CN111979196A - Separation and identification of peripheral blood CTC of biliary tract cancer by HSPG and downstream gene detection method - Google Patents
Separation and identification of peripheral blood CTC of biliary tract cancer by HSPG and downstream gene detection method Download PDFInfo
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- CN111979196A CN111979196A CN202010867780.5A CN202010867780A CN111979196A CN 111979196 A CN111979196 A CN 111979196A CN 202010867780 A CN202010867780 A CN 202010867780A CN 111979196 A CN111979196 A CN 111979196A
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Abstract
The invention provides a separation and identification method of peripheral blood CTC of biliary tract cancer by HSPG and a downstream gene detection method, the tumor cells are captured in peripheral blood CTC for separation and identification, a DNA extraction kit is adopted for DNA extraction of a sample, a high-throughput sequencing method is adopted for the whole genome amplification, library construction, sequencing data filtration and quality evaluation, sequencing depth coverage statistics, variation detection, key variation identification and other process methods of the source nucleic acid of the biliary tract cancer blood circulation tumor cells, a whole set of high-throughput sequencing analysis system is established, tumor specific molecular information is obtained for improving specificity, the tumor cells are captured in blood through specific sorting antibodies, and then obtaining spider silk traces of genetic information carried by the released DNA and carrying out detection analysis, thereby realizing the method for analyzing and detecting the DNA by utilizing the gene expression level change of the CTC-derived nucleic acid and the gene expression level change of the tissue-derived nucleic acid.
Description
Technical Field
The invention relates to the technical field of biological and medical detection, in particular to a separation and identification method of peripheral blood CTC of biliary tract cancer by HSPG and a downstream gene detection method.
Background
Circulating Tumor Cells (CTCs) in human peripheral blood refer to tumor cells that spread from a tumor lesion into the peripheral blood circulation and can develop into a metastatic lesion of the tumor under certain conditions. Since more than 90% of cancer deaths are caused by metastasis, CTCs are a direct source of tumor metastasis, the isolation and molecular detection of CTCs from blood is becoming increasingly important. The peripheral blood composition is complex, wherein the red blood cell count is 5.0-6.0X 1012/mL, the white blood cell count is 4.0-10.0X 106/mL, and the number of circulating tumor cells may be only 0-100. The detection of CTCs has been expanded from simple cell counting to various directions of downstream cluster analysis, molecular typing, gene, transcription and protein level detection, and has entered clinical practice in breast cancer, colorectal cancer, prostate cancer and lung cancer, but there is still a great deal of blank in the diagnosis and treatment of biliary tract cancer.
High-throughput sequencing (also known as "next generation" sequencing) has had a great impact on basic research and is revolutionizing clinical practice. NGS technologies are widely used in routine clinical practice, including non-invasive prenatal testing (NIPT) and pre-embryo implantation genetic diagnosis/screening (PGD/PGS), genetic diseases, tumors, drug genomes, etc. NGS applications have shifted from gene assembly to Whole Exon Sequencing (WES) and Whole Genome Sequencing (WGS). The NGS gene detection of tumors is currently mainly aimed at two types of specimens, one is the mutant genome panel sequencing of solid tumor tissues, and the other is the liquid biopsy of peripheral blood and urine. The solid tumor tissue NGS is limited in that the tumor material is limited to a certain point, and is difficult to repeat for many times, and the detection result has significance for the medication guidance of patients who are initially diagnosed and treated, but the tumor development, the treatment response, the tumor re-development and even the tumor metastasis can be continuously and dynamically changed along with the time. In addition, some metastatic patients may have difficulty in obtaining tissue materials, or may only come from a part of lesions, and cannot reflect the overall tumor condition of the patients; therefore, there is a strong clinical significance in developing CTC-NGS based on fluid biopsy.
Heparan Sulfate Proteoglycan (HSPG) is a group of glycoproteins bound to core proteins by heparan sulfate chains, distributed on the cell surface and in the extracellular matrix, the matrix HSPG being mainly associated with cellular infiltration and migration; the cytomembrane HSPG mainly comprises syndecano and Glypican, and the functions of the cytomembrane HSPG relate to: co-receptors as growth factors regulate signal transduction, co-regulate cell migration with adhesion molecules, bind to regulate extracellular matrix metalloproteinase activity, and the like. In tumor development and progression, the content, composition and function of HSPG are abnormal, and membrane HSPG can bind with various growth factors (such as FGF, PDGF, EGF, HGF, IGF and the like) and activate related channels. In poorly differentiated adenocarcinomas, HSPG levels are elevated, cellular differentiation is low, local metastasis is associated, HSPG sulfation levels are elevated in GBC tissues in advanced clinical stages, sulfated HSPG positive patients are less responsive to chemotherapy and have shorter survival, while less research is currently being conducted on overall HSPG expression in biliary tract cancers.
Tumor cells are captured in peripheral blood through a specific sorting antibody of the biliary tract cancer HSPG, so that spider silk trails of genetic information carried by released DNA are known and detected and analyzed, and an analysis method for timely detecting the DNA by utilizing a non-invasive liquid biopsy mode is realized, so that the method has great advantages and potential. The invention provides a method for selecting a specific recognition factor HSPG of biliary tract cancer, specifically sorting and identifying circulating tumor cells in peripheral blood of a patient with biliary tract cancer, extracting nucleic acid through obtained CTC, and completing high-throughput sequencing analysis of CTC-DNA in clinical research. Researching the correlation between the dynamic change index of CTC in peripheral blood of biliary tract tumor and clinical detection index, and the consistence and difference analysis between the gene expression level change of CTC source nucleic acid and the gene expression level change of tissue source nucleic acid.
Disclosure of Invention
The invention aims to provide a method for specifically sorting, identifying and detecting CTC in biliary tract tumor serum, which comprises the steps of selecting a specific recognition factor HSPG of biliary tract cancer, specifically sorting and identifying circulating tumor cells in peripheral blood of a patient with biliary tract cancer, extracting nucleic acid through the obtained CTC, and completing high-throughput sequencing analysis of CTC-DNA. Researching the correlation between the dynamic change index of CTC in peripheral blood of biliary tract tumor and clinical detection index, and the consistence and difference analysis between the gene expression level change of CTC source nucleic acid and the gene expression level change of tissue source nucleic acid.
