Method and device for obtaining a tcr index of a plasma sample

Abstract

The application belongs to the field of biological detection and biological information, and provides a method and device for obtaining a TCR index of a plasma sample. + The application uses a method for analyzing the plasma sample, and takes the tumor-specific CD8 + As a comparison basis, the lung cancer tumor-specific exhausted T cell CDR3 beta sequence consistency is taken as a comparison index, the TCR index of the plasma sample is taken as an index for evaluating the lung cancer risk by calculating the average proportion (AvgFreq) and clonality (Clonality), the plasma sample from the lung cancer patient and the healthy person can be effectively distinguished, the efficiency of the lung cancer population screening is improved, and the application has important scientific significance and good application prospect.

Description

Technical Field

[0001] This invention relates to a method and apparatus for detecting plasma samples, and more particularly to a method and apparatus for obtaining the TCR index of plasma samples, belonging to the fields of biological detection and bioinformatics. Background Technology

[0002] Lung cancer is the most common cause of cancer death. According to the GLOBOCAN 2020 estimate, there were approximately 2.2 million new cases of lung cancer and 1.8 million lung cancer deaths in 2020. Of the new lung cancer cases globally in 2020, approximately 37% were from China, and of lung cancer deaths, approximately 39.8% were from China. Despite rapid advancements in lung cancer treatment in recent years, the overall prognosis has not significantly improved. Although the five-year survival rate for stage I lung cancer patients can reach 68%–92%, only 20.8% of lung cancer patients in my country are diagnosed with stage I lung cancer. Currently, LDCT screening is recommended for high-risk groups for lung cancer. However, the proportion of patients detected by LDCT with pulmonary nodules is relatively high (average approximately 20%), and the high false-positive rate (96.4%) of LDCT can lead to over-treatment and unnecessary psychological burden. Therefore, there is an urgent need for an auxiliary method to help assess the malignant potential of lung cancer and reduce the false-positive rate.

[0003] Liquid biopsy is a non-invasive screening technique that, compared to LDCT, provides quantifiable results and involves no radiation exposure. This technique involves detecting and analyzing biomarkers, particularly circulating tumor cells (CTCs) and circulating tumor nucleic acids (ctDNA), from bodily fluids such as blood and urine. Several studies have reported that T-cell repertoires (TCRs) can be used as biomarkers in cancer treatment. However, few studies have used TCR repertoire data for lung cancer identification.

[0004] Therefore, there is an urgent need in this field for a new liquid biopsy marker for lung cancer that can be used to assess the risk of lung cancer and identify lung cancer patients. Summary of the Invention

[0005] This invention addresses the aforementioned deficiencies in the prior art by providing a method for obtaining the TCR index of a plasma sample, comprising the following steps:

[0006] 1. Obtain known tumor-specific CD8 + The types of T cell receptors depleted; the types and abundance of plasma T cell receptors obtained from plasma samples;

[0007] 2. Statistical analysis of plasma T-cell receptor (P) and tumor-specific CD8+ in plasma samples. + The types and abundance (T~P) of T cell receptors with a concordance of greater than 50%, preferably greater than 60%;

[0008] 3. Calculate plasma T-cell receptor and tumor-specific CD8+. + The average proportion (AvgFreq) of T cell receptors (T-P) with a concordance of greater than 50%, preferably greater than 60%, of all T cell receptor (P) types in the plasma sample is used to calculate the clonality of plasma T cell receptors in the plasma sample. The average proportion and clonality are then integrated to obtain the TCR index (TCR_index) of the plasma sample.

[0009] The plasma sample is preferably derived from the peripheral blood of a lung cancer patient or a healthy individual.

[0010] Studies have shown that exhausted T cells in tumors originate from tumor-specific T cells, while T cells responding to acute infection do not produce exhausted T cells. Therefore, exhausted T cells can represent tumor-reactive T cells.

[0011] In a preferred embodiment of the present invention, the T cell receptor is characterized by the CDR3 region (CDR3β sequence) of the T cell receptor β chain.

[0012] In a further preferred embodiment of the present invention, the TCR index of a plasma sample is obtained by sequencing the CDR3β sequence of plasma T cells in the plasma sample.

[0013] In a more preferred embodiment of the present invention, the method includes the following steps:

[0014] 1. Obtaining tumor-specific CD8 from known single-cell sequencing data + The CDR3 region (CDR3β sequence) of the plasma T cell receptor β chain was depleted. The CDR3 region (CDR3β sequence) of the plasma T cell receptor β chain in the plasma sample was sequenced to obtain the sequencing read length. The selected read lengths were spliced ​​to form multiple spliced ​​sequences. The multiple spliced ​​sequences were subjected to CDR3β sequence feature identification to obtain the type and abundance of plasma T cell receptor CDR3β sequences.

[0015] 2. Statistical analysis of plasma T cell receptors and tumor-specific CD8+. + The types and abundance of CDR3β sequences with greater than 50% sequence identity and preferably greater than 60% for exhausted T cell receptor sequences;

[0016] 3. Calculate plasma T cell receptor and tumor-specific CD8 using formula ① + The average proportion of CDR3β sequences with greater than 50% sequence identity and preferably greater than 60% among all CDR3β sequences in plasma T cells:

[0017]

[0018] T is tumor-specific CD8. + The CDR3β sequence of exhausted T cells;

[0019] P is the CDR3β sequence of the plasma sample;

[0020] T to P are CDR3β sequences in P that have greater than 50% similarity to T, preferably greater than 60%.

[0021] N is the number of T to P;

[0022] P i Freq is the proportion of the i-th CDR3β sequence in T to P to the total number of CDR3β sequences in P;

[0023] Calculate plasma T cell CDR3β sequence clonality using formula ②:

[0024]

[0025] P is the CDR3β sequence of the plasma sample;

[0026] R is the number of P;

[0027] P j Freq is the proportion of the j-th CDR3β sequence in P to the total number of CDR3β sequences in P;

[0028] Integrating the average proportion and clonality, the TCR index of the sample is obtained using formula ③:

[0029] TCR_index=36973.350×AvgFreq+13.958×Clonality-6.474③.

[0030] The above-mentioned tumor-specific CD8 of the present invention + The CDR3 region (CDR3β sequence) of the exhausted T cell receptor β chain was obtained from single-cell sequencing data from Liu et al. (Liu B, Hu X, Feng K, Gao R, Xue Z, Zhang S, Zhang Y, Corse E, Hu Y, Han W et al: Temporal single-cell tracing reveals clonal revival and expansion of precursor exhausted T cells during anti-PD-1 therapy in lung cancer. Nat Cancer 2022, 3(1):108-121.).

[0031] In a preferred embodiment of the present invention, the method further includes:

[0032] 4. The TCR index of the plasma sample is compared with a threshold to assess the risk of lung cancer in the plasma sample. The threshold for the TCR index is preferably -1.326. The higher the TCR index, the higher the risk of lung cancer in the plasma sample.

