A method for identifying the source of tumor tissue based on a second-generation sequencing platform

Abstract

The present application relates to the technical field of forensic evidence, and particularly relates to a tumor tissue source identification method based on a second-generation sequencing platform. The present application uses the second-generation sequencing technology to count and statistically analyze the typing results of 15 STR markers. Compared with the commonly used STR marker combination based on capillary electrophoresis, the present application has better stability, higher accuracy and is more suitable for precise tracing of tumor tissues. The analysis method is simple, the influence of background signal does not need to be considered, and the result is easier to interpret. The present application can be used for tumor tissue source identification based on the second-generation sequencing platform.

Description

Technical Field

[0001] This invention relates to the field of forensic biological evidence technology, specifically to a method for identifying the origin of tumor tissue based on a next-generation sequencing platform. Background Technology

[0002] Cancer is a major public health problem worldwide. The incidence and mortality rates of cancer remain high, making it one of the most common causes of death. Medical disputes caused by problems such as incorrect, chaotic, and contaminated tumor tissue samples are becoming increasingly frequent, and judicial identification cases involving the tracing of tumor tissue origins are also increasing. Therefore, the identification of the origin of tumor tissues has become more important. How to accurately identify the origin of tumor tissues remains a challenge in the field of judicial identification, mainly in the following two aspects: (1) Tumor tissue samples are mixed samples of tumor cells and normal cells, making it difficult to detect the lower components; (2) Many tumors have widespread STR level mutations during their growth, which makes it impossible to identify them using conventional individual identification criteria, which is also the most significant identification challenge at present. Although many mature systems based on capillary electrophoresis (CE) platform STR typing technology have been developed for individual identification of ordinary samples, its application in individual identification of tumor tissues is still relatively lacking.

[0003] Next-generation sequencing (NGS) technology is currently the mainstream genotyping method in the field of biology, characterized by high throughput and high sensitivity. Compared to the traditional CE genotyping platform, it is more suitable for the detection of molecules with low copy numbers. According to the molecular clonal theory, tumor tissue is a mixture of normal cells and tumor cells; therefore, using a more sensitive detection method is more conducive to the detection of target genes. Although patents CN117219162A and CN101921851A have established methods for identifying the origin of tumor tissue, these methods are limited to the CE platform. With the innovation of molecular biology technology, NGS technology has been further developed and is widely used in various aspects of forensic evidence tracing research. Due to the significant difference in sensitivity between the two platforms, NGS often identifies a greater number of alleles. Therefore, detection methods previously established based on traditional platforms cannot meet the application requirements of the new mainstream technology platform, and there is an urgent need to establish new tumor tissue origin identification methods based on the next-generation sequencing platform.

[0004] Currently, NGS-based methods for identifying the origin of tumor tissue have been reported, but most of these methods target single nucleotide polymorphism (SNP) markers. Since most forensic population databases target STR markers, SNP-based methods for identifying the origin of tumor tissue have limited applicability; STR markers remain the most practical molecular markers in forensic identification. The ForenSeq DNA Signature Prep Kit system is currently the most commonly used NGS technology platform, and studies have shown that tumor tissue identification can be performed based on the 27 STR markers available on this platform. However, this detection method is limited to the 27 STR combinations available on the platform, ignoring the influence of different STR molecules on the detection results. This allows markers with poor stability to be included in the calculation, reducing the system efficiency of the identification method.

[0005] Therefore, it is necessary to develop a more accurate method for identifying the origin of tumor tissue based on a next-generation sequencing platform. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for identifying the origin of tumor tissue based on a next-generation sequencing platform. This method can be used to accurately trace the origin of human tumor tissue samples.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention discloses a method for identifying the origin of tumor tissue based on a next-generation sequencing platform, the steps of which are as follows:

[0009] a) Subtype detection of tumor tissue samples and normal samples to be tested;

[0010] b) Compare the results of step a) with those of tumor tissue samples and normal samples to be tested, and analyze the genotyping results of locus markers D3S1358, FGA, D8S1179, vWA, D7S820, CSF1PO, D2S1338, D19S433, TH01, TPOX, D12S391, D6S1043, PentaE, D4S2408 and D20S482.

[0011] c) Count the number of common alleles on the 15 STR markers between the tumor tissue sample and the normal sample to be tested in step b);

[0012] d) Calculate the judgment result;

[0013] e) Determine the origin of tumor tissue samples based on the tumor tissue sample origin identification method.

[0014] Preferably, in step c), the number of loci with two identical subtypes among 15 STR markers in the tumor tissue sample and the normal sample to be tested is counted and denoted as A2; when A2≥9, the tumor tissue sample and the normal sample to be tested are excluded from originating from unrelated individuals; when A2≥12, the tumor tissue sample and the normal sample to be tested are determined to originate from the same individual.

[0015] The present invention has the following beneficial effects:

[0016] This invention provides a tumor tissue origin identification method based on a next-generation sequencing platform. It can use next-generation sequencing technology to perform simple counting and statistical analysis on the typing results of 15 STR markers, which has good versatility and is more convenient to calculate. Compared with traditional STR marker detection data, it has higher sensitivity and is more suitable for typing detection of forensic micro-samples (especially micro-puncture tissue samples). Compared with existing STR marker combinations, the 15 STR marker combinations used in this invention are more stable and have higher accuracy. Attached Figure Description

[0017] Figure 1 This is the A2 distribution under 15 STR marker combinations in Embodiment 1 of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Unless otherwise specified, the technical means used in the implementation examples are conventional means well known to those skilled in the art.

[0020] This invention provides a combination of tumor origin identification markers based on the commonly used second-generation sequencing platform ForenSeq DNA Signature Prep Kit system: D3S1358, FGA, D8S1179, vWA, D7S820, CSF1PO, D2S1338, D19S433, TH01, TPOX, D12S391, D6S1043, Penta E, D4S2408, and D20S482.

[0021] This invention also provides a method for tumor origin identification based on the above 15 markers, the specific steps of which are as follows:

[0022] a) Tumor tissue samples and normal samples to be tested were classified and detected using the ForenSeq DNA Signature Prep Kit system;

[0023] b) Statistical analysis of the typing results of tumor tissue samples and normal samples under test in D3S1358, FGA, D8S1179, vWA, D7S820, CSF1PO, D2S1338, D19S433, TH01, TPOX, D12S391, D6S1043, Penta E, D4S2408 and D20S482;

[0024] c) Count the number of common alleles on a single STR marker between tumor tissue samples and normal samples to be tested;

[0025] It should be noted that, according to the genetic characteristics of human cells, normal tissue samples are diploid, which is reflected in the presence of two alleles at one gene locus marker; tumor tissue is a mixture of normal cells and tumor cells, and tumor cells may have genomic variations. Their typing results often deviate from the diploid characteristics, which is reflected in the presence of two or more alleles at one gene locus marker. Therefore, the above-mentioned common alleles, as the number of alleles that are the same in a single marker between the tumor tissue sample and the normal sample to be tested, should have the following three situations: (1) 0 identical alleles: indicating that there are no identical alleles between the tumor tissue sample typing and the normal sample typing; (2) 1 identical allele: indicating that there is 1 identical allele between the tumor tissue sample typing and the normal sample typing; (3) 2 identical alleles: indicating that there are 2 identical alleles between the tumor tissue sample typing and the normal sample typing.

[0026] d) Calculation and judgment results: Based on the above calculation of the common alleles of a single locus, the number of loci with 2 identical subtypes in the tumor tissue sample subtype and the normal sample to be tested is counted (denoted as A2).

[0027] e) Determine the origin of tumor tissue samples according to the tumor tissue sample origin identification method. Specifically, when A2≥9, exclude tumor tissue samples and normal samples to be tested from originating from unrelated individuals; when A2≥12, determine that tumor tissue samples and normal samples to be tested originate from the same individual.

[0028] It should be noted that although existing technologies CN117219162A (Evidence Strength Assessment Method for Identification of Origin of Tumor Tissue STR Maps) and CN101921851A (A Method for Identifying the Origin of Tumor Tissue Based on the Identifiler System) have also established methods for identifying the origin of tumor tissue, the above inventions are based on the identification method of the CE system. Due to the high sensitivity of the second-generation sequencing platform, the background data generated is higher than that of the CE system, and it is not suitable for the current mainstream second-generation sequencing platforms. In addition, the innovation of the tumor tissue origin identification method described in this invention is also reflected in: (1) it adopts data from the mainstream second-generation sequencing platform, and the data distribution is more in line with the result characteristics of high-throughput data platforms, making it more suitable for tumor tissue origin identification on the second-generation sequencing platform; (2) it uses a second-generation sequencing platform with better sensitivity, which is more suitable for genotyping analysis of trace tissues; (3) it only selects A2 as the judgment standard, which reduces the amount of calculation and simplifies the identification method; (4) it selects 15 STR markers, which are more universal and have higher accuracy than other published second-generation sequencing platform markers.

[0029] The present invention will be further described below with reference to specific embodiments.

[0030] Example 1

[0031] This example compares the efficacy of different STR marker combinations in identifying the origin of tumor tissue, including accuracy, sensitivity, and specificity. Details are as follows:

[0032] This embodiment selected nine autosomal STR marker combinations, specifically including eight STR combinations from commonly used STR detection kits, and the 15 STR combinations selected in this invention. DNA extraction and typing were performed on 78 tumor tissue samples and their corresponding peripheral blood samples. Of the 78 samples, 55 were randomly selected for establishing the tumor tissue origin identification method, and the remaining 23 were used to verify the accuracy of this identification method (e.g., ...). Figure 1 (As shown in Table 1). The results show that the sensitivity was 100% for different STR marker combinations, while the specificity and accuracy varied, ranging from 97.56% to 99.88% and 97.59% to 99.89%, respectively. Among them, the 15 STR combinations used in this invention showed the best accuracy, reaching 99.89%.

[0033] Table 1. Accuracy, sensitivity, and specificity of tumor tissue origin identification under different STR marker combinations.

[0034]

[0035]

[0036] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for identifying the origin of tumor tissue based on a next-generation sequencing platform, characterized in that: The steps are as follows: a) Subtype detection of tumor tissue samples and normal samples to be tested; b) Compare the genotyping results of tumor tissue samples and normal samples from step a) with those of the locus markers D3S1358, FGA, D8S1179, vWA, D7S820, CSF1PO, D2S1338, D19S433, TH01, TPOX, D12S391, D6S1043, Penta E, D4S2408, and D20S482. c) Count the number of common alleles on the 15 STR markers between the tumor tissue sample and the normal sample to be tested in step b); d) Calculate the judgment result; e) Determine the origin of tumor tissue samples based on the tumor tissue sample origin identification method.

2. The method for identifying the origin of tumor tissue based on a next-generation sequencing platform according to claim 1, characterized in that: In step c), the number of loci with two identical subtypes among 15 STR markers in the tumor tissue sample and the normal sample to be tested is counted and denoted as A2. When A2≥9, the tumor tissue sample and the normal sample to be tested are excluded from originating from unrelated individuals. When A2≥12, the tumor tissue sample and the normal sample to be tested are determined to originate from the same individual.

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