Peripheral blood cfDNA detection system
By optimizing probe design, UMI design and bio-information analysis processes, the shortcomings in existing cfDNA detection technologies in terms of sensitivity and specificity are solved, and higher molecular recovery and detection sensitivity are achieved.
Patent Information
- Application Number
- CN202311818482.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-27
- Publication Date
- 2025-06-27
AI Technical Summary
Existing cfDNA detection techniques have shortcomings in sensitivity and specificity, especially when capturing highly fragmented cfDNA and reducing false positives caused by UMI.
Optimize probe design, UMI design and bioinformatic analysis processes, design probes according to the breaking method of cfDNA, select UMI sequences with 3-10 bases differential, and improve detection accuracy through liquid phase hybrid capture enrichment and sequencing technology.
The molecular recovery rate and detection sensitivity of cfDNA are significantly improved. The detection rate of traditional methods when detecting mutations of 0.1-0.5% is 50-80%, while the present invention reaches 92%, and the detection rate of mutations of 0.05% at lower level also reaches 79%.
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Figure CN120210359A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biological detection technologies, and more particularly to peripheral blood liquid biopsy detection, and specifically to a peripheral blood cfDNA detection system and a corresponding detection method. Background Art
[0002] Circulating free DNA (cfDNA) in blood is a liquid biopsy marker that has received extensive attention in recent years. It mainly originates from apoptosis and necrosis of cells, or release from active cells. Tumor-related signals can be detected by detecting cfDNA in blood samples. Due to its unique non-invasive characteristics, cfDNA has occupied an important position in cancer research. In the past decade, researchers have revealed the potential of cfDNA in cancer diagnosis, prognosis, and efficacy monitoring. CfDNA can carry tumor-specific mutations, which can be used for early detection of cancer and monitoring of minimal residual disease. In addition, the relationship between the concentration of cfDNA and tumor burden indicates that it can be used as a prognostic marker. 【1、2、3】 .
[0003] However, despite the broad application prospects of cfDNA, its application in clinical practice is still limited, and there are many challenges. For example, the amount of detection material is small, and there are approximately 10,000 copies of genomic cfDNA in 10 ml of peripheral blood; the content of tumor-related circulating tumor DNA (ctDNA) is even lower, usually around 0.1%; in addition, cfDNA is highly fragmented DNA, and the commonly used molecular detection technology PCR in clinical practice will be affected. Therefore, cfDNA liquid biopsy has extremely high requirements for detection technology, such as requiring a sensitivity of 0.1% or even lower.
[0004] To improve the sensitivity of detection technology, scientists have invented many detection methods, such as Duplex-Seq, iDES-Seq, CAPP-Seq, etc. Based on these technologies, a series of commercial detection products have been developed, such as AVENIO ctDNA (Expanded Kit) (Roche), TruSight Tumor 170+UMI (Illumina), xGen Non-small Cell Lung Cancer (IDT), etc. 【4、5、6】。However, these technologies all have some deficiencies: First, some steps in the detection system do not take into account the particularity of cfDNA, and the detection sensitivity, etc. will be affected to a certain extent. For example, the Duplex-Seq method uses 12 random bases as single-molecule tags (UMIs). In bioinformatics analysis, some UMIs will repeat multiple bases, such as GCAGGGGGGAAA. Such UMIs are prone to errors during sequencing, and this part of the data can only be discarded, affecting the final detection sensitivity. In addition, since random bases will result in only 1-2 base differences between two UMIs, and there will also be 1-2 base errors during the detection process, some data will be incorrect due to the excessive similarity of UMIs, introducing false positive detection results. Second, the liquid hybridization capture probes of these products and methods are designed based on the characteristics of DNA fragments in tissues and do not take into account the particularity of cfDNA. Highly fragmented cfDNA is related to the nucleosome organizational structure. When detecting tissues, the entire DNA strand in the tissue is first extracted, and then enzymes or ultrasound are used to break the DNA into a series of DNA fragments. This process is a random fragmentation process. The probes of these products are all designed based on the random fragmentation method for tissue detection and do not consider the influence of the nucleosome organizational structure. Therefore, when using these probes to capture cfDNA in blood, the result will be lower than that of tissue detection and the best effect cannot be achieved. These products can only stably detect gene mutations above 0.5%. 【7】 Therefore, there is still room for improvement in these current cfDNA liquid biopsy technologies. Summary of the Invention
[0005] The present invention addresses the above problems and optimizes the probe design, UMI design, and bioinformatics analysis process in view of the defects of traditional cfDNA detection technologies. First, according to the fragmentation method of cfDNA, probes are laid specifically, and at the same time, according to the sequence characteristics of the target region, the lengths and densities of different probes are adjusted to achieve a better capture effect. Second, the UMI is redesigned, and sequences with 3-10 base differences from each other are selected as UMIs, which do not contain repetitive sequences and sequences prone to sequencing errors, minimizing data loss due to inappropriate UMIs. At the same time, due to the large differences between UMIs, the false positives caused by UMI confusion are also reduced.