[0033] Another aspect of the present invention provides an apparatus for obtaining the TCR index of a plasma sample, comprising the following modules:

[0034] 1. Acquisition module, used to acquire known tumor-specific CD8. + The types of T cell receptors depleted; the types and abundance of plasma T cell receptors obtained from plasma samples;

[0035] 2. Statistical module, used to statistically analyze plasma T-cell receptor (P) and tumor-specific CD8+ in plasma samples. + The types and abundance (T~P) of T cell receptors with a concordance of greater than 50%, preferably greater than 60%;

[0036] 3. Calculation module, used to calculate plasma T cell receptor and tumor-specific CD8. + The average proportion (AvgFreq) of T cell receptors (T-P) with a concordance of greater than 50%, preferably greater than 60%, of all T cell receptor (P) types in the plasma sample is used to calculate the clonality of plasma T cell receptors in the plasma sample. The average proportion and clonality are then integrated to obtain the TCR index (TCR_index) of the plasma sample.

[0037] The plasma sample is preferably derived from the peripheral blood of a lung cancer patient or a healthy individual.

[0038] In a preferred embodiment of the present invention, the T cell receptor is characterized by the CDR3 region (CDR3β sequence) of the T cell receptor β chain.

[0039] In a further preferred embodiment of the present invention, the TCR index of a plasma sample is obtained by sequencing the CDR3β sequence of plasma T cells in the plasma sample.

[0040] In a more preferred embodiment of the present invention, the device includes the following modules:

[0041] 1. Sequencing information acquisition module, used to obtain tumor-specific CD8 from known single-cell sequencing data. +The CDR3 region (CDR3β sequence) of the plasma T cell receptor β chain was depleted. The CDR3 region (CDR3β sequence) of the plasma T cell receptor β chain in the plasma sample was sequenced to obtain the sequencing read length. The selected read lengths were spliced ​​to form multiple spliced ​​sequences. The multiple spliced ​​sequences were subjected to CDR3β sequence feature identification to obtain the type and abundance of plasma T cell receptor CDR3β sequences.

[0042] 2. Statistics module, used to analyze plasma T cell receptors and tumor-specific CD8+. + The types and abundance of CDR3β sequences with greater than 50% sequence identity and preferably greater than 60% for exhausted T cell receptor sequences;

[0043] 3. Calculation module, used to calculate plasma T cell receptor and tumor-specific CD8 using formula ①. + The average proportion of CDR3β sequences with greater than 50% sequence identity and preferably greater than 60% among all CDR3β sequences in plasma T cells:

[0044]

[0045] T is tumor-specific CD8. + The CDR3β sequence of exhausted T cells;

[0046] P is the CDR3β sequence of the plasma sample;

[0047] T to P are CDR3β sequences in P that have greater than 50% similarity to T, preferably greater than 60%.

[0048] N is the number of T to P;

[0049] P i Freq is the proportion of the i-th CDR3β sequence in T to P to the total number of CDR3β sequences in P;

[0050] Calculate plasma T cell CDR3β sequence clonality using formula ②:

[0051]

[0052] P is the CDR3β sequence of the plasma sample;

[0053] R is the number of P;

[0054] P j Freq is the proportion of the j-th CDR3β sequence in P to the total number of CDR3β sequences in P;

[0055] Integrating the average proportion and clonality, the TCR index of the sample is obtained using formula ③:

[0056] TCR_index=36973.350×AvgFreq+13.958×Clonality-6.474③.

[0057] In a preferred embodiment of the present invention, the device further includes:

[0058] 4. Judgment module, used to compare the TCR index of plasma sample with a threshold to assess the risk of lung cancer in plasma sample. The threshold of TCR index is preferably -1.326. The higher the TCR index, the higher the risk of lung cancer in plasma sample.

[0059] Another aspect of the present invention provides the application of a reagent for obtaining the TCR index in the preparation of a formulation or kit for obtaining the TCR index of a plasma sample.

[0060] In another aspect, the present invention provides the application of the device described herein in obtaining the TCR index of plasma samples.

[0061] By adopting the above technical solution, the present invention achieves the following beneficial effects:

[0062] 1. Compared with LDCT screening, this invention uses a method of analyzing plasma samples, which has the advantages of liquid biopsy.

[0063] 2. This invention uses tumor-specific CD8. + Using exhausted T cell receptors as the basis for comparison, and taking the consistency of CDR3β sequence in lung cancer tumor-specific exhausted T cells as the comparison indicator, the accuracy of the TCR index obtained by this invention is greatly improved.

[0064] 3. This invention uses average proportion (AvgFreq) and clonalness as model features and employs Logistic regression analysis, which takes into account both the uniformity and Shannon index of the model without causing redundancy, thus greatly improving the accuracy of the TCR index obtained by this invention.

[0065] In summary, this invention uses the TCR index of plasma samples as an indicator to assess the risk of lung cancer, which can effectively distinguish plasma samples from lung cancer patients and healthy individuals, improve the efficiency of lung cancer screening, and has important scientific significance and good application prospects. Attached Figure Description

[0066] Figure 1 This is an overall flowchart of the method for obtaining the present invention;

[0067] Figure 2 This is a preferred flowchart of the method for obtaining the present invention;

[0068] Figure 3This is an overall structural diagram of the acquisition device of the present invention;

[0069] Figure 4 This is a preferred structural diagram of the acquisition device of the present invention;

[0070] Figure 5 This is a distribution diagram of the TCR index in plasma samples from healthy individuals and lung cancer patients according to the present invention;

[0071] Figure 6 This is a graph showing the ROC curves and TCR indexes for the training and validation sets of this invention.

[0072] Figure 7 For this invention, data graphs with a consistency greater than 50% were selected;

[0073] Figure 8 For this invention, data plots with a consistency greater than 70% were selected;

[0074] Figure 9 For this invention, data graphs with a consistency greater than 80% were selected;

[0075] Figure 10 For this invention, data graphs with a consistency greater than 90% were selected. Detailed Implementation

[0076] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. Through these descriptions, the features and advantages of the present application will become clearer and more apparent.

[0077] Example 1: Obtaining TCR index from plasma samples of 81 lung cancer patients and 200 healthy individuals

[0078] (I) Extraction and Information Analysis of Plasma Samples

[0079] Peripheral blood was collected from 81 lung cancer patients and 200 healthy individuals from the blood bank. After extracting plasma from each peripheral blood sample, gDNA was extracted and a library was constructed. For samples with a library concentration ≥0.15 ng / μl, sequencing was performed. The quality-controlled paired-end sequencing data generated from the sequencing were analyzed.

[0080] 1. Plasma extraction from peripheral blood samples

[0081] (1) First centrifugation

[0082] Peripheral blood samples were centrifuged at room temperature for 10 minutes at a speed of 2910 rpm and a centrifugal force of 1608 rcf.

[0083] (2) First separation

[0084] At room temperature, aspirate the upper plasma layer into a 5ml plasma centrifuge tube, and aspirate 1ml of the middle white membrane layer into a 2ml leukocyte centrifuge tube.

[0085] (3) Second centrifugation

[0086] The plasma sample obtained from the first separation was centrifuged at 16000 rcf at room temperature for 10 min.

[0087] (4) Second separation

[0088] At room temperature, collect all the plasma into a 5ml plasma centrifuge tube.

