Patent Application
20210130916
This application claims priority to and the benefit of Korean Patent Application No. 10-2019-0139264, filed on Nov. 4, 2019, the disclosure of which is incorporated herein by reference in its entirety.
The present invention relates to a probe for detecting hepatitis B virus and a method for detecting an insertion site of hepatitis B virus at high efficiency based on the analysis method of next-generation sequencing using the probe.
Hepatitis B virus (HBV) is a disease which is the main cause of liver cancer, and approximately 300 million people worldwide are affected by HBV. Hepatitis B virus (hereinafter, referred to as ‘HBV’) is a virus belonging to the Hepadnaviridae family and infects only liver cells of humans specifically. Symptoms of hepatitis are fatigue for mild cases, and jaundice may appear in severe cases. In the late stage of the disease, complications of cirrhosis such as, ascites, edema, gastroesophageal variceal bleeding, hepatic encephalopathy, blood coagulation abnormality, and hepatorenal syndrome can appear.
In the case of patients who have been infected in childhood, the period of immune tolerance occurs continuously for 10 to 30 years in which the proliferation of virus occurs but no symptoms of hepatitis appear, but when these healthy carriers reach a certain period (15 to 30 years old), hepatocytes are damaged by the action of the immune system and develop into hepatitis. When e-antigen seroconversion (HBeAg seroconversion) occurs quickly, viral proliferation is suppressed and symptoms of hepatitis do not develop any further, but when the proliferation of virus is not effectively suppressed, and it develops into chronic hepatitis and liver cirrhosis, and in severe cases, it develops into liver cancer.
Hepatitis B virus can be inserted (integration) into the human genome during viral proliferation and life cycle, and although this step is not essential for viral replication, integration of the HBV DNA into a host genome contributes to the occurrence of liver cancer by inducing genomic instability and altering the expression of cancer-related genes. Until recently, the existence of this genomic insertion phenomenon has traditionally been discovered by polymerase chain reaction (PCR), but this method has a limitation in finding all of HBV-inserted molecules in the entire human genome because it biases detection of only the inserted virus localized in the human genome region designated by a specific primer. Therefore, a new method was necessary to investigate HBV insertion in the entire human genome.
Recently, with the introduction of next-generation sequencing (NGS) technology, it is possible to overcome the limitations of traditional PCR-based studies and to attempt non-biased detection of HBV insertion sites across the entire human genome. The present invention provides a method for analyzing HBV insertion sites at high efficiency based on NGS and a probe applied thereto.
The present invention provides a probe for detecting hepatitis B virus and a method for detecting an insertion site of hepatitis B virus at high efficiency based on the analysis method of next-generation sequencing using the probe.
The present invention provides a probe composition for detecting hepatitis B virus (HBV) consisting of sequences of SEQ ID NO: 1 to SEQ ID NO: 215.
In addition, the present invention may provide a kit for detecting hepatitis B virus (HBV) including the probe composition.
In addition, the present invention may provide a method for detecting hepatitis B virus (HBV), wherein the method is a method for detecting hepatitis B virus (HBV) through next-generation sequencing (NGS), the method including hybridizing a target sample with a probe composition for detecting hepatitis B virus (HBV) consisting of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 215 to capture a target gene.
In addition, the present invention may provide a method for providing information for the diagnosis of liver cancer using the method.
According to the present invention, a probe may be provided that is capable of confirming an insertion site of HBV in the human genome with a possibility of developing into liver cancer. In addition, by applying the probe to the analysis method of next-generation sequencing, HBV insertion sites in the human genome can be analyzed at low cost and high efficiency.
The above and other objects, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which:
Hereinafter, the present invention will be described in detail.
The present invention may detect an insertion site of hepatitis B virus (HBV) located in the human genome at high efficiency based on next-generation sequencing (NGS). Specifically, in a DNA library constructed from a patient's liver tissue, an HBV sequence may be captured with a probe complementary to the self-constructed HBV. Based on this, HBV and breakpoints of the human genome may be detected (refer to
As used herein, the term “probe” refers to a nucleic acid fragment corresponding to several bases to several hundred bases for specific binding to DNA or RNA, and afterwards, the presence or absence of specific DNA or RNA may be confirmed by amplification, separation, and detection.
The present invention provides a probe for detecting hepatitis B virus (HBV) consisting of nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 215.
The probe may detect an insertion site of hepatitis B virus in the human genome. More specifically, the probe may detect an insertion site of hepatitis B virus (HBV) using the analysis method of next-generation sequencing.
The probe may be applied to the detection of hepatitis B virus of Koreans, and more specifically, it may be applied to the detection of genotype hepatitis C virus.
The length of the probe is 120 nucleotides. When the length of the probe is too short or too long, false hybridization increases and the likelihood of a decrease in specificity increases. In the present invention, hybridization efficiency was maximized by optimizing the length of a probe as above.
In addition, the probe is based on the complete genome sequences of 8 prototypes of hepatitis B virus (HBV) of Koreans, and by allowing each HBV nucleotide sequence to overlap, it is designed to have almost 100% coverage for hepatitis B virus (HBV) of Koreans.
In addition, the present invention provides a composition for detecting hepatitis B virus (HBV), including the probe. The composition may include deoxynucleoside triphosphate (dNTP), heat-resistant polymerase, and a metal ion salt such as magnesium chloride and the like, in addition to the probe.
In addition, the present invention provides a kit for detecting hepatitis B virus (HBV), including the composition.
The kit may include a barcoding primer in which an adapter suitable for the NGS device to be used is combined with a barcode sequence.
In addition, the kit may further include a reagent commonly used in a method for detecting nucleic acid. For example, it may include deoxynucleoside triphosphate (dNTP), heat-resistant polymerase, and a metal ion salt such as magnesium chloride and the like that are required for PCR reaction, and may include dNTP, sequenase, and the like that are required for sequencing. In addition, the kit may take the form of a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, and these may be partially or entirely formed from plastic, glass, paper, foil, wax, and the like. The container may be equipped with a completely or partially removable plug, which is initially part of a container or may be attached to the container by mechanical, adhesive, or other means. The container may be equipped with a stopper that may allow access to the contents by an injection needle. The kit may include an external package, and the external package may include instructions for use of the components.
The present invention provides a method for detecting hepatitis B virus (HBV), wherein the method is a method for detecting hepatitis B virus (HBV) through next-generation sequencing (NGS), the method including hybridizing a target sample with a probe for detecting hepatitis B virus (HBV) composed of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 215 to capture a target gene.
As used in, the term “hybridization” means that complementary single-stranded nucleic acids form double-stranded nucleic acids. The degree of complementarity required for hybridization may vary depending on the hybridization conditions, and in particular, if it can be optimized at temperature, it may be preferably optimized to a temperature described in the protocol that can be specified by the probe manufacturer.
As used herein, the term “target gene” refers to a gene sequence to be detected, and it is hybridized with a probe under hybridization, annealing, or amplification conditions.
As used herein, the term “target gene” is not different from the terms used in the present specification such as “target gene”, “target gene sequence”, or “target sequence”, and these terms are used interchangeably in the present specification.
As used herein, a target sample refers to a sample including a gene region to be detected, and it may be collected from at least one selected from the group consisting of tissue, blood, serum, saliva, urine, semen, and body fluid, and specifically, it may be liver tissue derived from a patient.
In addition, the present invention provides a method for detecting hepatitis B virus (HBV), including (a) hybridizing a target sample including a target gene with a probe for detecting hepatitis B virus (HBV) composed of a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 215 to capture a target gene and amplifying to create a library; and (b) sequencing the library to map the produced nucleotide sequence in the human and HBV reference sequences for analysis to confirm an insertion site of hepatitis B virus (HBV) in the human genome.
The hybridizing may be performed at a temperature of 65° C. for 16 hours to 24 hours.
Since it is a temperature and time condition that optimizes the efficiency of probe hybridization, the hybridization efficiency may be lowered when an experiment outside this range is performed.
The target gene may be a hepatitis B virus (HBV) gene of Koreans.
In addition, the present invention may provide a method for providing information for the diagnosis of liver cancer, using the method.
Hereinafter, the present invention will be described in more detail through exemplary embodiments. Objects, features, and advantages of the present invention will be easily understood through the following exemplary embodiments. The present invention is not limited to the exemplary embodiment described herein, and may be embodied in other forms. The exemplary embodiments introduced herein are provided in order to sufficiently convey the spirit of the present invention to those of ordinary skill in the technical field to which the present invention pertains. Therefore, the present invention should not be limited by the following exemplary embodiments.
In order to perform next-generation sequencing analysis for the detection of an HBV insertion site, a probe for HBV capture was prepared based on the following complete genome sequences of 8 representative Korean HBV types. Complementary probes were prepared such that each HBV nucleotide sequence overlapped with each other. The probe was synthesized through the HPLC purification method, and the concentration and purity of the synthesized probe were confirmed using the BioAnalyzer device.
(Sequence Information)
The probe targets the following 8 viruses.
complete genome“,”AY641559.1 Hepatitis B virus isolate He53 (https://www.ncbi.nlm.nih.gov/nuccore/AY641559.1)
complete genome“,”DQ683578.1 Hepatitis B virus from South Korea (https://www.ncbi.nlm.nih.gov/nuccore/DQ683578.1)
complete genome“,”GQ872211.1 Hepatitis B virus (https://www.ncbi.nlm.nih.gov/nuccore/GQ872210.1)
complete genome“,”JN315779.1 Hepatitis B virus genotype C2 (https://www.ncbi.nlm.nih.gov/nuccore/JN315779)
complete genome“,”KR184660.1 Hepatitis B virus isolate SS_3_22 (https://www.ncbi.nlm.nih.gov/nuccore/KR184660.1)
complete sequence, isolate 22Y04HCC“,”AB014381.1 Hepatitis B virus genomic DNA (https://www.ncbi.nlm.nih.gov/nuccore/3582357)
complete sequence, isolate 36Y18HCC“,”AB014395.1 Hepatitis B virus genomic DNA (https://www.ncbi.nlm.nih.gov/nuccore/3551389)
D23680.1 Hepatitis B virus (B4-HBVST1) complete genome sequence (https://www.ncbi.nlm.nih.gov/nuccore/D23680.1)
Based on the above contents, it was prepared by Tilling density 1X, Boosting: balanced, probe group size: 25.595 kbp, Total probe: 215. The sequence information of each designed probe was shown in Table 2 below.
2-1. DNA Shearing
1) Extract genomic DNA from liver tissue of a patient with hepatitis and crush (sonication) it into nucleotides of about 100 to 120 base pairs in length. After diluting 1 μg of gDNA passed through Quality Control (QC) on a 96-well plate with 60 μL, transfer it to a Covaris strip tube and seal with sealing tape.
2) Transfer the strip tube to a steel rack and mount it on a device.
3) As Table 3 below, shear it after setting Covaris (Covaris LE200).
2) Sample Purification
1) Transfer the sheared sample into a new 1.5 mL tube.
2) Place 90 μL of AMPure beads, vortex it for 5 seconds, and perform incubation at room temperature for 5 minutes.
3) Place the sample in a magnetic particle concentrator (MPC), and after 3 minutes, discard the supernatant.
4) Add 200 μL of 70% ethanol while the sample is in MPC, and after 1 minute, discard the supernatant (repeat twice).
5) Completely dry the beads (5 minutes to 10 minutes).
6) Remove the sample tube from MPC, add 50 μL of nuclease-free water, and resuspend AMPure beads.
7) After incubating at room temperature for 2 minutes to 3 minutes, spin it down.
8) Place the sample in MPC, and after 2 minutes, transfer 48 μL of the supernatant into a new 1.5 mL tube.
2-3. Repairing the Ends
1) After mixing all of the components of Table 4 below, lid off in PCR and perform at 20° C. for 30 minutes.
2) Sample Purification
{circle around (1)} Place the sample performed in 3. 1) above into a new 1.5 mL tube.
{circle around (2)} Place 180 μL of AMPure beads (1.8×), vortex it for 5 seconds, and perform incubation for 5 minutes at room temperature.
{circle around (3)} Place the sample in a magnetic particle concentrator (MPC), and after 3 minutes, discard the supernatant.
{circle around (4)} While the sample is in MPC, add 200 μL of 70% ethanol, and after 1 minute, discard the supernatant (repeat twice).
{circle around (5)} Completely dry the beads (5 minutes to 10 minutes).
{circle around (6)} Remove the sample tube from MPC, add 32 μL of nuclease-free water, and resuspend AMPure beads.
{circle around (7)} After incubating for 2 minutes to 3 minutes at room temperature, spin it down.
{circle around (8)} Place the sample in MPC, and after 2 minutes, transfer 30 μL of the supernatant into a new 1.5 mL tube.
2-4. Addition of A′ Base to the 3′ End of DNA Fragment
1) After adding all of the components of Table 5 below, lid off in PCR and perform at 37° C. for 30 minutes.
2) Sample Purification
{circle around (1)} Place the sample performed in 4. 1) above into a new 1.5 mL tube.
{circle around (2)} Place 180 μL of AMPure beads (1.8×), vortex it for 5 seconds, and perform incubation for 5 minutes at room temperature.
{circle around (3)} Place the sample in a magnetic particle concentrator (MPC), and after 3 minutes, discard the supernatant.
{circle around (4)} While the sample is in MPC, add 200 μL of 70% ethanol, and after 1 minute, discard the supernatant (repeat twice).
{circle around (5)} Completely dry the beads (5 minutes to 10 minutes).
{circle around (6)} Remove the sample tube from MPC, add 15 μL of nuclease-free water, and resuspend AMPure beads.
{circle around (7)} After incubating for 2 minutes to 3 minutes at room temperature, spin it down.
{circle around (8)} Place the sample in MPC, and after 2 minutes, transfer 13 μL of the supernatant into a new 1.5 mL tube.
2-5. Adapter Ligation to DNA Fragment
1) After adding all of the components of Table 6 below, lid off in PCR and perform at 20° C. for 15 minutes.
2) Sample Purification
{circle around (1)} Place the sample performed in 2-5. 1) above into a new 1.5 mL tube.
{circle around (2)} Place 180 μL of AMPure beads (1.8×), vortex it for 5 seconds, and perform incubation for 5 minutes at room temperature.
{circle around (3)} Place the sample in a magnetic particle concentrator (MPC), and after 3 minutes, discard the supernatant.
{circle around (4)} While the sample is in MPC, add 200 μL of 70% ethanol, and after 1 minute, discard the supernatant (repeat twice).
{circle around (5)} Completely dry the beads (5 minutes to 10 minutes).
{circle around (6)} Remove the sample tube from MPC, add 17 μL of nuclease-free water, and resuspend AMPure beads.
{circle around (7)} After incubating for 2 minutes to 3 minutes at room temperature, spin it down.
{circle around (8)} Place the sample in MPC, and after 2 minutes, transfer 15 μL of the supernatant into a new 1.5 mL tube.
2-6. Amplification of Adapter-Ligated Library
1) Prepare components in Table 7 below.
2) Amplify according to the Pre-LM PCR program below.
2-7. Sample Purification
{circle around (1)} Transfer the sample passed through the steps above into a new 1.5 mL tube.
{circle around (2)} Place 180 μL of AMPure beads (1.8×), vortex it for 5 seconds, and perform incubation for 5 minutes at room temperature.
{circle around (3)} Place the sample in a magnetic particle concentrator (MPC), and after 3 minutes, discard the supernatant.
{circle around (4)} While the sample is in MPC, add 200 μL of 70% ethanol, and after 1 minute, discard the supernatant (repeat twice).
{circle around (5)} Completely dry the beads (5 minutes to 10 minutes).
{circle around (6)} Remove the sample tube from MPC, add 17 μL of nuclease-free water, and resuspend AMPure beads.
{circle around (7)} After incubating for 2 minutes to 3 minutes at room temperature, spin it down.
{circle around (8)} Place the sample in MPC, and after 2 minutes, transfer 15 μL of the supernatant into a new 1.5 mL tube.
2-8. Assessment of Quality and Quantity
In order to confirm whether the library size was made within the intended range to optimize the efficiency of hybridization and to confirm the concentration to check if the amount at which hybridization could be attempted was achieved, the size and concentration of a library were measured using Agilent 4200 Tape Station and D1000 Screen Tape, and the result was shown in
2-9. Hybridization
1) Drill a hole in a 1.5 mL tube lid and dispense 200 ng or more and 500 ng or less of the prepped library.
2) Completely dry using SpeedVac (45° C.) (60 minutes).
3) After making a block mix as below, place 5.6 μL each into a dried tube, vortex lightly, and resuspend the library (prepped library).
4) After making a hybridization buffer with the composition of Table 10 below, dispense 0.2 mL into a PCR tube.
5) Perform RNase block dilution as Table 11 below.
6-1) The volume used for hybridization is different depending on the total size of a probe. It is because the concentration of the probe itself is different. Since the volume is different, the dilution ratio and the used volume of the RNase block should be different. The final concentration of RNase block is the same as 6-1 and 6-2. As a result, in the case of a general bait of 3 MB or more, it is applied to a large-sized probe targeting the entire exome.
6-2) The volume used for hybridization is different depending on the total size of a probe. It is because the concentration of the probe itself is different. Since the volume is different, the dilution ratio and the used volume of the RNase block should be different. The final concentration of RNase block is the same as 6-1 and 6-2. In the case of a general bait of 3 MB or less, it was applied in this experiment.
7) For gDNA library+block mix plate or a strip tube (prepped library), set up the PCR program as below and perform.
8) When the temperature of a prepped library (an entire set of libraries made available for NGS sequencing of gDNA samples used in the experiment) sample reaches 65° C., place the prepped library sample in a capture library (a set of probes including a target area of the size of 120 nt) and a hybridization mix (a reagent (buffer) to enable hybridization conditions) prepared above, and mix well by pipetting up and down for 3 to 5 times.
9) Close the lid well and hybridize for 24 hours at 65° C. (lid 105° C.) (up to 72 hours is possible).
2-10. Preparation of Magnetic Beads
1) Preheat SureSelect Wash Buffer #2 in a water bath (65° C.).
2) Vortex well Dynal MyOne Streptavidin T1 (Invitrogen) magnetic beads.
3) Dispense 50 μL per sample into a 1.5 mL tube.
4) Wash the beads as the following.
a. Place 200 μL of SureSelect Binding buffer and vortex lightly.
b. After spinning down, place it in DynaMag-2 device for 1 minute, and remove the supernatant.
c. Repeat the above process for a total of 3 times.
5) Resuspend the beads washed in 200 μL of SureSelect Binding buffer.
2-11. Hybridization Capture Selection with SureSelect
1) After mixing a hybridization mixture and a bead solution, mount it on a rotator and perform a reaction at room temperature for 30 minutes (check if the sample in the tube is mixed well).
2) After spinning down, place it in DynaMag-2 device for 3 minutes and remove the supernatant. 3) Place 200 μL of SureSelect Wash Buffer #1 and vortex until the beads are completely resuspended.
4) Incubate at room temperature for 15 minutes. Lightly vortex every 5 minutes to mix the beads well.
5) After spinning down, place it in DynaMag-2 device for 3 minutes and remove the supernatant.
6) Wash the beads as the following.
a. Place 200 μL of prewarmed SureSelect Wash Buffer #2 and vortex until the beads are completely resuspended.
b. Incubate at 65° C. for 10 minutes. Lightly vortex every 5 minutes to mix the beads well.
c. After spinning down, place it in DynaMag-2 device and remove the supernatant.
d. Repeat the above process for a total of 3 times. 7) Place 30 μL of nuclease-free water in MPC and resuspend.
2-12. Addition of Index Tags by Amplification After Hybridization (Post-Hybridization)
1) Prepare reagents as in Table 15 below.
2) Perform amplification according to the PCR program below.
2-13. Purification of Sample Using Agencourt AMPure XP beads
1) Vortex 50 μL of the amplified DNA library and 90 μL of AMPure beads (1.8×) and mix.
2) Incubate at room temperature for 5 minutes.
3) Place the sample in a magnetic particle concentrator (MPC), and after 3 minutes, discard the supernatant.
4) Add 500 μL of 70% ethanol while the sample is in MPC, and after 1 minute, discard the supernatant (repeat twice).
5) Completely dry the beads (5 minutes to 10 minutes).
6) Remove the sample tube from MPC, add 15 μL of nuclease-free water, and resuspend AMPure beads.
7) After incubating at room temperature for 2 minutes to 3 minutes, spin it down.
8) Place the sample in MPC, and after 2 minutes, place 30 μL of the supernatant into a new 1.5 mL tube.
2-14. Confirmation of Library
It is a library state after hybridization has been performed and only a target region has been amplified. In
2-15. Analysis of HBV Gene Insertion Site
The sequenced reads were mapped to the reference sequence (HBV+Human genome) to create a BAM file, which is a binary of the Sequence Alignment map (SAM) file. Among the mapped reads, the chimeric read that was split-mapped to HBV and the human genome was selected to identify break points. Next, for each point, a region that satisfied read count >10, average mapping quality (MQ) >20 was defined as an HBV-human integration site, and the location of HBV and the human genome was searched. Recurrently inserted human genes were collected, gene-annotation was performed and analyzed to discover the overall biological function of each gene, and the results were shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0139264 | Nov 2019 | KR | national |
