Method and apparatus for identifying microsatellite instability using primer combination

Abstract

The present application provides a method for identifying microsatellite instability using a primer combination, the method comprising the steps of: (1) setting a plurality of MSI target sequences; (2) for a plurality of gene reads including one or more sequences among the plurality of MSI target sequences, forming a primer combination group for each gene read having the same start point and end point; (3) for each MSI target sequence included in the primer combination group, identifying whether gene deletion is present; and (4) for the MSI target sequence in which the presence of gene deletion has been identified, determining whether "False shortening" or "True shortening" is present through comparisons in the primer combination group.

Description

Method and device for detecting microsatellite instability using a combination of start and stop sequences

[0001] This application claims priority to Korean Patent Application No. 10-2024-0097990, filed July 24, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a method and device for identifying microsatellite instability using a combination of start and stop conditions.

[0003] Microsatellites are short repetitive DNA sequences scattered throughout chromosomes, and the length of these microsatellites varies from person to person. Microsatellite instability refers to differences in length between normal and cancerous tissues in the same person due to insertions or deletions of repetitive sequences in the microsatellites. In other words, microsatellite instability is a phenomenon in which errors that occur during DNA replication are not corrected due to abnormalities in the DNA mismatch repair system, which accelerates the accumulation of point mutations, resulting in changes in the length of repetitive microsatellite sequences ubiquitously distributed throughout all genes. When the gene repair system is paralyzed due to microsatellite instability, the ability to relieve stress caused by chronic inflammation is reduced, which can lead to tumor development.

[0004] Microsatellite instability is generally divided into two types: high-level MSI (MSI-H), in which more than 30% of the measured microsatellite markers have microsatellite instability, also called replication error positive (RER+); low-level MSI (MSI-L), in which less than 30% of the measured microsatellite markers have microsatellite instability; and microsatellite stable (MSS), in which there is no microsatellite instability at all. According to the International Guideline for Evaluation of MSI in Colorectal Cancer adopted at the 1997 NCI workshop, BAT26, BAT25, D5S346, D2S123, and D17S250 should be included as a reference panel for colorectal cancer. However, there is no specific consensus on the number or types of microsatellite markers to be measured for gastric cancer. Since cancer cells with positive microsatellite instability have unique clinical and pathological characteristics and different prognoses compared to those with negative microsatellite instability, many studies are being conducted to apply this to clinical practice and use it as an indicator for early detection of cancer and predicting prognosis or response to treatment.

[0005] However, although techniques and algorithms to replace existing MSI tests have been introduced through recent NGS analysis, the sensitivity of most techniques and algorithms for MSI testing is not high.

[0006] The problem that our center wants to solve includes presenting a method that can distinguish between MSS, MSI-L, and MSI-H, and increase the reliability of the instability determination results. Specifically, one of the reasons for the low sensitivity of the existing MSI test is that many sequencing errors occur due to the repetition of the same sequence in the MSI target region. Therefore, our center uses start-and-stop combination to determine whether the gene deletion confirmed in each MSI target sequence is a deletion due to a PCR error (false shortening) or a deletion that actually exists in the original gene sequence (true shortening), and by utilizing the determination results of multiple MSI target sequences, it is possible to effectively determine whether the original gene sequence is MSI, thereby providing a method that can increase the sensitivity of the MSI determination result.

[0007] The above tasks are merely examples, and there may be additional tasks that can be understood by a person skilled in the art through this document.

[0008] The first aspect of the present invention is a method for confirming microsatellite instability using a sequence combination, wherein the method

[0009] (1) Step of setting multiple MSI target sequences

[0010] (2) A step of forming a start-end combination group for each gene read having the same start point and end point for a plurality of gene reads including one or more sequences among the plurality of MSI target sequences.

[0011] (3) A step of checking whether there is a gene deletion for each MSI target sequence included in the above-mentioned sequence combination group, and

[0012] (4) A method is provided, including a step of determining whether the MSI target sequence in which the above gene deletion is confirmed is 'false shortening' or 'true shortening' through comparison within the above sequence combination group.