[0006] To achieve the above object, the specific technical solutions adopted by the present invention are as follows:
[0007] The peripheral blood cfDNA detection system provided by the present invention includes the following modules:
[0008] (1) Probe design module. According to the cfDNA sequencing statistics results of at least hundreds of target tumors, find the different cfDNA fragmentation methods in each target region, and then lay probes according to the fragmentation method of each target region.
[0009] The fragmentation pattern of cfDNA in blood is the fragmentation between two nucleosome organizational structures, which is different from the random fragmentation pattern of long DNA chains in tissues under the action of external forces such as enzymes or ultrasound. Among these cfDNA fragments, the majority of a cfDNA fragment carries a fragment of a nucleosome organizational structure, accounting for about 50% - 80%, and the rest mostly have 2 - 4 nucleosome organizational structures in a cfDNA fragment.
[0010] The rules followed for probe laying are as follows: More probes are laid in the regions where cfDNA is concentrated, and relatively fewer probes are laid in the regions where cfDNA is dispersed. After the probe design is completed, DNA probes are first synthesized and then transcribed into RNA probes.
[0011] (2) UMI design module. According to the principle that the base differences between UMIs are 2 - 8 bases, hundreds of UMI adapters are designed, and each adapter consists of a 7 - 8 base sequence. Each UMI adapter is synthesized separately for cfDNA library construction;
[0012] The sequences of the UMI adapters described in the present invention are shown in Table 1 below.
[0013] (3) cfDNA library construction module. Using the above UMI adapters as ligation adapters, a cfDNA library is constructed, and the constructed library is subjected to liquid hybridization capture enrichment and sequenced simultaneously.
[0014] When constructing the cfDNA library, it is constructed by existing methods. When ligating the adapters, the adapters in the original kit are replaced with the above UMI adapters; the constructed library is subjected to liquid hybridization capture enrichment, and the hybridization capture reagent uses a commercial reagent (such as the Agilent SureSelect XT target enrichment kit), and the probes are the probes prepared by the probe design module; when sequencing, an existing sequencing platform, such as the Illumina Novaseq sequencing platform, is used to sequence the library.
[0015] (4) Bioinformatics analysis module. Based on the UMI analysis data in the sequencing data, it is compared with the reference gene, and after processing the data, gene mutations in the sample are identified. The specific steps are as follows:
[0016] First, the data is quality - checked, then the UMIs in the data are extracted, and then the data is compared with the human reference genome hg19, and the data is processed according to the principle of Duplex - Seq to identify gene mutations in the sample.
[0017] (5) Result analysis module. According to the existing database, the gene mutation results are interpreted and the analysis results are output.
[0018] The detection system of the present invention is applicable to the detection of various tumors, including lung cancer, digestive system tumors (gastric cancer, intestinal cancer, pancreatic cancer, esophageal cancer, gastroesophageal junction tumor, gastrointestinal stromal tumor), brain tumors (glioma), urinary system tumors (renal cancer, bladder cancer, urothelial tract tumor), liver cancer, cholangiocarcinoma, gallbladder cancer, ampullary tumor, breast cancer, thyroid cancer, endometrial cancer, ovarian cancer, cervical cancer, prostate cancer, otolaryngological tumors, acute myeloid leukemia, multiple myeloma, etc.
