Primers and kit for detecting HPV integration sites

Abstract

This invention discloses a kit for detecting HPV integration sites, belonging to the field of genetic engineering. Using this invention, targeted sequencing of partially unknown sequences can be performed at very low sequencing throughput (approximately 400K-600K) to obtain sufficient data (approximately 50X coverage depth) to yield reliable results. It can detect HPV integration in samples including, but not limited to, cervical cancer samples. Another objective of this invention is to verify the integration status of primer-corresponding regions using methods such as hybridization, quantitative fluorescence sequencing, or first-generation sequencing, employing single or partial primers provided by this invention. The presence of HPV integration strongly suggests that the patient's condition is CIN and cervical cancer. This invention provides more data support for the clinical research and diagnosis of cervical cancer from the perspective of HPV integration.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, and relates to medicine and biotechnology. Specifically, it relates to a primer and kit for detecting HPV integration sites. Background Technology

[0002] Cervical cancer is one of the most common gynecological malignancies. High-risk HPV infection is the most important risk factor for cervical cancer.

[0003] Studies have shown that HPV integration into the human genome (hereinafter referred to as HPV integration) is positively correlated with the course of cervical cancer. The incidence of HPV integration in cervical cancer patients is significantly higher than that in CIN patients, while HPV integration is rarely found in cervicitis patients.

[0004] High-throughput sequencing technology, also known as "next-generation" sequencing technology (NGS), is characterized by its ability to sequence hundreds of thousands to millions of DNA molecules simultaneously and its generally shorter read lengths, propelling scientific research forward. With its rapid development, the scientific community has also begun to increasingly apply first-generation sequencing technology to solve biological problems. Currently, HPV integration detection primarily uses whole-genome sequencing based on high-throughput sequencing, but this method is very expensive, and the sequencing data is mostly from the human genome. Furthermore, targeted sequencing based on target regions must target known sequences. This invention can perform high-throughput sequencing on sequences with only partially known information, targeting and capturing the target region for subsequent detection; we call this method semi-conservative target region sequencing (STRS). Summary of the Invention

[0005] The purpose of this invention is to provide primers and a kit for HPV integration detection that are highly specific, sensitive, and have a wide range of gene detection coverage. The technical solution to achieve the above objectives is as follows.

[0006] A primer for detecting HPV integration, the nucleotide sequence of which is shown in the sequence listing (see sequence listing); the human HPV integration detection method is high-throughput sequencing;

[0007] Another objective of this invention is to provide an HPV integration detection kit, comprising: 1. Enzyme digestion and end repair reagents: Fragmentation Buffer, FERA Solution, Fragmentation Enzyme Mix; 2. Ligation reaction reagents: Ligation Buffer, LT-BC# adapter, DNA Ligase, Ligation solution; 3. Target region amplification reagents: TEPCR buffer, LT-Forward Primer, HotStarTaq DNA Polymerase; 4. Universal primer amplification reagents: UPCR buffer, LT-Universal Primer, LT-P1 Primer, HotStarTaq DNA Polymerase. Primer sequences are shown in the sequence listing. sequence list

[0008] The beneficial effects of this invention are:

[0009] The primers and kit for detecting HPV integration of this invention can be used to provide a comprehensive assessment of HPV integration status in patients. This invention features low throughput and high accuracy, making it suitable for research and clinical applications in breast cancer. Attached Figure Description

[0010] Figure 1 The core principle diagram of the HPV integration site in this invention.

[0011] Figure 2 Examples of using this invention to detect HPV integration sites. Specific implementation methods

[0012] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0013] The HPV integrated detection method includes the following steps:

[0014] Genomic DNA was extracted from the sample;

[0015] Primers for amplifying HPV integration fragments (see sequence listing) and library construction reagents are provided. The human genomic DNA extracted in the previous step is used as a template for library construction.

[0016] The library constructed in the above steps is subjected to high-throughput sequencing to obtain sequencing data;

[0017] The high-throughput sequencing data obtained in the previous step were analyzed using Biomedical Genomics Workbench 5 software to obtain information on HPV integration fragments in the samples.