[0089] (5) Sample processing after separation

[0090] The remaining peripheral blood after separation is stored in a peripheral blood temporary storage box and put into storage within one week; after storage, it is stored at -80℃ for long-term storage. The obtained cells are stored at 4℃ and put into storage within one week; after storage, they are stored at -80℃ for long-term storage. The obtained plasma is stored at -20℃ and put into storage within one week; after storage, it is stored at -80℃ for long-term storage.

[0091] 2. Extraction of gDNA

[0092] DNA was extracted from whole blood or cell samples using the CWE9600 Blood DNA Kit. The specific procedures are as follows.

[0093] (1) Preparations before operation

[0094] Briefly centrifuge Buffer ML, KL, CW1, GW2, MW3, and EB before opening. Unopened reagents can be stored at 15-25℃ for one year. Shake Buffer ML and Buffer KL well before use and check for precipitation. If precipitation is found, heat in a 56℃ constant temperature mixer or dry heater and gently shake to dissolve the precipitate.

[0095] Dissolve the proteinase K powder in the specified amount of proteinase K storage buffer and store at -20°C. Do not leave the prepared proteinase K at room temperature for extended periods, and avoid repeated freeze-thaw cycles to prevent affecting its activity. Proteinase K is stable for one year at -20°C.

[0096] For the preparation of the isopropanol and Magbeads mixture, use 300 μL of isopropanol and 10 μL of Magbeads (mixed at 110%) per sample. The isopropanol and Magbeads mixture should be vortexed for 20 seconds before use to make it a homogeneous solution.

[0097] Check the temperature of the thermostatic mixer and set the heating temperature to 56°C, or use a digital display dry heater and set the temperature to 56°C in advance.

[0098] After being taken out of storage at -20℃, blood collection tubes need to be temporarily thawed at 2-8℃. They should not be thawed directly at room temperature to prevent the glass blood collection tubes from bursting due to a sudden temperature rise.

[0099] (2) Experimental Operation

[0100] Arrange the samples in order;

[0101] Add 20 μl of proteinase K to the bottom of a labeled 1.5 ml EP tube;

[0102] Invert the whole blood sample to mix thoroughly. Add 150 μl of whole blood sample to the 1.5 ml EP tube containing the protease. (If cells are extracted or the sample volume is less than 150 μl, add PBS (1×, pH 7.4) to make up to 150 μl beforehand. The maximum cell count is 5 × 10⁻⁶.) 6 Add 200 μl of Buffer ML to the EP tube, cap it, vortex for 15 seconds to mix thoroughly, and then briefly centrifuge to collect the droplets adhering to the inner cap into the tube.

[0103] Place the EP tube in a 56°C constant temperature mixer or dry heat bath for 30 minutes; during this time, prepare a 1.5 ml EP tube for the final DNA elution buffer.

[0104] After removing the EP tube from the thermostatic mixer or dry heater, centrifuge briefly, let it cool to room temperature, add 310 μl of thoroughly mixed isopropanol and Magbeads mixture, vortex mix for 5 seconds, and then place the EP tube on a thermostatic mixer at 25°C and 1600 rpm and vortex mix for 5 minutes.

[0105] After a brief centrifugation, collect the droplets adhering to the inner tube cap into the tube. Transfer the reaction samples from the EP tube sequentially into a 96-well plate and add a negative control. Use NF H2O as the negative control and add it to the 96-well plate. Then place the 96-well plate on a magnetic rack and let it stand for 1 minute. After the Magbeads are completely adsorbed onto the sidewall of the 96-well plate, discard the solution completely.

[0106] Remove the 96-well plate from the magnetic rack, add 750 μl of Buffer KL, and repeatedly aspirate and desorb the liquid 20 times using a pipette, let it stand for 5 seconds, then aspirate and desorb the liquid 10 times to ensure that the Magbeads are in a well mixed state, and let it stand for 1 minute.

[0107] Place the 96-well plate on the magnetic rack and let it stand for 1 minute. Once the Magbeads have completely adsorbed onto the sidewalls of the 96-well plate, discard the solution completely.

[0108] Remove the 96-well plate from the magnetic rack, add 750 μl of Buffer GW1, and repeatedly aspirate and desorb the liquid 20 times using a pipette, let it stand for 5 seconds, aspirate and desorb the liquid 10 times to ensure that the Magbeads are in a well mixed state, and let it stand for 1 minute.

[0109] Place the 96-well plate on the magnetic rack and let it stand for 1 minute. Once the Magbeads have completely adsorbed onto the sidewalls of the 96-well plate, discard the solution completely.

[0110] Remove the 96-well plate from the magnetic rack, add 750 μl of Buffer GW2, and repeatedly aspirate and desorb the solution 20 times using a pipette, then let it stand for 5 seconds. Repeat the aspiration and desorption process 10 times to ensure the Magbeads are well mixed, and let it stand for 1 minute.

[0111] Place the 96-well plate on the magnetic rack and let it stand for 1 minute. Once the Magbeads have completely adsorbed onto the sidewalls of the 96-well plate, discard the solution completely.

[0112] Keep the 96-well plate in the magnetic rack, add 750 μl of Buffer WM3, let it stand for 1 min, and then completely discard the solution;

[0113] Remove the 96-well plate from the magnetic rack, add 100 μl of Buffer EB, and repeatedly aspirate and desorb the liquid 20 times using an adjustable-gap pipette. Transfer all the magnetic bead mixture to the corresponding standard tubes, and place the EP tubes on a constant temperature mixer at 56°C and 1600 rpm for 10 min of shaking and elution.

[0114] Place the abbreviated EP tube on the magnetic rack and let it stand for 2 minutes. After the Magbeads are completely adsorbed onto the side wall of the EP tube, use a pipette to transfer the elution buffer to the same numbered EP tube for collecting the elution buffer and perform DNA quality control testing.

[0115] Concentration was determined using a BioTek quantitative ELISA reader or Qubit. If fragment quality control was required, agarose gel electrophoresis was performed.

[0116] When the concentration of the anion exchange agent is <0.5 ng / μL, it is considered qualified and subsequent experiments can be carried out.

[0117] DNA quality should be determined according to the following criteria:

[0118] Table 1. Classification of DNA Grades for Conventional Products

[0119]

[0120] Table 2 OncolR Product DNA Grade Classification

[0121]

[0122] Of these, Level 1 and Level 2 samples can be tested normally; Level 3 samples are risky samples; and Level 4 samples are unqualified samples.

[0123] Fragment size was determined by 1% agarose gel electrophoresis. The electrophoresis was performed at a constant voltage of 130V for about 30 minutes. The gel image was then edited after electrophoresis.

[0124] Store the extracted gDNA samples at -20 to -80°C and store them in the database within one week.

[0125] 3. Construction of TCR libraries for immune repertoire

[0126] This immune repertoire TCR library is suitable for constructing peripheral blood DNA samples and tissue DNA samples (including fresh surgical tissue, paraffin sections, and puncture tissue).

[0127] An immune repertoire (IR) refers to the sum of all functionally diverse T cells and B cells in the circulatory system of an individual at any given time. Immune repertoire sequencing (IR-Seq) is a technique that uses multiplex PCR to amplify the complementarity-determining region (CDR3 region) of the TCR, targeting T / B lymphocyte receptors, and then combines this with high-throughput sequencing to comprehensively assess the diversity of the immune system and deeply explore the relationship between the immune repertoire and disease.

