CN102373288A - Method and kit for sequencing target areas - Google Patents

Abstract

The present invention relates to the field of genetic engineering, and provides a method and a kit for sequencing a target region. The method includes the following steps: A. using specific primers to amplify multiple target regions in a sample to be tested, and based on Amplifying the product to construct a sequencing library; B. performing single-molecule amplification on the sequencing library to obtain a plurality of single-molecule amplification products corresponding to the multiple target regions; C. simultaneously performing a single-molecule amplification on the multiple single-molecule amplification products High-throughput gene sequencing to obtain sequence information of the multiple target regions. This method and its corresponding kit use high-throughput sequencing technology to simultaneously perform regional sequencing on multiple target regions, and can accurately obtain the sequence information of these regions, including the variation of known mutations and unknown mutation sites, with high detection sensitivity , and can further detect a large number of samples at the same time.

Description

A method and kit for sequencing a target region

technical field

The invention relates to the field of genetic engineering, more specifically, to a method and a kit for sequencing a target region.

Background technique

Since the advent of the draft nucleotide sequence of the human genome and many other draft genomes, as well as the discovery of a large number of single nucleotide polymorphisms (SNPs) and small insertion/deletion polymorphisms in the draft genomes of humans and many other organisms, people began to Interested in nucleic acid determination. Studies have shown that nucleic acid sequence variations at specific positions contain a large amount of biological information, which may be related to certain traits of humans or animals and plants. These information will have a wide impact on all aspects of medicine and agriculture. For example, it is anticipated that developments in this field may enable personalized medicine.

At present, the common method for sequencing the target region (specific position) is the Sanger sequencing method. The Sanger sequencing method can detect the target region, but the Sanger sequencing method can only sequence a certain region of a sample at a time. The cost is high, the sensitivity is low (20%), and accurate data on the variation of each mutation site cannot be obtained.

Therefore, there is a need for a new method for regional detection of multiple target regions of the sample to be tested at the same time. This method can accurately obtain the sequence information of these regions, including the variation of each mutation site of known mutations and unknown mutations, and detect The sensitivity is high, and it can further detect a large number of samples at the same time.

Contents of the invention

The object of the present invention is to provide a method and a kit for sequencing target regions, which can detect multiple target regions of a sample to be tested at the same time, and accurately obtain the sequence information of these regions, including various mutations of known mutations and unknown mutations. The variation of the mutation site has high detection sensitivity, and it can further detect a large number of samples at the same time.

The present invention is achieved in this way, a method for sequencing the target region, comprising the following steps:

A. Use specific primers to amplify multiple target regions in the sample to be tested, and construct a sequencing library based on the amplified products;

B. performing single-molecule amplification on the sequencing library to obtain multiple single-molecule amplification products corresponding to the multiple target regions;

C. Simultaneously performing high-throughput gene sequencing on the multiple single-molecule amplification products to obtain sequence information of the multiple target regions.

Wherein, described step A comprises the following steps:

A1. using specific primers to amplify multiple target regions in the sample to be tested, and obtain amplification products corresponding to the multiple target regions;

A2. Use adapter elements to connect the amplified products corresponding to the multiple target regions to obtain a sequencing library; the adapter elements use blunt end adapters, protruding end adapters, adapters with stem-loop structures, and bifurcated adapters. at least one.

Wherein, step A2 includes the following steps:

A21. Fragmenting the amplified products corresponding to the multiple target regions to obtain fragmented products;

A22. Use the adapter element to connect the fragmented product to construct a sequencing library.

The length of the target region sequencing library described in step A is not particularly limited, and is preferably 25bp-500bp. More preferably, it is 50bp to 200bp, and more preferably, it is 70bp to 130bp.

Wherein, said step A22 includes the following steps:

A221. Using the first linker to connect the two ends of the fragmentation product to obtain the first ligation product;

A222. cyclization of the first connection product to obtain a cyclization product;

A223. II s type restriction endonuclease digests the cyclization product to obtain the digested product;

A224. Connect the second linker and the third linker to both ends of the restriction product to obtain a sequencing library.

Wherein, said step A22 includes the following steps:

A221'. Use the fourth linker to connect with the fragmentation product to obtain the second connection product;

A222'. II s type restriction endonuclease digests the second connection product to obtain an enzyme-cut fragment with the fourth linker;

A223'. The digested fragment with the fourth linker is connected with the fifth linker to form a sequencing library.

Wherein, at least one of the linker elements in the step A2 includes a first tag sequence, which is used to tag the sequencing libraries of different samples to be tested during the library construction process. The first tag sequence is preferably a nucleic acid molecule with a specific base sequence, and the number of bases is not limited.

Further, the number of bases in the first tag sequence is 3-20, more preferably 4-10.

Wherein, the specific primer described in step A is fully or partially complementary to the target region.

Further, among the specific primers corresponding to each target region, at least one primer is partially complementary to the target region, and the 5' end of the partially complementary primer contains a second tag sequence, which is used in the process of amplifying the target region, The amplification products of target regions of different samples to be tested are labeled. The second tag sequence is preferably a nucleic acid molecule with a specific base sequence, and the number of bases is not limited.

Further, the number of bases in the second tag sequence is 3-20, more preferably 4-10.

Wherein, the amplification of each target region of the same sample in the step A is carried out simultaneously or partly simultaneously or independently.

Wherein, the single-molecule amplification method described in step B is at least one of emulsion PCR and bridge PCR.

Wherein, the high-throughput sequencing technology described in step C is a polymerase-based sequencing-by-synthesis method or a ligase-based sequencing-by-ligation method.

The present invention also provides a kit that can be used in any of the sequencing methods of the present invention, the present invention is achieved in this way, a kit for sequencing the target region, comprising:

Specific primers for amplifying multiple target regions in the sample to be tested;

Linker elements are used to combine with amplification products to construct sequencing libraries.

Wherein, the joint element adopts at least one of a blunt end joint, a protruding end joint, a joint with a stem-loop structure and a bifurcated joint.

Wherein, at least one adapter in the adapter element includes a first tag sequence, which is used to mark the sequencing libraries of different samples to be tested during the library construction process. The first tag sequence is preferably a nucleic acid molecule with a specific base sequence, and the number of bases is not limited, preferably 3-20.

Wherein, the specific primer is fully or partially complementary to the target region.

Further, among the specific primers corresponding to each target region, at least one primer is partially complementary to the target region, and the 5' end of the partially complementary primer contains a second tag sequence, which is used in the process of amplifying the target region, The amplification products of target regions of different samples to be tested are labeled. The second tag sequence is preferably a nucleic acid molecule with a specific base sequence, and the number of bases is not limited, preferably 3-20, more preferably 4-10.