[0013] The second aspect of the present invention relates to a device for determining microsatellite instability using a sequence combination, comprising at least one processor and at least one memory (storage device / storage) including a computer program code,

[0014] The at least one memory and the computer program code are configured to cause the device to operate via the at least one processor.

[0015] (1) A step of setting multiple MSI target sequences;

[0016] (2) a step of forming a start-end combination group for each gene read having the same start point and end point for a plurality of gene reads including at least one sequence among the plurality of MSI target sequences;

[0017] (3) a step of checking whether there is a gene deletion for each MSI target sequence included in the above seed combination group; and

[0018] (4) A device is provided configured to perform a process including a step of determining whether the MSI target sequence in which the above gene deletion is confirmed is 'false shortening' or 'true shortening' through comparison within the above sequence combination group.

[0019] The effect of the present invention includes reducing the influence of sequencing errors in MSI determination, thereby increasing the determination sensitivity.

[0020] The above effects are merely examples, and there may be additional effects that can be understood by a person skilled in the art through this disclosure.

[0021] Figure 1 is a flowchart of a method according to the present invention.

[0022] Figure 2 is a block diagram of a device according to the present invention.

[0023] Figure 3 illustrates a process for confirming gene deletion using a seed combination according to the present invention according to one embodiment of the present invention.

[0024] Figure 4 illustrates a process for confirming gene deletion using a seed combination according to the present invention according to one embodiment of the present invention.

[0025] Figure 5 illustrates an example of false shortening according to the present invention according to one embodiment of the present invention.

[0026] Figure 6 illustrates an example of True shortening according to the present invention according to one embodiment of the present invention.

[0027] Figure 7 shows the results of comparing the performance of determining MSI using various methods according to Example 1.

[0028] Below, with reference to the attached drawings, embodiments of the present invention are described in detail to facilitate easy implementation by those skilled in the art. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0029] Throughout this specification, whenever a part is said to 'include' a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0030] Throughout this specification, the terms “step of” or “step of” do not mean “step for”.

[0031] Throughout this specification, the term 'combination(s) thereof' included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0032] Throughout this specification, references to 'A and / or B' mean 'A or B, or A and B.'

[0033] Throughout this specification, a "start-end pair" refers to a combination of genetic reads that have the same starting and ending points when mapped to a reference sequence. For example, when multiple DNA fragments (genes) are fragmented during the sequencing process, the starting and ending points are theoretically random. A start-end pair utilizes the extremely low probability that any two fragmented genes will have the same starting and ending points.

[0034] Throughout this specification, the term "microsatellite" refers to a short DNA sequence that is repeated and arranged throughout a gene. The length of the DNA sequence may range from 1 to 10, for example.

[0035] Throughout this specification, "MSI (microsatellite instability)" refers to a condition in which a mutation or other problem occurs in a microsatellite, causing a change in the number of repeats of a DNA sequence, resulting in a difference in the length of the microsatellite compared to the normal length. MSI is primarily tested by examining the length of microsatellites after sequencing, and is typically confirmed using multiple microsatellites (MSI target sequences) as markers.

[0036] Throughout this specification, the term "false shortening" indicates that the DNA deletion identified in the MSI target sequence within the gene read is an error that occurred during the sequencing process, such as PCR. Conversely, throughout this specification, the term "true shortening" indicates that the DNA deletion identified in the MSI target sequence within the gene read is not an error but actually exists in the original gene sequence.

[0037] The functions realized by the components described in this specification may be implemented in processing circuitry including general purpose processors, special purpose processors, integrated circuits, Application Specific Integrated Circuits (ASICs), Central Processing Units (CPUs), circuits and / or combinations thereof programmed to realize the described functions. A processor includes transistors or other circuits and is considered a circuit or processing circuit. The processor may be a programmed processor that executes a program stored in a memory.