[0128] T cell receptors (TCRs) are specific receptors on the surface of mature T cells that can recognize homologous antigens. Their encoding genes are highly diverse, with the CDR3 hypervariable region exhibiting the highest diversity. A set of primers was designed using the conserved regions at both ends of the CDR3 region, and a portion of the Adaper sequence was added to the 5' end of the primers. Multiplex PCR was used to capture the CDR3 region, thereby obtaining the target region product. The target region product was then used to construct a library suitable for high-throughput sequencing using PCR.

[0129] The TCR library for immune repertoire was constructed using the QIAGEN MμL tiplex PCR Kit. The specific steps are as follows.

[0130] (1) Preparations before operation

[0131] Aliquot 2×QIAGEN MμL tiplex PCR MasterMix and 5×Q solution from the QIAGEN MμL tiplex PCR Kit, and aliquot 2×KAPA HiFi HotStart Ready Mix. After vortexing and centrifuging the reaction mixture, aliquot it into 1.5mL centrifuge tubes.

[0132] Samples with excessively high concentrations (peripheral blood DNA concentration greater than 600 ng / μl, tissue DNA concentration greater than 500 ng / μl) should be diluted, while samples with excessively low concentrations (peripheral blood DNA concentration less than 110 ng / μl, tissue DNA concentration less than 80 ng / μl) should be concentrated. The diluted or concentrated DNA samples should be requantified. The requantified concentration of peripheral blood DNA using Qubit-HS should be between 110 ng / μl and 600 ng / μl, and the requantified concentration of tissue DNA using Qubit-HS should be between 80 ng / μl and 500 ng / μl.

[0133] (2) Experimental Operation

[0134] ①Multiplex PCR

[0135] Take out the pre-packaged 2×QIAGEN MμL tiplex PCR Master Mix, TCR DNA 2-3TJ-L long primers and positive control (mixed plasmid) and thaw them in a 4℃ freezer. Prepare the samples, take out 5×Q solution and place it on an ice box for later use. Shake and centrifuge the reagents, primers and samples.

[0136] Prepare 0.2 mL PCR tubes (one PCR tube for each sample + positive control P and negative control N), and label the samples sequentially.

[0137] Add Nuclease-Free Water and DNA sample according to the sample replenishment volume and sampling volume. For samples that require concentration or dilution, calculate the sampling volume and replenishment volume according to the requantified concentration and the initial library construction volume shown in the table below before sampling (the total volume of sampling volume and replenishment volume should be 18 μl).

[0138] Table 3 Starting quantity for different sample types

[0139]

[0140] Instructions for use of the positive / negative control are shown in the table below.

[0141] Table 4 Instructions for use of positive / negative controls

[0142]

[0143] Prepare the PCR reaction PreMix according to the table below on an ice box. After thoroughly shaking and centrifuging, add 32 μl of PreMix to each PCR reaction tube.

[0144] Table 5. PreMix system for multiplex PCR reaction

[0145] 2×QIAGEN MμLtiplex PCR Master Mix 25 5×Q solution 5 TCR-2000 primers 2 Total volume 32

[0146] Shake the PCR reaction tube thoroughly to mix and centrifuge. Start the corresponding program on the PCR instrument. After confirming that the program is correct according to the table below, run the PCR instrument and wait for the temperature to rise and reach 95°C before removing it from the machine.

[0147] Table 6 Multiplex PCR reaction procedure

[0148]

[0149] ② Three-step purification of multiplex PCR products

[0150] Step 1 Purification: Purify using 1.2 times the volume (1.2 times the volume of the multiplex PCR product) of purification magnetic beads.

[0151] Take out the required amount of magnetic beads according to the number of samples to be constructed, and let them stand at room temperature for at least 30 minutes. Turn on the dry heater and adjust its temperature to 37°C.

[0152] Shake the magnetic beads thoroughly. Prepare three sets of shallow well plates in advance, each containing 1.2 times the volume (60 μL), 0.5 times the volume (15 μL), and 0.7 times the volume (28 μL) of magnetic beads, according to the number of samples to be constructed. Mark them as “1.2×”, “0.5×”, and “0.7×”, respectively.

[0153] Add 50 μL of PCR product to 1.2 times the volume (60 μL) of magnetic beads, mix well by pipetting, and let stand at room temperature for 10 min to allow DNA to bind to the magnetic beads. During the standing period, prepare 70% ethanol (15 mL ultrapure water + 35 mL anhydrous ethanol).

[0154] Place the “1.2×” shallow well plate on the magnetic rack and let it adsorb until the liquid becomes clear. Discard the supernatant.

[0155] Keep the shallow well plate on the magnetic rack, add 200 μL of 70% ethanol to each well, let stand for 30 seconds, and discard the supernatant.

[0156] Repeat the previous step once, and use a 10μL eight-channel pipette to remove the remaining liquid.

[0157] To dry the magnetic beads, place the shallow-hole plate on a 37°C dry heater and heat it until the surface of the magnetic beads no longer reflects light. Then remove the shallow-hole plate from the 37°C dry heater.

[0158] Add 31 μL of Nuclease-Free Water to the shallow well plate, mix well by pipetting, and incubate at room temperature for 5 min to fully dissolve the DNA in the Nuclease-Free Water.

[0159] Place the shallow well plate on a magnetic rack until the liquid becomes clear. Pipette 30 μL of the clear liquid into the shallow well plate containing 0.5 times (15 μL) of magnetic beads. During the transfer, make sure to match each bead to the next one to avoid mixing them up.

[0160] Second step purification: 0.5 times the volume of the purified product from the previous step, purified with magnetic beads.

[0161] After the first step of purification and the final step of transferring the purified product, mix thoroughly by pipetting and let stand at room temperature for 10 minutes to allow the DNA to bind to the magnetic beads.

[0162] Place the “0.5×” shallow well plate on the magnetic rack and let it adsorb until the liquid is clear. Then, take 40 μL of the clear liquid into the shallow well plate containing 0.7 times (28 μL) of magnetic beads. When transferring the liquid, make sure to match each bead to the next one to avoid mixing them up.

[0163] Third step purification: 0.7 times the volume of the purified product from the previous step, purified with magnetic beads.

[0164] After transferring the purified product in the final step of the second purification step, mix thoroughly by pipetting and let stand at room temperature for 10 minutes to allow the DNA to bind to the magnetic beads.

[0165] Prepare a set of 8-tube PCR arrays according to the number of samples to be used for library construction, and label them sequentially (1, 2, 3, etc.).

[0166] Place the “0.7×” shallow well plate on the magnetic rack and let it adsorb until the liquid becomes clear. Discard the supernatant.

[0167] Keep the shallow well plate on the magnetic rack, add 200 μL of 70% ethanol to each well, gently blow and agitate 5-8 times, and discard the supernatant.

[0168] Repeat the previous step once, and use a 10μL eight-channel pipette to remove the remaining liquid.