Compared with the prior art, the method and kit of the present invention perform deep sequencing on multiple target regions of the sample to be tested simultaneously through high-throughput sequencing technology, and accurately obtain the sequence information of these target regions, including known mutations and unknown mutations The variation of each mutation site in each sample can be accurately obtained, and the frequency of each mutation site of each sample can be accurately obtained. The detection sensitivity is high, and it can further perform multi-region sequencing on a large number of samples at the same time.

Description of drawings

FIG. 1 is a flowchart of a method for sequencing a target region in one embodiment of the present invention;

Figure 2 is a joint schematic diagram of a single protruding end joint in one embodiment of the present invention;

Fig. 3 is a structural schematic diagram of a double protruding end joint in an embodiment of the present invention;

Fig. 4 is a structural schematic diagram of a joint with a stem-loop structure in one embodiment of the present invention;

Fig. 5 is a schematic structural view of a bifurcated joint in an embodiment of the present invention;

FIG. 6 is a schematic structural view of a T-terminal bifurcation adapter in an embodiment of the present invention;

Fig. 7 is a schematic structural view of a bifurcated joint in another embodiment of the present invention;

Figure 8 is a schematic structural view of a dideoxy bifurcated linker in one embodiment of the present invention;

Fig. 9 is a flowchart of a method for constructing a sequencing library using fragmentation products and linker elements in an embodiment of the present invention;

Fig. 10 is a flowchart of a method for constructing a sequencing library using fragmentation products and linker elements in another embodiment of the present invention;

Fig. 11 is a flowchart of a method for constructing a sequencing library using fragmentation products and linker elements in another embodiment of the present invention.

Detailed ways

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments.

The target region of the present invention is any sequence on any genome, which can be selected according to needs, including but not limited to the internal sequence of the gene, the external regulatory region of the gene, the internal sequence of the gene, including but not limited to the internal sequence of the gene Intron regions, exon regions, regions containing both introns and exons.

Fig. 1 shows a kind of method flow process of sequencing target region of the present invention, and this method comprises the following steps:

S1. Use specific primers to amplify multiple target regions in the sample to be tested, and construct a sequencing library based on the amplified products;

S2. performing single-molecule amplification on the sequencing library to obtain multiple single-molecule amplification products corresponding to the multiple target regions;

S3. Simultaneously performing high-throughput gene sequencing on the multiple single-molecule amplification products to obtain sequence information of the multiple target regions.

This method uses high-throughput sequencing technology to perform deep sequencing on the target region of the sample to be tested. This method can detect multiple target regions of the sample to be tested at the same time, and accurately obtain the sequence information of these regions, including various mutations of known mutations and unknown mutations. The variation of the mutation site, the detection sensitivity is high, and the frequency of the mutation of each mutation site in each sample can be accurately obtained. In addition, by controlling the size of the amplified product of each target region, the fragmentation step can be eliminated, the experimental steps can be reduced, the experimental efficiency can be improved, and the cost can be reduced.

The sequence information obtained by this method can be used in various scientific researches, including but not limited to population sequence analysis, gene function research, and protein function research.

It should be noted:

The sample to be tested in step S1 is any form of sample that can extract nucleic acid, including but not limited to: whole blood, serum, plasma and tissue samples; the tissue samples include but not limited to: paraffin-embedded tissue, fresh tissue and frozen slice.

In the sequencing library obtained in step S1, there are multiple sequencing library molecules, and the single-molecule amplification of the sequencing library means that the various library molecules in the sequencing library are spaced in the form of a very small amount (or even a single molecule). Isolate (but these library molecules still belong to the same reaction system as a whole), and achieve amplification in their respective spaces to improve the signal of various molecules in subsequent sequencing reactions.

In the prior art, the Sanger sequencing technology can only sequence a certain region of a sample at a time, and the sequencing of multiple target regions can only be achieved through multiple reactions. However, in the present invention, after each molecule in the sequencing library undergoes single-molecule amplification, each molecule of the sequencing library forms a single-molecule copy array, and each single-molecule copy array is in a different position during high-throughput gene sequencing, so that The hybridization between the sequencing primer and the single-molecule copy array, and the extension reaction under the action of the enzyme can be carried out simultaneously without mutual interference. Therefore, a large number (millions, tens of millions, or even more) of single-molecule copy arrays can be sequenced at the same time, and then the required sequence information can be obtained by collecting the corresponding signals, and the sensitivity of sequencing is higher than that of Sanger higher.

Wherein, the specific primer described in step S1 is fully or partially complementary to the target region.

Further, in each pair of primers for the target region, at least one primer is partially complementary to the target region, and the 5' end of the primer has a second tag sequence. The second tag sequence is used to mark the amplified products of the target region of different samples to be tested during the process of amplifying the target region.

The second tag sequence is preferably a nucleic acid molecule with a specific sequence, and its number of bases is not limited. The base number of the second tag sequence is preferably 3-20, more preferably 4-10.

In addition, the specific primers can also carry other labels, including but not limited to: biotin label, polyhistidine label, antigen, antibody, so that the purification of the amplification product of the target region is very convenient.

In addition, the amplification of different target regions of the same sample to be tested can be performed simultaneously or independently or partly simultaneously. In the specific experimental process, any of the above-mentioned schemes can be selected according to the needs.

If each target region is amplified separately, the amount of the amplified product can be determined to ensure that the number of molecules of the amplified product of the target region used to construct the target region sequencing library in step S1 remains consistent, and there will be no possibility of amplification due to the amplification step. As a result, the copy numbers of different target regions of the same sample to be tested are different, which in turn affects the subsequent sequencing reaction results.

Of course, when the size of each target region is similar and the GC content is also similar, by rationally designing primers and using multiplex PCR technology, step S1 can simultaneously amplify multiple target regions, and ensure the amplification between target regions. The increasing efficiency remains basically the same, so that the experimental efficiency can be effectively improved and the cost of the reaction can be reduced.

When there are multiple samples to be tested, the amplification of target regions of different samples must be performed separately.

In one embodiment, the specific implementation process of step S1 is:

S11. Using specific primers, amplify multiple target regions in the sample to be tested to obtain amplification products corresponding to the multiple target regions;

S12. Using adapter elements to connect the amplified products corresponding to the multiple target regions to obtain a sequencing library; the adapter elements use blunt end adapters, protruding end adapters, adapters with stem-loop structures, and bifurcated adapters. at least one.

It should be noted:

In step S11, the amplification of multiple target regions in the same sample to be tested can be performed simultaneously or independently or partly simultaneously. Corresponding adjustments can be made according to the actual situation, such as: the annealing temperature of the amplification-specific primers, the size of the amplified target region, the GC content, the number of amplified target regions, etc. Amplification of target regions for different samples to be tested must be performed separately.

In step S12, the connection of the adapter element and the amplification product can be realized in various ways, including direct connection of the adapter element and the amplification product, or connection after the amplification product is processed.

The linker element in step S12 is used to construct the sequencing library, and may include one or more linkers.

Wherein, at least one adapter among the adapter elements described in step S12 includes a first tag sequence, and the first tag sequence is used to tag the sequencing libraries of different samples to be tested during the library construction process. In this way, after the target region sequencing libraries are respectively obtained, the target region sequencing libraries of different samples to be tested can be mixed in the same reaction system for single-molecule amplification reaction, and then high-throughput sequencing can be performed at the same time. Improve the efficiency of the sequencing reaction and reduce the cost of sample detection.

The first tag sequence is preferably a nucleic acid molecule with a specific base sequence, and the number of bases is not limited. Further, the number of bases in the first tag sequence is 3-20, so that at least ⁴³ samples can be detected simultaneously at a time. After comprehensive consideration of various conditions, such as: specificity of the tag, cost of the linker, length of the linker, etc., the base number of the first tag sequence is preferably 4-10. Through the combination of the second label and the first label, the invention of the present invention can detect at least 4 ³ × 4 ³ samples each time, that is, 4096 samples.

Linkers can be modified in various ways, including but not limited to: being biotinylated or methylated, or both biotinylated and methylated. In one embodiment, the linker is biotinylated and connected to non-biotinylated fragmentation products, and the presence of biotin facilitates the separation and purification of the constructed sequencing library. In another embodiment, the adapter is methylated and ligated to unmethylated fragments, and the ligation products are then digested with a restriction enzyme that cleaves only methylated DNA, since only successfully ligated ligation Only the product can be cleaved, so only the successfully ligated linker is cleaved, thus ensuring the uniqueness of the digested product.

There are also various structural forms of linkers, including but not limited to: blunt-ended linkers, protruding-end linkers, linkers with stem-loop structures, and bifurcated linkers. One or more adapters can be used in the construction of a sequencing library. Among them, protruding end joints, joints with stem-loop structures and bifurcated joints can effectively prevent the self-connection of multiple joints during the connection process. Regarding the above joint forms of the joint, several embodiments will be provided below.

In a first embodiment, the linker element employs a blunt-ended linker, which is a double-stranded fully complementary nucleic acid molecule.

In a second embodiment, the adapter element is an overhanging end adapter, which is a double-stranded nucleic acid molecule including at least one overhanging end. The number of bases at the protruding end is not particularly limited, and is preferably 1 to 10 bases. According to the structure of the double-stranded nucleic acid molecule, the overhanging end adapters can be divided into two types, namely single overhanging end adapters and double overhanging end adapters.

A single protruding end fitting as shown in Figure 2 has a blunt end at one end and a protruding end at the other end. Single protruding end fittings with bifurcated fittings prevent self-ligation of the fittings. In order to prevent self-ligation of the single protruding end adapter with a blunt end, the 3'OH on the blunt end can be modified (including but not limited to blocking the hydroxyl with an amino group), or the 5' phosphate group on the blunt end can be removed.

A double overhang adapter as shown in Figure 3, which contains two overhangs, which can be on one nucleotide strand (Figure 3a) or on different nucleotide strands (Figure 3b). When the two overhanging ends are on different nucleic acid strands, they are not complementary to each other to prevent adapter self-ligation during ligation.

In the third embodiment, the joint element adopts a joint with a stem-loop structure, as shown in FIG. 4 . The linker is a single-stranded nucleic acid molecule, which comprises a first complementary pairing region 1, a stem-loop region 2 and a second complementary pairing region 3 (Fig. 4a), and the first complementary pairing region 1 can be combined with the second complementary matching region 3 Complementary pairing, and the complementary pairing region formed by them includes at least one restriction endonuclease recognition site, and through the restriction endonuclease recognition site, a specific enzyme can cut or excise the stem-loop region, so that the single-stranded nucleic acid Molecules become double-stranded nucleic acid molecules for subsequent manipulation. As shown in Figure 4b, the linker with a stem-loop structure can also have a protruding end 4, which can be located at the 5' end or 3' end of the single-stranded nucleic acid molecule. The existence of the protruding end 4 can further prevent the self-connection phenomenon of the joint. The protruding end is preferably T.

In the fourth embodiment, the joint element adopts a bifurcated joint, as shown in Figure 5, the joint is a double-stranded nucleic acid molecule, including a complementary region and a bifurcated region, and each of the two single strands of the bifurcated region contains at least one Amplify primer binding sites. Preferably, the complementary region of the bifurcation adapter includes at least one restriction endonuclease recognition site, which can be digested to form a terminal during the library construction process, so as to facilitate subsequent operations.

The bifurcated design of the bifurcated adapter can avoid the phenomenon of self-connection of multiple adapters during the library construction process; the amplification primer binding site contained on the bifurcated region can be directly used to bind the amplification primer for amplification reaction

Wherein, each strand of the bifurcation region of the bifurcation adapter contains N nucleotides; preferably, 9≤N≤30. Wherein, there is no limit to the number of nucleotide pairs in the pairing region of the bifurcated junction; preferably, the number of nucleotide pairs in complementary pairing is 7 to 15, and more preferably, the number of nucleotide pairs in complementary pairing is 9~13.

Wherein, the 3' end of the pairing region of the bifurcated linker is a protruding end or a blunt end. Preferably, the 3' end of the pairing region of the bifurcated adapter is a protruding end, and the protruding end can be complementary paired with the sticky end of the fragmented product, which improves the ligation efficiency and facilitates the smooth progress of the sequencing library construction reaction.

Preferably, the bifurcated joint is a T-terminal bifurcated joint, the 3' end of the pairing region of the joint is a protruding end, and the last base of the protruding end is T; for example, the T-terminal bifurcated joint shown in Figure 6, N in the figure is any one of A, T, C, and G bases.

Preferably, the 3' end of the pairing region of the bifurcation linker is an overhang end, and the nucleotides at the overhang end include universal bases. Among them, the number of bases at the protruding end is not particularly limited, but is preferably 1-4. For example, in the bifurcated linker shown in Figure 7, N in the figure is any one of A, T, C, and G bases, and X is a universal base.

Preferably, the bifurcated linker is a dideoxy linker, the 3' end of the pairing region of the linker is a blunt end, and the last nucleotide at the 3' end is a nucleotide with a dideoxy base; for example, Figure 8 In the dideoxy bifurcated linker shown, N in the figure is any one of A, T, C, and G bases, and dd indicates that the last nucleotide at the 3' end is a cytosine nucleus with a dideoxy base glycosides.

It should be noted that the above joint components are only some examples, and are not intended to limit the protection scope of the present invention.

About the implementation of step S12:

In one embodiment, step S12 constructs a sequencing library by directly connecting adapter elements with amplification products.