The technical scheme adopted by the invention is as follows: a method for separating and identifying peripheral blood CTC of biliary tract cancer by HSPG comprises the following steps:
(1) sampling: taking 7.5mL of peripheral blood from a biliary tract cancer patient, placing the peripheral blood in an anticoagulation tube, and uniformly mixing a whole blood sample;
(2) removing plasma protein and plasma (serum) nucleic acid from the blood sample collected in the step (1), adding a buffer solution into the whole blood, removing supernatant through layered centrifugation, and then, shaking a centrifugal tube to uniformly mix and precipitate cells;
(3) removing red blood cells: after mixing, adding lysis solution into a centrifuge tube, placing the centrifuge tube into a vertical mixing instrument for mixing, centrifuging to remove supernatant, slightly shaking the centrifuge tube for mixing, precipitating cells, and adding buffer solution;
(4) layering and centrifuging: adding a layering liquid into a new centrifugal tube, superposing all the liquids in the step (3) to the top layer of the layering liquid, then cleaning the tube wall of the centrifugal tube by using a buffer solution, and simultaneously transferring the cleaning liquid to the top layer of the buffer solution for centrifugation;
(5) and (3) removing white blood cells: after centrifugation, 3 layers of solution can be seen, the solution on the top 2 layers is gently absorbed into a new centrifugal tube, buffer solution is respectively added, after inversion and uniform mixing, centrifugation is carried out, supernatant fluid is discarded, buffer solution is added, and cells are uniformly mixed and precipitated;
(6) washing the magnetic particles: sucking a proper amount of immune lipid magnetic ball (combined with HSPG antibody) suspension into an EP tube, standing, sucking and discarding the solution, adding a buffer solution, mixing uniformly, standing, discarding the supernatant, repeating for 3 times, and then resuspending the magnetic particles to the original volume by using the buffer solution;
(7) antibody incubation: slowly adding a certain amount of immune lipid magnetic beads (combined with HSPG antibodies) into the sample treated in the step (6), adjusting the shaking speed of a shaking table, obliquely fixing a centrifugal tube on the shaking table, and shaking at room temperature;
(8) removing free magnetic particles: transferring the liquid of the centrifuge tube in the step (7) into a new centrifuge tube, standing the centrifuge tube on a magnetic frame, transferring the liquid into the new centrifuge tube respectively, arranging the centrifuge tube at the upper end of the centrifuge tube, centrifuging the liquid, discarding supernatant, and coating the supernatant on glass slides respectively;
(9) air-drying the glass slide at room temperature, dropwise adding 4% paraformaldehyde to fix cell surface antigens, and dropwise adding 1% fetal calf serum to seal cell surface nonspecific sites;
(10) adding an anti-working solution to the slide glass obtained after the blood sample is enriched in the step (9), incubating in a dark place, and fully washing;
(11) adding a second antibody working solution into the sample obtained in the step (10), incubating in a dark place, fully washing, adding a DAPI working solution for dyeing, and slightly washing;
(12) dripping glycerol on the sample in the step (11), and reading the cover plate; and performing CTC source circulating tumor cell-based DNA extraction on the sample by adopting a DNA extraction kit.
Preferably, the DNA extraction kit can adopt QIAGEN kit.
Preferably, the antibody incubation and free magnetic particle elimination in the steps (7) and (8) are carried out for capturing the circulating tumor cells of the peripheral blood specifically:
s1: processing the received blood sample: adding 4mL of blood into a 5mL centrifuge tube, adding 40 μ l of HSPG immune lipid magnetic beads, reversing and mixing uniformly, and incubating at room temperature for 20 min;
s2: and (3) standing the sample on a magnetic frame, adsorbing the immune lipid magnetic ball for capturing the CTC on one side of the magnetic frame, inverting the magnetic frame and the sample, and washing the sample on the centrifugal tube cover. Standing for 10 minutes, and sucking and removing the clear liquid;
s3: adding 1mLddH2O to wash the immune lipid magnetic spheres;
s4: adding 100ul CF1 for smear, and oven drying at 37 deg.C;
s5: dropping 4% paraformaldehyde, fixing for 8min, and preheating in a water bath at 37 deg.C containing 2 XSSC for 10 min;
s6: respectively selecting 75%, 85% and anhydrous ethanol for dehydration, and respectively performing dehydration for 2 min. And air-dried at room temperature;
s7: adding 10ul of fluorescent probe, sealing, hybridizing at 76 ℃ for 10min in a hybridization instrument, and hybridizing at 37 ℃ for 1.5 h;
s8: tearing off the mounting glue by using a forceps, putting the mounting glue into formamide (43 ℃), peeling off a cover glass, soaking and washing for 10min, then soaking and washing for 15min by adopting 2 XSSC, and shaking once every 5 min;
s9: coating 100ul of CK fluorescent antibody staining solution on the sample area, and incubating for 1h under the condition of a wet box at 37 ℃ and keeping out of the sun;
s10: CK fluorescent antibody staining solution was washed away, washed twice with 0.2% BSA, and 10ul of DAPI coverslip was added.
Preferably, the process of extracting DNA of circulating tumor cells by using the DNA extraction kit in step (13) is specifically:
(a) adding 2.5 times volume of Solutiona into 10ml of blood, reversing, mixing uniformly, centrifuging at 8000rpm for 5min, and discarding supernatant;
(b) repeating step a for 1-2 times, and discarding the supernatant;
(c) preparing a mixed solution of solution B and solution C according to information provided by an attached table of the DNA extraction kit;
(d) adding 5ml of mixed solution of solution B and solution C, blowing and beating uniformly by using a disposable plastic straw, incubating in a water bath or incubator at 60 ℃ for 10 minutes, and turning over and mixing uniformly once during incubation, wherein the solution is changed from red to yellow green or brown;
(e) adding isopropanol with the same volume, fully reversing and uniformly mixing until filamentous or flocculent DNA appears;
(f) transferring the floccule into a 1.5ml EP tube added with 800ul of 75% alcohol by using a pipette, inverting for a plurality of times, centrifuging for 1 minute at 12000prm, discarding the supernatant, and drying for 10-15 minutes at room temperature;
(g) adding 1000ul of eluent, incubating at 60 ℃ for 10-15 minutes or overnight to dissolve DNA, and blowing and beating uniformly by a pipette or a disposable plastic straw, and storing the DNA at-20 ℃.