[0169] To dry the magnetic beads, place the shallow-hole plate on a 37°C dry heater and heat until cracks appear on the surface of the magnetic beads (dry the magnetic beads until slight cracks appear on the surface; do not over-dry, as over-drying will lead to product loss). Then remove the shallow-hole plate.

[0170] Add 24 μL of Nuclease-Free Water to the shallow well plate to reconstitute the DNA. Mix well by pipetting and incubate at room temperature for 5 min to fully dissolve the DNA in Nuclease-Free Water.

[0171] Place the shallow well plate on a magnetic rack until it becomes completely clear. Pipette 23 μL of the clear liquid into an eight-tube PCR array. When transferring, make sure to match the tube labels one by one to avoid confusion.

[0172] ③PCR introduces index

[0173] According to the index number corresponding to each sample, take out the 30p double index primers, dissolve them at room temperature, shake them thoroughly to mix, and centrifuge.

[0174] Take the required amount of 2×KAPA HiFi HotStart Ready Mix from the -20℃ freezer, thaw it in the 4℃ freezer, shake it thoroughly to mix, centrifuge it, and place it on an ice box.

[0175] Prepare the PCR reaction PreMix according to the table below on an ice box, vortex to mix, and centrifuge.

[0176] Table 7 PCR Reaction PreMix System

[0177] Index 1 (30p) 1 Index 2 (30p) 1 2xKAPA HiFi HotStart ReadyMix 25 Total volume 27

[0178] Add 27 μL of PreMix to each PCR tube containing the purified product.

[0179] Add 1 μL of the corresponding Index (30p) to each well in turn.

[0180] Shake the PCR reaction tube thoroughly to mix and centrifuge. Start the corresponding program on the PCR instrument and run it according to the table below. Once the temperature rises and reaches 98°C, remove it from the machine.

[0181] Table 8 PCR reaction procedure

[0182]

[0183] ④Purification after PCR

[0184] Purify according to the method used for library construction.

[0185] Take the required amount of magnetic beads according to the number of samples for library preparation and let them stand at room temperature for at least 30 minutes. Turn on the dry heater and adjust the temperature to 37°C. While the magnetic beads are equilibrating, prepare two sets of PCR eight-tube strips according to the number of samples. Label one set of tubes and keep the other set as a spare.

[0186] Shake the magnetic beads thoroughly. Take a shallow-well plate and add 50 μL of purified magnetic beads to each well. Transfer the PCR product obtained in the above steps into the corresponding wells, mix by pipetting, and let stand at room temperature for 10 min to allow the DNA to bind to the magnetic beads. During the standing period, prepare 70% ethanol (15 mL ultrapure water + 35 mL anhydrous ethanol).

[0187] Place the “1.2×” shallow well plate on the magnetic rack and let it adsorb until the liquid becomes clear. Discard the supernatant.

[0188] Keep the shallow well plate on the magnetic rack, add 200 μL of 70% ethanol to each well, let stand for 30 seconds, and discard the supernatant.

[0189] Repeat the previous step once, and use a 10μL eight-channel pipette to remove the remaining liquid.

[0190] To dry the magnetic beads, place the shallow-hole plate on a 37°C dry heater and heat it until the surface of the magnetic beads no longer reflects light. Then, remove the shallow-hole plate from the 37°C dry heater.

[0191] Dissolve the DNA completely in 23 μL of Nuclease-Free Water.

[0192] Take a set of PCR eight-tube strips, label only the first six wells of each row, and add 2.5 μL of 10×SampleLoading Buffer to each well. Add 25 μL of Sizing DNA Ladder to the seventh well.

[0193] Place the shallow-well plate on a magnetic rack until the liquid is completely clear. Pipette 22.5 μL of the clear liquid into the PCR eight-tube array prepared in step 9.5, vortex to mix, and centrifuge. During transfer, ensure each tube matches its label carefully to avoid confusion.

[0194] ⑤ Product E-Gel Power Snap Recycling

[0195] Prepare a set of 1.5mL EP tubes, mark the serial number on the tube cap, print the label, and stick it on the corresponding EP tube;

[0196] Connect the Invitrogen E-Gel Power Snap electrophoresis apparatus to a power source, turn on the switch, install the E-Gel™ 2% agarose gel, and check that the gel is properly connected to the apparatus.

[0197] Add 50 μL of Nuclease-Free Water to each of the two rows of wells on the gel. Add the sample and marker mixture to the spot wells. Select the program "Size Select 2%" and set the electrophoresis time (approximately 16-18 min, which can be adjusted according to the size of the recovered fragments). Secure the instrument cap and start the program.

[0198] Turn on the blue light transmitter and observe the migration of the sample within the gel.

[0199] When the target band with the fastest migration rate (approximately 250 bp, varying between samples) migrates to the top of the recovery well among the 6 samples, pause the program, remove and discard the liquid from the 6 sample recovery wells using a pipette, add 30 μL of Nuclease-Free Water back into the 6 sample recovery wells, and continue running the program.

[0200] When the brightest main band of the target band (approximately 250 bp, varying between samples) migrates to the recovery well and becomes diffuse, pause the procedure and transfer the sample from the recovery well to a new PCR reaction tube using a pipette. Due to slight variations in the main band position between samples, all 6 samples must be recovered separately to avoid sample loss.

[0201] Transfer the recovered sample into the prepared 1.5 mL tube, making sure to match each tube correctly during the transfer.

[0202] ⑥ Document Quality Control

[0203] The constructed library samples were quantified using an ELISA reader or Qubit-HS, and all constructed library samples were sent for quality control testing. The fragment size was determined using Labchip assays, and the quality control standards were as follows.

[0204] Table 9 Quality Control Table

[0205] Library Concentration Library concentration ≥0.15 ng / μl Re-extract DNA sample

[0206] 4. Sequencing

[0207] This section describes the sequencing of the circularized library using the DNBSEQ-T7RS platform.

[0208] The DNBSEQ-T7RS uses combined probe anchoring polymerization (cPAS) technology, which polymerizes DNA molecular anchors and fluorescent probes onto DNA nanospheres (DNBs). A high-resolution imaging system then acquires the light signals, which are digitized to obtain high-quality, high-accuracy sample sequence information. This technology enables large-scale parallel sequencing while maintaining sequencing accuracy.

[0209] (1) Preparations before operation

[0210] Before using the instrument, restart it. Click the icon in the upper right corner of the instrument interface, then click "Shut Down." After the instrument shuts down, turn the power switch to the "off" position. Wait 2 minutes, then turn the power switch to the "on" position and wait for the instrument to start up.

[0211] Prepare the DNBSEQ-T7RS high-throughput sequencing reagent kit in advance. Thaw in a water bath at room temperature (5 hours), or thaw in a refrigerator 18 hours in advance.

[0212] (2) Experimental Operation

[0213] ① DNB preparation

[0214] Remove the library and place it on an ice box for later use. Remove the DNB preparation buffer, TE buffer, and DNB termination buffer and thaw them at room temperature. Place the DNB polymerase buffer I on an ice box for about 0.5 hours. After thawing, vortex the mixture for 5 seconds, then briefly centrifuge and place it on an ice box for later use.

