Chip capable of firstly capturing and then sequencing and sequencing method thereof
By using chips and circular processing technologies that capture first and then sequence in NGS technology, the problems of low capture efficiency and high cost in the existing technology are solved, and efficient and low-cost targeted sequencing are achieved, which significantly improves target capture and Sequencing depth.
Patent Information
- Application Number
- CN202410869096.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-01
- Publication Date
- 2024-12-20
AI Technical Summary
Existing next-generation sequencing technologies (NGS) have problems with low capture efficiency, high cost and complex operation in targeted post-capture sequencing methods, especially multiplex PCR and hybrid probe capture technologies that are limited by primer binding and reagent costs.
Using a chip that captures first and then sequences, the P5 sequence and the P7' sequence with hybridization probe are linked by enzyme cleavage sites, combined with circularization treatment and rolling ring amplification technology to achieve efficient capture and sequencing of the target sequence. Reduce sequencing costs.
It significantly improves the target species and specificity, reduces sequencing costs, enhances anti-pollution ability, and improves capture efficiency. It has higher capture efficiency and lower cost compared with conventional tNGS methods.
Smart Images
Figure CN119161968A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of molecular biology, in particular to gene sequencing, and specifically relates to a chip capable of first capturing and then sequencing and a sequencing method thereof. Background Art
[0002] With the development of gene sequencing technology, the application of next generation sequencing (NGS) technology is becoming more and more extensive in clinical practice, such as targeted treatment and drug resistance monitoring of tumors, screening and diagnosis of genetic diseases, pathogen diagnosis in the field of critical and severe diseases, and many other aspects. So far, NGS mainly has two technical routes: mNGS and tNGS. mNGS sequences all nucleic acid sequences in the sample, with a wide coverage. The disadvantage is that a large number of sequences other than the target sequence will be measured, which will cause data waste and have a negative impact on the result judgment. tNGS captures the target sequence in the sample before sequencing. The target is clear, and the influence of non-target sequences on sequencing can be eliminated, greatly reducing costs.
[0003] Currently, the widely used tNGS routes include multiplex PCR and hybridization probe capture. Multiplex PCR is limited by the fact that primers may bind to each other, PCR products will compete for resources, and the number of targets that can be captured simultaneously is limited. Hybridization probes can capture a large number of different targets at the same time, but hybridization probes are prone to capture fragments containing part of the target sequence, and the reagent cost and operation complexity are much higher than multiplex PCR. Therefore, it is urgent to develop a sequencing method that is simple to operate and reduces costs. Summary of the invention
[0004] The present invention provides a new t-NGS sequencing chip that can be adapted to a variety of application scenarios, such as pathogen detection, tumor diagnosis, genetic testing, etc., and cooperates with a new sequencing method Reci pe to complete capture enrichment followed by sequencing, which greatly increases the types of targets and improves specificity while reducing sequencing costs.
[0005] One aspect of the present invention provides a chip capable of capture before sequencing, characterized in that: the chip comprises two upper and lower substrates, the inner surfaces of the two substrates are connected with a P5 sequence and a P7' sequence with a hybridization probe; the hybridization probe and the P7' sequence are connected by a restriction site.
[0006] In some embodiments, the hybridization probe is a specific sequence for hybridization capture target sequence, and the 3' end of the last base of the hybridization probe is -OH.
[0007] In some embodiments, the material of the substrate is selected from glass, PDMS, silicon wafer, polymethyl methacrylate or polytetrafluoroethylene.
[0008] In some embodiments, the restriction enzyme cleavage site is an EcoR1 restriction enzyme cleavage site.
[0009] Another aspect of the present invention provides a sequencing method, which uses the chip as described above for sequencing, characterized in that the method comprises the following steps:
[0010] Step 1: Circularize the single-stranded library;
[0011] Step 2: The circularized library enters the chip, and the hybridization probe captures the target sequence;
[0012] Step 3: The target sequence is subjected to rolling circle amplification to form a long chain containing n copies of the target sequence on P7';
[0013] Step 4: adding an enzyme that recognizes the restriction site, releasing n independent libraries containing the target sequence by restriction digestion, and removing the hybridization probe on P7';
[0014] Step 5: The library containing the target sequence is re-hybridized with the P5 sequence on the chip, bridge amplified, and sequenced.