A downstream gene detection method of HSPG for peripheral blood CTC of biliary tract cancer adopts a high-throughput sequencing method to carry out whole genome amplification of biliary tract cancer blood circulation tumor cell source nucleic acid, and comprises the following specific steps:
(1) thawing and lysing the frozen CTC PCR plates on ice, and adding 0.4M HCl to each well after lysing;
(2) performing whole genome amplification by adopting multiple replacement amplification, preparing a reaction mixture, mixing sterilized water, 10 multiplied reaction buffer solution in a copy genome amplification kit, BSA, DTT, dNTPs, random hexamer and the like for each reaction according to the quantity, and then incubating at 30 ℃ on a thermal cycler;
(3) paramagnetic beads in nucleic acid purification kit were used to terminate the subsequent MDA reaction, 100 μ L of paramagnetic beads in nucleic acid purification kit was added to each reaction sample and incubated at room temperature for 5 min;
(4) then placing the sample on a 96-hole magnetic separation frame for incubation for 5 min;
(5) removing the supernatant of each sample, adding 100 mu L of 70% ethanol into each well, removing the ethanol, repeating the operation once, and cleaning the paramagnetic beads;
(6) after complete removal of ethanol, drying at room temperature for 10min, then adding 60 μ L of Tris-EDTA buffer, setting at pH 8, resuspending the paramagnetic beads, incubating at room temperature for 5min, then returning to the magnetic separation rack for 5min, and transferring the purified product to a new PCR plate;
(7) quantitative comparison of MDA products was performed using dsDNA quantitative analysis kit, and samples with higher nucleic acid content than negative controls were selected for whole genome sequencing.
Preferably, the replication genome amplification kit may employ a RepliPHIkit (Epicentre) kit.
Preferably, the paramagnetic beads in the nucleic acid purification kit can adopt AmpureXPbeads (Beckman Coulter) kit.
Preferably, the dsDNA quantitative analysis kit can adopt a Quant-ITPicoGreendsDNAsalaykit (Invitrogen) kit.
Preferably, the downstream gene detection method can be used for constructing a library of the biliary tract cancer blood circulation tumor cell derived nucleic acid, and comprises the following specific steps:
s1: preparing a whole genome sequencing library using the NGS sample solution;
s2: quantifying the library using a library quantification kit;
s3: whole genome sequencing was performed using a sequencing platform.
Preferably, NexteraDNAamplePrepKit (Illumina) is used as the NGS sample solution.
Preferably, the library quantification kit can be a library quantification kit for illumina (Kapa Biosystems).
Preferably, the sequencing platform can adopt an IlluminaHiSeqXTen platform.
Preferably, the downstream gene detection method can be used for filtering sequencing data and evaluating quality of the nucleic acid derived from the biliary tract cancer blood circulation tumor cells, and comprises the following specific steps:
(a) data filtration mainly includes pairs of three cases of Reads, containing linker sequences of Reads; (ii) Reads in which the number of bases in the single-ended Read exceeds 10% of the total number of bases in the Read; the number of bases in the single-ended Read with a mass value below 5 exceeds 50% of the Reads length proportion of the strip;
(b) evaluating whether the library building sequencing meets the standard or not by counting the sequencing error rate, the data volume, the comparison rate and the like;
(c) according to the sequencing characteristics of a sequencing platform, the proportion of Q30 is required to be more than 80%, the average error rate is required to be less than 0.1%, and then subsequent analysis is carried out.
Preferably, the downstream gene detection method can be used for sequencing depth coverage statistics of the biliary tract cancer blood circulation tumor cell-derived nucleic acid, and comprises the following specific steps:
(1) comparing the effective sequencing data to a reference genome through BWA to obtain an initial comparison result in a BAM format;
(2) marking and repeating the BAM file to obtain a final comparison result in the BAM format;
(3) and (3) counting the coverage of the final comparison result in the step (2).
Preferably, the downstream gene detection method can be used for detecting the variation of the nucleic acid derived from the biliary tract cancer blood circulation tumor cells, and comprises the following specific steps:
s1: detecting (GATK) and annotating SNP variation information, and counting the distribution of SNP on each gene functional element;
s2: detecting (GATK) and annotating InDel variant information;
s3: counting the distribution of InDel on each gene functional element;
s4: detecting (CNVnator) and annotating CNV variation information, and counting the distribution of CN on each gene functional element;
s5: SV variation information was detected (Crest, Breakdancer) and annotated.
Preferably, the downstream gene detection method can identify key variation of the nucleic acid from the biliary tract cancer blood circulation tumor cells, and identify genomic variation related to each clinical characteristic by comparing and analyzing different variation in CTCs of biliary tract cancer patients with different tumor grading stages, chemotherapy sensitivity, drug resistance and radiotherapy sensitivity.
Has the advantages that: the invention relates to a method for separating and identifying peripheral blood CTC of biliary tract cancer and detecting downstream genes, which comprises the steps of selecting a specific recognition factor HSPG of the biliary tract cancer, carrying out specific separation and identification on circulating tumor cells in peripheral blood of a patient with biliary tract cancer, extracting DNA through the obtained CTC, and completing high-throughput sequencing analysis of CTC-DNA. The method comprises the steps of selecting and identifying CTC in peripheral blood through HSPG immune lipid magnetic balls, improving the selection efficiency, adopting a high-throughput sequencing method to perform the process methods of whole genome amplification, library construction, sequencing data filtration and quality evaluation, sequencing depth coverage statistics, variation detection, key variation identification and the like of the biliary tract cancer blood circulation tumor cell source nucleic acid, establishing a whole set of high-throughput sequencing analysis system, obtaining tumor specific molecular information for improving the specificity, capturing tumor cells in blood through specific separation antibodies, comprehensively analyzing all results by combining tumor parts, stages and transfer type clinical data, performing pairing analysis on CTC-NGS and tissue NGS of biliary tract cancer, and achieving the purpose of analyzing and detecting by using the gene expression level change of the CTC source nucleic acid and the gene expression level change of the tissue source nucleic acid.
Drawings
FIG. 1 is a schematic representation of heparan sulfate proteoglycan structure (HSPG) of the present invention;
FIG. 2 is a flow chart of the preparation of HSPG immune lipid magnetic beads of the invention;
FIG. 3 is an immunofluorescence plot of peripheral blood CTCs isolated via HSPG immunoliposome magnetic beads according to the present invention;
FIG. 4 is a flow chart of the present invention for identification of CTC by HSPG immune lipid magnetic bead sorting;
FIG. 5 is an analysis flowchart of the downstream gene detection method (high throughput sequencing method) of the present invention.
Detailed Description
The specific sorting, identifying and detecting method of CTC in serum of biliary tract tumor of the present invention is further described in detail below.