[0215] Each DNB preparation system has a volume of 100 μL, requires 40 fmol of ssDNA library, and requires 270 μL of DNB to be loaded onto one sequencing slide.

[0216] Take 0.2 mL of an eight-tube or PCR tube and prepare the reaction mixture on ice.

[0217] Table 10. DNB preparation system 1

[0218] ssDNA Library Total input volume X TE buffer 20-X DNB Preparation Buffer / App-A DNB Preparation Buffer 20 Total volume 40

[0219] Mix the reaction mixture using a vortex mixer and then place it in a PCR instrument for reaction.

[0220] Table 11 DNB Reaction Conditions 1

[0221] 95℃ 1min 65℃ 1min 40℃ 1min 4℃ ∞

[0222] After the reaction is complete, the centrifuge is briefly used to centrifuge the liquid on the tube wall to the bottom of the tube, and then it is immediately placed on an ice box.

[0223] Remove DNB polymerase buffer II (LC) and place it on an ice box. Centrifuge briefly for 5 seconds and place on an ice box for later use.

[0224] Prepare the enzyme mix, and after preparation, shake thoroughly to mix and centrifuge.

[0225] Table 12 Preparation of DNB System 2

[0226] DNB polymerase buffer I 40 DNB Polymerase Buffer II (LC) 4 Total volume 44

[0227] Add the cyclization reaction mix to the reaction tube, 44 μL / tube, mix thoroughly and centrifuge.

[0228] Perform the reaction on a PCR instrument under the following conditions.

[0229] Table 13 DNB Reaction Conditions 2

[0230]

[0231] After the reaction is complete, immediately add 20 μL of DNB stop buffer and slowly mix by pipetting with a wide-mouth pipette tip 5-8 times. Do not shake or vigorous pipetting. It can be stored at 4°C for later use (within 48 hours).

[0232] After the DNB preparation is complete, take 2 μL for use. The concentration was determined using the ssDNA Assay Kit. A concentration of 8 ng / μL or higher is considered acceptable; otherwise, the sample must be prepared again. If the concentration exceeds 40 ng / μL, it needs to be diluted to 20 ng / μL with DNB loading buffer I before use.

[0233] ②DNB mixture

[0234] The plasma and cell libraries were pooled, circularized, and DNBs were prepared separately, and then pooled and mixed according to the amount and concentration of data.

[0235] ③ DNB loading

[0236] A. DNB Loading Preparation

[0237] Remove the sample loading reagent plate from the DNBSEQ-T7RS DNB loading kit and thaw it in a water bath at room temperature for 1-2 hours. After complete thawing, store it in a refrigerator at 2-8℃ for later use, or thaw it in the refrigerator one day in advance. Before use, gently invert and mix 5 times, then centrifuge.

[0238] Take out a 0.5 mL cryovial, add the reagents as shown in the table below, and gently pipette to mix 5-8 times. Do not shake, centrifuge, or vigorously pipette.

[0239] Table 14 DNB Loading System 1

[0240] DNB Loading Buffer II 90 DNB Polymerase Buffer II (LC) 1 DNB 270 Total volume 361

[0241] Remove the sequencing slide from the DNBSEQ-T7RS DNB loading kit, open the outer packaging, check that the slide is intact, and use an air can to blow clean the back of the slide.

[0242] B. DNB loading

[0243] Launch MGIDL-T7 to enter the program, enter your username and password, and click "Login" to enter the main interface.

[0244] Choose either A or B to proceed, and then click Load.

[0245] Open the loading compartment door.

[0246] Use the RFID scanning area to scan the ID information of the sample loading reagent plate. Remove the sealing film from the sample loading reagent plate and add 4 mL of 0.1 M NaOH to well #11. If using App-A sequencing, completely aspirate and discard the reagent in well #1, wash 3-5 times with ultrapure water, and then add 2 mL of App-A sequencing primer working solution 1. Place the prepared sample loading reagent plate oriented correctly on the reagent plate holder of the MGIDL-T7; the interface will indicate that the reagent plate is in place.

[0247] Enter the DNB ID. Place the 0.5ml cryovial containing the DNB loading system into the DNB reagent tube well of the MGIDL-T7. The interface will display that the DNB tube has been placed.

[0248] Use the RFID scanning area to scan the sequencing slide ID information. Hold the slide by both sides, align the positioning protrusions on the slide upwards with the positioning grooves on the slide platform, and place the slide frame against the lower frame of the slide platform. Gently press down on the edges of the slide with both hands. Press the slide attachment button on the slide platform to ensure it is fully attached to the platform; the interface will display that the slide has been placed.

[0249] Close the loading compartment door, click "Load," and select "Yes." Slide loading begins.

[0250] When the interface displays that the slide has been loaded, press the slide attachment button on the slide download platform to remove the loaded slide and proceed with sequencing. If not used immediately, please store the slide in a 4°C refrigerator (loaded slides should be used within one week).

[0251] After loading is complete, place the cleaning substrate, click "Post-Cleaning," and select "Yes" in the pop-up dialog box to begin MGIDL-T7 cleaning. Once cleaning is complete, click "Back" to load the substrate again.

[0252] ④Preparation before sequencing

[0253] A. Preparation of sequencing reagent tank

[0254] Open the reagent tank cover and wipe away any condensation with a lint-free paper towel.

[0255] Using a clean 1mL pipette tip, gently poke a sample application well with a diameter of about 2cm at the edge of wells #9 and #10.

[0256] Add 5.0 ml (PE100) or 6.8 ml (PE150) dNTPs mixture IV to well #9, and add 13.5 ml (PE100) or 18.3 ml (PE150) dNTPs mixture II to well #10.

[0257] Add 5.0 ml (PE100) or 6.8 ml (PE150) of DNA polymerase mixture to well #9, and add 6.8 ml (PE100) or 6.1 ml (PE150) of DNA polymerase mixture to well #10.

[0258] Use the provided transparent sealing film to seal sample wells #9 and #10, being careful not to cover the center of the wells to avoid affecting the descent of the reagent needle. Place the reagent trough horizontally on the table, hold both sides with both hands, and shake clockwise 10-20 times, then counterclockwise 10-20 times, ensuring that a vortex is visible to the naked eye, until the color of the upper and lower layers of reagent #9 is uniform, to ensure thorough mixing of the reagents.

[0259] Use a 1 mL pipette to transfer 600 μL of PE MDA polymerase mixture into the MDA reagent tube. Vortex to mix, then add the mixture to well #8, ensuring there are no air bubbles at the bottom of the tube during addition.

[0260] If using App-A sequencing, puncture wells #5, #12, and #14, remove and discard the reagents, rinse with ultrapure water 3-5 times, and then add the reagents according to the table below, ensuring there are no air bubbles at the bottom of the tube when adding them.

[0261] Table 15 App-A Primer Addition Table

[0262] 5 App-A Barcode Primer Working Solution 2 5.0 12 App-A sequencing primer working solution 2 5.0 14 App-A MDA primer working solution 5.0

[0263] For double-barcode sequencing at both ends, puncture well #3 and add 3.5 mL of barcode primer 3 working solution or App-A barcode primer 3 working solution, ensuring there are no air bubbles at the bottom of the tube when adding.

[0264] B. Preparation for cleaning the reagent tank

[0265] Shake well to clean the reagent tank and wipe it clean with lint-free paper.

[0266] Select any well #2, puncture the film with a clean 1mL pipette tip, and add 45mL of 0.1M NaOH.

[0267] C. Filling of pure water reagent barrels

[0268] Fill the pure water tank with laboratory-grade water, and be sure to open the vent hole of the pure water tank.

[0269] Each chip consumes 3 liters of pure water.

[0270] The purified water is used for sequencing, so it must be kept clean and the purified water in the container must be completely replaced once a week. Before filling with new purified water, spray the inside of the purified water container lid and the surface of the purified water tube with 75% alcohol, then wipe it clean with a clean, lint-free cloth, and rinse the purified water container three times with clean purified water.

[0271] ⑤ Sequencing.

[0272] Enter your username and password, and click "Login" to enter the main interface.

[0273] Select A / B / C / D according to your sequencing needs, click the "Sequencing" option on the interface, and then select "New Sequencing".

[0274] After loading the sequencing slide, blow it clean with an air canister to ensure that there is no visible dust on the surface and back of the slide. Then, place the slide into the core drive and press the core drive button to retract the core drive.

[0275] Open the reagent compartment door, moisten a lint-free paper or cloth with laboratory-grade water, and use it to wipe the bottom and sides of the refrigerator, keeping them clean and dry. Place the sequencing reagent tank into the upper low-temperature compartment and the cleaning reagent tank into the lower ambient temperature compartment. Close the low-temperature compartment door and the ambient temperature compartment door, and finally close the reagent compartment door.

[0276] After the RFID system automatically identifies the sequencing reagent tank, cleaning reagent tank, and slide ID, the ID information will be displayed in the corresponding text box (if it cannot be automatically identified, it can be manually entered).

[0277] Click "Next" to review the information. After confirming that everything is correct, click "Start," select "Yes," and begin sequencing.

[0278] ⑥ Quality control standards

[0279] After sequencing is completed, check the corresponding sequencer task number in the LIMS system / information analysis / data quality control section. Perform quality control on the sequencer's operating parameter Q30 to confirm the sequencing status: Q30 is above 85%, total reads are ≥4500M / Run, and the pre-sorting of the quality control sample (190420-ICD2-49) is fixed at 0.3G. The actual data output should be between 0.3-0.6G. This sequencing is considered qualified. If the quality control fails to control the sequence, the cause needs to be investigated and a re-sequencing should be arranged.

[0280] (II) Information Analysis

[0281] The following analysis will focus on the quality-controlled paired-end sequencing data generated from the sequencing process.

[0282] The sequencing data for each sample were analyzed using steps 1-3 as follows.

[0283] 1. By comparing the read lengths with the PCR primer sequences, sequencing reads containing specific primer sequences were selected. Specific information for primers 1-45 is shown in Table 16.

[0284] Table 16 Information on Primers 1-45

[0285]

[0286]

[0287]

[0288]

[0289] 2. Concatenate the selected read lengths to form multiple concatenation sequences;

[0290] 3. Perform CDR3β sequence feature identification on the multiple spliced ​​sequences to obtain the types of CDR3β sequences and the number of occurrences of each type of CDR3β, i.e., the types and number of TCR sequences.

[0291] For example, a total of 7107 CDR3β sequences were obtained from the plasma sample of lung cancer SM108, of which the sequence CASSTRDRGVDEQYF appeared 722 times. Therefore, the TCR sequence types of SM108 are 7107, the number of sequences CASSTRDRGVDEQYF is 722, and the clonality of SM108 is 0.295.

[0292] For each peripheral blood T cell sample (P), 377 tumor-specific CD8+ lines from the single-cell sequencing data were selected. + The number of CDR3β cells with a sequence homology greater than 60% (N) in exhausted T cells was calculated as the average proportion of all CDR3β sequences in plasma T cells. This proportion was then integrated with the CDR3β sequence clonality of plasma samples using a logistic regression model to obtain the corresponding TCR index.

[0293] TCR index = 36973.350 × average proportion + 13.958 × clonalness - 6.474

[0294] Taking the plasma sample from the lung cancer sample SM108 as an example, the 7107 CDR3β sequences of SM108 were compared with 377 tumor-specific CD8 sequences. + Blast sequence alignment was performed on exhausted T cell sequences, and 44 CDR3β sequences with greater than 60% identity were found, such as the SM108 sequence CASSTRDRGVDEQYF, which is consistent with tumor-specific CD8. +The sequence identity of the exhausted T cell sequence CASSVTDRGLDEQYF was 80%. The total clonal proportion of these 44 CDR3β sequences in the SM108 sample was 0.003955, therefore the average proportion was 0.003955 ÷ 44 = 8.99E-05. Consequently, the TCR index = 36973.350 × 8.99E-05 + 13.958 × 0.295 - 6.474 = 0.963. Since 0.963 > -1.326, the SM108 prediction result is consistent with plasma samples from lung cancer patients.

[0295] The results from plasma samples from 81 lung cancer patients and 200 healthy individuals are shown in Table 17.

[0296] Table 17 Results Statistics Table

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303] Example 2: Predictive value analysis of TCR index in identifying plasma samples from lung cancer patients

[0304] The TCR index of plasma samples obtained above from 81 lung cancer patients and 200 healthy individuals was plotted. Figure 5 As can be seen, there was a significant difference in the TCR index between plasma samples from lung cancer patients and healthy individuals (P < 0.0001). The TCR index was significantly higher in plasma samples from lung cancer patients than in plasma samples from healthy individuals. Therefore, the TCR index can be used as a predictive indicator for identifying plasma samples from lung cancer patients and healthy individuals.

[0305] ROC curves were plotted for the samples in the training and validation sets respectively. Figure 6 It can be seen that the AUC of the ROC curve on the training set is 0.885, the optimal cut-off value is -1.028, the sensitivity is 80.5%, and the specificity is 85.0%. The AUC of the ROC curve on the validation set with five-fold cross-validation is 0.919, the optimal cut-off value is -1.326, the sensitivity is 85.7%, and the specificity is 93.0%. Therefore, the critical value for determining the TCR index is -1.326.

[0306] Example 3: Validating the model and predictive value of other consistency thresholds

[0307] To determine the relationship between plasma T cell receptor (P) and tumor-specific CD8 + To determine the consistency index of exhausted T cell receptors (T), the present invention conducted the following gradient experiments.

[0308] 1. The consistency index is greater than 50%.

[0309] When the concordance index is set to greater than 50%, the average proportion of CDR3β sequences with greater than 50% concordance in SM108 plasma samples to all CDR3β sequences in plasma T cells is 8.99E-05, with a clonality of 0.295. The formula for calculating the TCR index is:

[0310] TCR_index=44193.237×AvgFreq+9.702×Clonality-6.248

[0311] The TCR index was calculated to be 0.586.

[0312] from Figure 7 As can be seen, there was a significant difference in the TCR index between plasma samples from lung cancer patients and healthy individuals (P < 0.0001). The TCR index was significantly higher in plasma samples from lung cancer patients than in plasma samples from healthy individuals. The TCR index can serve as a predictive indicator for distinguishing between lung cancer and plasma samples from healthy individuals.