[0019] Beneficial guarantees and effects of the present invention:
[0020] After optimizing the probe design, UMI design and corresponding bioinformatics analysis of the present invention, the molecular recovery rate has been improved compared with the existing reagents. By statistically analyzing the cfDNA detection results of 172 cases, it is found that the cfDNA molecular recovery rate of the traditional kit varies from 20% to 63%, while the median value of the molecular recovery rate of the present invention reaches 65% ( Figure 4 ). Since more molecules can be recovered, the sensitivity of the whole detection of the present invention has also been improved. When detecting mutations of 0.1 - 0.5% by the traditional method, the detection rate varies from 50% to 80%. The detection rate of 0.1% mutation of the present invention is 92%, and for mutations of the lower level of 0.05%, the detection rate is also 79% ( Figure 5 ).
[0021] It can be seen that the optimized cfDNA liquid biopsy method in the present invention can improve the detection sensitivity, making the detection more sensitive and accurate. Brief Description of the Drawings
[0022] The following further illustrates the present disclosure in conjunction with the drawings, where these displays are only for illustrating the embodiments of the present disclosure and not for limiting the scope of the present disclosure.
[0023] Figure 1 Shows the structural block diagram of the peripheral blood cfDNA detection system of the present invention;
[0024] Figure 2 Shows the schematic diagram of laying probes according to the cfDNA fragmentation pattern;
[0025] Figure 3 Shows the comparison between the traditional UMI and the UMI adopted by the present invention;
[0026] Figure 4 Shows the comparison result of the cfDNA recovery rate between the present invention and the existing detection methods;
[0027] Figure 5 Shows the comparison result of the low-frequency mutation detection rate between the present invention and the existing detection methods. Detailed Embodiments
[0028] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include detailed descriptions of traditional methods, such as PCR methods, those for constructing vectors and plasmids, methods for inserting genes encoding proteins into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in many publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 nd edition, Cold spring Harbor Laboratory Press。
[0029] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described may be applied to the present invention, and the preferred implementation methods and materials described in the specific implementation manners are for illustrative purposes only.
[0030] According to Figure 1 , the peripheral blood cfDNA detection system 100 of the present invention includes a probe design module 1, a UMI design module 2, a cfDNA library construction module 3, a bioinformatics analysis module 4, and a result analysis module 5.
[0031] The probe design module 1 finds different cfDNA cleavage patterns in each target region according to the cfDNA sequencing statistical results of at least hundreds of target tumors, and then lays probes according to the cleavage pattern of each target region.
[0032] The cleavage pattern of cfDNA in blood is cleavage between two nucleosome organizational structures, which is different from the random cleavage pattern of long DNA chains in tissues under the action of external forces such as enzymes or ultrasound. Figure 2 Shows the difference between the cfDNA cleavage pattern and randomly fragmented tissue gDNA. The left two figures show that the cfDNA cleavage pattern is related to the nucleosome organization, and the breakpoints are mainly located between two nucleosomes, while the breakpoints of randomly fragmented gDNA in the right figure show a normal distribution and have no significant correlation with nucleosomes.
[0033] Among the free cfDNA fragments in blood, the majority of cfDNA fragments carry a fragment of a nucleosome organizational structure, accounting for about 50% - 80%, and the rest mostly carry 2 - 4 nucleosome organizational structures per cfDNA fragment, and a small number of cfDNA carry more nucleosome organizational structures.
[0034] The rules for probe laying are as follows: more probes are laid in the regions where cfDNA is concentrated, and relatively fewer probes are laid in the regions where cfDNA is dispersed. After the probe design is completed, DNA probes are first synthesized and then transcribed into RNA probes.
[0035] The UMI design module 2 designs hundreds of UMI adapters according to the principle that the base differences between UMIs are 2 - 8 bases. Each adapter consists of a 7 - 8 base sequence, and each UMI adapter is synthesized separately for cfDNA library construction.
[0036] The sequences of the UMI adapters described in the present invention are shown in Table 1 below:
[0037] Table 1 Summary of UMI adapter sequences
[0038]
[0039]
[0040] Figure 3 shows the sequence comparison between traditional UMI and the UMI of the present invention. Duplesx - Seq uses 12 random bases as UMI ( Figure 3 a); in iDES - Seq UMI, only 2 - 4 random bases ( Figure 3 b); the present invention uses a selected fixed - sequence UMI with the number of random bases reaching 2 - 8 ( Figure 3 c).
[0041] The cfDNA library construction module 3 uses the above - mentioned UMI adapter as the ligation adapter to construct a cfDNA library, and performs liquid - phase hybridization capture enrichment on the constructed library, and simultaneously performs sequencing.
[0042] When constructing the cfDNA library, it is constructed by using the existing method. When ligating the adapter, the adapter in the original kit is replaced with the above - mentioned UMI adapter; the constructed library is subjected to liquid - phase hybridization capture enrichment, and the hybridization capture reagent uses commercial reagents (such as Agilent SureSelect XT target enrichment kit), and the probes are the probes prepared by the probe design module; when sequencing, an existing sequencing platform is used, such as the Illumina Novaseq sequencing platform to sequence the library.
[0043] The bioinformatics analysis module 4 analyzes the data alignment of the reference gene based on the UMI analysis data in the sequencing data, and identifies gene mutations in the sample after processing the data. The specific steps are as follows: first, perform quality control on the data, then extract the UMI in the data, and then align the data to the human reference genome hg19, and process the data and identify gene mutations in the sample according to the principle of Duplex - Seq.
[0044] The result analysis module 5 interprets the gene mutation results according to the existing database and outputs the analysis results.
[0045] The above modules can be integrated together to form a large system, or can be separately set up for cooperative use.
[0046] The detection system of the present invention is applicable to the detection of various tumors, including lung cancer, digestive system tumors (gastric cancer, intestinal cancer, pancreatic cancer, esophageal cancer, gastroesophageal junction tumor, gastrointestinal stromal tumor), brain tumors (glioma), urinary system tumors (renal cancer, bladder cancer, urothelial tract tumor), liver cancer, cholangiocarcinoma, gallbladder cancer, ampullary tumor, breast cancer, thyroid cancer, endometrial cancer, ovarian cancer, cervical cancer, prostate cancer, otolaryngological tumors, acute myeloid leukemia, multiple myeloma, etc.
[0047] Taking lung cancer as an example below, the specific detection steps of the present invention are described, and the detection processes of other tumors are similar.
[0048] 1. Probe design: Statistically analyze the cfDNA sequencing results of 172 cases of lung cancer, find different cfDNA cleavage patterns in each target region ( Figure 2 ), and then lay probes according to the cleavage pattern of each target region. More probes are laid in the regions where cfDNA is concentrated or, and relatively fewer probes are laid in the regions where cfDNA is dispersed.
[0049] 2. Probe preparation: Synthesize DNA probes according to the probe design results, and then transcribe them into RNA probes for subsequent hybridization capture experiments.
[0050] 3. UMI design: Select the UMI adapters shown in Table 1 above. The base differences between UMIs are 2 - 8 bases. Synthesize UMI adapters respectively and then mix them together for cfDNA library construction.
[0051] 4. cfDNA library construction: Use a commercial kit to construct a cfDNA library (such as NEB UltraII DNA library preparation kit). Replace the original kit adapter with a UMI adapter when connecting the adapter.
[0052] 5. Perform liquid hybridization capture enrichment on the constructed library. The hybridization capture reagent uses a commercial reagent (such as Agilent SureSelect XT target enrichment kit), and the probes are the probes prepared in step 2.
[0053] 6. Sequence the library using the Illumina Novaseq sequencing platform.
[0054] 7. Bioinformatics analysis: First, perform quality control on the data, then extract UMI from the data, and then align the data to the human reference genome hg19. Process the data according to the principle of Duplex-seq and identify gene mutations in the samples.
[0055] Statistically analyze the test results and interpret the gene mutations in the results based on the corresponding databases.
[0056] Compare the method of the present invention with existing detection methods. The results show that the cfDNA molecular recovery rate of traditional kits ranges from 20% to 63%, while the median molecular recovery rate of the present invention reaches 65% ( Figure 4 ). Since more molecules can be recovered, the sensitivity of the entire detection of the present invention is also improved. When detecting mutations of 0.1 - 0.5% by traditional methods, the detection rate ranges from 50% to 80%. The detection rate of 0.1% mutations of the present invention is 92%, and for mutations at a lower level of 0.05%, the detection rate is also 79% ( Figure 5 ).