Example 1
A method for separating and identifying peripheral blood CTC of biliary tract cancer by HSPG comprises the following steps:
(1) sampling: taking 7.5mL of peripheral blood from a biliary tract cancer patient, placing the peripheral blood in an anticoagulation tube, and uniformly mixing a whole blood sample;
(2) removing plasma protein and plasma (serum) nucleic acid from the blood sample collected in the step (1), adding a buffer solution into the whole blood, removing supernatant through layered centrifugation, and then, shaking a centrifugal tube to uniformly mix and precipitate cells;
(3) removing red blood cells: after mixing, adding lysis solution into a centrifuge tube, placing the centrifuge tube into a vertical mixing instrument for mixing, centrifuging to remove supernatant, slightly shaking the centrifuge tube for mixing, precipitating cells, and adding buffer solution;
(4) layering and centrifuging: adding a layering liquid into a new centrifugal tube, superposing all the liquids in the step (3) to the top layer of the layering liquid, then cleaning the tube wall of the centrifugal tube by using a buffer solution, and simultaneously transferring the cleaning liquid to the top layer of the buffer solution for centrifugation;
(5) and (3) removing white blood cells: after centrifugation, 3 layers of solution can be seen, the solution on the top 2 layers is gently absorbed into a new centrifugal tube, buffer solution is respectively added, after inversion and uniform mixing, centrifugation is carried out, supernatant fluid is discarded, buffer solution is added, and cells are uniformly mixed and precipitated;
(6) washing the magnetic particles: sucking a proper amount of immune lipid magnetic ball (combined with HSPG antibody) suspension into an EP tube, standing, sucking and discarding the solution, adding a buffer solution, mixing uniformly, standing, discarding the supernatant, repeating for 3 times, and then resuspending the magnetic particles to the original volume by using the buffer solution;
(7) antibody incubation: slowly adding a certain amount of immune lipid magnetic beads (combined with HSPG antibodies) into the sample treated in the step (6), adjusting the shaking speed of a shaking table, obliquely fixing a centrifugal tube on the shaking table, and shaking at room temperature;
(8) removing free magnetic particles: transferring the liquid of the centrifuge tube in the step (7) into a new centrifuge tube, standing the centrifuge tube on a magnetic frame, transferring the liquid into the new centrifuge tube respectively, arranging the centrifuge tube at the upper end of the centrifuge tube, centrifuging the liquid, discarding supernatant, and coating the supernatant on glass slides respectively;
(9) air-drying the glass slide at room temperature, dropwise adding 4% paraformaldehyde to fix cell surface antigens, and dropwise adding 1% fetal calf serum to seal cell surface nonspecific sites;
(10) adding an anti-working solution to the slide glass obtained after the blood sample is enriched in the step (9), incubating in a dark place, and fully washing;
(11) adding a second antibody working solution into the sample obtained in the step (10), incubating in a dark place, fully washing, adding a DAPI working solution for dyeing, and slightly washing;
(12) dripping glycerol on the sample in the step (11), and reading the cover plate; and performing CTC source circulating tumor cell-based DNA extraction on the sample by adopting a DNA extraction kit.
Preferably, the DNA extraction kit can adopt QIAGEN kit.
Wherein, the processes of antibody incubation and free magnetic particle elimination in the steps (7) and (8) for capturing the circulating tumor cells of the peripheral blood are as follows:
s1: processing the received blood sample: adding 4mL of blood into a 5mL centrifuge tube, adding 40 μ l of HSPG immune lipid magnetic beads, reversing and mixing uniformly, and incubating at room temperature for 20 min;
s2: and (3) standing the sample on a magnetic frame, adsorbing the immune lipid magnetic ball for capturing the CTC on one side of the magnetic frame, inverting the magnetic frame and the sample, and washing the sample on the centrifugal tube cover. Standing for 10 minutes, and sucking and removing the clear liquid;
s3: adding 1mLddH2O to wash the immune lipid magnetic spheres;
s4: adding 100ul CF1 for smear, and oven drying at 37 deg.C;
s5: dropping 4% paraformaldehyde, fixing for 8min, and preheating in a water bath at 37 deg.C containing 2 XSSC for 10 min;
s6: respectively dehydrating with 75%, 85% and anhydrous ethanol for 2min, and air drying at room temperature;
s7: adding 10ul of fluorescent probe, sealing, hybridizing at 76 ℃ for 10min in a hybridization instrument, and hybridizing at 37 ℃ for 1.5 h;
s8: tearing off the mounting glue by using a forceps, putting the mounting glue into formamide (43 ℃), peeling off a cover glass, soaking and washing for 10min, then soaking and washing for 15min by adopting 2 XSSC, and shaking once every 5 min;
s9: coating 100ul of CK fluorescent antibody staining solution on the sample area, and incubating for 1h under the condition of a wet box at 37 ℃ and keeping out of the sun;
s10: CK fluorescent antibody staining solution was washed away, washed twice with 0.2% BSA, and 10ul of DAPI coverslip was added.
Wherein, the process of extracting the DNA of the circulating tumor cells by adopting the QIAGEN kit in the step (13) specifically comprises the following steps:
(a) adding 2.5 times volume of Solutiona into 10ml of blood, reversing, mixing uniformly, centrifuging at 8000rpm for 5min, and discarding supernatant;
(b) repeating step a for 1-2 times, and discarding the supernatant;
(c) preparing a mixed solution of solution B and solution C according to information provided by an attached table of a QIAGEN kit;
(d) adding 5ml of mixed solution of solution B and solution C, blowing and beating uniformly by using a disposable plastic straw, incubating in a water bath or incubator at 60 ℃ for 10 minutes, and turning over and mixing uniformly once during incubation, wherein the solution is changed from red to yellow green or brown;
(e) adding isopropanol with the same volume, fully reversing and uniformly mixing until filamentous or flocculent DNA appears;
(f) transferring the floccule to a 1.5ml EP tube added with 800ul of 75% alcohol by using a pipette, inverting for a plurality of times, centrifuging for 1 minute at 12000prm, discarding the supernatant, and drying for 10-15 minutes at room temperature;
(g) adding 1000ul of Elutionbuffer, incubating at 60 ℃ for 10-15 minutes or overnight to dissolve DNA, blowing and beating uniformly by using a pipette or a disposable plastic pipette, and storing the DNA at-20 ℃.
The preparation process of the HSPG immune lipid magnetic bead comprises the following steps of: (i) iron oxide (Fe3O4) nanoparticles, (ii) cholestrol, (iii) GHDC, (iv) DOPC, (v) HSPG. FA acts as a biological ligand to specifically capture subsets of cancer cells that overexpress the FA Receptor (FR) on their membrane. Superparamagnetic Fe3O4 nanoparticles allow magnetic separation after capture of HSPG cells, and isolated CTCs can be identified with immunofluorescence. The GHDC can react with the protein antibody, and the HQCMC can increase the grafting amount of the magnetic microsphere and the antibody through the active group contained in the HQCMC, and has the functions of emulsification and dispersion and a surfactant.