In another embodiment, when the amplified product of the target region is large or the sequencing library of the target region to be constructed is small, the amplified product is fragmented, and then the fragmented product is ligated with adapter elements to construct a sequencing library. Library, as shown in Figure 9, step S12 comprises the following steps:

S121. Fragment the amplified products corresponding to the multiple target regions to obtain fragmented products;

S122. Using the adapter element to connect the fragmented product to construct a sequencing library.

Through the fragmentation step S121, the amplified product of the target region is converted into smaller fragmented products, thereby facilitating further deep sequencing of the target region. In addition, different amplification products can be transformed into fragmented products of similar length through fragmentation processing, which can facilitate subsequent unified sequencing.

It should be noted:

In step S121, there are various methods for fragmenting the amplified product of the target region, including but not limited to: ultrasonic method, spray method, chemical shearing method and enzyme cleavage method. According to the actual situation, the experiment can be carried out with an appropriate method.

After the fragmentation treatment, the steps of separation and purification of fragmentation products and terminal modification may also be included. According to the length of the sequenced fragments, for the fragmented nucleic acid fragments, separate and purify the target nucleic acid fragments. The separation methods can be commonly used methods, such as gel electrophoresis, sucrose gradient or cesium chloride gradient sedimentation, column chromatography separation, etc. According to the fragmentation method used, the further terminal modification of the obtained target nucleic acid fragments includes but not limited to: phosphorylation or dephosphorylation, end filling and A at the end, so as to facilitate subsequent linker element ligation. The above-mentioned target nucleic acid fragment is not limited in length, preferably 25bp-500bp, more preferably 30bp-200bp, more preferably 40-100bp.

The length of the fragmented product in step S121 is not limited, preferably 25bp-500bp, more preferably 30bp-200bp, more preferably 40bp-100bp. On the premise of realizing the sequencing of the target region, as the length of the target region fragments contained in the target region sequencing library molecule shortens, the sequencing depth of the target region of the high-throughput sequencing technology will be deepened; and the deeper the sequencing depth, that is The more times of sequencing for each base position of the target region, the more accurate the sequencing result, and the more sensitive the detection of a small number of mutations in the sample; this can effectively prevent the low proportion of the target region with mutations in the sample , and the absolute value of the sequencing signal leading to the mutation is low, resulting in inaccurate sequencing results.

In order to limit the size of the sequencing library of the target region, after step S121 or S122, the fragmented products or the sequencing library of the target region can be separated and purified. There are many separation and purification methods, including but not limited to: gel method, sucrose gradient or cesium chloride gradient sedimentation and column chromatography separation. According to the actual situation, the experiment can be carried out with an appropriate method.

According to the above joint components, with regard to step S122, this step will be further described below through multiple embodiments and drawings.

In one embodiment of the present invention, adapters are directly connected to both ends of the fragmented products to form a sequencing library.

The joint can be at least one of the above-mentioned blunt end joint, protruding end joint, joint with stem-loop structure and bifurcated joint.

In another embodiment of the present invention, as shown in FIG. 10, step S122 can be specifically implemented by the following steps:

S1221. Using the first linker to connect the two ends of the fragmented product to obtain a first ligated product;

S1222. Cyclizing the first ligated product to obtain a cyclized product;

S1223. Type II s restriction endonuclease digests the cyclization product to obtain the digested product;

S1224. Connecting a second linker and a third linker to both ends of the digested product to obtain a sequencing library.

In step S1221, the first linker can be one of a blunt end linker, a protruding end linker, a linker with a stem-loop structure, and a bifurcated linker, and the first linker contains a type II s restriction endonuclease Enzyme cutting recognition site; the type II s restriction endonuclease is a restriction endonuclease whose cutting site is outside the recognition sequence, including but not limited to: Acu I, Alw I, Bbs I, BbV I, Bcc I, BceA I, BciV I, BfuA I, Bmr I, Bpm I, BpuE I, Bsa I, BseM II, BseR I, Bsg I, BsmA I, BsmB I, BsmF I, BspCN I, BspM I, BspQ I , BtgZ I, Ear I, Eci I, EcoP15 I, Fau I, Fok I, Hga I, Hph I, HpyAV, MboⅡ, Mly I, Mme I, Mnl I, NmeAIII, Ple I, Sap I, SfaN I, and TspDT I, preferably Acu I, Bsg I, EcoP15 I or Mme I.

When the first linker is a bifurcated linker, the restriction endonuclease recognition site of type II s is located in the complementary region; when the first linker is a linker with a stem-loop structure, the restriction endonuclease The distance between the recognition site and the stem-loop structure is shorter than the distance between the recognition site for restriction endonuclease digestion of type II s and the stem-loop structure.

If the fragmented product is repaired by an end repair enzyme and A is added to the end, the first linker is preferably a bifurcated linker with a T-terminal. If the fragmented product is only repaired by an end repair enzyme to fill in the ends of the fragmented product, the first linker is preferably a dideoxy linker.

In step S1222, there are many ways to realize the cyclization of the first ligation product.

In an embodiment of the present invention, step S1222 includes the following steps:

S12221. Amplifying the first ligation product with enzyme-cutting primers to obtain an amplification product;

S12222. Enzymatic digestion is performed on the amplified product, so that the amplified product forms a cohesive end and circularizes itself into a circularized product.

The 3' ends of the restriction primers are respectively complementary to the two ends of the first ligation product, and the 5' ends both contain restriction endonuclease recognition sites. Both ends of the amplified product formed by amplification in step S12221 contain restriction endonuclease recognition sites, and then under the action of the corresponding enzyme, the two ends of the amplified product form sticky ends, and the two The cohesive ends are complementary and capable of self-circularization.

In another embodiment of the present invention, the first linker contains two enzyme recognition sites, one of which is a restriction endonuclease recognition site, which is used to make the two ends of the first ligation product formed in step S1222 Under the action of the corresponding enzymes, the cohesive ends are formed, and they are complementary to each other and can undergo self-circularization. The other is a type II s restriction endonuclease recognition site, which is used in step S1223 to recognize the cyclization product with an enzyme that recognizes the restriction endonuclease, perform digestion, and then obtain a digestion product.

It should be noted that the above two examples are only two implementations of the present invention for realizing the cyclization of the first ligation product, and no specific limitation is imposed on the protection scope of the present invention.

In step S1223, an enzyme that can recognize the restriction recognition site on the first linker and cut the circularization product (DNA) but not the first linker is used for digestion. The restriction recognition sites on the first adapter include but are not limited to: Mme I restriction recognition sites, Acu I restriction recognition sites, and Bsg I restriction recognition sites.

In step S1224, the second linker can be one of a blunt end linker, a protruding end linker, a linker with a stem-loop structure and a bifurcated linker, and can be labeled with biotin. The third joint may be one of a blunt end joint, a protruding end joint, a joint with a stem-loop structure and a bifurcated joint. The second linker and the third linker can be the same or different. Preferably, the second joint and the third joint are the same and are bifurcated joints. More preferably, the bifurcated junction is a T-terminal bifurcated junction or a dideoxy bifurcated junction.