[0313] Furthermore, the AUC of the training set ROC curve was 0.874, the optimal cut-off value was -1.497, the sensitivity was 76.0%, and the specificity was 87.8%. The AUC of the validation set five-fold cross-validation ROC curve was 0.879, the optimal cut-off value was -1.574, the sensitivity was 79.1%, and the specificity was 85.7%.

[0314] 2. Consistency greater than 70%

[0315] When the concordance index is set to greater than 70%, the average proportion of CDR3β sequences with greater than 70% concordance in SM108 plasma samples to all CDR3β sequences in plasma T cells is 8.99E-05, with a clonality of 0.295. The formula for calculating the TCR index is:

[0316] TCR_index=43277.129×AvgFreq+9.644×Clonality-6.176

[0317] The TCR index was calculated to be 0.559.

[0318] from Figure 8 As can be seen, there was a significant difference in the TCR index between plasma samples from lung cancer patients and healthy individuals (P < 0.0001). The TCR index was significantly higher in plasma samples from lung cancer patients than in plasma samples from healthy individuals. The TCR index can serve as a predictive indicator for distinguishing between lung cancer and plasma samples from healthy individuals.

[0319] Furthermore, the AUC of the training set ROC curve was 0.872, the optimal cut-off value was -1.498, the sensitivity was 75.0%, and the specificity was 87.8%. The AUC of the validation set five-fold cross-validation ROC curve was 0.875, the optimal cut-off value was -1.572, the sensitivity was 79.1%, and the specificity was 85.7%.

[0320] 3. Consistency greater than 80%

[0321] When the concordance index is set to greater than 80%, the average proportion of CDR3β sequences with greater than 80% concordance in SM108 plasma samples to all CDR3β sequences in plasma T cells is 7.74E-05, with a clonality of 0.295. The formula for calculating the TCR index is:

[0322] T TCR_index=37312.498×AvgFreq+9.226×Clonality-5.617

[0323] The TCR index was calculated to be -0.009.

[0324] from Figure 9 As can be seen, there was a significant difference in the TCR index between plasma samples from lung cancer patients and healthy individuals (P < 0.0001). The TCR index was significantly higher in plasma samples from lung cancer patients than in plasma samples from healthy individuals. The TCR index can serve as a predictive indicator for distinguishing between lung cancer and plasma samples from healthy individuals.

[0325] Furthermore, the AUC of the training set ROC curve was 0.864, the optimal cut-off value was -1.531, the sensitivity was 75.0%, and the specificity was 85.4%. The AUC of the validation set five-fold cross-validation ROC curve was 0.860, the optimal cut-off value was -1.414, the sensitivity was 81.4%, and the specificity was 78.6%.

[0326] 4. Consistency greater than 90%

[0327] When the concordance index is set to greater than 90%, the average proportion of CDR3β sequences with greater than 90% concordance in SM108 plasma samples to all CDR3β sequences in plasma T cells is 6.64E-05, with a clonality of 0.295. The formula for calculating the TCR index is:

[0328] TCR_index=16101.096×AvgFreq+7.941×Clonality-3.629

[0329] The TCR index was calculated to be -0.217.

[0330] from Figure 10 As can be seen, there was a significant difference in the TCR index between plasma samples from lung cancer patients and healthy individuals (P < 0.0001). The TCR index was significantly higher in plasma samples from lung cancer patients than in plasma samples from healthy individuals. The TCR index can serve as a predictive indicator for distinguishing between lung cancer and plasma samples from healthy individuals.

[0331] Furthermore, the AUC of the training set ROC curve was 0.800, the optimal cut-off value was -1.123, the sensitivity was 79.0%, and the specificity was 73.2%. The AUC of the validation set five-fold cross-validation ROC curve was 0.826, the optimal cut-off value was -0.992, the sensitivity was 88.4%, and the specificity was 67.9%.

[0332] The gradient experiments described above show that when plasma T cell receptor (P) interacts with tumor-specific CD8... + When the T-cell receptor (T) concordance was greater than 50%, it was sufficient to predict the difference between plasma samples from lung cancer patients and healthy individuals. Specifically, there was a significant difference in the TCR index between plasma samples from lung cancer patients and healthy individuals (P < 0.0001). The TCR index was significantly higher in plasma samples from lung cancer patients than in plasma samples from healthy individuals. Therefore, the TCR index can be used as a predictive indicator for distinguishing between lung cancer and healthy individuals. Furthermore, the AUC of the five-fold cross-validation ROC curves on both the training and validation sets was greater than 0.800.

[0333] Taking all factors into consideration, the consensus threshold corresponding to the optimal prediction value ultimately selected by this invention is 60%.

[0334] The present application has been described above with reference to preferred embodiments; however, these embodiments are merely exemplary and illustrative. Various substitutions and modifications can be made to the present application based on these embodiments, all of which fall within the protection scope of the present application.

Claims

1. A device for obtaining the TCR index of a plasma sample, comprising the following modules: (1) Sequencing information acquisition module, used to obtain tumor-specific CD8 from known single-cell sequencing data. + The CDR3 region of the plasma T cell receptor β chain was depleted. The CDR3 region of the plasma T cell receptor β chain in the plasma sample was sequenced to obtain the sequencing read length. The selected read lengths were spliced ​​to form multiple spliced ​​sequences. The multiple spliced ​​sequences were subjected to CDR3β sequence feature identification to obtain the types and abundance of plasma T cell receptor CDR3β sequences. (2) Statistical module, used to count plasma T cell receptors and tumor-specific CD8. + The types and abundance of CDR3β sequences with greater than 60% receptor sequence identity in exhausted T cells; (3) Calculation module, used to calculate plasma T cell receptor and tumor-specific CD8 using formula ① + The average proportion of CDR3β sequences with greater than 60% receptor sequence identity in exhausted T cells out of all CDR3β sequences in plasma T cells: ① T It is a tumor-specific CD8 + The CDR3β sequence of exhausted T cells; P It is a CDR3β sequence from a plasma sample; yes P Zhongyu T CDR3β sequences with greater than 60% consistency; N yes Quantity; yes The Middle The CDR3β sequence accounts for P The proportion of total CDR3β sequences; Calculate plasma T cell CDR3β sequence clonality using formula ②: ② P It is a CDR3β sequence from a plasma sample; R yes P Quantity; yes The Middle The CDR3β sequence accounts for P The proportion of total CDR3β sequences; Integrating the average proportion and clonality, the TCR index of the sample is obtained using formula ③: ③。 2. The apparatus according to claim 1, further comprising: (4) Judgment module, which compares the TCR index of plasma sample with the threshold to assess the risk of lung cancer in plasma sample. The higher the TCR index, the higher the risk of lung cancer in plasma sample.

3. The apparatus according to claim 2, wherein the threshold value of the TCR index is -1.

326.

4. The apparatus of claim 1, wherein the plasma sample is derived from a peripheral blood sample from a lung cancer patient or a healthy individual.

5. Use of the reagent for obtaining the TCR index in the preparation of the apparatus according to any one of claims 1-4.

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