A method and device for automatic review of mutation sites

Abstract

The present invention discloses a method and device for automatic review of mutation sites. The method includes: (1) obtaining sequencing reads as sites to be reviewed; (2) determining whether the sequencing reads supporting the mutation are high-quality sequencing reads; (3) determining the authenticity of the mutation sites. The method of the present invention has high accuracy, does not rely on the subjective judgment of reviewers, and ensures the consistency of review. At the same time, the review efficiency is high, saving nearly 100 man-hours per month and saving training costs. The present invention solves the problems of easy human misjudgment, subjective dependence and low efficiency due to manual inspection. Therefore, the present invention realizes fast, accurate and consistent review of mutation sites.

Description

Technical Field

[0001] The present invention relates to the field of bioinformatics processing, and particularly to a method and device for automated review of mutation sites. Background Art

[0002] When analyzing clinical samples by NGS, to avoid filtering out meaningful positive mutations and causing missed detections, mutation detection software (such as freebayes, vardict, varscan, mutect2) usually relaxes the detection threshold to increase the number of detected mutations. However, relaxing the mutation detection quality control threshold will introduce false positive mutations, and an amplified false positive rate is also intolerable in clinical practice. Therefore, after the software automatically detects, a large number of mutation sites still need to be manually verified.

[0003] Manual review has the following limitations: ① Due to differences in experience and other aspects among different people, when reviewing the same sample, inconsistent results often occur, and subjective misjudgments are prone to occur. ② The training cost of reviewers is relatively high, and it is time-consuming and laborious to train professional reviewers. ③ The efficiency of manual review is relatively low. Especially when there are many urgent samples, the workload of reviewers is large, resulting in a huge labor cost.

[0004] Therefore, there is still a need to develop a method and device for automated review of mutation sites to avoid the problem of detecting false positive sites in NGS analysis of clinical samples, so as to achieve fast, accurate, and consistent site review. Summary of the Invention

[0005] To solve at least some of the problems in the prior art, the present invention provides a method and device for automated review of mutation sites. The method of the present invention has high accuracy, does not rely on the subjective judgment of reviewers, and ensures the consistency of review. At the same time, the review efficiency is high, saving nearly 100 man-hours per month and saving training costs. Specifically, the present invention includes the following content.

[0006] In a first aspect of the present invention, there is provided a method for automated review of mutation sites, which includes the following steps:

[0007] (1) Obtain sequencing reads as sites to be reviewed;

[0008] (2) Determine whether the sequencing reads supporting the mutation are high-quality sequencing reads;

[0009] (3) Determine the truth or falsehood of the mutation site.

[0010] In some embodiments, according to the method for automated review of mutation sites of the present invention, when it is necessary to determine the truth or falsehood of somatic mutations, the method further includes: (4) Determine whether the mutation site is true in the tumor control sample.

[0011] Those skilled in the art know how to obtain sequencing reads as sites to be reviewed, for example, using sequencing data or sequencing reads obtained from a sequencer or a sequencing platform as sites to be reviewed.

[0012] In certain embodiments, according to the method for automated review of mutation sites of the present invention, in step (2), the following metrics are used to determine whether the sequencing reads supporting the mutation are high-quality sequencing reads:

[0013] a. The alignment quality of the sequencing reads;

[0014] b. Whether the mutation is located at the end of the sequencing read;

[0015] c. The length of nucleotide editing on the sequencing read, including nucleotide substitution, insertion, and / or deletion;

[0016] d. The base quality of the sequencing read supporting the mutation.

[0017] In certain embodiments, according to the method for automated review of mutation sites of the present invention, in step (2), when the score of the alignment quality of the sequencing read is higher than 30, the base quality of the sequencing read supporting the mutation is higher than 20, the distance from the mutation to the end of the sequencing read is more than 5 bp, and the length of nucleotide editing on the sequencing read is less than 3, the sequencing read supporting the mutation is determined to be a high-quality sequencing read.

[0018] In certain embodiments, according to the method for automated review of mutation sites of the present invention, in step (3), the following metrics are used to determine the authenticity of the mutation site:

[0019] i. Whether there is a preference for high-quality sequencing reads supporting the mutation, which is calculated using the ratio of the number of positive and negative sequencing reads;

[0020] ii. Whether the mutation is located in a repetitive region of the genome;

[0021] iii. Whether the number of high-quality sequencing reads exceeds a threshold.