Separating and identifying the peripheral blood CTC of the biliary tract cancer by using HSPG immune lipid magnetic beads.
Firstly, collecting 7.5mL anticoagulated blood of a tumor patient, centrifuging at 1000rpm/min for 10min, carefully taking the supernatant, placing the supernatant in an EP tube, adding PBS (phosphate buffer solution) with the same amount as the supernatant into the EP tube, fully mixing the supernatant and the supernatant, adding 10 mu L HSPG immune lipid magnetic spheres, incubating the mixture at room temperature for 30min, and mixing the mixture once every 10 min; inserting the EP tube into a magnetic separation frame for adsorption for 5min, and removing the supernatant to obtain the CTC-magnetic ball precipitate.
Immunofluorescence identification of CTC captured by immune lipid magnetic spheres.
Firstly, adding 10 mu L of 4% paraformaldehyde to fix cells for 10 min; PBS washing 3 times; adding 30 mu L, CK19 DAPI staining solution, 10 mu L, CD45 FITC staining solution and 10 mu L PE staining solution, mixing uniformly and staining for 15min in dark; PBS washing 3 times; adding 10 mu L of deionized water into an EP tube for resuspension, uniformly coating the mixture on an anti-falling glass slide, and observing and counting the number under a fluorescence microscope after the liquid drops are dried.
And (3) extracting the DNA of the circulating tumor cells of the CTC nucleic acid by adopting a DNA extraction kit.
Preparing a reagent required for checking and extracting according to a DNA extraction kit, completing the extraction of nucleic acid from peripheral hematoma cells, drying for 10-15 minutes at room temperature, adding 1000ul of buffer solution (Elutionbuffer), incubating for 10-15 minutes at 60 ℃ or dissolving DNA overnight, uniformly blowing by using a pipette or a disposable plastic straw, measuring the concentration of the nucleic acid and recording, and storing the DNA at-20 ℃ for subsequent sequencing detection.
Example 2
A downstream gene detection method of HSPG for peripheral blood CTC of biliary tract cancer adopts a high-throughput sequencing method to carry out whole genome amplification of biliary tract cancer blood circulation tumor cell source nucleic acid, and comprises the following specific steps:
(1) thawing and lysing the frozen CTC PCR plates on ice, and adding 0.4M HCl to each well after lysing;
(2) performing whole genome amplification by adopting multiple replacement amplification, preparing a reaction mixture, mixing sterilized water, 10 multiplied reaction buffer solution in a copy genome amplification kit, BSA, DTT, dNTPs, random hexamer and the like for each reaction according to the quantity, and then incubating at 30 ℃ on a thermal cycler;
(3) paramagnetic beads in nucleic acid purification kit were used to terminate the subsequent MDA reaction, 100 μ L of paramagnetic beads in nucleic acid purification kit was added to each reaction sample and incubated at room temperature for 5 min;
(4) then placing the sample on a 96-hole magnetic separation frame for incubation for 5 min;
(5) removing the supernatant of each sample, adding 100 mu L of 70% ethanol into each well, removing the ethanol, repeating the operation once, and cleaning the paramagnetic beads;
(6) after complete removal of ethanol, drying at room temperature for 10min, then adding 60 μ L of Tris-EDTA buffer, setting at pH 8, resuspending the paramagnetic beads, incubating at room temperature for 5min, then returning to the magnetic separation rack for 5min, and transferring the purified product to a new PCR plate;
(7) quantitative comparison of MDA products was performed using dsDNA quantitative analysis kit, and samples with higher nucleic acid content than negative controls were selected for whole genome sequencing.
Wherein, the replication genome amplification kit can adopt a RepliPHIkit (Epicentre) kit.
The paramagnetic beads in the nucleic acid purification kit can adopt AmpureXPbeads (BeckmanCoulter) kit.
Wherein, the dsDNA quantitative analysis kit can adopt a Quant-ITPicoGreendsDNAsalaykit (Invitrogen) kit.
The downstream gene detection method can be used for constructing a library of the biliary tract cancer blood circulation tumor cell-derived nucleic acid, and comprises the following specific steps:
s1: preparing a whole genome sequencing library using nexteradnasampleprephit (Illumina);
s2: quantification of the library using library quantification kit for illumina (KapaBiosystems);
s3: the IlluminaHiSeqXTen platform performs whole genome sequencing.
The downstream gene detection method can be used for filtering sequencing data and evaluating quality of the biliary tract cancer blood circulation tumor cell-derived nucleic acid, and comprises the following specific steps:
(a) data filtration mainly includes pairs of three cases of Reads, containing linker sequences of Reads; (ii) Reads in which the number of bases in the single-ended Read exceeds 10% of the total number of bases in the Read; the number of bases in the single-ended Read with a mass value below 5 exceeds 50% of the Reads length proportion of the strip;
(b) evaluating whether the library building sequencing meets the standard or not by counting the sequencing error rate, the data volume, the comparison rate and the like;
(c) according to the sequencing characteristics of a sequencing platform, the proportion of Q30 is required to be more than 80%, the average error rate is required to be less than 0.1%, and then subsequent analysis is carried out.
The downstream gene detection method can be used for sequencing depth coverage statistics of the biliary tract cancer blood circulation tumor cell source nucleic acid, and comprises the following specific steps:
(1) comparing the effective sequencing data to a reference genome through BWA to obtain an initial comparison result in a BAM format;
(2) marking and repeating the BAM file to obtain a final comparison result in the BAM format;
(3) and (3) counting the coverage of the final comparison result in the step (2).
The downstream gene detection method can be used for detecting the variation of the source nucleic acid of the biliary tract cancer blood circulation tumor cells, and comprises the following specific steps:
s1: detecting (GATK) and annotating SNP variation information, and counting the distribution of SNP on each gene functional element;
s2: detecting (GATK) and annotating InDel variant information;
s3: counting the distribution of InDel on each gene functional element;
s4: detecting (CNVnator) and annotating CNV variation information, and counting the distribution of CN on each gene functional element;
s5: SV variation information was detected (Crest, Breakdancer) and annotated.
The downstream gene detection method can identify key variation of the biliary tract cancer blood circulation tumor cell source nucleic acid, and identify genome variation related to each clinical characteristic through comparative analysis of different tumor grading stages, chemotherapy sensitivity, drug resistance and difference variation in CTC of biliary tract cancer patients with radiotherapy sensitivity.
The downstream gene detection method of the HSPG on the peripheral blood CTC of the biliary tract cancer adopts a high-throughput sequencing method to amplify the whole genome of the nucleic acid from the biliary tract cancer blood circulation tumor cells.
PCR plates of frozen CTCs were lysed and whole genome amplification was performed and incubated on a thermal cycler (Eppendorf) at 30 ℃. As in the same manner as in example 2, the MDA product was quantified using Quant-ITPicoGreendsDNAsalaykit (Invitrogen), and samples with higher nucleic acid content than the negative control were selected for Whole Genome Sequencing (WGS).
The downstream gene detection method can be used for constructing a library of the biliary tract cancer blood circulation tumor cell-derived nucleic acid. First, a whole genome sequencing library was prepared using nexteradnasampleprephit (Illumina); the Library was then quantified using Library quantification kit for illumina (KapaBiosystems); and then carrying out whole genome sequencing on the IlluminaHiSeqXTen platform.
The downstream gene detection method can be used for sequencing data filtration and quality evaluation of the CTC source nucleic acid of the biliary tract cancer blood circulation tumor cells. Data filtration mainly includes pairs of three cases of Reads, containing Adapter sequence (Adapter); reads in which the number of bases of N (N indicates that base information cannot be determined) in the single-ended Read exceeds 10% of the total number of bases in the single-ended Read; and (3) evaluating whether the database construction sequencing meets the standard or not by counting the sequencing error rate, data quantity, comparison rate and the like.
The downstream gene detection method can be used for sequencing depth coverage statistics of the CTC-derived nucleic acid of the biliary tract cancer blood circulation tumor cells. Valid sequencing data were aligned to the reference genome by BWA, resulting in initial alignment in BAM format. And carrying out marking repetition and other processing on the BAM file so as to obtain a final comparison result in a BAM format, and carrying out coverage statistics by using effective data compared to a reference genome.
The downstream gene detection method can be used for detecting the variation of the CTC-derived nucleic acid of the blood circulating tumor cells of the biliary tract cancer. S1: detecting (GATK) and annotating SNP variation information, and counting the distribution of SNP on each gene functional element; detecting (GATK) and annotating InDel variant information; counting the distribution of InDel on each gene functional element; detecting (CNVnator) and annotating CNV variation information, and counting the distribution of CN on each gene functional element; SV variation information was detected (Crest, Breakdancer) and annotated.
The downstream gene detection method can be used for carrying out key variation identification on the CTC source nucleic acid of the biliary tract cancer blood circulation tumor cells. And (3) comparing and analyzing the differential variation in CTC of different tumor grading stages, chemotherapy sensitivity, drug resistance and radiotherapy sensitivity biliary tract cancer patients, and identifying the genomic variation related to each clinical characteristic.
Example 3
(1) Determining the sample size:
(a) 100 biliary tract cancer patients are organized in the early stage, a detection system and indexes are evaluated, and the subsequent sample amount is measured and calculated.
(b) According to the results of the previous research, the statistics of the number of CTC identified by sorting the specific HSPG antibody of the biliary tract cancer is preliminarily calculated by a statistician, the sorting efficiency and the difference among single individuals are compared, the sensitivity and the specificity of a marker are counted, the total sample size required is further calculated, and about 500 patients are expected to be grouped.
(c) From the results of the previous studies, the number and frequency of mutations in the gene derived from the CTC nucleic acid were further counted to calculate the total sample size required, which was expected to be included in approximately 500 patients.
(2) And (3) inclusion standard:
the study object is a biliary tract cancer patient diagnosed by imaging of a hepatobiliary tumor cooperation group in a certain surgical hospital. The following is the standard of receiving peripheral blood liquid biopsy, and the detection results of patients in the group are all subjected to diagnosis stage and metastasis type correlation analysis. The patients in the group are in accordance with the individual treatment subgroup conditions discussed in the MDT cooperative group, and then enter each treatment subgroup for detection after treatment and correlation analysis of treatment effect and prognosis.
Grouping standard:
a. the biliary tract cancer patient identified after multidisciplinary discussion has no history of relevant anti-tumor treatments such as operation, radiotherapy or chemotherapy;
b. the age is more than or equal to 18 years old;
c. measurable or evaluable tumor lesions (according to RECIST1.1 criteria) by CT or MRI examination;
kps score greater than 60;
e. good blood function: the hemoglobin is more than or equal to 8g/dL, the absolute count of the neutrophils is more than or equal to 1.5 multiplied by 109/L, and the platelet count is more than or equal to 100 multiplied by 109/L;
f. good liver and kidney function storage: liver function Child-PughA grade, creatinine clearance >60 ml/min; creatinine <120 μmol/L;
g. no heart failure and uncontrollable chest pain; myocardial infarction and cerebral infarction did not occur within 12 months before the start of the study;
h. signing the informed consent.
(3) Exclusion criteria:
a. no evidence of pathology of biliary tract cancer;
b. patients who have previously suffered from other malignancies;
c. active periods of infection or other serious infections that may interfere with the test results of the subject;
d. patients with severe anemia or other organ dysfunction;
e. pregnant or lactating women;
f. an individual who is receiving corrective monitoring or monitoring;
g. other conditions may interfere with the detection of the item.
After the patient or the client signed the informed consent, 7.5ml of peripheral blood (with the proviso that 7.5ml of each of hydrothorax, ascites and bile is retained) is retained before the start of treatment, and CTC counting, sorting and typing and immunofluorescence imaging are carried out.
(4) The research process comprises the following steps:
1) registering the selected biliary tract cancer patient into a group;
2) sorting identification and gene mutation detection of CTC are carried out by sorting factors specific to HSPG;
3) obtaining the clinical pathological result of a patient, receiving the information of operation diagnosis and treatment and the like;
4) and (3) receiving the sorting identification and gene mutation detection of the CTC of the postoperative patient for 1 month reexamination.
(5) The downstream gene detection method and the process of the peripheral blood CTC of the biliary tract cancer are as follows:
1) enrichment of CTC in peripheral blood of biliary tract cancer
CTC detection is used as simple blood detection, can capture and evaluate circulating tumor cells at any time to determine the prognosis of a patient, is easy to obtain a peripheral blood sample, has small wound, can be repeatedly collected, is an ideal sample source for clinical routine detection, and realizes the primary condition for detection development by separation and enrichment of CTC. At present, the separation of CTC mainly comprises ultracentrifugation, magnetic bead immunocapture, precipitation or filtration and other methods. An antigen-antibody combined positive capture method is adopted, separation and enrichment of CTC are carried out through immunomagnetic beads, and HSPG antibodies are combined with immunomagnetic beads to capture HSPG positive CTC.