It should be noted that between steps S1222 and S1223, step S1222A: rolling circle amplification of the circularized product may also be included to obtain a rolling circle amplification product. Through step S1222A, it is possible to ensure sufficient raw materials for the subsequent enzyme digestion step S1223.

Alternatively, between steps S1221 and S1222, step S1221A may also be included: using amplification primers to amplify the first ligation product to obtain an amplification product. The amplification primers are respectively complementary to the linker sequences at both ends of the first ligation product. Through step S1221A, sufficient raw materials can be ensured for the subsequent cyclization step.

This protocol can avoid rolling circle amplification after step S1222, and replace it with ordinary PCR amplification in step S1221A before step S1222, which can effectively reduce the amount of restriction endonuclease II s in the subsequent enzyme digestion step.

Wherein, the first joint is preferably a bifurcated joint.

Wherein, the complementary region of the bifurcated linker may contain at least one enzyme recognition site. The restriction endonuclease recognition site can be a common restriction endonuclease recognition site, and can also be an IIs type restriction endonuclease recognition site.

Wherein, the amplification primer described in step S1221A is preferably a biotinylated primer, which is beneficial to the recovery and purification of the amplification product.

Wherein, the amplification primer in step S1221A has at least one specific restriction enzyme recognition site.

If the specific enzyme recognition site carried on the amplification primer is a uracil base, then in step S1222, the uracil-specific excision reagent is used for enzyme digestion, and then ligation and cyclization are performed.

If the specific restriction endonuclease recognition site carried by the amplification primer is a restriction endonuclease recognition site, then in step S1222, the corresponding restriction endonuclease is used for digestion, and then ligation and circularization are performed.

In another embodiment of the present invention, as shown in FIG. 11, step S122 can be specifically implemented by the following steps:

S1221'. Use the fourth linker to connect with the fragmented product to obtain a second ligated product;

S1222'. II s type restriction endonuclease digests the second connection product to obtain a fragment with the fourth linker;

S1223'. The enzyme-cut fragment with the fourth linker is connected to the fifth linker to form a sequencing library.

It should be noted:

In step S1221', the fourth linker can be one of a blunt end linker, a protruding end linker, a linker with a stem-loop structure, and a bifurcated linker, and the fourth linker contains a type II s restriction Enzyme digestion recognition site; when the fourth joint is a bifurcated joint, the II s type restriction endonuclease recognition site is located in the complementary region; when the fourth joint is a joint with a stem-loop structure When , the distance between the restriction endonuclease recognition site and the stem-loop structure is shorter than the distance between the II s type restriction endonuclease recognition site and the stem-loop structure.

In step S1222', an enzyme that can recognize the enzyme recognition site on the fourth linker and cut the circularization product (DNA) but not the fourth linker is used for enzyme digestion. The restriction recognition sites on the fourth adapter include but are not limited to: Mme I restriction recognition sites, Acu I restriction recognition sites, and Bsg I restriction recognition sites. If the fragmented product is repaired by an end-repair enzyme and A is added to the end, the fourth linker is preferably a bifurcated linker with a T-terminal. If the fragmented product is only repaired by an end repair enzyme, the fourth linker is preferably a dideoxy linker.

In step S1223', the fifth linker can be one of a blunt end linker, a protruding end linker, a linker with a stem-loop structure and a bifurcated linker, and can be labeled with biotin.

In another embodiment of the present invention, step S122 specifically includes the following steps:

S1221". Directly connect adapters with a stem-loop structure to both ends of the fragmented product to form a fragmented product with a stem-loop structure;

S1222". Use restriction endonuclease to cut or excise the stem-loop region of the fragmented product with stem-loop structure, so as to form a sequencing library.

The technical scheme utilizes the joint with the stem-loop structure, which prevents the self-connection phenomenon of multiple joints.

When the number of sequencing libraries formed by the above-mentioned schemes is too small, which is not conducive to the subsequent single-molecule amplification steps, the following steps can also be performed:

Using primers complementary to the adapters at both ends of the sequencing library in the target region, amplifying the obtained sequencing library as a template to obtain an amplified sequencing library. This step can meet the requirements for the amount of the target sequencing library in subsequent experiments.

Wherein, the single-molecule amplification described in step S2 refers to the sequencing of the molecules in the target region in the library, and spatial isolation in the form of a very small amount (even a single molecule) (but these library molecules still belong to the same reaction system as a whole) , and achieve amplification in their respective spaces to enhance the signals of various molecules in subsequent sequencing reactions. The method of single molecule amplification includes but not limited to: emulsion PCR (Emulsion PCR, EPCR), bridge PCR.

The biggest feature of the EPCR is that it can form a large number of independent reaction spaces for DNA amplification. The basic process is that before the PCR reaction, the aqueous solution containing all the reaction components of the PCR is injected onto the surface of the mineral oil rotating at high speed, and the aqueous solution instantly forms countless small water droplets wrapped by the mineral oil. These small water droplets constitute an independent PCR reaction space. Ideally, each droplet contains only one DNA template (target region sequencing library molecule) and one magnetic bead, which contains a primer complementary to the consensus sequence (introduced by the adapter element) of the target region sequencing library molecule. After the reaction, the amplified product of the same source DNA template with a huge copy number is immobilized on the surface of the magnetic beads. For the specific steps of EPCR, please refer to PCR amplification from single DNA molecules on magnetic beads in emulsion: application for high-throughput screening of transcription factor targets, Takaaki Kojima, Yoshiaki Takei, Miharu Ohtsuka et al, Nucleic Acids 3, Nool. Research, 2 .17; Dual primer emulsion PCR for nextgeneration DNA sequencing, MingYan Xu, Anthony D. Aragon, Monica R. Mascarenas et al, BioTechniques48: 409-412 (May 2010); BEAMing: single-molecule PCR on microparticles inwater-in-oil emulsions, Frank Diehl, Meng Li, Yiping He, nature methods, Vol.3, No.7, July 2006, etc.

The basic principle of the bridge-type PCR is that the primers of the bridge-type PCR are fixed on the solid-phase carrier, and the PCR amplification product will be fixed on the solid-phase carrier during the PCR process, and the PCR amplification product can be combined with the solid-phase carrier. The primers of the complementary pairs are complementary to form a bridge, and then the complementary paired primers are amplified with the bridged amplification product as a template. By controlling the amount of initial template added, after the bridge PCR reaction is completed, the amplified products exist in clusters on the solid-phase support, and the amplified products of each cluster are amplified products of DNA templates from the same source. For its specific principles and implementations, please refer to the following documents: CN20061009879.X, US6227604.

