A bile cfDNA library construction method and application

Abstract

The present invention provides a bile cfDNA library construction method, comprising the following steps: ultrasonically interrupting the extracted bile cfDNA to obtain bile cfDNA fragments, wherein the ultrasonic interruption power is 50-60W and the interruption time is 80-100s; the bile cfDNA fragments are sequentially end-repaired and tailed, connector-connected and purified, fragment screened, library amplified and purified to construct a bile cfDNA library. The present invention constructs a library system by detecting the total amount of bile cfDNA and the distribution of bile cfDNA fragments, determining the bile cfDNA fragment interruption conditions, and then using LP-WGS combined with bioinformatics analysis to detect the whole genome copy number variation of bile cfDNA of BTC patients and analyze and annotate, providing reliable technical means for the discovery of biomarkers, early diagnosis of patients with malignant biliary tumors, and prognosis monitoring related research.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a bile cfDNA library construction method and application. Background Art

[0002] Biliary tract cancer (BTC) is a series of invasive malignant tumors, mainly including ampullary cancer, bile duct cancer and gallbladder cancer, among which bile duct cancer can also be divided into intrahepatic, hilar and distal bile duct cancer. Although the only possible cure for bile duct cancer is radical surgical resection, it is difficult to observe obvious symptoms in most BTC in the early stage, and it is not until the late stage that it is possible to be diagnosed. In addition, about 10%-30% of BTC patients initially identified as resectable are found to be unable to be resected after laparotomy, resulting in BTC patients missing the best time for surgical resection. In addition, the overall survival of most patients with advanced BTC after surgical resection is less than 12 months, and the survival rate of patients with advanced and metastatic disease is less than 10%, resulting in poor prognosis for BTC patients. Therefore, early diagnosis is an effective means to improve the prognosis of BTC patients.

[0003] At present, imaging, routine cytology and biopsy are the methods for diagnosing malignant biliary tumors. Among them, imaging methods mainly include US (ultrasound), CT (computed tomography), MRI (magnetic resonance imaging), 18-fluorodeoxyglucose positron emission tomography and ERCP (endoscopic retrograde pancreaticocholangiopancreatography). Ultrasound can be used as the first imaging examination for cross-sectional detection, but this technology cannot be used for the staging of malignant tumors. CT is the main method for diagnosing and staging biliary cancer, but it cannot detect abnormal tumor tissue with a diameter of less than 1 cm or smaller. MRI has an advantage in resolution over CT when detecting soft tissue, but it is not effective in detecting tumors with a diameter of less than 5 mm. 18-fluorodeoxyglucose positron emission tomography has a low specificity and is not enough to diagnose primary lesions, but it can assist MRI and CT. As an invasive means of evaluating biliary cancer, ERCP has an imaging diagnostic sensitivity and specificity of only 74% and 70%, respectively. At the same time, ERCP can be used as a sampling method for conventional cytology and biopsy, but its sensitivity is only 20%-40%, and it is easy to cause tumor spread and metastasis. Current clinical guidelines do not recommend this technology for the diagnosis of biliary tract cancer.

[0004] As a key technology for early screening, early diagnosis and treatment of tumors, liquid biopsy can be used for multiple and convenient sampling and examination of the examinee in a minimally invasive or non-invasive manner for auxiliary diagnosis of tumors, efficacy evaluation, postoperative monitoring and guidance of clinical medication, etc. The sampling types mainly include blood, urine, ascites and pleural lavage fluid. At present, blood is the main liquid biopsy fluid, and cancer components such as circulating tumor DNA (ctDNA), extracellular vesicles, circulating RNA and circulating tumor cells can be detected and analyzed to achieve early diagnosis, prognosis and recurrence monitoring of cancer. However, most BTC components may cause a decrease in concentration after flowing through the blood throughout the body, increasing the difficulty of detection. Studies have shown that some blood markers such as CA19-9 can be used for the diagnosis of BTC, but their specificity is limited. Based on the anatomical location specificity of the gallbladder and bile duct, bile is secreted by hepatocytes and transferred from the bile duct to the gallbladder for concentration and storage, promoting the entire enterohepatic circulation. It can directly contact the liver, gallbladder and digestive system, and contains a large number of components including cfDNA, RNA, extracellular vesicles, various metabolites and salts that can be used for cancer research. Perhaps bile is more suitable than blood for liquid biopsy to diagnose BTC. Studies have shown that the sensitivity and specificity of bile cfDNA in detecting tumor gene mutations are better than those in blood (PMID: 33375555).