[0015] In some embodiments, the enzyme that recognizes the restriction site is EcoR1 enzyme.
[0016] In some embodiments, the step 4 further includes adding enzyme cleavage probe 1 and enzyme cleavage probe 2.
[0017] Another aspect of the present invention provides use of the chip in preparing a t-NGS sequencing kit.
[0018] Compared with the prior art, the advantages and positive effects of the present invention are:
[0019] 1. The present invention performs t-NGS on a chip, which has the potential to have higher capture efficiency compared to conventional tNGS (multiple PCR, probe capture). Multiplex PCR and probe capture are limited by primer and probe design and the number of probes used.
[0020] 2. The capture probe of the present invention is simple in design and can significantly reduce sequencing costs because it has the same capture capability as conventional tNGS.
[0021] 3. The present invention uses conventional libraries, which are more resistant to contamination than conventional t-NGS that requires PCR. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.
[0023] Figure 1is a schematic diagram of the sequencing chip of the present invention;
[0024] Figure 2 A schematic diagram of the probes and sequences on the surface of the sequencing chip of the present invention;
[0025] Figure 3 The sequencing process of the present invention;
[0026] Figure 4 Schematic diagram for the construction of circularized libraries;
[0027] Figure 5 Schematic diagram of hybridization capture, rolling circle amplification, and target sequence enrichment on the chip;
[0028] Figure 6 Schematic diagram of enzyme digestion to remove hybridization probes and release N target library sequences;
[0029] Figure 7 Comparison results of the amount of library data for mNGS, multiplex PCR-tNGS, probe capture-tNGS, and the method of the present invention;
[0030] Figure 8 Comparison results of mNGS, multiplex PCR-tNGS, probe capture-tNGS and the method of the present invention Spike RPM;
[0031] Fig. 9 The results of Spike depth comparison of mNGS, multiplex PCR-tNGS, probe capture-tNGS and the method of the present invention are shown. DETAILED DESCRIPTION
[0032] The present invention is further described below in conjunction with specific embodiments:
[0033] To facilitate a better understanding of the present invention, but not to limit the present invention, the experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.
[0034] Example 1
[0035] A sequencing chip of the present invention comprises the following components: Figure 1 As shown in the figure, two glass slides are combined into a chip, and the upper and lower surfaces of the chip are planted with probes for capturing the library at a certain density. Figure 2As shown, the glass surface is seeded with a P5 sequence and a P7' sequence with a hybrid capture target. The P5 sequence is mainly used for capturing the enriched library bridge amplification. The P7' sequence is connected to a specific sequence for hybrid capture (the 3' end of the last base is -OH), which is used for hybrid capture of a specific target sequence. The P7' sequence and the specific capture sequence are connected by an EcoR1 restriction site, which is used to remove the specific capture sequence before the library bridge amplification.
[0036] Example 2
[0037] Another object of the present invention is to provide a sequencing method for the above chip. The sequencing process is as shown in the attached Figure 3 .
[0038] The difference from the conventional library is that the 3' end of the P7' sequence of the library of the present invention is connected to an EcoR1 restriction site. Before loading the library, a circularization primer is used to circularize the single-stranded library. Figure 4 Circularization is the key to capture and enrich the target sequence on the chip. The circularized library enters the chip. If there is a sequence in the library that can complement the capture probe, it will be captured by the capture probe and form a long chain containing n copies of the target sequence on P7' through rolling circle amplification. Figure 5 . Add enzyme probe 1, enzyme probe 2 and EcoR1 enzyme, the enzyme probe binds to the long chain to form EcoR1 enzyme site, enzyme cleavage releases n independent libraries containing target sequence and removes the capture probe on P7', the library containing target sequence is hybridized with P5 sequence on the chip again, and after bridge amplification, the target sequence can be sequenced conventionally, as shown in the attached Figure 6 The corresponding sequences of P5, P7, circularization primer, restriction probe 1 and restriction probe 2 are shown in Table 1.