As mentioned above, in the prior art, Sanger sequencing technology can only sequence a certain region of a sample at a time due to its own technical limitations. In order to realize the simultaneous detection of multiple regions in the sample at one time, the present invention adopts a high-throughput gene sequencing method in the sequencing method. Compared with Sanger sequencing, high-throughput gene sequencing is more convenient and sensitive to detect sequence information. After each molecule in the sequencing library is amplified by single molecule, each molecule of the sequencing library forms a single-molecule copy array. Throughput gene sequencing is in different positions, so that the hybridization between the sequencing primer and the single-molecule copy array, and the extension reaction under the action of the enzyme can be carried out simultaneously without mutual interference. Therefore, a large number (millions, tens of millions, or even more) of single-molecule copy arrays can be sequenced at the same time, and then the required sequence information can be accurately obtained by collecting the corresponding signals, and the sensitivity of sequencing Higher than Sanger. In particular, the fragmentation of the amplification products of multiple target regions is equivalent to an increase in the number of sequencing of each base of the target region molecules of the same sequence, which can further improve the sensitivity of sequencing.

Wherein, the high-throughput sequencing technology described in step S3 includes, but is not limited to: polymerase-based sequencing-by-synthesis and ligase-based sequencing-by-ligation.

Polymerase-based sequencing-by-synthesis methods are based on removably labeled nucleotides. In each synthesis reaction, each template strand can only be extended once at most. The general flow of the polymerase-based sequencing-by-synthesis method is as follows:

a. Sequencing primers bind to the known sequence shared by the single-molecule amplification product through complementary pairing (the single-molecule amplification product is fixed on the primer-solid phase carrier complex), and under the action of DNA polymerase, the Remove the labeled nucleotides to perform a single base extension synthesis reaction, collect the labeling signal of the added nucleotides, and then obtain a single-molecule amplification product complementary to the 3'most base of the sequencing primer (fixed on the primer-solid The base sequence information of the next bit on the phase-carrier complex).

b. Excise the removable marker, and then under the action of DNA polymerase, continue the single-base extension synthesis reaction with the nucleotide with the removable marker, collect the marker signal of the added nucleotide, and then obtain the sequence primer 3 'The base sequence information of the next two bits of the single-molecule amplification product with complementary terminal bases.

Step b is repeated until the synthesis reaction cannot be continued, so as to obtain all sequence information of the single-molecule amplification product.

Ligase-based ligation sequencing methods are all based on fluorescently labeled oligonucleotide probes. One of the ligase ligation sequencing methods is based on a fluorescently labeled oligonucleotide probe with n bases at a specific position, counting from its 5' end. The a position has a fluorescent label, where different bases correspond to specific fluorescent labels, because the 3' end of the oligonucleotide probe has been specifically modified, and the oligonucleotide probes cannot be directly connected to each other. For each ligation reaction, only one oligonucleotide probe can be ligated to each single-molecule amplification product. The general flow of the ligation sequencing method is as follows:

A. Sequencing primers are bound by complementary pairing on the known sequence shared by the unimolecular amplification product (the unimolecular amplification product is immobilized on the primer-solid phase carrier complex), using the above-mentioned oligonucleotide probes, Under the action of ligase, connect the nucleic acid probe to the above-mentioned oligonucleotide chain, and then collect the fluorescent signal to obtain the a-th base sequence after the 3' end of the known sequence shared by the single-molecule amplification product information.

B. Cut off the fluorescent label on the oligonucleotide, under the action of ligase, use the above-mentioned oligonucleotide probe as the raw material, continue the ligation reaction, and then collect the fluorescent signal, so as to obtain the common value of the single-molecule amplification product The base sequence information at the 2a position after the 3' end of the known sequence.

Repeat step B until the ligation reaction cannot be continued, so as to obtain the base sequence information of positions a, 2a, 3a, 4a... after the 3' end of the known sequence shared by the unimolecular amplification products.

Then, the sequencing primer and the oligonucleotide probe connected thereto are denatured and eluted from the single-molecule amplification product, and the above reaction is repeated with a primer that has one base less at the 3' end than the previous sequencing primer, thereby Obtain the base sequence information of positions a-1, 2a-1, 3a-1, 4a-1... after the 3' end of the known sequence shared by the unimolecular amplification products. Repeat this step to finally obtain the a-(a-1), 2a-(a-1), 3a-(a-1), 4a-(a -1) The base sequence information of ..., so as to obtain the entire sequence information of the single-stranded amplification product.

Another ligase ligation sequencing method is also based on fluorescently labeled oligonucleotide probes, the oligonucleotide probes have n bases and are divided into h (h≤n) groups, Different fluorescent labels of the same group of oligonucleotide probes correspond to different base sequences at the same specific position, and the difference between different groups is that the specific positions corresponding to different fluorescent labels are different, because the 3' of the oligonucleotide probe Specific modification is carried out at the end, and the oligonucleotide probes cannot be directly connected to each other. In each ligation reaction, only one oligonucleotide probe can be connected to each single-molecule amplification product. The general flow of the ligation sequencing method is as follows:

a. The sequencing primer is bound to the known sequence shared by the single molecule amplification product (the single molecule amplification product is immobilized on the primer-solid phase carrier complex) by complementary pairing, using the above-mentioned oligonucleotide probe One group (the base position corresponding to the fluorescent label is x, x≤h), under the action of ligase, connect the nucleic acid probe to the above-mentioned oligonucleotide chain, and then collect the fluorescent signal to obtain the The base sequence information at position x after the 3' end of the known sequence common to the amplified products is used to denature and elute the sequencing primers and their connected oligonucleotide probes from the single-molecule amplified products.

b. Then re-combine the sequencing primers on the single-molecule amplification product, and use an oligonucleotide probe set different from that in step a (the base position corresponding to the fluorescent label is y, y≤h), in the ligase Under the action, the nucleic acid probe is connected to the above-mentioned oligonucleotide chain, and then the fluorescent signal is collected to obtain the y-th base sequence information after the 3' end of the known sequence shared by the single-stranded amplification product, and the sequencing Primers and their attached oligonucleotide probes are denatured and eluted from single-molecule amplification products.

c. Repeat step b until the oligonucleotide probes of group h have undergone a ligation reaction respectively, so as to obtain the 1st, 2nd... , The base sequence information of the h position.

Replace the primers with one or more universal bases at the 3' end compared with the previous sequencing primers to carry out the reaction according to the above principle, which can extend the reading of the 3' end base sequence of the known sequence shared by the obtained single molecule amplification products long.

For the principle and specific implementation of this ligase-based ligation sequencing method, please refer to CN200710170507.1.

Taking the detection of exons 19 and 21 of the EGFR gene as an example, the present invention proposes an embodiment, designing specific primers for amplifying exons 19 and 21 of the EGFR gene: 19F (SEQ ID NO: 1), 19R ( SEQ ID NO: 2), 21F (SEQ ID NO: 3), 21R (SEQ ID NO: 4).