[0005] Low-depth whole-genome sequencing (LP-WGS) is based on the next-generation sequencing (NGS) technology to detect genomic copy number variations. It has shown more advantages in prenatal diagnosis than other technologies such as chromosome microarray analysis (CMA), such as wide detection range, high throughput, simple operation, good compatibility, better detection of low-proportion mosaicism and low DNA sample volume detection (PMID: 30950010), and has significant advantages in serving early clinical diagnosis. A large number of studies have shown that LP-WGS can be used for early diagnosis of tumors: the use of ULP-WGS to analyze cfDNA fragment size and plasma cfDNA copy number changes found that the tumor score level was significantly correlated with the tumor burden of malignant peripheral nerve sheath tumors, and it has the potential to be used for early detection of high-risk cancer susceptible populations (PMID: 34464388); other studies have found that LP-WGS successfully detected CNVs in plasma cfDNA with tumor scores ≥ 10%, accounting for about 30% of first-line NSCLC patients, which may provide value in the early detection of cancer and progression (PMID: 30617129).

[0006] So far, there are still few studies on bile cfDNA in human diseases, and bile cfDNA has not been used as an effective technical means for liquid biopsy in clinical practice. Liquid biopsy is based on individual body fluid samples, and the sampling is non-invasive or minimally invasive, easy to collect, and the components can well reflect the patient's systemic tumor burden, which can well supplement the shortcomings of tissue biopsy, which is the most widely used in clinical research. Bile exists in large quantities in the human enterohepatic system, and is anatomically closer to the digestive system than the blood flowing throughout the body. In the diagnosis of related digestive system diseases such as BTC based on liquid biopsy, bile may be better than blood. In recent years, a large number of studies have shown that LP-WGS technology has shown obvious advantages in prenatal diagnosis and tumor diagnosis, but there is currently no research on the application of bile cfDNA combined with LP-WGS in liquid biopsy to ultimately assist clinical early diagnosis. Due to the lack of a method system specifically for bile cfDNA LP-WGS, the application of bile cfDNA LP-WGS technology in the early diagnosis of BTC is limited. Therefore, there is an urgent need for an LP-WGS method system suitable for bile cfDNA to facilitate the clinical transformation of BTC biomarkers such as early diagnosis and early treatment. Summary of the invention

[0007] The purpose of the present invention is to provide a bile cfDNA library construction method and application. The present invention extracts cfDNA from the bile of patients with biliary tract cancer, detects the total amount of bile cfDNA and the distribution of bile cfDNA fragments, determines the conditions for the fragmentation of bile cfDNA fragments, and then constructs a library system. Subsequently, LP-WGS combined with bioinformatics analysis is used to detect the whole genome copy number variation of bile cfDNA in BTC patients and analyze and annotate, providing a reliable technical means for the discovery of biomarkers, early diagnosis of patients with malignant biliary tumors, and prognosis monitoring related research.

[0008] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0009] The present invention provides a bile cfDNA library construction method, comprising the following steps: S1, a shearing process: ultrasonically shearing the extracted bile cfDNA to obtain bile cfDNA fragments, wherein the ultrasonic shearing power is 50W-60W, and the shearing time is 80s-100s; S2, a library construction process: the bile cfDNA fragments obtained in step S1 are sequentially subjected to end repair and 3' end A-tailing, linker connection and purification, fragment screening to obtain bile cfDNA library fragments, library amplification and purification, and a bile cfDNA library is constructed.

[0010] As a preferred implementation scheme, the following steps are also included: S3, quality control: quality control of the bile cfDNA obtained in step S1 and the bile cfDNA and bile cfFNA library fragments after library construction in step S2; S4, sequencing analysis: LP-WGS combined with bioinformatics analysis technology is used to detect the whole genome CNV of bile cfDNA, and perform analysis and annotation.