[0018] The extraction of genomes involved in the above methods can be performed using conventional operating methods in this field.

[0019] This invention provides a method for detecting HPV integration sites. Because high-throughput sequencing methods provide more comprehensive detection results, the accuracy and precision of the study are enhanced.

[0020] In the implementation of this invention, the design principle comprehensively considers the impact of primer pairs on the sensitivity, specificity, and amplification efficiency of PCR amplification. Primers are designed according to the principle of complementary base pairing, with multiple primers covering the HPV genome sequence and not interfering with each other. After primer design, BLAST detection is performed separately to ensure that they are not complementary to other genes. Based on this, primer sequences listed in the primer sequence list are selected.

[0021] Primers for capturing the HPV integration site are listed in the sequence listing.

[0022] The principle of library construction is shown in the figure below. After DNA molecules are broken by enzyme digestion, a specific fragment (UMI) is added to each DNA molecule in the sample to prevent mismatches caused by PCR amplification, which would lead to errors in data analysis. At the same time, by using UMI to analyze fragments, the original proportion of heterozygous fragments can be accurately analyzed, increasing the accuracy and reliability of the results. The purity of the DNA to be tested should be between A260 / A280 = 16-20, the paraffin genome concentration should be 20 ng / ul, and the fresh tissue genome concentration should be greater than 8 ng / ul. However, fresh tissue is difficult to obtain, so paraffin sections are generally chosen for DNA extraction.

[0023] The specific embodiments of the present invention will be described in detail below, but it should be emphasized that the present invention is not limited to the specific embodiments described.

[0024] In a specific embodiment of the present invention, the specific steps for detecting HPV integration information are as follows:

[0025] Extract the human genomic DNA template to be tested, wherein the human genomic DNA template is DNA extracted from human cervical lesion tissue.

[0026] The aforementioned DNA was used as a template for library construction.

[0027] The library constructed in the previous step is then subjected to high-throughput sequencing to obtain sequencing data.

[0028] The high-throughput sequencing data obtained in the previous step were analyzed using Biomedical Genomics Workbench 5 software to assess HPV integration in the samples.

[0029] The following is a detailed description of each of the main steps mentioned above:

[0030] Materials and Methods

[0031] Main reagents and consumables

[0032] The main reagents, main equipment, and online analysis platforms used in the study are shown in Tables 2 and 3, respectively. The target region amplification primers used in the library construction amplified the full sequence of the HPV genome, totaling 7906 bp, using 76 primers.

[0033] Table 2. Major Reagents and Manufacturers

[0034]

[0035] Table 3 Major Instruments and Manufacturers

[0036]

[0037] Genomic DNA extraction: DNA was extracted from the sample using a commercially available genomic DNA extraction kit.

[0038] Genomic DNA quality control: DNA purity and concentration identification (nucleic acid protein quantification instrument): A 260 / 280 ratio between 1.6 and 2.0 indicates high DNA purity and no protein contamination. (260 represents the absorbance of DNA at 260 nm, and 280 represents the absorbance of proteins bound to DNA at 280 nm; a ratio between 1.6 and 20 indicates good DNA purity.)

[0039] Gene library construction: The principle of library construction is shown in the figure below. After DNA molecules are fragmented by enzyme digestion, a specific fragment (UMI) is added to each DNA molecule in the sample to prevent mismatches caused by PCR amplification, which could lead to errors in data analysis. At the same time, using UMI analysis fragments can accurately analyze the original proportion of heterozygous fragments, increasing the accuracy and reliability of the results. The specific steps are as follows:

[0040] Genomic concentration equilibration of samples before library construction: Based on the quantitative results, the concentration of extracted genomic samples was equilibrated to 8 ng / ul.

[0041] Genome fragmentation and end repair: Prepare the enzyme digestion and end repair reaction system according to the table below. The enzyme mixture corresponding to 5 μL needs to be added last. Gently pipette 7-8 times to ensure thorough mixing.

[0042] Table 4 Enzyme digestion and end-repair response system

[0043]

[0044] The reaction conditions are as follows.

[0045] Table 5. Reaction conditions for enzyme digestion and terminal repair

[0046]

[0047] Specific connector connection reaction

[0048] Prepare the Mix according to the table below and record the numbers. Finally, add 5 μL of the corresponding enzyme solution and gently pipette 7-8 times to mix.

[0049] Table 6. Specific Connector Connection Reaction System

[0050]

[0051] Place the above reaction system into a PCR instrument and react at 20°C for 5 minutes.

[0052] Ligation product purification

[0053] Add 50 μL of nuclease-free water to each reaction tube, then add 100 μL of Beads magnetic beads, mix well with a pipette, incubate at room temperature for 5 min, add to a magnetic rack and incubate for 10 min, then carefully wash away the supernatant with a pipette; add 200 μL of 80% ethanol to each reaction tube, move the 96-well plate one row on the magnetic rack, move the 96-well plate one row on the magnetic rack after 10 seconds, and move it in this way 5 times, remove the ethanol with a pipette to complete one ethanol wash, add 200 μL of 80% ethanol to the reaction tube again, repeat the ethanol wash once, and air dry. Add 52 μL of nuclease-free water to the reaction tube, mix well with a pipette, place the reaction tube on a magnetic rack for about 3 minutes until the solution becomes clear, and transfer 50 μL of supernatant to a new PCR reaction tube to complete the first elution. Add 52 μL of Beads magnetic beads to the new reaction tube and incubate at room temperature for 5 minutes. After adding the beads to the magnetic rack for 10 minutes, carefully wash away the supernatant with a pipette. Add 200 μL of 80% ethanol to each reaction tube, move the 96-well plate one row on the magnetic rack, and move it another row after 10 seconds. Repeat this movement 5 times. Remove the ethanol with a pipette to complete one alcohol wash. Add another 200 μL of 80% ethanol to the reaction tube and repeat the alcohol wash once more. Let the tubes air dry. Add 12 μL of nuclease-free water to the reaction tube, mix with a pipette, place the reaction tube on a magnetic rack for about 3 minutes until the solution becomes clear, and then transfer 9.4 μL of the supernatant to a new PCR reaction tube to complete the second elution.

[0054] Target region amplification reaction

[0055] Prepare the mix according to the table below and dispense it into the 96-well PCR plate purified in the previous step.

[0056] Table 9 Target Region Amplification Reaction System

[0057]

[0058] Perform the amplification reaction of the target region according to the conditions in the table below.

[0059] Table 10 Target Region Amplification Reaction Conditions

[0060]

[0061] Purification of target region amplification products

[0062] Add 60 μL of nuclease-free water to each reaction tube, then add 104 μL of magnetic beads. Mix well with a pipette and incubate at room temperature for 5 minutes. Place the tubes on a magnetic rack and incubate for 10 minutes. Carefully wash away the supernatant with a pipette. Add 200 μL of 80% ethanol to each reaction tube. Move the 96-well plate one row on the magnetic rack, wait 10 seconds, then move it another row. Repeat this process 5 times. Remove the ethanol with a pipette to complete one ethanol wash. Add another 200 μL of 80% ethanol to the reaction tubes and repeat the ethanol wash once more. Allow the tubes to air dry. Add 16 μL of nuclease-free water to each reaction tube and mix well with a pipette. Place the tubes on a magnetic rack for approximately 3 minutes until the solution becomes clear. Transfer 13.4 μL of the supernatant to a new PCR reaction tube to complete the elution.

[0063] Universal primer amplification reaction

[0064] Prepare the mix according to the table below, and dispense it into the purified 96-well PCR plates from the previous step.

[0065] Table 11 Universal primer amplification reaction system

[0066]

[0067] Perform the amplification reaction of the target region according to the conditions in the table below.

[0068] Table 12 Universal primer amplification reaction conditions

[0069]

[0070] Purification of target region amplification products

[0071] Add 60 μL of nuclease-free water to each reaction tube, followed by 104 μL of magnetic beads. Mix well with a pipette and incubate at room temperature for 5 minutes. Place the tubes on a magnetic rack and incubate for 10 minutes. Carefully wash away the supernatant with a pipette. Add 200 μL of 80% ethanol to each reaction tube. Move the 96-well plate one row on the magnetic rack, wait 10 seconds, then move it another row. Repeat this process 5 times. Remove the ethanol with a pipette to complete one alcohol wash. Add another 200 μL of 80% ethanol to the reaction tubes and repeat the alcohol wash once more. Allow the tubes to air dry. Add 30 μL of nuclease-free water to each reaction tube and mix well with a pipette. Place the tubes on the magnetic rack for approximately 3 minutes until the solution becomes clear. Transfer 28 μL of the supernatant to a new PCR reaction tube to complete the elution. Library construction is complete.

[0072] Library Quantitative

[0073] Reagent preparation

[0074] Calculate the required volume of working solution based on the number of samples to be tested. One sample preparation = 199 μL Qubit dsDNA HS Buffer + 1 μL Qubit dsDNA HS Reagent*200X solution. The sample quantity calculation = number of samples to be tested + 1 S0 + 1 S10 + 1 S standard + loss (one or two extra samples may be needed during calculation). After calculation, prepare the working solution, mix well, and aliquot. Aliquot 198 μL for samples and S standard per tube, and 190 μL for S0 and S10 per tube, and label accordingly. After labeling, add 2 μL of sample to each labeled tube, 2 μL of dsDNA HS Standard#2 solution to each labeled tube, and 10 μL of dsDNA HS Standard#1 and dsDNA HS Standard#2 solution to each labeled tube. Mix well, centrifuge briefly, and incubate in the dark for 3 minutes. After incubation, S0 and S10 are first detected on the instrument to create a standard curve, then S standard is detected, and finally the concentration of the sample to be tested is detected on the instrument and the sample concentration is recorded.

[0075] Qubit 3.0 detects library concentration

[0076] On the main interface, select the analysis type (DNA, RNA, protein, etc.) for which the standard is to be run. Press DNA, select the dsDNA High Sensitivity option, and enter the standard interface. For the selected analysis, if calibration has been performed before, the Qubit 3.0 fluorometer will prompt to select whether to read a new standard or use the previous calibration. Press Yes to read a new standard. A prompt to insert Standard#1 appears on the interface. Insert Standard#l (S0) into the sample well and press Read to ensure that Standard#1 suitable for the analysis is used. The reading process takes about 3 seconds. Insert Standard#2 (S10) and press Read to ensure that the data points of the sample are shown on the screen. Select Sample to enter the sample interface. Insert the sample into the sample well and press Read. The reading process takes about 3 seconds. Once the measurement is completed, the result will be shown on the interface. The displayed number is the concentration of nucleic acid in the analysis tube. If you want to read the next sample, take out the sample from the sample well, insert the next sample, and press Read Next Sample to repeat the sample reading until all samples are read and record the results. Samples with a concentration greater than 0.2 ng / ul are qualified.

[0077] Emulsion PCR (OT)

[0078] Mixed library

[0079] Correctly compile the on-machine record form according to the concentration and specific tags of the sample library. Note that libraries with the same specific tags cannot be arranged in the same run.

[0080] Select Standard#2 suitable for the analysis. After reading Standard#2, the calibration is completed. The new standard curve and the standards connected by straight lines

[0081] Sort the sample libraries according to the record form, shake and mix well for 30 seconds, and centrifuge for 2 seconds. Samples that need to be diluted are diluted to a final concentration of 100 pM, and the diluted samples are shaken and mixed well for 30 seconds and centrifuged for 2 seconds.

[0082] According to the mixing volume on the record form, add the mixing amount of each sample to a new labeled 1.5 mL EPP tube respectively. Shake and mix well for 30 seconds, briefly centrifuge for 2 seconds, and place on an ice box.

[0083] Template preparation

[0084] Turn on One Touch 2 and select the "Clean Instrument" program; fill the template preparation reaction oil I to 1 / 2 full and the demulsifier I to 1 / 3 full; ensure that the old amplification plate and template preparation cleaning connector are installed correctly; empty the waste bottle and place a waste collection bottle under the needle to collect the waste; confirm each step again on the screen and click "Next" to start the cleaning program, which will take 15 minutes.

[0085] After cleaning with One Touch 2, remove the old amplification plate; install the new amplification plate, collection tube (with 150 μL of demulsifier II pre-added to the collection tube), and connector; remove template preparation reaction oil I, invert the solution in the bottle 3 times to mix, add template preparation reaction oil I to 1 / 2 of the bottle, and demulsifier I to 1 / 3 of the bottle.

[0086] The recommended volume is 10 μL for 100 pM sequencing. However, the actual volume may need to be adjusted based on the sequencing results.

[0087] Reagent preparation: Equilibrate the emulsion PCR buffer at room temperature (if it is the first time using it, thaw it on an ice box in advance and store it at 2℃~8℃ after use), vortex for 30 seconds, and centrifuge for 2 seconds; briefly centrifuge the emulsion PCR enzyme mixture for 2 seconds and place it on an ice box; place the template vector solution at room temperature, vortex for 1 minute before use to mix thoroughly, and centrifuge for 2 seconds.

[0088] According to the table below, add the reagents sequentially to the emulsion PCR buffer tube at room temperature, and mix thoroughly by repeatedly pipetting.

[0089] Table 13 Emulsion PCR Reaction System

[0090]

[0091] Precautions: Prepare the reaction solution at room temperature (15-30℃); the prepared reaction solution must be reacted on the OneTouch2 instrument within 15 minutes.

[0092] Take a new template preparation filter and place it on the shelf with the sample well facing upwards. Vortex the amplification reaction solution for 5 seconds, centrifuge for 2 seconds, and immediately mix it several times with a 1000 μL pipette. Add the amplification reaction solution slowly and vertically into the template preparation filter in three portions through the sample well. Before the third addition, centrifuge the emulsion PCR buffer tube at low speed for a few seconds to reduce air bubbles, being careful not to create any. After all the amplification reaction solution has been added, continue to slowly and vertically add 200 μL of reaction oil II through the sample well.

[0093] Remove the cleaning connector from the One Touch 2, and slowly flip the template preparation filter from the previous step so that the sample dispensing port is facing down, and install it on the One Touch 2.

[0094] On the One Touch 2 screen, select "Run" and then select the corresponding program: Proton: Ion PI TM For Hi-QOT2200Kit, select "Assisted" and confirm each step again. Click "Next" to run the program, which takes 45 hours.

[0095] After OneTouch2 finishes running, click the Next command and centrifuge for 10 minutes. After centrifugation, click Open Lid, remove the collection tube, and place it on the double-sided panel. If no further operation is performed within 15 minutes after centrifugation, click the Final Spin command and centrifuge again for 10 minutes.

[0096] Pipettes the solution from each of the two collection tubes down to 100 μL. Mix the remaining 100 μL thoroughly and then transfer it to a new 1.5 mL EP tube, labeled OT. (Note: When aspirating the supernatant, the pipette tip should not touch the bottom of the collection tube on the handle side.)

[0097] Pipette 100 μL of nuclease-free water into each of the original two collection tubes, vortex several times, and then transfer to the tube labeled OT. Pipette another 600 μL of nuclease-free water into the OT tube to bring the volume to 1 mL. Vortex for 30 seconds, then centrifuge at 15,500 × g for 8 minutes.

[0098] Reduce the solution to 100 μL, add 900 μL of nuclease-free water, vortex for 30 seconds, and centrifuge at 15,500×g for 8 minutes.

[0099] Reduce the solution to 20 μL. Add template resuspension buffer to a final volume of 100 μL. Mix well for 30 seconds, then centrifuge for 2 seconds. (Note: The template solution obtained in this step must be used for ES operations on the same day.)

[0100] Clean the One Touch 2 device and turn off the power.

[0101] Template enrichment (ES)

[0102] Prepare the Melt-off Solution solution using the following preparation system:

[0103] Table 14 Template Enrichment Melt-off Solution Solution Preparation System

[0104]

[0105] Cleaning C1 magnetic beads: Vortex the C1 magnetic beads for 30 seconds. Pipette 100 μL of the magnetic beads into a 15 mL EP tube, place it on a magnetic rack for 2 minutes, and discard the supernatant. Pipette 1 mL of the C1 magnetic bead cleaning solution into the tube, vortex for 30 seconds, place it on a magnetic rack for 2 minutes, and discard the supernatant. Pipette 130 μL of the C1 magnetic bead resuspension into the tube, and gently agitate to mix the magnetic beads, being careful not to generate excessive air bubbles. (Note: The 1M NaOH solution should be used within 7 days. If used after 7 days, it must be prepared fresh or repackaged.)

[0106] The 100 μL sample obtained in

[00121] , 130 μL of magnetic beads, 300 μL × 3 of template cleaning solution, and 300 μL of Melt-off Solution were sequentially added to an eight-tube bundle.

[0107] Place the eight-tube strip on the ES, attach the special pipette tip, place the 200μL EP tube, and click the "start" button. The instrument will start running, which will take approximately 35 minutes.

[0108] After the instrument has finished running, the sample will be automatically collected in a 200 vL EP tube. Remove the EP tube and cap it. Centrifuge the EP tube at 15,500 × g for 5 minutes, then remove it and observe for any magnetic beads remaining.

[0109] After centrifugation, remove the EP tube, aspirate until only 10 μL of solution remains, add 200 μL of water, and blow the tube 10 times.

[0110] Centrifuge at 15,500×g for 5 minutes. After centrifugation, remove the EP tube and aspirate until only 10 μL of solution remains. Add 90 μL of water to bring the final volume to 100 μL. Pipette 10 times. Vortex to mix for 5 seconds, then centrifuge briefly for 2 seconds. The template solution at this point can be stored at 2–8℃ for no more than 3 days.

[0111] On-machine sequencing

[0112] Instrument cleaning

[0113] Open the nitrogen cylinder and adjust the pressure to 25 psi. Turn on the DA8600, enter the main interface, click "Cleaning," and select the water wash or chlorine wash program according to the usage of the DA8600.

[0114] Chlorine washing: Take chlorine washing reagent tube II, rinse the chlorine washing bottle and chlorine bottle twice with 18MΩ pure water, and fill the chlorine bottle with 1L of 18MΩ pure water. Add one chlorine tablet to the chlorine bottle containing 1L of 18MΩ pure water, dissolve for 10 minutes, add 1mL of 1mol / L NaOH, and mix by inverting. Filter 100mL of washing solution into chlorine washing reagent tube II using a 0.45μm filter. Install the two chlorine washing reagent tubes II in positions C1 and C2. On the DA8600 screen, select the "Cleaning" option, install the chlorine washing chip, and click "Next" according to the on-screen prompts until the cleaning program starts running. The cleaning time is approximately 18 minutes. After chlorine washing is complete, perform a water rinse.

[0115] Water washing: Take the dedicated water washing reagent tube II, label it C1 and C2, and wash twice with 18MΩ pure water. Add 100mL of 18MΩ pure water to each of bottles C1 and C2, and install them in the C1 and C2 positions. On the DA8600 screen, select the "Cleaning" option, install the water washing chip, and click "Next" according to the on-screen prompts until the cleaning program starts running. The cleaning time is approximately 18 minutes.

[0116] Instrument initialization

[0117] Empty the Sequencing Solution II vial, label it W2, and wash it three times with 18MΩ pure water. Add 1920mL of 18MΩ pure water, add one vial of Sequencing Solution II, add 12µl of 1M NaOH solution, cap the vial, and invert to mix 8 times. Take out two new vials of Sequencing Solution II, label them W1 and W3 respectively. Add 32µL of 1mol / L NaOH to W1, and add 50mL of 1× Sequencing Solution III to W3, then cap the vials. (Note: If precipitation occurs, incubate 1× Sequencing Solution III at 37°C for 30 minutes before first use; Sequencing Solution II should be stored at room temperature away from light, and the Sequencing Solution II vial should be replaced after 20 uses.) On the DA8600 screen, select "Initialize," replace the pipettes at positions W1, W2, and W3, install the vials in the corresponding positions, and tighten the caps. Install the chip for initialization. Follow the on-screen prompts to confirm the instrument status. Click "Next" until the program starts running; the first stage takes approximately 30 minutes. (Note: Change gloves when replacing pipettes, and ensure gloves do not touch the pipette body during installation. Besides chlorine-washed chips, water-washed chips and chips that have already been initialized can be used for initialization (the initialized chip should be recently used or new). Take out dGTP, dCTP, dATP, and dTTP beforehand, place them on an ice box, and shake to mix thoroughly after they have fully dissolved. Take four new reagent tubes II, label them "G," "C," "A," and "T" respectively, and add 70 μL of the corresponding dNTP solution. After the first stage of initialization is complete, replace the pipette with the one for the dNTP position, install the reagent tube in the corresponding position, and tighten the screw. Follow the on-screen prompts to continue initialization, which takes approximately 30 minutes. After initialization, return to the main interface. (Note: Change gloves when replacing pipettes, and ensure gloves do not touch the pipette body during installation.)

[0118] Configure Plan

[0119] Log in to the DA8600 server, click "Plan", and then click "Templates" in the "Plan" drop-down menu. Under the corresponding application, select "Plan new run", or select "Plan Run" from the existing templates. Follow the prompts to select DNA for Application and other for Target Technique in "Application", then click "Next (Kits)". Select the Kits information according to the table below, leaving the rest blank, and then click "Next (Monitor)".

[0120] Table 15 Sequencing Plan Settings

[0121]

[0122] The Monitor sequencing real-time monitoring parameters are at their default values ​​and should not be modified. Then click "Next (Reference)". Select hg19 for the Reference Library option, leave the other two options blank, and click "Next (Plugins)". Do not select any Plugins options; click "Next (Output)". In the Output selection, choose T21 and click "Next (Save)". Enter the Run Plan Name and Barcode usage. Run Name naming convention: SEQ + last two digits of the year + month + number of times the sample was run in that month, e.g., SEQ1502088. Barcode usage: Select the corresponding Barcode number and enter the corresponding sample number after it. After entering, you must verify that the barcode number and sample number are correct. Click "Plan Run" to save the Plan settings.

[0123] Preparing for computer lab session

[0124] Pre-prepare the quality control template, sequencing primers, and sequencing enzyme solution on ice. Prepare the library for PCR near the end of DA8600 initialization. Vortex the quality control template for 30 seconds to mix, then briefly centrifuge for 2 seconds. Add 5 μL of quality control template to the template solution and vortex for 5 seconds. Centrifuge at 15,500×g for 5 minutes, carefully aspirating the supernatant with the pipette tip pointing towards the non-precipitated area, reserving 10 μL of solution in the tube. Add 15 μL of annealing buffer to the template solution from the previous step, bringing the total volume to 25 μL. After thawing the sequencing primers on ice, vortex for 5 seconds to mix, then centrifuge for 2 seconds. Add 20 μL of sequencing primers to the sample solution obtained from the previous step, ensuring a total volume of 45 μL. Vortex the sample solution for 10 seconds to mix, then briefly centrifuge for 2 seconds. Anneal the sample solution on the PCR instrument according to the following program: 95℃ for 2 min, 37℃ for 2 min, 25℃ hold. After annealing, add 10 μL of loading buffer to the sample solution obtained in the previous step, vortex for 5 seconds to mix, centrifuge briefly for 2 seconds, and place at room temperature.

[0125] Sample loading and sequencing

[0126] Pipe 55 μL of the sample solution obtained in the previous step and inject it into the sample inlet of the chip from the previous step. Place the chip on a centrifuge with the notch facing outwards, aligning it with the old chip. Centrifuge for 10 minutes. During centrifugation, prepare the following reagents:

[0127] Prepare a 50% annealing buffer by mixing 0.5 mL of annealing buffer and 0.5 mL of nuclease-free water in a 1.5 mL EP tube. This solution can be used within 7 days after preparation.

[0128] Prepare a 50% wash solution by mixing 0.5 mL of 100% isopropanol solution and 0.5 mL of annealing buffer in a 1.5 mL EP tube. Prepare this solution on the same day it is to be used.

[0129] Mix 6 μL of sequencing enzyme solution and 60 μL of 50% annealing buffer in a 1.5 mL EP tube to prepare the enzyme reaction solution. Place the prepared solution on an ice box.

[0130] Prepare a foaming agent by mixing 49 μL of 50% annealing buffer and 1 μL of foaming agent in a 1.5 mL EP tube.

[0131] Inject 100 μL of air into the foaming agent using a pipette, and rapidly and repeatedly blow the liquid for 5 seconds to break down large bubbles into smaller ones. Repeat this process once to create dense, small bubbles. After centrifugation, remove the chip and inject 100 μL of fine bubbles into the sample wells at a uniform speed, removing any liquid overflowing from the sample outlet. Add 55 μL of 50% annealing buffer to the sample inlet, return the chip to the centrifuge, and centrifuge for 30 seconds. Repeat the foaming agent preparation process once more. Add 100 μL of 50% wash buffer vertically and slowly twice into the sample wells of the chip. After each addition of solution, remove the overflowing liquid from the other well. Add 100 μL of 50% annealing buffer vertically and slowly three times into the sample wells of the chip, avoiding the formation of air bubbles. After each addition of solution, remove the overflowing liquid from the other well. Add 65 μL of enzyme reaction solution vertically and slowly into the sample wells of the chip, avoiding the formation of air bubbles. Remove the overflowing liquid from the other well.

[0132] After 5 minutes of enzyme incubation at room temperature, install the chip into the Proton chip slot, run the program, select the pre-set plan, and after verifying that everything is correct, begin sequencing. The sequencing time is 2.5 hours. Within 72 hours after the program finishes running, return to the main menu and perform a water wash. If more than 72 hours have passed, perform a chlorine wash followed by a water wash. After water washing, turn off the instrument on the main menu and cover the dNTP well with a tube to prevent dust accumulation. If the instrument will not be used for an extended period, close the nitrogen cylinder valve.

[0133] Data analysis and result judgment

[0134] Sequencing loading results

[0135] During the sequencing process, the chip loading rate is above 90%, the single-clone reads are above 60%, the usable reads are above 50%, the utilization rate is above 50%, the average fragment distribution is around 110bp, and the data is qualified, as shown in the figure below. Then, according to the sample label (barcode) information, all the original sample data are matched and downloaded from the server.

[0136] Raw sequencing data processing

[0137] Filtering of raw sequencing data

[0138] The sequencing data were analyzed using Biomedical Genomics Workbench 5 software. First, the sample barcode was removed, and duplicate reads were removed based on the unique molecular marker (UMI). Then, the deduplicated reads were compared with the human genome sequence (GRCh37 / hg19) as a reference sequence to analyze the location of HPV integration into the human genome.

[0139] Annotation of HPV integrated reads and other information in sequencing data

[0140] Below is an example of breast cancer sample analysis results implementing this invention:

[0141] The HPV gene detected multiple integration sites, which were then marked on the human genome. Figure 2 As shown.

[0142] The above description is merely a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto. Any person skilled in the art can easily obtain simple changes or equivalent substitutions to the technical solution within the scope of the technology disclosed in the present invention, and all such changes or substitutions fall within the protection scope of the present invention.

Claims

1. A primer for detecting HPV integration sites in the human genome, characterized in that: The sequences of the primers are shown in SEQ1-SEQ76.

2. A kit for detecting HPV integration sites in the human genome, characterized in that: The kit contains the primers as described in claim 1.

3. The kit according to claim 2, characterized in that: The kit also includes enzyme digestion and end repair reagents, ligation reaction reagents, target region amplification reagents, and universal primer amplification reagents.

4. A method for detecting HPV integration sites in the human genome using the kit described in claim 2 or 3, characterized in that: The method includes sample D-weasel extraction, construction of HPV integration fragment library, high-throughput sequencing, data analysis and result judgment.

5. The application of the primers as described in claim 1 in the preparation of reagents for detecting HPV integration sites in the human genome.

6. The application of the kit as described in any one of claims 2-3 in the preparation of reagents for detecting HPV integration sites in the human genome.

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