1. Extraction of DNA from samples to be tested

DNA was extracted from whole blood samples (1 to 10), serum samples (11 to 20), and paraffin tissue samples (21 to 30) using common nucleic acid extraction kits on the market, and marked accordingly.

2. Amplification of the target area

The amplification of exons 19 and 21 of the EGFR gene of the same sample was carried out in the same reaction system, and the reaction system was as follows:

19F (10μM) 2μL;

19R (10μM) 2μL;

21F (10μM) 2μL;

21R (10μM) 2μL;

dNTP (2.5mM each) 4μL;

Sample DNA to be tested 20ng;

Ex Taq(5U/μL) 0.25μL;

10×Ex Taq Buffer 5μL;

ddH ₂ O up to 50 μL.

The PCR reaction conditions are as follows:

95°C for 3 minutes;

94°C for 30s, 58°C for 30s, 72°C for 30s; repeat 25 cycles;

72°C for 7min.

The PCR cleaning kit was used to clean the amplified products of each sample, remove unamplified primers and dNTPs, and recover the amplified products.

3. Construction of target region sequencing library

This step can have multiple implementations, and in one embodiment of the present invention, comprises the following two steps:

1. Fragmentation of amplified products

Fragmentation was performed using sonication. The specific operation is as follows: add the amplified product (about 50 μL) recovered by PCR cleaning of each sample into 400 μL TE buffer, sonicate for 4 s under 430 W power, with an interval of 20 s, and repeat 5 times to obtain fragmented products. The fragmented products of each sample were separated and purified by using 1% agarose gel, and fragmented products with a size between 40 bp and 100 bp were recovered by gel cutting.

2. Construction of sequencing library

Before constructing the sequencing library of the target region, it is necessary to modify the ends of the fragmented products recovered from gel cutting to facilitate the connection of adapter elements. In this example, the end modification of the fragmented products recovered from gel cutting includes phosphorylation, end filling and A reaction at the end.

The specific implementation is as follows:

1) Phosphorylation and end-filling reactions

The system is:

20μL (about 2000ng) of the fragmented product recovered by gel cutting;

10mM dNTP 1.5μL;

T4 DNA polymerase (5U/μL) 1 μL;

Klenow DNA polymerase 0.1 μL;

T4 polynucleotide kinase (10U/μL) 0.5μL;

10m MATP 1.5μL;

10×T4 ligase buffer 10μL;

Add ddH ₂ O to 100 μL.

The reaction conditions are: incubate at 20°C for 20 min. After the reaction, the recovery kit was used for purification and recovery.

2) Add A tail at the end

The reaction system is:

60μL (about 1000ng) of the recovered product after phosphorylation and end-filling;

Klenow buffer (NEB Buffer2) 10μL;

10mM dATP 2μL;

Klenow enzyme (3'to 5'exo minus, 10U/μL) 1μL;

Add ddH ₂ O to 100 μL.

The reaction conditions are: incubate at 37°C for 30 min. After the reaction was completed, it was purified and recovered using a purification kit.

3) Connection connector 1

In this example, the T-terminal bifurcated adapter as shown in FIG. 6 is used as the adapter 1. The T-terminal bifurcated adapters used in the same sample are the same, and the T-terminal bifurcated adapters of different samples are different. The difference lies in the different tag sequences. The tag sequences corresponding to different samples are shown in the table below.

Under the action of T4 ligase, the above-mentioned T-terminal bifurcated adapters were respectively ligated with the products purified and recovered after adding A tails to form fragments with adapter 1. The connection system is:

Add 50μL (about 500ng) of the recovered product after adding A tail;

Connector 1 2μL (about 3000ng);

10mM ATP 5μL;

T4 DNA ligase (10U/μL) 1 μL;

10×T4 ligase buffer 10μL;

Add ddH ₂ O to 100 μL.

The reaction conditions are: incubate at 16°C for more than 4h. After the reaction was completed, it was purified and recovered using a purification kit.

4) PCR amplification of the fragment with adapter 1

The amplification system is:

The fragment with linker 1 is about 300ng;

10×Ex Taq Buffer 100μL;

Primer F (100 μM, SEQ ID NO: 11) 10 μL;

Primer R (100 μM, SEQ ID NO: 12) 10 μL;

Ex Taq(5U/μL) 7.5μL;

ddH ₂ O up to 1000 μL.

The PCR reaction conditions are as follows:

95°C for 3 minutes;

94°C for 30s, 58°C for 30s, 72°C for 30s; repeat 25 cycles;

72°C for 7min.

The PCR cleaning kit was used to clean the amplified products of each sample, remove unamplified primers and dNTPs, and recover the amplified products of the fragments with adapter 1.

5) Type II s restriction endonuclease digestion

The amplified product of the recovered fragment with adapter 1 was digested with Acu I, and the reaction system was as follows:

60 μL (3-5 μg) of the amplified product of the recovered fragment with adapter 1;

10×NEB Buffer 4 8μL;

Acu I(NEB) 2μL(10U);

SAM (3.2mM) 1μL (final concentration 50μM);

ddH ₂ O up to 80 μL.

Reaction conditions: incubate at 37°C for 1 hour. After the reaction, the digested product was purified and recovered using a purification kit.

6) Connect connector 2

Use the protruding end bifurcated adapter as shown in Figure 7 as the adapter 2, and connect with the recovered enzyme digestion product to obtain a sequencing library. The connection system is as follows:

Recovered digested product 2 μg;

Connector 2 1μL (10pM);

10T4 DNA ligase (3U/μL) 1 μL;

10×T4 ligase buffer 2μL;

Add ddH ₂ O to 100 μL.

In connection conditions, incubate at 14°C for 2h. After the reaction, use a purification kit to purify and recover, and then denature to form a single strand to obtain a sequencing library.

4. Single-molecule amplification of the target region sequencing library

Measure the respective concentrations of the 30 target region sequencing libraries obtained in step (3), then mix them at the same concentration, and then perform single-molecule amplification to obtain single-molecule amplification products. The single-molecule amplification method can be EPCR or bridge PCR.

It is preferably EPCR amplification, and the unimolecular amplification primers are preferably: SEQ ID NO: 5 and SEQ ID NO: 6.

5. High-throughput sequencing of single-molecule amplification products

The sequencing method may adopt a synthetase-based sequencing-by-synthesis method or a ligase-based sequencing-by-ligation method. By performing bioinformatics analysis on the sequencing results, the sequence information of exons 19 and 21 of the EGFR gene of the above-mentioned 30 samples can be obtained. Analysis of the sequence results shows that, except for the mutation in exon 19 of the EGFR gene in sample 23 (see SEQ ID NO: 8 for sequence information, the mutation rate is 6.2%), all exons in other samples are wild type.