[0011] As a preferred embodiment, the fragment screening process in step S2 includes first using 0.6X to 0.65X magnetic beads to purify to remove the DNA fragments adsorbed by the magnetic beads; and then using 0.77X to 0.8X magnetic beads to purify to obtain bile cfDNA library fragments.

[0012] As a preferred embodiment, the fragment screening process in step S2 includes first using 0.65X magnetic beads to purify to remove the DNA fragments adsorbed by the magnetic beads; and then using 0.77X magnetic beads to purify to obtain bile cfDNA library fragments.

[0013] As a preferred embodiment, the ultrasonic interruption power is 50W and the interruption time is 80s.

[0014] As a preferred embodiment, the fragmentation conditions for library construction include: the main peak of bile cfDNA after fragmentation is a single peak with a normal distribution between 250 bp and 450 bp.

[0015] As a preferred embodiment, the fragmentation conditions for library construction also include: the main peak of bile cfDNA after fragmentation accounts for no less than 60% between 200 bp and 500 bp.

[0016] As a preferred embodiment, step S1 also includes extraction of bile cfDNA, and the method for extracting bile cfDNA includes the following steps: S1. After cleaving and digesting the bile sample with a protease, the bile cfDNA is precipitated; S2. The bile cfDNA is enriched using a silica-based adsorption column; S3. The adsorption column enriched in step S2 is washed, and the bile cfDNA is eluted and collected.

[0017] As a preferred embodiment, the concentration of the bile cfDNA library is at least 3.0 ng / μL, and the total amount of the bile cfDNA library is at least 50 ng.

[0018] As a preferred embodiment, the concentration of the bile cfDNA library is 13.3 ng / μL to 21.2 ng / μL, and the total amount of the bile cfDNA library is 266 to 424 ng.

[0019] The present invention also provides a bile cfDNA detection kit, which uses the above-mentioned method to detect bile cfDNA.

[0020] The present invention also provides an application of the above method in detecting CNV changes in the whole genome of bile cfDNA in patients with biliary tract cancer.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The method provided by the present invention combines bile cfDNA with LP-WGS for the first time and verifies that bile cfDNA has the potential to become an important means of liquid biopsy.

[0023] 2. This invention explored and determined for the first time the fragmentation conditions suitable for bile cfDNA, providing strong evidence support for subsequent library construction.

[0024] 3. This invention is the first to establish a low-depth whole-genome library construction method system suitable for bile cfDNA.

[0025] 4. The present invention is based on bile samples from multiple real patients with malignant biliary tumors in clinical practice. The cfDNA isolated from them can better reflect clinical problems and is the basic prerequisite for applying bile cfDNA to liquid biopsy. It may subsequently provide effective clinical value for the diagnosis and detection of patients with malignant biliary tumors.

[0026] 5. The present invention verifies that bile cfDNA is mainly composed of large fragments, which provides guidance for subsequent research.

[0027] 6. The low-depth whole-genome sequencing method of bile cfDNA established in the present invention is combined with bioinformatics analysis for the first time, and it is shown that this method can be used to study CNV in patients with biliary tract tumors, providing strong technical support for the subsequent application of CNV changes of bile cfDNA to liquid biopsy, and for the early diagnosis, treatment and recurrence monitoring of BTC patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing the distribution of bile cfDNA fragments in 6 patients with malignant biliary tumors in the present invention, wherein 1-6 represent patients 1-6 with malignant biliary tumors, respectively.

[0029] Figure 2 This is a comparison result diagram of the proportion of small fragments after the interruption time is 100s and 130s when the interruption power is 50W.

[0030] Figure 3 This is a diagram showing the fragment distribution results of bile cfDNA fragmentation of 6 patients with malignant biliary tumors in the present invention, wherein 1-6 represent patients 1-6 with malignant biliary tumors, respectively.

[0031] Figure 4This is a diagram of the bile cfDNA library fragment distribution results of 6 patients with malignant biliary tumors in the present invention, wherein 1-6 represent patients 1-6 with malignant biliary tumors, respectively.

[0032] Figure 5 This is a graph of CNV changes in the whole genome of bile cfDNA LP-WGS of two patients with malignant biliary tract tumors in the present invention, wherein 1-2 represent patients 1-2 with malignant biliary tract tumors, respectively. DETAILED DESCRIPTION

[0033] The technical solution of the present invention is described in detail below in conjunction with the embodiments. The reagents and biological materials used below are all commercial products unless otherwise specified.

[0034] Example 1: Extraction, concentration and fragment distribution detection of bile cfDNA in patients with malignant biliary tumors

[0035] (1) Study cohort and experimental groups

[0036] A total of 6 patients with malignant biliary tumors were included in this study, including 3 cases of cholangiocarcinoma and 3 cases of gallbladder cancer. Bile samples were collected from all patients before surgery. Each patient with malignant biliary tumor was given an accurate pathological diagnosis after surgery. The bile samples of each patient with malignant biliary tumor were pretreated and bile cfDNA was extracted using the 3D-BCF method.

[0037] (2) Pretreatment of bile samples from patients with malignant biliary tumors

[0038] The bile samples collected from patients with malignant biliary tract tumors before surgery were stored in 10 mL free nucleic acid storage tubes (BEAVER Cell Free DNA Tubes, 43803, China) and shipped at room temperature. After receiving the bile samples, the bile samples were first centrifuged at 1,600 g for 10 min at 4 ° C to remove cell debris. After centrifugation, the supernatant was aspirated into a new 1.5 mL centrifuge tube, and then centrifuged again at 16,000 g for 15 min at 4 ° C. Finally, the supernatant was aspirated and divided into 1.5 mL centrifuge tubes at 1 mL per tube. The pretreated bile samples were stored in a -80 ° C refrigerator and subsequently used to extract bile cfDNA.

[0039] (3) 3D-BCF method for extracting cfDNA from bile samples of patients with malignant biliary tumors

[0040] The 3D-BCF method for extracting bile cfDNA is described in detail in patent CN202110835904.6. The specific steps are: after taking the bile sample out from -80°C, place it in a 37°C water bath until it is completely thawed, then centrifuge at 16,000g for 5 minutes at 4°C, and draw 300μL of bile supernatant into a new 1.5mL centrifuge tube. 1.68 μL Carrier RNA (1017647, Qiagen, Shanghai, China) was added to 240 μL of commercial lysis buffer ACL (939017, Qiagen, Shanghai, China), and after mixing, 240 μL of the mixture was added to the 1.5 ml centrifuge tube containing 300 μL of bile supernatant, and 30 μL of proteinase K (19133, Qiagen, Shanghai, China) was added to the centrifuge tube, inverted and mixed, and incubated in a metal bath at 60°C for 30 minutes. During the incubation period, the mixture was inverted and mixed every 10 minutes to make the incubation more complete. After the incubation was completed, 540 μL of precipitation reagent Buffer ACB (1069275, Qiagen, Shanghai, China) was added to promote the precipitation of cfDNA. In order to better precipitate cfDNA, the bile mixture was placed in a -20°C refrigerator for 5 minutes. 700 μL of bile mixture was added to the QIA-quick adsorption column (28115, Qiagen, Shanghai, China) that can capture a wider range. To ensure that the adsorption column can more fully enrich bile cfDNA, it was first allowed to stand at room temperature for 5 min after the first loading, and then the centrifugation operation was repeated until all samples passed through the adsorption column. 500 μL of Buffer PE (19065, Qiagen, Shanghai, China) was added to the adsorption column and centrifuged twice. After that, the remaining liquid in the adsorption column was removed by high-speed centrifugation and the adsorption column was placed in a new 1.5 mL centrifuge tube. 30 μL of elution buffer (19086, Qiagen, Shanghai, China) was added to the adsorption column. After incubation at room temperature for 3 min, the cfDNA was eluted and collected by centrifugation, and the aliquots were placed in a -20°C or -80°C refrigerator for long-term storage.

[0041] (4) Detection of bile cfDNA concentration and fragment size

[0042] The concentration of separated bile cfDNA was detected by Qubit 3.0 fluorescence quantification instrument and matching reagents (Q32854, Thermo Fisher, USA), and the total amount of bile cfDNA obtained in each group was calculated. The specific detection process is shown in the instructions of the kit. The results of bile cfDNA concentration determination are shown in Table 1. The 3D-BCF method can extract a sufficient amount of bile cfDNA, and the concentration of separated cfDNA varies between different bile samples. The bile cfDNA concentration is approximately between 6.44-206ng / μL. The total amount of bile cfDNA obtained is sufficient for subsequent fragmentation and library construction.

[0043] Table 1

[0044]

[0045] In order to detect the fragment size distribution of bile cfDNA after extraction, the Agilent 2100 analyzer and the matching chip and reagents (5067-4626, Agilent, USA) were used to analyze the fragment distribution of bile cfDNA extracted. For the specific detection process, please refer to the manufacturer's instructions of this kit. Figure 1 1 is a graph showing the distribution of bile cfDNA fragments in 6 patients with malignant biliary tract tumors in the present invention, wherein 1-6 represent patients with malignant biliary tract tumors No. 1-6, respectively. Figure 1 As shown in the figure, bile cfDNA isolated from the bile of 6 patients with malignant biliary tumors are mainly large fragments, and only a few samples have a certain proportion of small fragments. Among them, the main peak of most bile cfDNA is about 6000bp, which is a relative size of DNA fragments; the main peak of commonly used plasma cfDNA is about 167bp. In contrast, the main peak of bile cfDNA 6000bp is a large fragment. From the peak shape, it can be seen that some bile cfDNA also has fragments other than the main peak, such as small fragments of 100-600bp.

[0046] Example 2: Ultrasonic fragmentation and peak shape detection of bile cfDNA

[0047] Take the bile cfDNA sample extracted in Example 1 out of the -20°C or -80°C refrigerator and thaw at room temperature, take a total of 1 μg of bile cfDNA into a new EP tube, make up the volume to 52 μl with 1×TE, and then transfer 52 μl of bile cfDNA mixture to the interruption tube (covaris, 520077, Germany). Turn on the Covaris M220 DNA interruption instrument and software, install the equipment, put the bile cfDNA sample to be interrupted into the interruption fixture, and close the safety door. Set the software interruption parameters as shown in Table 2 below, and click the RUN button to interrupt the bile cfDNA sample. Then transfer the interrupted bile cfDNA to a new EP tube, and use Agilent 4150 software and its supporting chips and reagents (5067-5585, Agilent, USA) to detect the fragment distribution results after interruption. The results of different interruption conditions in Table 2 show that when the interruption power is 50W, the main peak of the fragment after interruption is greater than the main peak of the fragment after interruption when the interruption power is 30 or 40W.

[0048] Table 2

[0049]

[0050] Considering the problem of insufficient total amount of DNA for the same sample to be interrupted under different conditions, other subpackaged bile cfDNA samples to be tested were used, and the operation in Example 2 was followed to detect whether extending the interruption time would affect the main peak distribution results of the fragments after interruption when the interruption power was 50 W. The software interruption parameters were set as shown in Table 3 below, and the RUN button was clicked to interrupt the bile cfDNA sample. The interrupted bile cfDNA was then transferred to a new EP tube, and the fragment distribution results after interruption were detected using Agilent 4150 software and its supporting chips and reagents (5067-5585, Agilent, USA).

[0051] Table 3

[0052]

[0053] The results of different interruption conditions in Table 3 show that when the interruption power is 50W and the interruption time is 80s, 100s, 130s and 170s, the main peak size of the fragment after interruption is similar, and the main peak difference is not large. Therefore, the present invention selects 80s, which takes the shortest time, as the interruption time. Under the interruption conditions of 50W interruption power and 80s interruption time, the main peak generated is between 250-450bp, which meets the subsequent requirements. The study found that the interruption conditions that meet the subsequent library construction mainly depend on whether the main peak generated is between 250-450bp, whether it is a single peak with a normal distribution, and preferably 200-500bp accounts for ≥60%. In addition, it was also found during the experiment that the longer the interruption time, the greater the proportion of small fragments of 0-200bp. Figure 2 This is a comparison result of the proportion of small fragments after the interruption time is 100s and 130s when the interruption power is 50W. Figure 2 As shown in the figure, compared with the interruption power of 50W for 100s, the proportion of 0-200bp small fragments after the interruption power of 50W for 130s increased from 15.66% to 20.42%. Therefore, a longer or shorter interruption time will result in a higher proportion of small cfDNA fragments or a higher proportion of large cfDNA fragments after interruption, which is not conducive to the subsequent construction of bile cfDNA library and affects the analysis of sequencing results.

[0054] Figure 3 This is a fragment distribution result diagram of bile cfDNA fragments of 6 patients with malignant biliary tumors in the present invention, wherein 1-6 represent patients with malignant biliary tumors No. 1-6, respectively. Figure 3 The results showed that the bile cfDNA of 6 patients with malignant biliary tumors showed a normal distribution after fragmentation, with the main peak between 250-450bp, and could be used for subsequent bile cfDNA library construction.

[0055] Among them, the standards for cfDNA library construction are:

[0056] (1) The total amount of bile cfDNA is ≥200 ng. The main peak is a typical normal distribution with a single main peak between 250-450 bp. Occasionally, the main peak is too large. In this case, additional time is needed to supplement the peak. For example, if the main peak is 700 bp, an additional 60 s can be added under the original conditions.

[0057] (2) The total library volume was ≥50 ng, the library concentration was ≥3.0 ng / μl, there was a single main peak between 455-565 bp, there was no adapter dimer or the adapter (about 160 bp) accounted for ≤3%, which was considered qualified.

[0058] Example 3: Constant library construction and sequencing after bile cfDNA shearing

[0059] The bile cfDNA interrupted in Example 2 was constructed using the KAPA DNA HyperPrep library construction kit (KK8504, Roche, Switzerland). The detailed process is referred to the instructions of the kit. The main process is as follows: Take 400ng (minimum 200ng is also acceptable) of the interrupted bile cfDNA and dilute it to 100μl in a PCR tube, add 100μl of magnetic beads to the PCR tube and mix well, and incubate at room temperature for 5min. Then let it stand on the magnetic rack for about 5min until the solution is clear and transparent, discard the supernatant, add 80% ethanol to the PCR tube containing the magnetic beads and wash twice, add 52μl of elution buffer after the magnetic beads are dried, and draw 50μl of supernatant into a new PCR tube. Then refer to the library construction instructions in sequence for end repair and A-tailing, adapter connection, post-connection purification, fragment screening, library amplification and post-amplification purification. Among them, the detailed operation process for fragment screening in the library construction manual is: add 67.6μl magnetic beads (0.65X) to 104μl of the connection and purification product, mix with a pipette tip, and incubate at room temperature for 5min; after the incubation is completed, the PCR tube is separated instantly, placed on the magnetic rack for about 10min, and the solution is clear and transparent. Carefully transfer 165μl of supernatant to a new PCR tube (or 1.5ml centrifuge tube), and discard the magnetic beads. Add 12.4μl of mixed magnetic beads (0.77X) to the above PCR tube, mix with a pipette tip, and incubate at room temperature for 5min. After the incubation is completed, the PCR tube is separated instantly, placed on the magnetic rack for about 5min, and the solution is clear and transparent. Carefully aspirate and discard the supernatant, then use 200μl 80% ethanol to wash twice, and then elute with 23μl elution buffer for subsequent library amplification. After the fragment screening was put into the adapter connection purification, referring to the purification data of the magnetic bead manufacturer, the present invention mainly built the library for the main peak of about 300bp, and then selected 0.65X magnetic bead purification to remove the large fragments adsorbed by the magnetic beads after the adapter connection, and then selected 0.77X magnetic bead purification to remove the small fragments. The purified fragment range was 260-575bp. After the library was purified, the concentration and fragment distribution were measured by Qubit 3.0 and Agilent 4150 software respectively. Figure 4 1 is a diagram showing the distribution results of bile cfDNA library fragments of 6 patients with malignant biliary tumors in the present invention, wherein 1-6 represent patients with malignant biliary tumors No. 1-6, respectively; Figure 4 The results showed that the bile cfDNA of all 6 patients with malignant biliary tumors had a library fragment range of 300bp-700bp and no junction peaks, which could be well matched with Figure 2The results in Table 4 show that the library concentration of the six patients ranged from 13.3 to 22.4 ng / μL, and the total library volume ranged from 266 to 424 ng. The main peaks of all libraries were approximately between 455 and 565 bp, which can be used for subsequent sequencing analysis.

[0060] Table 4

[0061] Patient number Library concentration (ng / μL) Total library amount (ng) 1 20.8 416 2 18.7 374 3 22.4 448 4 13.3 266 5 17.1 342 6 21.2 424

[0062] Example 4: LP-WGS sequencing and bioinformatics analysis after bile cfDNA shearing

[0063] Based on the bile cfDNA of patients No. 1 and No. 2 with malignant biliary tumors in Example 3 above, LP WGS sequencing was performed using the Nova6000 platform, and the sequencing data volume of each sample was 10G. After the sequencing data was downloaded from the machine, QDNAseq bioinformatics analysis was used to detect the whole genome CNV changes of bile cfDNA in patients No. 1 and No. 2 with malignant biliary tumors; Figure 5 This is a graph of CNV changes in bile cfDNA LP-WGS genome of two patients with malignant biliary tumors in the present invention, wherein 1-2 represent patients with malignant biliary tumors No. 1-2, respectively. Figure 5 The results showed that in both patients, there was an obvious deviation in the log2ratio value of the DNA at the corresponding position on the specific chromosome, that is, CNV change (when log2ratio = 0, it indicates that the AF (variant allele ratio) of the site in tumor patients and normal people is consistent, but when log2ratio is greater than 0 or less than 0, it indicates that there is amplification or deletion of the site).

[0064] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

Claims

1. A method for constructing a bile cfDNA library, characterized in that: The following steps are involved: S1, interruption process: ultrasonically interrupt the extracted bile cfDNA to obtain bile cfDNA fragments, wherein the ultrasonic interruption power is 50W-60W and the interruption time is 80s-100s; S2, library construction process: The bile cfDNA fragments obtained in step S1 are subjected to end repair and 3' end A tailing, adapter ligation and purification, fragment screening to obtain bile cfDNA library fragments, library amplification and purification, and a bile cfDNA library is constructed.

2. The method according to claim 1, characterized in that The following steps are also included: S3, quality control: quality control of the bile cfDNA obtained in step S1 and the bile cfDNA and bile cfFNA library fragments after library construction in step S2; S4, sequencing analysis: LP-WGS combined with bioinformatics analysis technology was used to detect whole-genome CNVs of bile cfDNA for analysis and annotation.

3. The method according to claim 1, characterized in that The fragment screening process in step S2 includes first using 0.6X to 0.65X magnetic beads to purify to remove the DNA fragments adsorbed by the magnetic beads; and then using 0.77X to 0.8X magnetic beads to purify to obtain bile cfDNA library fragments.

4. The method according to claim 3, characterized in that The fragment screening process in step S2 includes first using 0.65X magnetic beads to purify to remove the DNA fragments adsorbed by the magnetic beads; and then using 0.77X magnetic beads to purify to obtain bile cfDNA library fragments.

5. The method according to claim 1, characterized in that: The power of the ultrasonic interruption is 50W, and the interruption time is 80s.

6. The method according to claim 1, characterized in that The shearing conditions used for the library construction include: the main peak of the sheared bile cfDNA is a single peak with a normal distribution between 250 bp and 450 bp.

7. The method according to claim 6, characterized in that The fragmentation conditions for constructing the library also include: the main peak of the fragmented bile cfDNA between 200bp and 500bp accounts for no less than 60%.

8. The method according to claim 1, characterized in that: Before step S1, the step also includes extraction of bile cfDNA. The method for extracting bile cfDNA includes the following steps: S1. After the bile sample is cleaved and digested with protease, the bile cfDNA is precipitated; S2, enrichment of bile cfDNA using a silica-based adsorption column; S3. Wash the adsorption column enriched in step S2, elute and collect bile cfDNA.

9. The method according to claim 1, characterized in that: The concentration of the bile cfDNA library is at least 3.0 ng / μL, and the total amount of the bile cfDNA library is at least 50 ng.

10. The method according to claim 1, characterized in that The concentration of the bile cfDNA library is 13.3 ng / μL to 21.2 ng / μL, and the total amount of the bile cfDNA library is 266 to 424 ng.

11. A bile cfDNA detection kit, characterized in that: The kit detects bile cfDNA using the method described in any one of claims 1 to 10.

12. Use of the method according to any one of claims 1 to 10 in detecting whole genome CNV changes in bile cfDNA of patients with biliary tract cancer.

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