HK1240263B - Isolated oligonucleotide and use thereof in nucleic acid sequencing - Google Patents

Description

Isolated oligonucleotides and their use in nucleic acid sequencing

Technical Field

The present invention relates to the field of biotechnology. Specifically, it relates to isolated oligonucleotides and their use in nucleic acid sequencing. More specifically, it relates to an isolated oligonucleotide, a kit, a method for adding linkers to both ends of a double-stranded DNA fragment, a method for constructing a sequencing library for double-stranded DNA fragments, and a nucleic acid sequencing method.

Background Art

High-throughput sequencing has become a cornerstone of modern molecular biology, biotechnology, medicine, and other fields. In recent years, research on rapid, accurate, and cost-effective methods for determining gene expression levels and nucleotide sequences has continuously advanced. Second-generation high-throughput sequencing technology, based on the sequencing-by-synthesis principle, has matured, and major sequencing companies have focused on developing new sequencing products, shortening sequencing processes, and reducing costs. Currently, sequencing products based on second-generation sequencing include whole-genome resequencing, whole-transcriptome sequencing, and small RNA sequencing. In particular, target capture sequencing, a derivative of second-generation sequencing combined with microarray technology, uses a large number of oligonucleotide probes to bind complementary sequences to specific regions of the genome, enriching for specific segments. These segments are then sequenced using second-generation sequencing, enabling whole-exome sequencing (WES). This sequencing method offers significant advantages over whole-genome sequencing, as it reduces the data analysis burden.

However, the current technologies related to nucleic acid sequencing still need to be improved.

Summary of the Invention

The present invention aims to solve at least one of the technical problems existing in the prior art.

First, it should be noted that the present invention is based on the following discoveries made by the inventors:

Complete Genomics (sometimes referred to as "CG" in this article) currently has a set of second-generation sequencing technologies independently developed, which are suitable for human whole genome sequencing. Its library construction process mainly includes: genomic DNA fragmentation, first adapter ligation, double-strand circularization and enzyme digestion, second adapter ligation, and single-strand separation and circularization. Among them, the two adapter ligations are very important in the entire library construction process. The adapter is a DNA sequence that is fixed at both ends of the DNA fragment by connection. During sequencing, it can be recognized and used as the starting point for sequencing, so that the instrument can read the subsequent sequence information. In order to ensure that the read sequence information is easy to analyze, two different adapters need to be added to both ends of a DNA fragment (5' end and 3' end); in order to achieve this specific directional connection and avoid mutual connection between adapters, a sticky end adapter connection method can be used; however, this method requires fragments with sticky ends, and it is difficult to avoid the problem of mutual connection between fragments. The sequencing library construction of Complete Genomics adopts a method of adding adapters at both ends in multiple steps. To obtain fragments with adapters attached to both ends, five steps are required: ligating an adapter to one end of the DNA fragment, denaturing and annealing the DNA fragment, ligating an adapter to the other end of the DNA fragment, gap filling, and polymerase chain reaction. The multiple extension reactions require expensive reagents, and multiple purification and recovery steps are required between these steps, resulting in high overall costs and inefficiency. Furthermore, in current library construction protocols, this adapter ligation process must be performed twice.

To this end, the present invention proposes a method for adding linkers to both ends of a DNA fragment.

In the first aspect of the present invention, the present invention provides a separated oligonucleotide. According to an embodiment of the present invention, the oligonucleotide includes: a first chain, wherein the 5' terminal nucleotide of the first chain has a phosphate group, and the 3' terminal nucleotide of the first chain is a dideoxynucleotide; and a second chain, wherein the 5' terminal nucleotide of the second chain does not have a phosphate group, and the 3' terminal nucleotide of the second chain is a dideoxynucleotide, wherein the length of the first chain is greater than the length of the second chain, and a double-stranded structure is formed between the first chain and the second chain. Since the 3' ends of the first and second chains in the oligonucleotide are both dideoxynucleotides, and the 5' terminal nucleotide of the second chain does not have a phosphate group, these ends will not be able to connect with other nucleic acid fragments, thereby preventing the oligonucleotides from connecting with each other. Thus, the separated oligonucleotide can be used as a linker for constructing a sequencing library, and when constructing a sequencing library, different linkers can be connected to both ends of the nucleic acid fragment at the same time, while avoiding the connection between the linkers, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library.

According to a second aspect of the present invention, the present invention proposes a test kit. According to an embodiment of the present invention, the test kit comprises: a first joint and a second joint, wherein the first joint and the second joint are both the aforementioned separated oligonucleotides, wherein the first joint is different from the second joint. As previously mentioned, since the 3' ends of the first and second chains in the oligonucleotides according to an embodiment of the present invention are both dideoxynucleotides, and the 5' terminal nucleotides of the second chain do not have a phosphate group, these ends will not be able to interconnect with other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Thus, the test kit can be used as a joint for constructing a sequencing library, and when constructing a sequencing library, different joints can be connected at both ends of the nucleic acid fragments at the same time, while avoiding the mutual connection between the joints, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library. The above description of the features and advantages of the separated oligonucleotides according to an embodiment of the present invention is equally applicable to the test kit and will not be repeated here.

In the third aspect of the present invention, the present invention provides a method for adding linkers at both ends of a double-stranded DNA fragment. According to an embodiment of the present invention, the double-stranded DNA fragment has two blunt ends, and the four terminal nucleotides of the double-stranded DNA fragment do not have a phosphate group, and the method comprises: connecting the double-stranded DNA fragment with a first linker and a second linker to obtain a first connection product, wherein the first linker and the second linker are different, and the first linker and the second linker are both the separated oligonucleotides described above; using a first single-stranded DNA to replace the second chain of the first linker, and using a second single-stranded DNA to replace the second chain of the second linker, wherein the first single-stranded DNA can specifically match the first chain of the first linker to form a double-stranded structure, and the second single-stranded DNA can specifically match the second chain of the second linker to form a double-stranded structure. The first strand of the head specifically matches to form a double-stranded structure; the first single-stranded DNA and the second single-stranded DNA are respectively connected to the double-stranded DNA fragment to obtain a second connection product; and the second connection product is amplified using a first primer and a second primer to obtain an amplified product, wherein the amplified product is a DNA fragment with a linker connected at both ends, wherein the first primer contains the same sequence as one of the first single-stranded DNA and the second single-stranded DNA, and the second primer contains the same sequence as the other of the first single-stranded DNA and the second single-stranded DNA, and has an additional biotin at the 5' end compared to the other of the first single-stranded DNA and the second single-stranded DNA. As mentioned above, since the 3' ends of the first and second strands in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the 5' terminal nucleotide of the second strand does not have a phosphate group, these ends will not be able to connect to each other with other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Thus, the oligonucleotide can be used as a linker to connect different linkers at both ends of the nucleic acid fragment when constructing a sequencing library, while avoiding the connection between the linkers, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library. The aforementioned description of the features and advantages of the isolated oligonucleotides according to the embodiments of the present invention also applies to this method and will not be repeated here. Furthermore, when constructing a sequencing library, the first and second single-stranded DNAs can be used to replace the second strands of the two linkers, respectively, and form a more stable double-stranded structure with the first strand. Furthermore, by using the first and second single-stranded DNAs as primers for PCR amplification, DNA fragments with stable linkers at both ends can be generated.

In a fourth aspect of the present invention, the present invention proposes a method for constructing a sequencing library for double-stranded DNA fragments. According to an embodiment of the present invention, the double-stranded DNA fragment has two blunt ends, and none of the four terminal nucleotides of the double-stranded DNA fragment has a phosphate group, and the method comprises: according to the method for connecting a linker at both ends of the double-stranded DNA fragment described above, connecting a linker at both ends of the double-stranded DNA fragment to obtain a DNA fragment with a linker connected at both ends; separating a single-stranded DNA fragment from the DNA fragment with a linker connected at both ends; and circularizing the single-stranded DNA fragment to obtain a single-stranded DNA ring, which constitutes the sequencing library. As mentioned above, since the 3' ends of the first and second chains in the oligonucleotides according to the embodiment of the present invention are both dideoxynucleotides, and the 5' terminal nucleotide of the second chain does not have a phosphate group, these ends will not be able to connect to other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Thus, when constructing a sequencing library, the oligonucleotide can be used as a linker to connect different linkers at both ends of the nucleic acid fragment, while avoiding the linkers from connecting to each other, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library. The above description of the features and advantages of the isolated oligonucleotides according to the embodiments of the present invention is also applicable to this method and will not be repeated here. In addition, when constructing a sequencing library, the first single-stranded DNA and the second single-stranded DNA can be used to replace the second chains of the two connectors respectively, and form a more stable double-stranded structure with the first chain. Further, by using the first single-stranded DNA and the second single-stranded DNA as primers and performing PCR amplification, a DNA fragment with stable connectors at both ends can be formed. Further, by isolating the single-stranded DNA and performing a single-stranded circularization reaction, a sequencing library can be effectively obtained, such as a sequencing library for a CG sequencing platform.

In a fifth aspect, the present invention provides a method for nucleic acid sequencing. According to an embodiment of the present invention, the method comprises: constructing a sequencing library according to the method for constructing a sequencing library for double-stranded DNA fragments described above; and sequencing the sequencing library. As described above, since the 3' ends of the first and second strands of the oligonucleotides according to the embodiments of the present invention are both dideoxynucleotides, and the 5' terminal nucleotide of the second strand does not have a phosphate group, these ends cannot ligate with other nucleic acid fragments, thereby preventing ligation between oligonucleotides. