Method and apparatus for detecting type of human papillomavirus (HPV) by using amplicon-based next-generation sequencing technique

Abstract

A method for detecting a type of human papillomavirus (HPV) by using an amplicon-based next-generation sequencing technique, according to an aspect, comprises the steps of: obtaining a read by using a next-generation sequencing technique; aligning the obtained read to a reference to derive an alignment score; comparing the derived alignment score with at least one cutoff value to select a read to be used for reading; and detecting a type of human papillomavirus (HPV) on the basis of the selection.

Description

Method and device for detecting human papillomavirus (HPV) types using amplicon-based next-generation sequencing technology

[0001] A method and device for detecting the type of human papillomavirus (HPV) using an amplicon-based next-generation sequencing technique are disclosed.

[0002] Human papillomavirus (HPV) is a virus transmitted through sexual contact, and its significance lies in two key factors. First, HPV infection is the most common sexually transmitted infection in humans. It is the most prevalent sexually transmitted infection (STI) by a single factor, with an estimated 80% of all women being infected at least once in their lifetime. Therefore, regular HPV testing is essential for adult women, and HPV testing is a fundamental part of STI screening. Second, HPV infects epithelial cells at the site of human contact and causes hyperproliferation. This hyperproliferation often results in simple warts, genital warts, genital warts, and benign tumors such as condyloma accuminatum. In more severe cases, HPV is known to cause almost all cervical cancers, a significant number of oral, pharynx, and laryngeal cancers, and a large number of anal cancers.

[0003] Meanwhile, HPV testing can lead to early diagnosis of cancer and precancerous lesions of the cervix and anus, and it has been revealed that the HPV test has a better predictive sensitivity for cervical cancer than the Papanicolaou smear, which is the standard test for early detection of cervical cancer. Accordingly, the HPV test is recognized as a cervical cancer screening test in many countries, including the U.S. FDA.

[0004] Currently, about 120 types of HPV are known according to their subtypes or genotypes, and the entire genetic sequence and structure of 83 of them have been identified. About 40 types of HPV are so-called anogenital or genital type HPV, which invade the skin and mucous membranes of the anus and vulva, that is, the vagina, cervix, urethra, and penis. Most HPV infections are latent and asymptomatic, but some cause warts. Others cause precancerous lesions such as high-grade squamous intraepithelial lesions (HSIL) or cervical intraepithelial neoplasms, some of which progress to cancer. HPV types that cause precancerous lesions and cancer are called high-risk HPV, while those that do not are called low-risk HPV. Some researchers also categorize HPV into high-risk, intermediate-risk, and low-risk groups.

[0005] High-risk HPV types include HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 82. Low-risk HPV types include HPV types 6, 11, 34, 40, 42, 43, 44, 54, 55, 61, 62, 72, and 81. Probable high-risk types include HPV types 26, 53, 66, 67, 69, 70, and 73. Other types that have not been precisely classified include HPV types 7, 10, 27, 30, 32, 57, 83, 84, and 91. Worldwide, it is reported that 49.9% of cervical cancer patients are infected with HPV type 16, 13.7% with HPV type 18, 7.2% with HPV types 31, 33, and 35, and 8.4% with HPV type 45.

[0006] HPV infection is difficult to diagnose through culture, staining, tissue biopsy, or immunological tests, and only genetic testing can provide an accurate diagnosis. Recently, so-called genotyping analysis, which identifies not only the presence of HPV but also its type, has become widely used for HPV infection testing. The so-called "gold standard" test involves genotyping the product using an HPV DNA microarray after PCR. However, research on compositions, kits, and methods capable of simultaneously detecting genotypes for various HPV types while testing for HPV is limited.

[0007] The present invention aims to solve the above-mentioned problems and other problems.

[0008] The present invention provides a method and device for detecting human papillomavirus (HPV) types using amplicon-based next-generation sequencing technology. Furthermore, the present invention provides a computer-readable recording medium containing a program for executing the method on a computer. The technical challenges to be addressed are not limited to the technical challenges described above, and other technical challenges may exist.

[0009] One embodiment of the present invention for realizing the above-described task is a control method performed by a processor in a terminal including a communication unit and a processor.

[0010] The above control method comprises: a step of obtaining a new power generation profile PP(k) composed of power generation generated by a power generation system during a monitoring period; a step of obtaining a cluster index CI(k) of the new power generation profile PP(k) by performing hierarchical binary clustering (hereinafter referred to as 'HBC'); a step of inputting the cluster index CI(k) into an artificial intelligence that has learned a time series change of the cluster index to predict a future cluster index CI(k+1); a step of obtaining a future power generation profile PP(k+1) using the future cluster index CI(k+1); And a step of predicting future power generation to be generated in the power generation system using the future power generation profile PP(k+1), and in the step of obtaining the cluster index CI(k), if the new power generation profile PP(k) satisfies a predetermined condition, the number of clusters classified by the hierarchical binary clustering (HBC) is a method for detecting a type of human papillomavirus (HPV) using an amplicon-based next-generation sequencing technique according to one aspect of the new power generation, the method comprising: a step of obtaining a read using a next-generation sequencing technique; a step of aligning the obtained read to a reference to derive an alignment score; a step of selecting a read to be used for reading by comparing the derived alignment score with at least one cutoff value; and a step of detecting a human papillomavirus (HPV) type based on the selection.

[0011] A computer-readable recording medium according to another aspect includes a recording medium having recorded thereon a method for executing the above-described method on a computer.

[0012] According to another aspect, a computing device comprises at least one memory; and at least one processor; wherein the processor aligns reads obtained through a next-generation sequencing technique to a reference to derive an alignment score, compares the derived alignment score with at least one cutoff value to select reads to be used for reading, and detects a human papillomavirus (HPV) type based on the selection.

[0013] According to the present invention, by detecting the type of human papillomavirus (HPV) using an amplicon-based next-generation base sequence analysis technique, increased data analysis efficiency and accuracy can be provided.

[0014] Additionally, as the speed and accuracy of data analysis increases, it can help select information about the type of human papillomavirus (HPV) quickly and accurately.

[0015] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0016] FIG. 1 is a diagram illustrating an example of a system for detecting a type of human papillomavirus (HPV) according to one embodiment.

[0017] FIG. 2 is a configuration diagram illustrating an example of a user terminal according to one embodiment.

[0018] FIG. 3 is a flowchart illustrating an example of a method for detecting a type of human papillomavirus (HPV) according to one embodiment.

[0019] FIG. 4 is a diagram illustrating an example of a read obtained through a next-generation base sequence analysis technique according to one embodiment.

[0020] FIG. 5 is a diagram illustrating an example of an alignment score derived by aligning a lead to a reference according to one embodiment.

[0021] FIG. 6 is a diagram illustrating an example of a lead designating a human papillomavirus (HPV) type according to one embodiment.

[0022] The terms used in the examples are selected from widely used, current terms, as much as possible. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Furthermore, in certain cases, the applicant may arbitrarily select terms, in which case their meanings will be described in detail in the relevant description. Therefore, the terms used in the specification should be defined based on their intended meaning and the overall content of the specification, rather than simply their names.

[0023] Terms that include ordinal numbers, such as first, second, etc., may be used to describe various components, but the components are not limited by these terms. These terms are used solely to distinguish one component from another.

[0024] When a component is said to be "connected" or "connected" to another component, it means that any part of the life cycle that is directly connected or connected to that other component is said to "include" that component, unless otherwise stated, this does not exclude other components, but rather may include other components.

[0025] In the embodiments of the present disclosure, terms such as "module," "unit," "part," etc. are terms used to refer to components that perform at least one function or operation, and such components may be implemented as hardware or software, or a combination of hardware and software. In addition, a plurality of "modules," "units," "parts," etc. may be integrated into at least one module or chip and implemented as at least one processor, except in cases where each needs to be implemented as a separate, specific hardware.

[0026] The term "next-generation sequencing" or "NGS" or "next-generation sequencing" as used herein refers to any sequencing method that determines the nucleotide sequence of either individual nucleic acid molecules (e.g., in single-molecule sequencing) or clonally expanded proxies of individual nucleic acid molecules in a high-throughput manner (e.g., 10, 100, 1000, or more molecules are sequenced simultaneously). In one embodiment, the relative abundance of a nucleic acid species in a library can be estimated by counting the relative number of occurrences of its cognate sequences in data generated by a sequencing experiment. Next-generation sequencing methods are well known in the art and are described, for example, in [Metzker, M. (2010) Nature Biotechnology Reviews 11:31-46]. Next-generation sequencing can detect variants present in less than 5% of the nucleic acids in a sample.

[0027] The term "acquiring" in the present invention refers to obtaining possession of a physical entity or value, such as a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Indirectly acquiring" refers to performing a process to obtain the physical entity or value (e.g., performing a synthesis or analysis method). "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value).

[0028] Indirectly obtaining a physical entity involves performing a treatment that involves a physical change in a physical substance, such as a starting material. Representative changes include forming a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, or performing a chemical reaction that involves breaking or forming a covalent or non-covalent bond. Indirectly obtaining a value involves performing a treatment that involves a physical change in a sample or other substance, such as performing an analytical procedure that involves a physical change in a substance, such as a sample, an analyte, or a reagent, or performing an analytical method, such as a method that involves one or more of the following: separating or purifying a substance, such as an analyte or a fragment or other derivative thereof, from another substance; combining the analyte or a fragment or other derivative thereof with another substance, such as a buffer, solvent, or reactant; or changing the structure of the analyte or a fragment or other derivative thereof, for example by breaking or forming a covalent or non-covalent bond between first and second atoms of the analyte; Alternatively, it includes changing the structure of the reagent or a fragment or other derivative thereof, for example by breaking or forming a covalent or non-covalent bond between the first and second atoms of the reagent.

[0029] The term "obtaining a lead" in the present invention refers to obtaining possession of a nucleotide sequence or amino acid sequence by "directly obtaining" or "indirectly obtaining" the lead. "Directly obtaining" a lead means performing a process (e.g., performing a synthesis or analysis method) to obtain the sequence, such as performing a sequencing method (e.g., performing a next-generation sequencing (NGS) method). "Indirectly obtaining" a lead means receiving the sequence or information or knowledge of the sequence from another party or source (e.g., a third-party laboratory that directly obtained the sequence). The obtained lead need not be a complete sequence; for example, sequencing at least one nucleotide or obtaining information or knowledge that identifies one or more of the alterations disclosed herein, such as those present in a subject, constitutes obtaining the sequence.

[0030] Directly obtaining a lead involves performing a process involving a physical change in a physical material, such as a starting material, such as a tissue or cell sample, such as a biopsy or an isolated nucleic acid (e.g., DNA or RNA) sample. Representative changes include shearing or fragmenting two or more starting materials, such as creating physical entities from genomic DNA fragments (e.g., isolating a nucleic acid sample from tissue); combining two or more distinct entities into a mixture; performing a chemical reaction involving breaking or forming covalent or non-covalent bonds. Directly obtaining a value involves performing a process involving a physical change in a sample or other material, such as those described above.

[0031] The term 'detection' used in the present invention means being able to identify human papillomavirus in a sample and simultaneously isolate and identify the genotype of human papillomavirus.

[0032] Human papillomavirus (HPV) types can be broadly classified into low-risk and high-risk types, and viruses belonging to these genetically distinct types are collectively referred to as "human papillomaviruses" or "HPV." "Low-risk" HPV types include, but are not limited to, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV61, HPV70, HPV72, and HPV81. "High-risk" HPV types include, but are not limited to, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV68, HPV73, and HPV82.

[0033] Below, embodiments are described in detail with reference to the attached drawings. However, the embodiments may be implemented in various different forms and are not limited to the examples described herein.

[0034] FIG. 1 is a diagram illustrating an example of a system for detecting a type of human papillomavirus (HPV) according to one embodiment.

[0035] Referring to FIG. 1, the system (1) includes a user terminal (10) and a server (20). For example, the user terminal (10) and the server (20) may be connected via wired or wireless communication to transmit and receive data (e.g., reads obtained through a next-generation base sequence analysis technique) between each other.

[0036] For convenience of explanation, FIG. 1 illustrates that the system (1) includes a user terminal (10) and a server (20), but is not limited thereto. For example, the system (1) may include other external devices (not shown), and the operations of the user terminal (10) and the server (20) described below may be implemented by a single device (e.g., the user terminal (10) or the server (20)).

[0037] The user terminal (10) may be a computing device equipped with a display device and a device for receiving user input (e.g., a keyboard, a mouse, etc.), and including memory and a processor. For example, the user terminal (10) may be, but is not limited to, a notebook PC, a desktop PC, a laptop, a tablet computer, a smart phone, etc.

[0038] The server (20) may be a device that communicates with external devices (not shown), including a user terminal (10). For example, the server (20) may be a device that stores various data, including information about leads and information about human papillomavirus (HPV), and in some cases, may be a device with its own computing capabilities. For example, the server (20) may be a cloud server, but is not limited thereto.

[0039] A system (1) according to one embodiment detects the type of human papillomavirus (HPV). Specifically, a user terminal (10) acquires reads using a next-generation sequencing technique, aligns the acquired reads to a reference, and derives an alignment score. Then, the user terminal (10) compares the derived alignment score with at least one cutoff value to select reads to be used for reading, and detects the type of human papillomavirus (HPV) based on the selection. Therefore, a user (30) can confirm the type of human papillomavirus (HPV) using an amplicon-based next-generation sequencing technique.

[0040] Hereinafter, with reference to FIGS. 2 to 9, examples of detecting the type of human papillomavirus (HPV) using an amplicon-based next-generation base sequence analysis technique by a user terminal (10) will be described. Meanwhile, as described above with reference to FIGS. 2 to 9, the operations described below may also be performed by a server (20).

[0041] FIG. 2 is a configuration diagram illustrating an example of a user terminal according to one embodiment.

[0042] Referring to FIG. 2, the user terminal (100) includes a processor (110) and a memory (120). Although not illustrated in the drawing, general-purpose components may be included. For example, input / output interfaces and / or communication modules may be included. For convenience of explanation, only components related to the present invention are illustrated in FIG. 2. Therefore, in addition to the components illustrated in FIG. 2, other general-purpose components may be included in the user terminal (100). Furthermore, it will be apparent to those skilled in the art that the processor (110) and memory (120) illustrated in FIG. 2 may be implemented as independent devices.

[0043] The processor (110) can process computer program commands by performing basic arithmetic, logic, and input / output operations. Here, the commands can be provided from memory (120) or an external device (e.g., a server (20), etc.). In addition, the processor (110) can generally control the operations of other components included in the user terminal (100).

[0044] In particular, the processor (110) detects the type of human papillomavirus (HPV) using an amplicon-based next-generation sequencing technique. Specifically, the processor (110) obtains reads using the next-generation sequencing technique. Then, the processor (110) aligns the reads to a reference to derive an alignment score. Then, the processor (110) compares the derived alignment score with at least one cutoff value to select reads to be used for reading. Then, the processor (110) detects the type of human papillomavirus (HPV) based on the selection.

[0045] Specific examples of how the processor (110) operates according to one embodiment are described with reference to FIGS. 3 to 9.

[0046] The processor (110) may be implemented as an array of a plurality of logic gates, or may be implemented as a general-purpose microprocessor and a memory storing a program that can be executed on the microprocessor. For example, the processor (110) may include a general-purpose processor, a central processing unit (CPU), a microprocessor, a digital signal processor (DSP), a controller, a microcontroller, a state machine, etc. In some environments, the processor (110) may include an application-specific integrated circuit (ASIC), a programmable logic device (PLD), a field programmable gate array (FPGA), etc. For example, the processor (110) may also refer to the creation of processing devices, such as the creation of a digital signal processor (DSP) and a microprocessor, the creation of a plurality of microprocessors, the creation of one or more microprocessors combined with a digital signal processor (DSP) core, or the creation of any other such configuration.

[0047] The memory (120) may include any non-transitory computer-readable recording medium. As an example, the memory (120) may include a non-permanent mass storage device such as a random access memory (RAM), a read only memory (ROM), a disk drive, a solid state drive (SSD), a flash memory, etc. As another example, the non-permanent mass storage device such as a ROM, an SSD, a flash memory, a disk drive, etc. may be a separate permanent storage device distinct from the memory. In addition, the memory (210) may store an operating system (OS) and at least one program code (e.g., code for the processor (110) to perform an operation to be described later with reference to FIGS. 3 to 9).

[0048] These software components may be loaded from a computer-readable recording medium separate from the memory (120). This separate computer-readable recording medium may be a recording medium that can be directly connected to the user terminal (100), and may include, for example, a computer-readable recording medium such as a floppy drive, a disk, a tape, a DVD / CD-ROM drive, a memory card, etc. Alternatively, the software components may be loaded into the memory (120) through a communication module other than a computer-readable recording medium. For example, at least one program may be loaded into the memory (120) based on a computer program (e.g., a computer program for the processor (110) to perform the operations described below with reference to FIGS. 3 to 9) that is installed by files provided by developers or a file distribution system that distributes installation files of applications through a communication module.

[0049] The input / output interface may be a means for interfacing with a device (e.g., a keyboard, a mouse, etc.) for input or output that may be connected to or included in the user terminal (100). The input / output interface may be configured separately from the processor (110) or may be configured to be included in the processor (110).

[0050] The communication module may provide a configuration or function for the server (20) and the user terminal (100) to communicate with each other via a network. In addition, the communication module may provide a configuration or function for the user terminal (100) to communicate with other external devices. For example, control signals, commands, data, etc. provided under the control of the processor (110) may be transmitted to the server (20) and / or the external device via the communication module and the network.

[0051] Meanwhile, although not shown in FIG. 2, the user terminal (100) may further include a display device. Alternatively, the user terminal (100) may be connected to an independent display device via wired or wireless communication to transmit and receive data between the two.

[0052] FIG. 3 is a flowchart illustrating an example of a method for detecting a human papillomavirus (HPV) type according to one embodiment.

[0053] Referring to FIG. 3, the method for detecting a human papillomavirus (HPV) type is composed of steps that are processed in time series in the user terminal (10, 100) or processor (110) illustrated in FIGS. 1 and 2. Therefore, even if the content is omitted below, the content described above with respect to the user terminal (10, 100) or processor (110) illustrated in FIGS. 1 and 2 can also be applied to the method for detecting a human papillomavirus (HPV) type of FIG. 3.

[0054] In step 310, the processor (110) acquires a read using a next-generation base sequence analysis technique. As an example, the processor (110) may acquire a read characterized by being stored in a fastq file format, but is not limited thereto.

[0055] Hereinafter, with reference to FIG. 4, an example of a processor (110) acquiring a lead will be described.

[0056] FIG. 4 is a diagram illustrating an example of a read obtained through a next-generation base sequence analysis technique according to one embodiment.

[0057] Referring to FIG. 4, the processor (110) acquires lead information stored in a fastq file format. The lead information stored in a fastq file format consists of four rows. The processor (110) can acquire this lead information from information input by the user or from information already set.

[0058] Referring back to FIG. 3, in step 320, the processor (110) aligns the reads to the reference to derive an alignment score. Specifically, the processor (110) aligns the reads obtained through the next-generation base sequence analysis technique to the reference, and derives an alignment score of the aligned reads and the reference. Here, the alignment score is a score assigned to base pairs corresponding to identical nucleic acid bases, and may be calculated by using a sequence alignment program such as MUMMER or BWA to calculate the number of aligned bases, but is not limited thereto. As an example, if 11 out of 12 base sequences match when comparing the reference sequence and the production sequence, the alignment score may be calculated as 11 points. As another example, if the base sequences do not match, the alignment score may be calculated by assigning a penalty score.

[0059] Hereinafter, with reference to FIG. 5, an example in which a processor (110) aligns a lead to a reference and derives an alignment score will be described.

[0060] FIG. 5 is a diagram illustrating an example of an alignment score derived by aligning a lead to a reference according to one embodiment.

[0061] Referring to FIG. 5, the processor (110) aligns reads obtained through a next-generation base sequence analysis technique to a reference to derive an alignment score. Specifically, the processor (110) derives an alignment score by aligning reads 1 and 2 to the reference, respectively. It can be seen that the alignment score of reference 2 for read 1 is the highest at 82 points, and the alignment score of reference 3 for read 2 is the highest at 72 points.

[0062] Referring again to FIG. 3, at step 330, the processor (110) compares the alignment score with a cutoff value to select a lead to use for reading.

[0063] A reference may refer to a target for comparison of reads to determine whether any changes have occurred from an existing genetic sequence. References include a human papillomavirus (HPV) type reference and an internal control reference. As an example, the reference may be a human papillomavirus (HPV) type reference. Here, the human papillomavirus (HPV) type reference may be a collection of sequences that distinguish 200 types of human papillomavirus (HPV), but is not limited thereto. As another example, the reference may be an internal control reference. Here, the internal control reference is a control group for confirming sequences that are amplified and detected as sequences similar to the human papillomavirus (HPV) sequence. Human sequences are used, and may be, but are not limited to, hg19 or hg38 of NCBI.

[0064] As an example, the processor (110) can align the reads to a human papillomavirus (HPV) type reference to derive an alignment score, and select reads exceeding a cutoff value for the human papillomavirus (HPV) type reference to be used for reading, and reads below the cutoff value for the human papillomavirus (HPV) type reference to be excluded from reading. Here, the cutoff value for the human papillomavirus (HPV) type reference is selected from the group consisting of 40 to 80 points. Preferably, the cutoff value for the human papillomavirus (HPV) type reference is 50 points, and more preferably, the cutoff value for the human papillomavirus (HPV) type reference is 60 points.

[0065] As another example, the processor (110) can align reads that are not aligned to a human papillomavirus (HPV) type reference to an Internal Control reference to derive an alignment score, and select reads that are above a cutoff value for the Internal Control reference to be used for reading, and reads that are below the cutoff value for the Internal Control reference to be excluded from reading. Here, the cutoff value for the Internal Control reference is selected from the group consisting of 20 to 60 points. Preferably, the cutoff value for the Internal Control reference is 30 points, and more preferably, the cutoff value for the Internal Control reference is 40 points.

[0066] Referring again to FIG. 3, at step 340, the processor (110) detects a human papillomavirus (HPV) type. Specifically, the processor (110) designates a human papillomavirus (HPV) type for the selected read, and detects the human papillomavirus (HPV) type based on the human papillomavirus (HPV) type designated for each read.

[0067] First, the processor (110) assigns a human papillomavirus (HPV) type to the selected read. Specifically, for a read whose alignment score exceeds a cutoff value for a human papillomavirus (HPV) type reference, the type of the human papillomavirus (HPV) type reference exceeding the cutoff value is assigned as the human papillomavirus (HPV) type of the read.

[0068] Hereinafter, with reference to FIG. 6, an example in which the processor (110) designates a human papillomavirus (HPV) type for each lead will be described.

[0069] FIG. 6 is a diagram illustrating an example of a lead designating a human papillomavirus (HPV) type according to one embodiment.

[0070] Referring to FIG. 6, the processor (110) aligns the reads to the human papillomavirus (HPV) type reference and selects reads having an alignment score exceeding 50 points. For the selected reads, the type of the human papillomavirus (HPV) type reference having an alignment score exceeding 50 points is designated as the human papillomavirus (HPV) type of the corresponding read. Accordingly, Read 1 is designated as HPV type 16, and the remaining reads are designated in the same manner. For Read 8, “IC-1” means that the read is not aligned to the human papillomavirus (HPV) type reference, so it is aligned to the Internal Control reference, and the type of the Internal Control reference having an alignment score exceeding 30 points is designated as the read’s type. For Read 11, “No Align / Under Score” means that the read is not aligned to the human papillomavirus (HPV) type reference and is not aligned to the Internal Control reference.

[0071] Referring again to FIG. 3, the processor (110) detects a human papillomavirus (HPV) type based on a human papillomavirus (HPV) type designated for each lead.

[0072] As an example, the processor (110) detects the corresponding type as a human papillomavirus (HPV) type when the number of leads designated as the same human papillomavirus (HPV) type is greater than or equal to a cutoff value. Here, the cutoff value may be selected from a group consisting of 30 to 60. Preferably, the number of leads designated as the same human papillomavirus (HPV) type is 40, and more preferably, the number of leads designated as the same human papillomavirus (HPV) type is 50.

[0073] As another example, the processor (110) detects the human papillomavirus (HPV) reading result as negative when the number of reads designated as the same human papillomavirus (HPV) type is less than a cutoff value. Here, the cutoff value can be selected from a group consisting of 30 to 60. Preferably, the number of reads designated as the same human papillomavirus (HPV) type is 40, and more preferably, the number of reads designated as the same human papillomavirus (HPV) type is 50.

[0074] As another example, when the processor (110) detects the reading result of human papillomavirus (HPV) as negative, if the total number of Internal Control reads is 5 or more, it detects it as a valid value, and if the total number of Internal Control reads is less than 5, it detects it as an invalid value. The Internal Control read refers to a read that is not aligned to the human papillomavirus (HPV) type reference, but is aligned to the Human reference, which is the Internal Control reference.

[0075] As another example, the processor (110) detects the read result of human papillomavirus (HPV) as negative when the value of MatchRead / TotalRead is less than the cutoff value of MatchRead / TotalRead. Here, the cutoff value of MatchRead / TotalRead can be selected from the group consisting of 0.1 to 0.4. Preferably, the cutoff value of MatchRead / TotalRead is 0.3, and more preferably, the cutoff value of MatchRead / TotalRead is 0.2. Here, MatchRead refers to the number of reads whose alignment score exceeds the cutoff value for the human papillomavirus (HPV) type reference when aligned to the human papillomavirus (HPV) type reference (TypeRead) and the number of reads whose alignment score exceeds the cutoff value for the internal control reference when aligned to the internal control reference (TotalICRead). Here, TotalRead refers to the total number of reads initially acquired through next-generation sequencing.

[0076] As another example, the processor (110) detects the read result of human papillomavirus (HPV) as negative when the value of TotalICRead / MatchRead exceeds the cutoff value of TotalICRead / MatchRead. Here, the cutoff value of TotalICRead / MatchRead can be selected from the group consisting of 0.6 to 0.9. Preferably, the cutoff value of TotalICRead / MatchRead is 0.7, and more preferably, the cutoff value of TotalICRead / MatchRead is 0.8. Here, MatchRead refers to the number of reads whose alignment score exceeds the cutoff value for the human papillomavirus (HPV) type reference when aligned to the human papillomavirus (HPV) type reference (TypeRead) and the number of reads whose alignment score exceeds the cutoff value for the internal control reference when aligned to the internal control reference (TotalICRead). Here, TotalICRead refers to the number of reads whose alignment score exceeds the cutoff value for the internal control reference when aligned to the internal control reference.

[0077] As described above, the processor (110) detects the type of human papillomavirus (HPV) using an amplicon-based next-generation sequencing technique. Accordingly, the user (30) can confirm the type of human papillomavirus (HPV) using an amplicon-based next-generation sequencing technique.

[0078] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0079] Example 1. Application of cutoffs for human papillomavirus (HPV) type reference alignment scores and internal control reference alignment scores.

[0080] Figure 3 shows a flowchart according to Example 1 of the present invention.

[0081] Specifically, next-generation sequencing (NGS) was performed on materials spiked with HPV DNA to obtain reads. The experiment used an S5 sequencer (Thermo Fisher), but this is not limited to this, and sequencers on the Illumina platform can also be used. The same procedure was followed in other examples and experimental examples below.

[0082] Acquired leads and human papillomavirus (HPV) type references and internal control references were prepared.

[0083] First, the acquired reads were aligned to the human papillomavirus (HPV) type reference and the internal control reference, and the reads to be used for reading were selected based on the alignment results. Here, the human papillomavirus (HPV) type reference corresponds to a reference having each human papillomavirus (HPV) type, and 200 human papillomavirus (HPV) type references were used in this experiment. The human papillomavirus (HPV) type references were downloaded and used from https: / pave.niaid.nih.gov. Here, the internal control reference is the internal control, and in this experiment, hg19 or hg38 was downloaded and used as the internal control reference from NCBI. The selection process is as follows.

[0084] First, the acquired reads were aligned to the human papillomavirus (HPV) typing reference. Aligned and unaligned reads were distinguished. Among the aligned reads, those with an alignment score exceeding 60 were included in Group 1, while those with a score below 60 were excluded from the HPV typing analysis.

[0085] And, human papillomavirus (HPV) type was assigned to the leads included in group 1. The lead is assigned to the type of human papillomavirus (HPV) type reference with an alignment score exceeding 60 points. If there are two or more human papillomavirus (HPV) type references with an alignment score exceeding 60 points for one lead, the human papillomavirus (HPV) type reference with the highest score was detected as the representative human papillomavirus (HPV) type reference and assigned the type.

[0086] Reads that did not align to the human papillomavirus (HPV) typing reference were aligned to the internal control reference. Reads with an alignment score exceeding 40 points were included in Group 2, while reads with an alignment score below 40 points were excluded from the human papillomavirus (HPV) typing analysis.

[0087] Here, the human papillomavirus (HPV) type is detected by reading the above groups 1 and 2. Specifically, using the reads included in group 1, a type with 50 or more aligned reads is detected as a human papillomavirus (HPV) type. At this time, if the number of reads included in group 2 is 5 or more, it is determined as a valid experimental value.

[0088] Example 2. Applying cutoffs to MatchRead / TotalRead and TotalICRead / MatchRead

[0089] Specifically, leads were obtained by performing amplicon-based next-generation sequencing (NGS) on materials spiked with HPV DNA.

[0090] First, the selected reads were aligned to the human papillomavirus (HPV) typing reference and the internal control reference. Here, if the number of reads with HPV typing exceeds 50, MatchRead / TotalRead and TotalICRead / MatchRead were calculated.

[0091] Here, MatchRead refers to the number of reads whose alignment score exceeds the cutoff value for the human papillomavirus (HPV) type reference when aligned to the human papillomavirus (HPV) type reference (TypeRead) and the number of reads whose alignment score exceeds the cutoff value for the internal control reference when aligned to the internal control reference (TotalICRead) combined.

[0092] Here, TotalRead refers to the total number of reads initially acquired through next-generation sequencing. If MatchRead / TotalRead is 0.2 or higher or TotalICRead / MatchRead is 0.8 or lower, the type with 50 or more reads is detected as the corresponding human papillomavirus (HPV) type. If there are two or more types with 50 or more reads, all types with 50 or more reads are detected as the corresponding human papillomavirus (HPV) type.

[0093] If the number of reads with assigned human papillomavirus (HPV) types is less than 50, and if MatchRead / TotalRead is less than 0.2 and TotalICRead / MatchRead is greater than 0.8, check whether there are 5 or more total Internal Control reads. Here, Internal Control reads refer to the number of reads (TotalICRead) that exceed the cutoff value for the Internal Control reference when aligned to the Internal Control reference. If there are 5 or more Internal Control reads, the HPV typing result is negative and classified as a valid value. If there are less than 5 reads, the HPV typing result is negative and classified as an invalid value.

[0094] Comparative Example 1.

[0095] In Comparative Example 1, a cutoff was not applied because a read was obtained through amplicon-based next-generation sequencing (NGS) on a material spiked in HPV DNA.

[0096] Experimental Example 1. Reading results of Examples 1, 2 and Comparative Example 1

[0097] The results of reading the human papillomavirus (HPV) type from the results of Examples 1 and 2 and Comparative Example 1 above, and showing the sensitivity, specificity, and accuracy, are shown in Table 1 below.

[0098] Accuracy (%) Comparative Example 124.35 Example 159.82~65.38 Example 259.82~66.23

[0099] According to Table 1 above, the accuracy of Comparative Example 1 was 24.35%, while the accuracy of Example 1 was 59.82 to 65.38%, and the accuracy of Example 2 was 59.82 to 66.23%. That is, it was found that the accuracy results of Examples 1 and 2 were significantly higher than that of Comparative Example 1. These results mean that efficient reading is possible when applying the cutoff of the human papillomavirus (HPV) type reference and the Internal Control reference alignment score, or applying the cutoff and the MatchRead / TotalRead and TotalICRead / MatchRead cutoffs.

[0100] Meanwhile, the above-described method can be written as a program that can be executed on a computer, and can be implemented on a general-purpose digital computer that runs the program using a computer-readable recording medium. In addition, the structure of the data used in the above-described method can be recorded on a computer-readable recording medium through various means. The computer-readable recording medium includes storage media such as magnetic storage media (e.g., ROM, RAM, USB, floppy disk, hard disk, etc.) and optical reading media (e.g., CD-ROM, DVD, etc.).

[0101] Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from the essential characteristics of the above-described invention. Therefore, the disclosed methods should be considered illustrative rather than restrictive. The scope of the claims, not the foregoing description, is defined by the scope of the patent, and should be interpreted to encompass all differences within the scope equivalent thereto.

Claims

1. Step of obtaining leads using next-generation base sequence analysis techniques; A step of aligning the acquired leads to a reference to derive an alignment score; A step of selecting a lead to be used for reading by comparing the alignment score derived above with at least one cutoff value; and A method for detecting a type of human papillomavirus (HPV) using an amplicon-based next-generation sequencing technique, comprising: a step of detecting a type of human papillomavirus (HPV) based on the above selection.

2. In paragraph 1, The above reference is a method including a human papillomavirus (HPV) type reference and an Internal Control reference.

3. In paragraph 1, The above selection step is, A method for selecting a readout read by comparing an alignment score derived from aligning the acquired read to a human papillomavirus (HPV) type reference and a cutoff value for the human papillomavirus (HPV) type reference.

4. In paragraph 3, A method of selecting a cutoff value for the above human papillomavirus (HPV) type reference as 50 points, in which reads having an alignment score exceeding 50 points are used for reading, and reads having an alignment score of 50 points or less are excluded from reading.

5. In paragraph 3, The above selection step is, A method of selecting by comparing an alignment score derived by aligning reads that are not aligned to the above human papillomavirus (HPV) type reference to the Internal Control reference and a cutoff value for the Internal Control reference.

6. In paragraph 5, The above selection step is, A method of selecting in which the cutoff value for the above Internal Control reference is 30 points, and leads having an alignment score exceeding 30 points are used for reading, and leads having an alignment score below 30 points are excluded from reading.

7. In paragraph 3, The above detection step is, A method of designating, for a read whose alignment score exceeds a cutoff value for a human papillomavirus (HPV) type reference, the type of the human papillomavirus (HPV) type reference exceeding the cutoff value as the human papillomavirus (HPV) type of the read.

8. In paragraph 7, The above detection step is, A method in which the cutoff value for the above human papillomavirus (HPV) type reference is 50 points.

9. In paragraph 7, The above detection step is, A method of designating the type of the human papillomavirus (HPV) type reference having the highest score as the human papillomavirus (HPV) type of the lead when there are two or more human papillomavirus (HPV) type references exceeding the above cutoff value.

10. In paragraph 7, The above detection step is, A method for detecting a type as a human papillomavirus (HPV) type when the number of leads designated as the same human papillomavirus (HPV) type is 40 or more.

11. In Article 10, The above detection step is, A method for detecting a negative human papillomavirus (HPV) reading result when the number of reads designated as the same human papillomavirus (HPV) type is less than 40.

12. In paragraph 11, The above detection step is, A method of detecting a negative result for human papillomavirus (HPV) by detecting a valid value if the total number of Internal Control reads is 5 or more, and detecting an invalid value if the total number of Internal Control reads is less than 5.

13. In paragraph 10, The above detection step is, A method for detecting a negative result for human papillomavirus (HPV) when the value of MatchRead / TotalRead is less than 0.

4. (MatchRead = The number of reads whose alignment score exceeded the cutoff value for the human papillomavirus (HPV) type reference when aligned to the human papillomavirus (HPV) type reference and the number of reads whose alignment score exceeded the cutoff value for the Internal Control reference when aligned to the Internal Control reference, TotalRead = The total number of reads obtained through the initial next-generation sequencing) 14. In paragraph 10, The above detection step is, A method for detecting a negative result for human papillomavirus (HPV) when the value of TotalICRead / MatchRead exceeds 0.

6. (MatchRead = The number of reads whose alignment score exceeds the cutoff value for the human papillomavirus (HPV) type reference when aligned to the human papillomavirus (HPV) type reference and the number of reads whose alignment score exceeds the cutoff value for the internal control reference when aligned to the internal control reference, TotalICRead = The number of reads whose alignment score exceeds the cutoff value for the Internal Control reference when aligned to the Internal Control reference.

15. In paragraph 13, The above detection step is, A method of detecting a negative result for human papillomavirus (HPV) by detecting a valid value if the total number of Internal Control reads is 5 or more, and detecting an invalid value if the total number of Internal Control reads is less than 5.

16. In paragraph 14, The above detection step is, A method of detecting a negative result for human papillomavirus (HPV) by detecting a valid value if the total number of Internal Control reads is 5 or more, and detecting an invalid value if the total number of Internal Control reads is less than 5.

17. A computer-readable recording medium having recorded thereon a program for executing the method according to Article 1 on a computer.

18. At least one memory; and comprising at least one processor; The above processor, A computing device that aligns reads obtained through a next-generation base sequence analysis technique to a reference to derive an alignment score, compares the derived alignment score with at least one cutoff value to select reads to be used for reading, and detects a human papillomavirus (HPV) type based on the selection.

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