2) Microscopic visualization of CTC
The CD45 antigen is a leukocyte specific antigen, and leukocytes are marked by fluorescent anti-CD 45 antibodies; cytokeratin8/Cytokeratin18/Cytokeratin19 are surface antigens unique to CTC cells, and the CTC cells are labeled with fluorescent anti-Cytokeratin antibodies; DAPI is a DNA dye used to stain the nucleus of the cell; and (3) observing whether the HSPG antigen is expressed and the expression quantity in the CTC cells by using an antibody aiming at the target HSPG protein, and observing and photographing under a fluorescence microscope and counting the quantity.
3) Nucleic acid extraction of CTC
DNA extraction based on circulating tumor cells was performed using QIAGEN kit, and the concentration and purity of the DNA were detected and recorded to obtain DNA suitable for use in downstream gene detection methods (high throughput sequencing).
(6) Downstream gene detection method (high throughput sequencing) analysis of peripheral blood CTC-DNA
The analysis of the genetic information of the CTC of the biliary tract cancer focuses on the large data analysis of the molecular and genetic detection layer of the circulating single cells of the biliary tract cancer identified by investigation and sorting, and comprises single cell genetic mutation detection, single cell transcriptome sequencing, single cell whole genome sequencing and the like, and all results are combined with clinical data of tumor parts, stages and transfer types to be comprehensively analyzed, and the CTC-NGS and the tissue NGS of the biliary tract cancer are subjected to pairing analysis to find factors influencing consistency.
The overall expression of HSPGs is currently rarely studied in biliary tract cancer, and results have been obtained experimentally including: 527 samples of gallbladder cancer tissues show that the total HSPG is mainly expressed on the surfaces of gallbladder cancer cells, and the positive rate of the total HSPG expression is 77.0% (406/527).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (9)
1. A method for separating and identifying peripheral blood CTC of biliary tract cancer by HSPG is characterized in that: the method comprises the following steps:
(1) sampling: taking 7.5mL of peripheral blood from a biliary tract cancer patient, placing the peripheral blood in an anticoagulation tube, and uniformly mixing a whole blood sample;
(2) removing plasma protein and plasma nucleic acid from the blood sample collected in the step (1), adding a buffer solution into the whole blood, removing supernatant through layered centrifugation, and then, shaking the centrifugal tube gently to mix uniformly to precipitate cells;
(3) removing red blood cells: after mixing, adding lysis solution into a centrifuge tube, placing the centrifuge tube into a vertical mixing instrument for mixing, centrifuging to remove supernatant, slightly shaking the centrifuge tube for mixing, precipitating cells, and adding buffer solution;
(4) layering and centrifuging: adding a layering liquid into a new centrifugal tube, superposing all the liquids in the step (3) to the top layer of the layering liquid, then cleaning the tube wall of the centrifugal tube by using a buffer solution, and simultaneously transferring the cleaning liquid to the top layer of the buffer solution for centrifugation;
(5) and (3) removing white blood cells: after centrifugation, 3 layers of solution can be seen, the solution on the top 2 layers is gently absorbed into a new centrifugal tube, buffer solution is respectively added, after inversion and uniform mixing, centrifugation is carried out, supernatant fluid is discarded, buffer solution is added, and cells are uniformly mixed and precipitated;
(6) washing the magnetic particles: sucking a proper amount of immune lipid magnetic sphere suspension into an EP (EP) tube, standing, sucking and discarding the solution, adding a buffer solution, uniformly mixing, standing, discarding a supernatant, repeating for 3 times, and then resuspending the magnetic particles to the original volume by using the buffer solution;
(7) antibody incubation: slowly adding a certain amount of immune lipid magnetic spheres into the sample treated in the step (6), adjusting the shaking speed of a shaking table, obliquely fixing a centrifugal tube on the shaking table, and shaking at room temperature;
(8) removing free magnetic particles: transferring the liquid of the centrifuge tube in the step (7) into a new centrifuge tube, standing the centrifuge tube on a magnetic frame, transferring the liquid into the new centrifuge tube respectively, arranging the centrifuge tube at the upper end of the centrifuge tube, centrifuging the liquid, discarding supernatant, and coating the supernatant on glass slides respectively;
(9) air-drying the glass slide at room temperature, dropwise adding 4% paraformaldehyde to fix cell surface antigens, and dropwise adding 1% fetal calf serum to seal cell surface nonspecific sites;
(10) adding an anti-working solution to the slide glass obtained after the blood sample is enriched in the step (9), incubating in a dark place, and fully washing;
(11) adding a second antibody working solution into the sample obtained in the step (10), incubating in a dark place, fully washing, adding a DAPI working solution for dyeing, and slightly washing;
(12) dripping glycerol on the sample in the step (11), and reading the cover plate; and performing CTC source circulating tumor cell-based DNA extraction on the sample by adopting a DNA extraction kit.
2. The method of claim 1 for the isolated identification of peripheral blood CTCs of biliary tract cancer by HSPGs, wherein: the process of capturing the circulating tumor cells of the peripheral blood by antibody incubation and free magnetic particle removal in the steps (7) and (8) is specifically as follows:
s1: processing the received blood sample: adding 4mL of blood into a 5mL centrifuge tube, adding 40 μ l of HSPG immune lipid magnetic beads, reversing and mixing uniformly, and incubating at room temperature for 20 min;
s2: and (3) standing the sample on a magnetic frame, adsorbing the immune lipid magnetic ball for capturing the CTC on one side of the magnetic frame, inverting the magnetic frame and the sample, and washing the sample on the centrifugal tube cover. Standing for 10 minutes, and sucking and removing the clear liquid;
s3: adding 1mL of ddH2O to wash the immune lipid magnetic spheres;
s4: adding 100ul CF1 for smear, and oven drying at 37 deg.C;
s5: dropping 4% paraformaldehyde, fixing for 8min, and preheating in a water bath at 37 deg.C containing 2 XSSC for 10 min;
s6: respectively selecting 75%, 85% and anhydrous ethanol for dehydration, and respectively performing dehydration for 2 min. And air-dried at room temperature;
s7: adding 10ul of fluorescent probe, sealing, hybridizing at 76 ℃ for 10min in a hybridization instrument, and hybridizing at 37 ℃ for 1.5 h;
s8: tearing off the mounting glue by using a forceps, putting the mounting glue into formamide (43 ℃), peeling off a cover glass, soaking and washing for 10min, then soaking and washing for 15min by adopting 2 XSSC, and shaking once every 5 min;
s9: coating 100ul of CK fluorescent antibody staining solution on the sample area, and incubating for 1h under the condition of a wet box at 37 ℃ and keeping out of the sun;
s10: CK fluorescent antibody staining solution was washed away, washed twice with 0.2% BSA, and 10ul of DAPI coverslip was added.
3. The method of claim 1 for the isolated identification of peripheral blood CTCs of biliary tract cancer by HSPGs, wherein: the process of extracting the DNA of the circulating tumor cells by adopting the DNA extraction kit in the step (13) is specifically as follows:
(a) adding 2.5 times volume of Solutiona into 10ml of blood, reversing, mixing uniformly, centrifuging at 8000rpm for 5min, and discarding supernatant;
(b) repeating step a for 1-2 times, and discarding the supernatant;
(c) preparing a mixed Solution of Solution B and Solution C according to information provided by an attached table of the DNA extraction kit;
(d) adding 5ml of the mixed Solution of Solution B and Solution C, blowing and beating uniformly by using a disposable plastic straw, incubating in a water bath or incubator at 60 ℃ for 10 minutes, and turning over and mixing uniformly once during incubation, wherein the Solution is changed from red to yellow green or brown;
(e) adding isopropanol with the same volume, fully reversing and uniformly mixing until filamentous or flocculent DNA appears;
(f) transferring the floccule to a 1.5ml EP tube added with 800ul of 75% alcohol by using a pipette, inverting for a plurality of times, centrifuging for 1 minute at 12000prm, discarding the supernatant, and drying for 10-15 minutes at room temperature;
(g) adding 1000ul of eluent, incubating at 60 ℃ for 10-15 minutes or overnight to dissolve DNA, and blowing and beating uniformly by a pipette or a disposable plastic straw, and storing DNA at-20 ℃.
4. A downstream gene detection method of peripheral blood CTC of biliary tract cancer by HSPG is characterized in that: the method adopts a high-throughput sequencing method to amplify the whole genome of the source nucleic acid of the biliary tract cancer blood circulation tumor cells, and comprises the following specific steps:
(1) thawing and lysing the frozen CTC PCR plates on ice, and adding 0.4M HCl to each well after lysing;
(2) performing whole genome amplification by adopting multiple replacement amplification, preparing a reaction mixture, mixing sterilized water, 10 multiplied reaction buffer solution in a copy genome amplification kit, BSA, DTT, dNTPs, random hexamer and the like for each reaction according to the quantity, and then incubating at 30 ℃ on a thermal cycler;
(3) paramagnetic beads in nucleic acid purification kit were used to terminate the subsequent MDA reaction, 100 μ L of paramagnetic beads in nucleic acid purification kit was added to each reaction sample and incubated at room temperature for 5 min;
(4) then placing the sample on a 96-hole magnetic separation frame for incubation for 5 min;
(5) removing the supernatant of each sample, adding 100 mu L of 70% ethanol into each well, removing the ethanol, repeating the operation once, and cleaning the paramagnetic beads;
(6) after complete removal of ethanol, drying at room temperature for 10min, then adding 60 μ L of Tris-EDTA buffer, setting at pH 8, resuspending the paramagnetic beads, incubating at room temperature for 5min, then returning to the magnetic separation rack for 5min, and transferring the purified product to a new PCR plate;
(7) quantitative comparison of MDA products was performed using dsDNA quantitative analysis kit, and samples with higher nucleic acid content than negative controls were selected for whole genome sequencing.
5. The method of claim 4, wherein the downstream gene detection of peripheral blood CTCs of biliary tract cancer by HSPG is characterized by: the downstream gene detection method can be used for constructing a library of the biliary tract cancer blood circulation tumor cell source nucleic acid, and comprises the following specific steps:
s1: preparing a whole genome sequencing library using the NGS sample solution;
s2: quantifying the library using a library quantification kit;
s3: whole genome sequencing was performed using a sequencing platform.
6. The method of claim 4, wherein the downstream gene detection of peripheral blood CTCs of biliary tract cancer by HSPG is characterized by: the downstream gene detection method can be used for filtering sequencing data and evaluating quality of the biliary tract cancer blood circulation tumor cell-derived nucleic acid, and comprises the following specific steps:
(a) data filtration mainly includes pairs of three cases of Reads, containing linker sequences of Reads; (ii) Reads in which the number of bases in the single-ended Read exceeds 10% of the total number of bases in the Read; the number of bases in the single-ended Read with a mass value below 5 exceeds 50% of the Reads length proportion of the strip;
(b) evaluating whether the library building sequencing meets the standard or not by counting the sequencing error rate, the data volume, the comparison rate and the like;
(c) according to the sequencing characteristics of a sequencing platform, the proportion of Q30 is required to be more than 80%, the average error rate is required to be less than 0.1%, and then subsequent analysis is carried out.
7. A downstream of a peripheral blood CTC of a biliary tract cancer treated with HSPG according to claim 4 wherein: the downstream gene detection method can be used for sequencing depth coverage statistics of the biliary tract cancer blood circulation tumor cell source nucleic acid, and comprises the following specific steps:
(1) comparing the effective sequencing data to a reference genome through BWA to obtain an initial comparison result in a BAM format;
(2) marking and repeating the BAM file to obtain a final comparison result in the BAM format;
(3) and (3) counting the coverage of the final comparison result in the step (2).
8. The method of claim 4, wherein the downstream gene detection of peripheral blood CTCs of biliary tract cancer by HSPG is characterized by: the downstream gene detection method can be used for detecting the variation of the source nucleic acid of the biliary tract cancer blood circulation tumor cells, and comprises the following specific steps:
s1: carrying out variation information detection and annotation on the SNP, and carrying out distribution statistics on the SNP on each gene functional element;
s2: detecting and annotating InDel variation information;
s3: counting the distribution of InDel on each gene functional element;
s4: detecting and annotating CNV variation information, and counting the distribution of CN on each gene functional element;
s5: SV variation information is detected and annotated.
9. The method of claim 4, wherein the downstream gene detection of peripheral blood CTCs of biliary tract cancer by HSPG is characterized by: the downstream gene detection method can be used for identifying key variation of the nucleic acid from the biliary tract cancer blood circulation tumor cells, and identifying genome variation related to each clinical characteristic by comparing and analyzing different variation among CTCs of biliary tract cancer patients with different tumor grading stages, chemotherapy sensitivity, drug resistance and radiotherapy sensitivity.
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