[0038] In this specification, a circuit, part, unit, or means refers to hardware programmed to realize the described function or hardware that executes the function. The hardware may be any hardware disclosed in this specification or any hardware known to be programmed or executed to realize the described function.

[0039] If the hardware is a processor considered to be a circuit type, the circuit, the part, means or unit is a combination of hardware and software used to configure the hardware and / or the processor.

[0040] Hereinafter, implementation examples and embodiments of the present invention will be described in detail with reference to the attached drawings. However, the present invention may not be limited to these implementation examples and embodiments and drawings.

[0041] The first aspect of the present invention provides a method for determining microsatellite instability using a sequence combination. The method

[0042] (1) Step of setting multiple MSI target sequences

[0043] (2) A step of forming a start-end combination group for each gene read having the same start point and end point for a plurality of gene reads including one or more sequences among the plurality of MSI target sequences.

[0044] (3) A step of checking whether there is a gene deletion for each MSI target sequence included in the above-mentioned sequence combination group, and

[0045] (4) A step of determining whether the MSI target sequence in which the above gene deletion is confirmed is false shortening or true shortening through comparison within the above sequence combination group.

[0046] In one specific example, the multiple MSI target sequences may refer to multiple microsatellite sequences used for MSI determination of the original gene sequence. Typically, MSI determination uses five microsatellite sequences as markers, but there is no limitation on the number of MSI target sequences used herein.

[0047] In one specific example, the plurality of gene reads may be generated through Next-Generation Sequencing (NGS) using the original gene sequence to determine MSI. In addition, the present inventors have devised a method of regarding a pair of gene reads whose start and end points match the human reference genome sequence, i.e., gene reads having the same start and end points, as a single molecular barcode, and this is referred to herein as a Start-End pair (SEP) (Figs. 3 and 4).

[0048] In one specific example, the present invention can determine whether each MSI target sequence is 'Stable', 'False shortening', or 'True shortening'. Specifically, if there is at least one group of sequence combinations that satisfies each of the determination conditions proposed in the present invention, the MSI target sequence included in the corresponding sequence combination is determined as 'Stable', 'False shortening', or 'True shortening'. However, 'Stable', 'False shortening', or 'True shortening' does not necessarily indicate whether the original gene sequence is MSS, MSI-L, or MSI-H.

[0049] In one specific example, step (3) is a step to first confirm whether a gene deletion exists in each MSI target sequence. MSI target sequences without gene deletion are determined to be 'Stable', and MSI target sequences with gene deletion are determined through step (4) to determine whether the gene deletion is due to a PCR error or the like (False shortening) or whether it is due to an actual deletion of the original gene sequence (True shortening).

[0050] In one specific example, the step (4) may further include a step of determining whether the 'false shortening' or 'true shortening' is performed using 'whether the same gene deletion pattern is shown' and 'whether the sense strand and the antisense strand are identical' in the MSI target sequence in which the gene deletion of the plurality of gene reads included in the seed combination group is confirmed.

[0051] Specifically, step (4) is a step of comparing all gene reads within a set of parental pairs that include the MSI target sequence identified as having a gene deletion in step (3). Since the set of parental pairs is assumed to be derived from the same gene sequence, all gene sequences included in the set of parental pairs should be identical unless an error occurs during the sequencing process, such as NGS. Therefore, if a gene deletion exists in the MSI target sequence of the original gene sequence, the MSI target sequences of all gene reads included in the set of parental pairs should also have the same pattern of gene deletion, i.e., the gene should be deleted by the same sequence.

[0052] Therefore, when comparing the sequence of the seed combination group and the standard sequence, if the MSI target sequence of all gene reads included in the seed combination group shows the same gene deletion pattern, the MSI target sequence can be determined as 'True shortening', otherwise it can be determined as 'False shortening'.

[0053] In addition, under the same assumption, if no PCR error occurs, the sense sequence and antisense sequence within the sequence combination group must match, so if the sense sequence and antisense sequence match, the MSI target sequence can be determined as 'True shortening', and if not, it can be determined as 'False shortening'.

[0054] An example of 'False shortening' determined by the method according to the present invention is shown in Figure 5, and an example of 'True shortening' is shown in Figure 6.

[0055] In one specific example, the method comprises:

[0056] (5) It may further include a step of determining microsatellite instability using the MSI target sequence determined as 'True shortening'. Generally, MSI is determined as MSS (Microsatellite stable; 0), MSI-L (low; 1), and MSI-H (high; 2 or more) based on the number of sequences in which instability is found among 5 microsatellite sequences. However, the present invention does not determine MSS, MSI-L, and MSI-H in the above manner since there is no limitation on the number of microsatellite sequences used for MSI determination.

[0057] In addition, the step (5) above may be to determine whether the original gene sequence is MSS, MSI-L, or MSI-H by using the ratio of the number of MSI target sequences determined by the 'True shortening' to the number of the plurality of MSI target sequences. Here, the above ratio can be compared with a preset ratio standard, and the preset ratio standard can be directly set by the user according to experimental conditions or clinical environments.

[0058] As mentioned above, 'Stable', 'True shortening', and 'False shortening' determined for MSI targets are for cases where there is at least one group of sequence combinations that satisfies each determination condition presented in the present invention. Therefore, in order to increase the reliability of the MSI determination result, the inventors of the present invention introduced the ratio of the number of MSI target sequences determined as 'True shortening' to the number of the plurality of MSI target sequences into the determination process. For example, among a total of 48 MSI target sequences, 13 sequences (27.3%) were determined as 'True shortening', and if the MSI-H determination standard set by the user is 20% or more, it is determined as MSI-H. Conversely, if the ratio of MSI target sequences determined as 'True shortening' is less than 20%, it is determined as MSI-L, and if it is 0%, it is determined as MSS.

[0059] The second aspect of the present invention relates to a device for determining microsatellite instability using a sequence combination, comprising at least one processor (10) and at least one memory (20) including a computer program code,

[0060] The at least one memory (20) and the computer program code are executed by the device through the at least one processor (10).

[0061] (1) A step of setting multiple MSI target sequences;

[0062] (2) a step of forming a start-end combination group for each gene read having the same start point and end point for a plurality of gene reads including at least one sequence among the plurality of MSI target sequences;

[0063] (3) a step of checking whether there is a gene deletion for each MSI target sequence included in the above seed combination group; and

[0064] (4) A device is provided configured to perform a process including a step of determining whether the MSI target sequence in which the above gene deletion is confirmed is 'false shortening' or 'true shortening' through comparison within the above sequence combination group.

[0065] The first and second aspects of the present invention share the same technical features.

[0066] Example 1. Verification experiment

[0067] We used 16 cell lines whose MSI results were known by conventional tests. NGS experiments were performed on these cell lines using the PAN100 panel of the applicant (Dexome Co., Ltd.), and the MSI determination method according to the present invention and four existing algorithms for MSI testing (Cellosaurus, MSIsensor2, MANTIS, MSIsensor-Pro) were used to compare the discrimination performance of MSI using each determination method. Here, the discrimination performance was expressed as a score, excluding Cellosaurus, as the percentage (%) of sequences determined as 'True shortening' among the entire MSI target sequences, and a higher score in the order of MSI-H, MSI-L, and MSS means that MSI was well determined.

[0068] As a result, when the method according to the present invention was used, a score close to 0 was given to the MSS cell line, a very low score was given to the MSI-L cell line, and a very high score was given to the MSI-H cell line, and in particular, compared to when other algorithms were used, high scores and low scores were clearly distinguished, confirming that MSS, MSI-L, and MSI-H could be more effectively distinguished (Table 1, Fig. 7).

[0069] MSI discrimination performance comparison (average score) Cell cycle Existing test results Cellsaurus MSIsensor 2 MANTI SMSIsensor-Pro Original invention 293 TMSI-H Unable to analyze 41.8 32.9 10.2 5 1.6 IM 9 5 m MSI-HMSI-L 90.7 95.9 32.4 96.3 NUGC 3 MSI-HMSI-H 7 5.6 5 8.7 19.1 5 9.4 SNU 1 MSI-HMSI-H 9 4.6 94.4 2 4.196.3 HCT 11 6MSI-HMSI-H97.994.832.1100.0YCC28MSI-LMSI-L17.526.15.44.9MKN7MSI-LMSI-L14.733.25.22.3OC UM1MSI-LMSI-L18.225.36.02.2MKN45MSSMSS14.625.70.00.0MKN28MSSMSS14.310.90.00.0MKN74MSSAnalysis Not possible9.80.00.00.0KATOIIIMSSMSS14.023.40.00.0Hs746TMSSMSS7.522.40.10.0SNU16MSSMSS10.525.30.00.0SNU5MSSMSS22.022.30.02.2NCIN87MSSMSS9.826.90.08.9

[0070] [Explanation of symbols]

[0071] 10: Processor

[0072] 20: Memory

Claims

1. In a method for confirming microsatellite instability using a sequence combination, The above method (1) A step of setting multiple MSI target sequences; (2) a step of forming a start-end combination group for each gene read having the same start point and end point for a plurality of gene reads including at least one sequence among the plurality of MSI target sequences; (3) a step of checking whether there is a gene deletion for each MSI target sequence included in the above seed combination group; and (4) A method comprising a step of determining whether the MSI target sequence in which the gene deletion is confirmed is ‘false shortening’ or ‘true shortening’ through comparison within the above-mentioned sequence combination group.

2. In paragraph 1, A method, wherein the step (3) above additionally includes a step of determining the MSI target sequence included in the group of seed combinations in which gene deletion is not confirmed as 'Stable'.

3. In paragraph 1, In the above step (4), in the MSI target sequence where the gene deletion of the plurality of gene reads included in the above seed combination group is confirmed, 'Whether they show the same genetic deletion pattern'; and A method further comprising a step of determining whether the 'false shortening' or 'true shortening' is performed using 'whether the sense strand and the antisense strand match'.

4. In paragraph 1, The above method (5) A method further comprising a step of determining microsatellite instability using an MSI target sequence determined as 'true shortening'.

5. In paragraph 4, The method according to claim 1, wherein the step (5) further includes a step of determining whether the MSI target sequence is MSS, MSI-L or MSI-H by using the ratio of the number of MSI target sequences determined as 'True shortening' to the number of the plurality of MSI target sequences.

6. In a device for checking microsatellite instability using a sequence combination, at least one processor; and comprising at least one memory containing computer program code, The at least one memory and the computer program code are configured to cause the device to perform, through the at least one processor, (1) A step of setting multiple MSI target sequences; (2) a step of forming a start-end combination group for each gene read having the same start point and end point for a plurality of gene reads including at least one sequence among the plurality of MSI target sequences; (3) a step of checking whether there is a gene deletion for each MSI target sequence included in the above seed combination group; and (4) A device configured to perform a process including a step of determining whether the MSI target sequence in which the gene deletion is confirmed is 'false shortening' or 'true shortening' through comparison within the above-mentioned sequence combination group.

7. In paragraph 6, A device, wherein the step (3) above additionally includes a step of determining the MSI target sequence included in the group of seed combinations in which no gene deletion has been confirmed as 'Stable'.

8. In paragraph 6, In the above step (4), in the MSI target sequence where the gene deletion of the plurality of gene reads included in the above seed combination group is confirmed, 'Whether they show the same genetic deletion pattern'; and A device further comprising a step of determining whether the 'false shortening' or 'true shortening' is performed using 'whether the sense strand and the antisense strand match'.

9. In paragraph 6, The above process (5) A device further comprising a step of determining microsatellite instability using an MSI target sequence determined as 'true shortening'.

10. In paragraph 9, The device, wherein the step (5) further includes a step of determining whether the MSI target sequence is MSS, MSI-L or MSI-H by using the ratio of the number of MSI target sequences determined as 'True shortening' to the number of the plurality of MSI target sequences.

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