μL of pure water, place at room temperature for 5 minutes, place on a magnetic rack, and retain the supernatant after clarification. The product concentration is approximately 100 ng.

[0080] (4) Adapter ligation: Take 500 ng of amplified DNA, add 5 μL of FEA Buffer V2 (Novizan, ND627), add water to make up to 40 μL, mix well, and then add 10 μL of FEA Enzyme Mix V2 for enzyme digestion and fragmentation (Novizan, ND627). Incubate at 37℃ for 5 min, then at 65℃ for 30 min, and maintain at 4℃. Add 10 μL of T4 ligation buffer (NEB, E7445L), 4 μL of T4 ligase (NEB, E7445L), and 5 μL of universal next-generation sequencing adapter (Novizan, N34201) to the above reaction system, add water to make up to 100 μL, incubate at 20℃ for 15 min, and maintain at 4℃.

[0081] (5) After purification, add 60 μL of DNA purification magnetic beads to each well of the ligation product from step (4). After incubating at room temperature for 5 min, place the well on a magnetic rack and discard the supernatant after clarification. Then add 180 μL of freshly prepared 80% ethanol to each well and pipette in the opposite direction of the magnetic beads 8 times. After incubation for 30 s, remove all supernatant. Repeat the process of adding 180 μL of freshly prepared 80% ethanol to each well and pipette in the opposite direction of the magnetic beads 8 times. After incubation for 30 s, remove all supernatant, air dry for 5 min, add 10 μL of pure water, incubate at room temperature for 5 min, place the well on a magnetic rack, and retain the supernatant after clarification. The MRD samples were sequenced using a high-throughput sequencer to obtain 6G of data, and the data output met the requirements.

[0082] (6) Bioinformatics analysis: The accuracy of the kit is determined by analyzing the detection of IGH rearrangements in the accurate samples.

[0083] (7) Results Analysis and Conclusions: Based on the existing experimental system, the virus was successfully detected in 10 MRD samples. IGH Rearrangement. The above results indicate that the product accuracy is 100% with an input of 1.5 μg of DNA, as shown in Table 9.

[0084] Table 9: Accuracy Samples and Proportion of Tumor Cells

[0085] In summary, the above embodiments verify that the one-step PCR reaction conditions and ligation method of the present invention are used for IGH amplification bias correction and tumor cell quantification, effectively avoiding secondary amplification that amplifies amplification bias and reducing experimental complexity. Simultaneously, the addition of specific standard library combinations assists in correcting amplification bias in lymphoid tumor immune repertoire libraries and quantifying tumor cells, avoiding the influence of different rearranged primary structures and lengths on the quantitative results. Furthermore, the present invention adds long molecular tags with fixed base structures to assist in correcting molecular tags for amplification bias correction in lymphoid tumor immune repertoire libraries and quantifying tumor cells, avoiding the influence of different rearranged primary structures and lengths on the quantitative results due to amplification bias.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A standard library for correcting IGH amplification bias, characterized in that, The standard library includes five standards, named VJ118, VJ311, VDJ428, VDJ510, and VJ741. Among them, VJ118 includes the sequence SEQ ID No: 093, VJ311 includes the sequence SEQ ID No: 094, VDJ428 includes the sequence SEQ ID No: 095, VDJ510 includes the sequence SEQ ID No: 096, and VJ741 includes the sequence SEQ ID No:

097.

2. The standard library according to claim 1, characterized in that, The standard library covers rearrangement regions of V1, V3, V4, and V5, amplification lengths of 149bp-338bp, VDJ rearrangement, and VJ rearrangement.

3. A kit for correcting IGH amplification bias, characterized in that, The primer set includes the sequence SEQ ID No: 001-SEQ ID No: 090, the long molecular tag primer with fixed base structure of sequence SEQ ID No: 092, and the standard library mixture of sequences SEQ ID No: 093-SEQ ID No:

097.

4. The reagent kit according to claim 3, characterized in that, It also includes 10×PCR Buffer, dNTPs, and AmpliTaqGold. ™ DNA polymerase, MgCl2, FEA Buffer V2, FEA Enzyme Mix V2, T4 ligation buffer, T4 ligase and universal adapter for next-generation sequencing.

5. A method of using the reagent kit according to claim 3 or 4, characterized in that, Includes the following steps: S1. Extract total DNA from the leukemia sample to be tested and mix it with a standard library mixture containing sequences SEQ ID No: 093-SEQ ID No: 097; S2. Multiplex PCR amplification of the mixed DNA product was performed using primers containing the primer set with the sequence SEQ ID No: 001-SEQ ID No: 090 and the long molecular tag primer with the fixed base structure of the sequence SEQ ID No: 092, to obtain the PCR-amplified DNA; S3. The PCR-amplified DNA is ligated with adapters, constructed into a library, and then sequenced to obtain the total amount of DNA library and sequencing data. S4. Perform bioinformatics filtering analysis, calculate reads caused by filtering amplification bias, and simultaneously determine the proportion of tumor cells based on a standard library of specific combinations with known copy numbers.

6. The method of use according to claim 5, characterized in that, The PCR reaction conditions in step S2 are as follows: Step 1: 95℃, 10 min; Step 2: 95℃, 45 sec; Step 3: 60℃, 45 sec; Step 4: 72℃, 90 sec; Steps 2 to 4 are repeated 35 times; Step 5: 72℃, 7 min; Step 6: Hold at 4℃.

7. The method of use according to claim 5, characterized in that, In step S3, adapter ligation involves ligating the PCR-amplified DNA with a second-generation universal adapter to obtain a ligation product that can be directly sequenced.

8. The method of use according to claim 5, characterized in that, In step S4, the calculation of reads due to amplification bias includes the following process: The same rearranged reads before and after deduplication are calculated using long molecular tag primer sequences with fixed base structures. 1 minus Mi equals the amplification Dup, calculated using the following formula: ； Here, the reads after deduplication are denoted as Xi, the reads before deduplication are denoted as Yi, the proportion of amplification preference after different rearrangement types is denoted as Mi, and i represents the number of tens of thousands of rearrangement types.

9. The method of use according to claim 5, characterized in that, In step S4, the tumor cell percentage is calculated as follows: the tumor cell percentage is calculated using a long molecular tag primer sequence with a fixed base structure and a standard library. The nanograms of sample input per case are known to be Ck, and the total number of cells per sample is E. The sample contained 30 ng of human genomic DNA, totaling 10,000 copies. The total copy number was calculated, and then divided by 2 to determine the total cell count. The copy number of a specific combination of standard library samples was denoted as Aj, the deduplicated reads of that standard library were denoted as Bj, and the deduplicated reads of tens of thousands of rearrangements were denoted as Xi. The number of tumor cells D in this sample was: Finally, the percentage of tumor cells in this sample (Mj) is calculated by dividing the number of tumor cells in this sample by the total number of cells in this sample and multiplying by 100%. Finally, the average tumor cell percentage Mj of each standard in the standard library is calculated to obtain the final tumor cell percentage of the sample, where k represents the number of samples, j represents the number of standard libraries for this specific combination, and i represents the number of tens of thousands of rearrangements detected.

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