[0057] References related to the background technology of the present invention:
[0058] [1] Cisneros-Villanueva, M., Hidalgo-Pérez, L., Rios-Romero, M. et al. Cell-free DNA analysis in current cancer clinical trials: a review. Br J Cancer 126, 391–400 (2022).
[0059] [2] Garcia, J., Kamps-Hughes, N., Geiguer, F. et al. Sensitivity, specificity, and accuracy of a liquid biopsy approach utilizing molecular amplification pools. Sci Rep 11, 10761 (2021).
[0060] [3] Cescon, D. W., Bratman, S., Chan, S. M. et al. Circulating tumor DNA and liquid biopsy in oncology. Nat Cancer 1, 276–290 (2020).
[0061] [4] Schmitt MW, Kennedy SR, Salk JJ, Fox EJ, Hiatt JB, Loeb LA. Detection of ultra-rare mutations by next-generation sequencing. Proc Natl Acad Sci U SA. 2012 Sep 4;109(36):14508-13.
[0062] [5] Newman AM, Lovejoy AF, Klass DM, Kurtz DM, Chabon JJ, Scherer F, Stehr H, Liu CL, Bratman SV, Say C, Zhou L, Carter JN, West RB, Sledge GW, Shrager JB, Loo BW Jr, Neal JW, Wakelee HA, Diehn M, Alizadeh AA. Integrated digital error suppression for improved detection of circulating tumor DNA. Nat Biotechnol. 2016 May;34(5):547-555.
[0063] [6] Newman, A., Bratman, S., To, J. et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med 20, 548–554 (2014).
[0064] [7] Deveson, I.W., Gong, B., Lai, K. et al. Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology. Nat Biotechnol 39, 1115–1128 (2021).
[0065] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art can also make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.
Claims
1. A peripheral blood cfDNA detection system, characterized in that Comprising: A probe design module, which finds different cfDNA cleavage patterns in each targeted region according to the sequencing statistics results of cfDNA from at least hundreds of target tumors, and then lays probes according to the cleavage pattern of each targeted region; A UMI design module, which designs hundreds of UMI adapters according to the principle that the base difference between UMIs is 2 - 8 bases. Each adapter consists of a 7 - 8 base sequence, and each UMI adapter is synthesized separately for cfDNA library construction; A cfDNA library construction module, which uses the above UMI adapter as a ligation adapter to construct a cfDNA library, performs liquid hybridization capture enrichment on the constructed library, and simultaneously performs sequencing; A bioinformatics analysis module, which analyzes the UMI analysis data in the sequencing data against the reference gene, and identifies gene mutations in the sample after processing the data; A result analysis module, which interprets the gene mutation results and outputs the analysis results.
2. The peripheral blood cfDNA detection system according to claim 1, wherein: Among them, cfDNA is cleaved between two nucleosome structures, and the majority of a cfDNA fragment carries a fragment of a nucleosome organizational structure.
3. The peripheral blood cfDNA detection system according to claim 1, wherein: Among them, The rules for probe laying are as follows: more probes are laid in the regions where cfDNA is concentrated, and relatively fewer probes are laid in the regions where cfDNA is dispersed.
4. The peripheral blood cfDNA detection system according to claim 1, wherein: Among them, When synthesizing the probe, first synthesize the DNA probe, and then transcribe it into an RNA probe.
5. The peripheral blood cfDNA detection system according to claim 1, wherein: Among them, The sequences of the UMI adapters are as follows:
6. The peripheral blood cfDNA detection system according to claim 1, wherein: Among them, When constructing the cfDNA library, it is constructed by an existing method, and the adapter in the original kit is replaced with the UMI adapter when ligating the adapter.
7. The peripheral blood cfDNA detection system according to claim 1, wherein: Among them, The steps for the bioinformatics analysis module to perform bioinformatics analysis are as follows: first perform quality inspection on the data, then extract the UMIs in the data, then align the data to the human reference genome hg19, and process the data and identify gene mutations in the sample according to the principle of Duplex - Seq.
8. The peripheral blood cfDNA detection system according to claim 1, wherein: Among them, The tumors include lung cancer, digestive system tumors, brain tumors, urinary system tumors, liver cancer, and cholangiocarcinoma.