[0022] In certain embodiments, according to the method for automated review of mutation sites of the present invention, in step (3), when the ratio of positive to negative strands of high-quality sequencing reads is less than 10, the mutation is not located in a repetitive region, and the number of high-quality sequencing reads is greater than or equal to 3, the mutation site is determined to be true.

[0023] In certain embodiments, for the method of automated review of mutation sites according to the present invention, when determining whether it is a positive somatic mutation, it further includes determining whether the site is false in the tumor control sample, and when the ratio of the positive and negative strands of high-quality sequencing reads is less than 10, the mutation is not located in the repetitive region, and the number of high-quality sequencing reads is greater than or equal to 3, if it is determined that the mutation site is false, then it is determined that the mutation site is a positive somatic mutation.

[0024] The second aspect of the present invention provides a device for automated review of mutation sites, which includes:

[0025] A unit for obtaining sites to be reviewed, which is used to obtain sequencing reads as sites to be reviewed;

[0026] A unit for determining high-quality sequencing reads, which is used to determine whether the sequencing reads supporting the mutation are high-quality sequencing reads according to the selected first index and the corresponding threshold;

[0027] A unit for determining the truth or falsehood of mutation sites, which is used to determine the truth or falsehood of mutation sites according to the selected second index and the corresponding threshold;

[0028] Preferably, the first index includes:

[0029] a. The alignment quality of the sequencing reads;

[0030] b. Whether the mutation is located at the end of the sequencing reads;

[0031] c. The length of nucleotide editing on the sequencing reads, including nucleotide substitution, insertion, and / or deletion;

[0032] d. The base quality of the sequencing reads supporting the mutation;

[0033] Preferably, the second index includes:

[0034] i. Whether there is a preference for high-quality sequencing reads supporting the mutation, which is calculated using the ratio of the number of positive and negative sequencing reads;

[0035] ii. Whether the mutation is located in the repetitive region of the genome;

[0036] iii. Whether the number of high-quality sequencing reads exceeds the threshold;

[0037] Preferably, the second index further includes determining whether the mutation site is true in the tumor control sample.

[0038] The third aspect of the present invention provides a computer device, including a memory, a processor, and a computer program stored on the memory and executable on the processor, wherein when the processor executes the program, it implements the method described in the first aspect.

[0039] In a fourth aspect of the present invention, there is provided a computer storage medium storing a computer program which, when executed by a computer, implements the method described in the first aspect.

[0040] The present invention locates mutation sites and obtains the reads alignment quality supporting the mutation, the total number of reads supporting the mutation, whether the base supporting the mutation is at the end of the reads, whether the number of mutations existing on the reads supporting the mutation exceeds the threshold, whether the positive and negative strands of the reads supporting the mutation are balanced, and whether the mutation is in a genomic repeat region. By integrating the above information, the authenticity of the site is comprehensively judged. In addition, for somatic mutations, the determination of whether it is a germline mutation is added.

[0041] The present invention avoids the problem that some mutation detection software will detect some false positive mutations, and requires reviewers to use the IGV (Integrative Genomics Viewer) tool for manual inspection to exclude the false positive sites detected by the program, and solves the problems of easy human misjudgment, subjective dependence and low efficiency due to manual inspection. Therefore, the present invention uses a python program to achieve fast, accurate and consistent review of mutation sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Shows a schematic diagram of the automated review logic of mutation sites. DETAILED DESCRIPTION OF THE INVENTION

[0043] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features and embodiments of the present invention.

[0044] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for the numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and each intermediate value therebetween are specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0046] Example 1

[0047] This example provides a method for automatic review of mutation sites, which includes the following steps:

[0048] (1) Obtain sequencing reads as the sites to be reviewed;

[0049] (2) Determine whether the sequencing reads supporting the mutation are high-quality sequencing reads;

[0050] (3) Determine the authenticity of the mutation site.

[0051] When it is necessary to determine the authenticity of somatic mutations, it further includes: (4) Determine whether the mutation site is true in the tumor control sample.

[0052] In step (2), according to the following indicators, determine whether the sequencing reads supporting the mutation are high-quality sequencing reads:

[0053] a. The alignment quality of the sequencing reads;

[0054] b. Whether the mutation is located at the end of the sequencing reads;

[0055] c. The length of nucleotide editing on the sequencing reads, including nucleotide substitution, insertion, and / or deletion;

[0056] d. The base quality of the bases supporting the mutation on the sequencing reads.

[0057] The sequencing reads involved in this article refer to the sequencing data obtained on a PE 100 sequencer. The threshold for determining high-quality reads is set as follows:

[0058] (1) The alignment quality is higher than 30 (score);

[0059] (2) The base quality is higher than 20 (i.e., Q20. The Q20 value refers to the error probability given to the identified base during the base calling process of the sequencing process. If the quality value is Q20, the error recognition probability is 1%, that is, the error rate is 1%, or the correct rate is 99%);

[0060] (3) The mutation distance is more than 5bp from the end of the reads;

[0061] (4) The nucleotide editing length on the reads is less than 3;

[0062] After determining the high-quality reads that meet the above conditions, then determine whether it is a positive somatic mutation, and the threshold is set as follows:

[0063] (1) The positive / negative strand ratio of the high-quality reads is less than 10, and the ratio of the positive strand to the negative strand is from 0.1 to 10;

[0064] (2) The mutation is not located in the repetitive region;

[0065] (3) The number of high-quality reads is greater than or equal to 3;

[0066] (4) The site in the control sample is false, germline.

[0067] The control sample can be a solid tumor or blood cfDNA, ctDNA. In this embodiment, the control sample is a blood sample - white blood cells.

[0068] As Figure 1 shown, when the determination conditions do not meet that the positive / negative strand ratio of the high-quality reads is less than 10, and the ratio of the positive strand to the negative strand is from 0.1 to 10, the mutation is not located in the repetitive region, and the number of high-quality reads is greater than or equal to 3, the determination result is output as false. When the determination conditions meet that the positive / negative strand ratio of the high-quality reads is less than 10, and the ratio of the positive strand to the negative strand is from 0.1 to 10, the mutation is not located in the repetitive region, and the number of high-quality reads is greater than or equal to 3, the determination result is output as true, and at the same time determine whether the mutation in the control sample is false. When the mutation in the control sample is determined to be true, it is determined as a false somatic mutation, true germline mutation result. When the mutation in the control sample is determined to be false, it is determined as a true somatic mutation.

[0069] To further illustrate the practicality of the method of the present invention, in this embodiment, 4680 mutation sites to be manually reviewed in April are used to verify the accuracy and efficiency of the tool.

[0070] The accuracy comparison results are shown in Table 1-2 below:

[0071] Table 1

[0072] Review is true Review is false Manual review is true 2315 8 Manual review is false 0 2357

[0073] Table 2

[0074]

[0075]

[0076] Among them, for Indels of multiple bases, due to the differences in left alignment or right alignment when detecting mutations, this part of the mutations is set to be confirmed.

[0077] The efficiency comparison results are shown in Table 3 below:

[0078] Table 3

[0079] Total number of reviews Number of reviews requiring manual review Total man-hours (h) August 5686 5686 94.7 September 6118 6118 101.9 October 5480 5480 91.3 November 7688 7688 128.1 December 6458 6458 107.6 January 5498 5498 91.6 February 5590 5590 93.2 March 4570 4570 76.2 April (automated tool review) 4680 455 7.5

[0080] According to the method of the present invention, those skilled in the art can use a Python script to run on a common Windows or Linux system, so as to realize automatic mutation site auditing.

[0081] Example 2

[0082] This example provides a device for automatic auditing of mutation sites, which includes:

[0083] A unit for obtaining sites to be audited, which is used to obtain sequencing reads as sites to be audited;

[0084] A unit for determining high-quality sequencing reads, which is used to determine whether the sequencing reads supporting mutations are high-quality sequencing reads according to the selected first index and the corresponding threshold;

[0085] A unit for determining the authenticity of mutation sites, which is used to determine the authenticity of mutation sites according to the selected second index and the corresponding threshold.

[0086] In this example, the first index includes:

[0087] a. The alignment quality of the sequencing reads;

[0088] b. Whether the mutation is located at the end of the sequencing reads;

[0089] c. The length of nucleotide editing on the sequencing reads, including nucleotide substitution, insertion and / or deletion;

[0090] d. The base quality of the sequencing reads supporting mutations.

[0091] The second index includes:

[0092] i. Whether there is a preference for high-quality sequencing reads supporting mutations, which is calculated by the ratio of the number of positive and negative sequencing reads;

[0093] ii. Whether the mutation is located in the repetitive region of the genome;

[0094] iii. Whether the number of high-quality sequencing reads exceeds the threshold;

[0095] The second index further includes determining whether the mutation site is true in the tumor control sample.

[0096] In the high-quality sequencing read determination unit, the thresholds for determining high-quality reads are set as follows:

[0097] (1) The alignment quality is higher than 30 (score);

[0098] (2) The base quality is higher than 20 (i.e., Q20. The Q20 value refers to the error probability given for the identified base during the base calling process in the sequencing process. If the quality value is Q20, the error recognition probability is 1%, that is, the error rate is 1%, or the correct rate is 99%);

[0099] (3) The mutation distance from the end of the read is higher than 5 bp;

[0100] (4) The nucleotide editing length on the read is less than 3;

[0101] In the true / false determination unit of the mutation site, the thresholds for determining whether it is a positive somatic mutation are set as follows:

[0102] (1) The positive / negative strand ratio of high-quality reads is less than 10, and the ratio of the positive strand to the negative strand is 0.1 to 10;

[0103] (2) The mutation is not located in the repetitive region;

[0104] (3) The number of high-quality reads is greater than or equal to 3;

[0105] (4) The site in the control sample is false, germline.

[0106] Those skilled in the art can understand that the various exemplary embodiments described in the present invention can be implemented by software or in a manner combining software with necessary hardware. Therefore, according to the specific embodiments of the present invention, it can be embodied in the form of a software product, which can be stored in a non-volatile storage medium or a non-transitory computer-readable storage medium (which can be a CD-ROM, a USB flash drive, a mobile hard disk, etc.) or on the network, including several instructions to enable a computing device (which can be a personal computer, a server, a mobile terminal, or a network device, etc.) to execute the method according to the present invention.

[0107] In an exemplary embodiment, the program product of the present invention may adopt any combination of one or more readable media. The readable media may be a readable signal medium or a readable storage medium. The readable storage medium may be, for example, but not limited to, an electrical, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any combination of the above. More specific examples of the readable storage medium include, but are not limited to: an electrical connection having one or more wires, a portable disk, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disk read-only memory (CDROM), an optical storage device, a magnetic storage device, or any suitable combination of the above.

[0108] Correspondingly, based on the same inventive concept, the present invention also provides an electronic device.

[0109] In an exemplary embodiment, the electronic device is presented in the form of a general-purpose computing device. The components of the electronic device may include, but are not limited to: at least one processor, at least one memory, and a bus connecting different system components (including the memory and the processor).

[0110] Wherein, the memory stores program code, and the program code can be executed by the processing unit, so that the processing unit executes the method of the present invention. The processor at least includes the high-quality sequencing read segment determination unit and the true / false determination unit (which can also be referred to as a "module") of the mutation site of the present invention. The memory may include a readable medium in the form of a volatile storage unit, such as a random access storage unit (RAM) and / or a cache storage unit, and may further include a read-only storage unit (ROM).

[0111] The memory of the present invention may also include a program / utilities having a set (at least one) of program modules. Such program modules include, but are not limited to: an operating system, one or more application programs, other program modules, and program data. Each or some combination of these examples may include the implementation of a network environment.

[0112] The bus may represent one or more of several types of bus structures, including a memory bus or a memory controller, a peripheral bus, a graphics acceleration port, a processing unit, or a local bus using any bus structure in a variety of bus structures.

[0113] The electronic device may also communicate with one or more external devices (such as a keyboard, a pointing device, a Bluetooth device, etc.), may also communicate with one or more devices that enable a user to interact with the electronic device, and / or communicate with any device that enables the electronic device to communicate with one or more other computing devices (such as a router, a modem, etc.).

[0114] Such communication can be carried out through an input / output (I / O) interface. Also, the electronic device can also communicate with one or more networks (such as a local area network (LAN), a wide area network (WAN), and / or a public network, such as the Internet) through a network adapter. The network adapter communicates with other modules of the electronic device through a bus. It should be understood that although not shown herein, other hardware and / or software modules can be used in conjunction with the electronic device, including but not limited to: microcode, device drivers, redundant processing units, external disk drive arrays, RAID systems, tape drives, and data backup storage systems, etc.

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features. And these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for automated review of mutation sites, characterized in that: It includes the following steps: (1) Obtain sequencing reads as sites to be reviewed; (2) Determining whether the sequencing reads supporting the mutation are high-quality sequencing reads, and determining whether the sequencing reads supporting the mutation are high-quality sequencing reads according to the following indicators: a. Alignment quality of the sequencing reads; b. Whether the mutation is located at the end of the sequencing reads; c. The length of nucleotide editing on the sequencing read, including nucleotide substitution, insertion and / or deletion; d. The quality of the bases supporting the mutation on the sequencing read. When the score of the alignment quality of the sequencing read is higher than 30, the quality of the bases supporting the mutation on the sequencing read is higher than 20, the mutation distance is higher than 5 bp from the end of the sequencing read, and the length of the nucleotide editing on the sequencing read is less than 3, the sequencing read supporting the mutation is determined to be a high-quality sequencing read; (3) Determine whether the mutation site is true or false based on the following indicators: i. Whether there is a preference for high-quality sequencing reads that support the mutation, which is calculated by the ratio of the number of positive and negative sequencing reads; ii. Whether the mutation is located in the repetitive region of the genome; iii. Whether the number of high-quality sequencing reads exceeds a threshold. When the positive-negative strand ratio of high-quality sequencing reads is between 0.1 and 10, the mutation is not located in the repetitive region, and the number of high-quality sequencing reads is greater than or equal to 3, the mutation site is determined to be true; (4) Determine whether it is a positive somatic mutation. When the judgment conditions are met, the positive / negative strand ratio of high-quality reads is less than 10, the positive / negative strand ratio is between 0.1 and 10, the mutation is not located in the repetitive region, and the number of high-quality reads is greater than or equal to 3, the judgment result is output as true. At the same time, determine whether the control sample mutation is false. When the control sample mutation is determined to be true, it is determined to be a false somatic mutation and a true germline mutation result. When the control sample mutation is determined to be false, it is determined to be a true somatic mutation.

2. A device for automated review of mutation sites, characterized in that: include: A pending review site acquisition unit, which is used to acquire sequencing reads as pending review sites; a high-quality sequencing read determination unit, which is used to determine whether the sequencing read supporting the mutation is a high-quality sequencing read according to the selected first indicator and the corresponding threshold value, and when the alignment quality score of the sequencing read is higher than 30, the base quality supporting the mutation on the sequencing read is higher than 20, the mutation distance is higher than 5 bp from the end of the sequencing read, and the nucleotide editing length on the sequencing read is less than 3, the sequencing read supporting the mutation is determined to be a high-quality sequencing read; A mutation site authenticity determination unit, which is used to determine the authenticity of the mutation site according to the selected second indicator and the corresponding threshold value, and when the positive and negative strand ratio of the high-quality sequencing read is 0.1 to 10, the mutation is not located in the repeat region, and the number of high-quality sequencing reads is greater than or equal to 3, the mutation site is determined to be true; The first indicator includes: a. Alignment quality of sequencing reads; b. Whether the mutation is located at the end of the sequencing read; c. The length of nucleotide edits on the sequencing reads, including nucleotide substitutions, insertions, and / or deletions; d. The quality of bases supporting mutations in sequencing reads; The second indicator includes: i. Whether there is a bias in high-quality sequencing reads supporting the mutation, calculated using the ratio of the number of positive and negative sequencing reads; ii. Whether the mutation is located in a repetitive region of the genome; iii. Whether the number of high-quality sequencing reads exceeds the threshold; the second indicator also includes determining whether it is a positive somatic mutation. When the determination conditions are met, the positive / negative strand ratio of high-quality reads is less than 10, and the positive / negative strand ratio is 0.1 to 10, the mutation is not located in the repetitive region, and the number of high-quality reads is greater than or equal to 3, the determination result is output as true, and at the same time, it is determined whether the control sample mutation is false. When the control sample mutation is determined to be true, it is determined to be a false somatic mutation and a true germline mutation result. When the control sample mutation is determined to be false, it is determined to be a true somatic mutation.

3. A computer device comprising a memory, a processor and a computer program stored in the memory and executable on the processor, characterized in that: When the processor executes the program, the method according to claim 1 is implemented.

4. A computer storage medium, characterized in that: The device stores a computer program, which implements the method according to claim 1 when executed by a computer.

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