It should be noted that this embodiment is only a specific embodiment of the present invention, and does not have any limiting effect on the present invention. For example, the specific primer of EGFR gene can be replaced by other rationally designed primers; in addition, this embodiment Many of the steps in can be replaced with reference to the aforementioned method, and will not be repeated here.

Regarding the sensitivity of the method for sequencing the target region of the present invention, the present invention uses the following examples to verify.

By conventional design, construct plasmids containing respectively EGFR gene exon 19 wild-type sequence (SEQ ID NO: 7) and mutant sequence (SEQ ID NO: 8); containing EGFR gene exon 21 wild-type sequence (SEQ ID NO : 9) and the plasmid of the mutant sequence (SEQ ID NO: 10).

Then prepare plasmid mixtures with mutation rates of 20%, 10%, 5%, 3%, 1%, and 0%, respectively. The above-mentioned plasmid mixture containing 1000 copies of the plasmid was used as a template, and then the detection was carried out according to the method referring to the above-mentioned examples. The test results are shown in the table below:

The results show that the minimum detection limit of the method for sequencing the target region of the present invention is about 3%, and the detection results of 5% and above are basically consistent with the actual situation.

A kit for sequencing a region of interest, comprising:

Specific primers for amplifying multiple target regions in the sample to be tested;

Linker elements are used to combine with amplification products to construct sequencing libraries.

The kit is used for the sequencing of target regions, and can perform deep sequencing on multiple target regions of the sample at the same time, and accurately obtain the sequence information of these target regions, including the variation of each mutation site with known mutations and positional mutations. The mutation frequency of each mutation site in each sample can be obtained, and the detection sensitivity is high, which can be used for large-scale population screening in the target area to determine the proportion of various genotypes in the target area.

Wherein, the linker element is used to construct a target region sequencing library, including at least one or more linkers.

Wherein, the joint element may adopt various forms, such as at least one of a blunt end joint, a protruding end joint, a joint with a stem-loop structure, and a bifurcated joint.

Wherein, the linker can be methylated or biotinylated, or biotinylated and methylated simultaneously.

Wherein, at least one adapter in the adapter element includes a first tag sequence, which is used to mark the sequencing libraries of different samples to be tested during the library construction process. The first tag sequence is preferably a nucleic acid molecule with a specific base sequence, and the number of bases is not limited, preferably 3-20, more preferably 4-20.

Wherein, the specific primer is complementary or partially complementary to the target region.

Further, among the specific primers corresponding to each target region, at least one primer is partially complementary to the target region, and the 5' end of the primer contains a second tag sequence, which is used to treat different target regions in the process of amplifying the target region. The amplified product of the target region of the test sample is labeled. The second tag sequence is preferably a nucleic acid molecule with a specific base sequence, and its base length is not limited.

Furthermore, the number of bases in the second tag sequence is 3-20, more preferably 4-10.

Wherein, the kit may also include PCR amplification reagents, and the PCR amplification reagents include PCR enzyme, dNTP, PCR buffer, and Mg ²⁺ solution.

Wherein, the kit may further include an enzyme for recognizing the restriction enzyme recognition site on the linker element.

It should be noted that the gene sequence information detected by the method or kit of the present invention can be used in various scientific research, including but not limited to population sequence analysis, gene function research, and protein function research. For example, combined with subsequent further molecular biology tests, clinical trials, clinical observations and statistical analysis of comprehensive data, etc., to achieve a variety of scientific research purposes, including but not limited to: genotype distribution of certain specific genes in a specific population, various The relationship between gene mutation types and related physiological activities, etc.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (11)

1. A method for sequencing a target region, comprising the following steps: A. Use specific primers to amplify multiple target regions in the sample to be tested, and construct a sequencing library based on the amplified products; B. performing single-molecule amplification on the sequencing library to obtain multiple single-molecule amplification products corresponding to the multiple target regions; C. Simultaneously performing high-throughput gene sequencing on the multiple single-molecule amplification products to obtain sequence information of the multiple target regions. 2. The method for sequencing the target region according to claim 1, wherein said step A comprises the following steps: A1. using specific primers to amplify multiple target regions in the sample to be tested, and obtain amplification products corresponding to the multiple target regions; A2. Use adapter elements to connect the amplified products corresponding to the multiple target regions to obtain a sequencing library; the adapter elements use blunt end adapters, protruding end adapters, adapters with stem-loop structures, and bifurcated adapters. at least one. 3. The method for sequencing the target region according to claim 2, wherein said step A2 comprises the following steps: A21. Fragmenting the amplified products corresponding to the multiple target regions to obtain fragmented products; A22. Using linker elements to connect with the fragmented products to construct a sequencing library. 4. The method for sequencing the target region according to claim 3, wherein said step A22 comprises the following steps: A221. Using the first linker to connect the two ends of the fragmentation product to obtain the first ligation product; A222. cyclization of the first connection product to obtain a cyclization product; A223. II s type restriction endonuclease digests the cyclization product to obtain the digested product; A224. Connect the second linker and the third linker to both ends of the restriction product to obtain a sequencing library. 5. The method for sequencing the target region according to claim 3, wherein said step A22 comprises the following steps: A221'. Use the fourth linker to connect with the fragmentation product to obtain the second connection product; A222'. II s type restriction endonuclease digests the second connection product to obtain an enzyme-cut fragment with the fourth linker; A223'. The digested fragment with the fourth linker is connected with the fifth linker to form a sequencing library. 6. The method for sequencing a target region according to any one of claims 2 to 5, characterized in that at least one adapter in the adapter elements described in step A2 contains a first tag sequence for use in library construction During the process, the sequencing libraries of different samples to be tested are marked. 7. The method for sequencing a target region according to any one of claims 1 to 5, characterized in that, among the specific primers corresponding to each target region, at least one primer is partially complementary to the target region, and the part The 5' end of the complementary primer contains a second tag sequence, which is used to mark the amplified products of the target region of different samples to be tested during the process of amplifying the target region. 8. A kit for sequencing a target region, comprising: Specific primers for amplifying multiple target regions in the sample to be tested; Linker elements are used to combine with amplification products to construct sequencing libraries. 9. The kit for sequencing the target region according to claim 8, wherein the adapter element is at least one of a blunt end adapter, a protruding end adapter, an adapter with a stem-loop structure, and a bifurcated adapter . 10. The kit for sequencing the target region according to claim 8, characterized in that at least one adapter in the adapter element contains a first tag sequence, which is used for different to-be-tested sequences during library construction. Sample the sequencing library for labeling. 11. The kit for sequencing a target region according to any one of claims 8 to 10, wherein at least one of the specific primers corresponding to each target region is partially complementary to the target region, the The 5' end of the partially complementary primer contains a second label sequence, which is used to mark the amplification products of the target region of different samples to be tested during the process of amplifying the target region.

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