[0039] Table 1. P5, P7, circularization primer, restriction probe 1 and restriction probe 2 sequences
[0040]
[0041]
[0042] According to the mass ratio, a simulated DNA sample was prepared with the ratio of artificially synthesized Spike: human DNA = 0.01:99.99 (see Table 2 for the Spike sequence). The simulated sample was used as the test object, and the library was built and loaded onto the machine using mNGS, multiplex PCR-tNGS, probe capture-tNGS and the method of the present invention (see Table 3 for the multiplex PCR primers, capture probes and the capture probe sequences of the present invention). The sequencing used a 75 bp read length, and each method was repeated three times. Under similar data volumes (see Appendix 1), the library was constructed and loaded onto the machine using mNGS, multiplex PCR-tNGS, probe capture-tNGS and the method of the present invention (see Table 3 for the multiplex PCR primers, capture probes and the capture probe sequences of the present invention). The library was constructed and loaded onto the machine using mNGS, multiplex PCR-tNGS, probe capture-tNGS and the method of the present invention. ... Figure 7), analyze Spike RPM and the sequencing depth of each Spike base. Figure 8 , the Spike RPM of the present invention is significantly higher than that of the other three methods. Fig. 9 The coverage, uniformity and sequencing depth of Spike by the method of the present invention are superior to the other three.
[0043] Table 2. Spike sequence
[0044]
[0045] Table 3. Multiplex PCR primers, capture probes and capture probe sequences of the present invention
[0046]
[0047]
[0048]
[0049] The above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.
[0050] Although some specific embodiments of the present invention have been described in detail by way of example, it will be appreciated by those skilled in the art that the above examples are for illustration only and are not intended to limit the scope of the present invention. It will also be appreciated by those skilled in the art that various modifications may be made to the embodiments without departing from the scope and spirit of the present invention. The scope of the present invention is defined by the appended claims.
Claims
1. A chip capable of capturing first and sequencing later, characterized in that: The chip comprises two upper and lower substrates, and the inner surfaces of the two substrates are connected with a P5 sequence and a P7' sequence with a hybridization probe; the hybridization probe and the P7' sequence are connected via a restriction site.
2. The chip according to claim 1, characterized in that: The hybridization probe is a specific sequence for hybridization capture of a target sequence, and the 3' end of the last base of the hybridization probe is -OH.
3. The chip according to claim 1, characterized in that: The material of the substrate is selected from glass, PDMS, silicon wafer, polymethyl methacrylate or polytetrafluoroethylene.
4. The chip according to claim 1, characterized in that: The restriction enzyme cleavage site is the EcoR1 restriction enzyme cleavage site.
5. A sequencing method, using the chip according to any one of claims 1 to 4 for sequencing, characterized in that: The method comprises the following steps: Step 1: Circularize the single-stranded library; Step 2: The circularized library enters the chip, and the hybridization probe captures the target sequence; Step 3: The target sequence is subjected to rolling circle amplification to form a long chain containing n copies of the target sequence on P7'; Step 4: Add an enzyme that recognizes the restriction site, release n independent libraries containing the target sequence by restriction digestion, and remove the hybridization probe on P7'; Step 5: The library containing the target sequence is re-hybridized with the P5 sequence on the chip, bridge amplified, and sequenced.
6. The sequencing method according to claim 5, characterized in that The enzyme that recognizes the restriction site is EcoR1 enzyme.
7. The sequencing method according to claim 5, characterized in that In step 4, restriction enzyme probe 1 and restriction enzyme probe 2 are also added.
8. Use of the chip as described in any one of claims 1 to 4 in preparing a t-NGS sequencing kit.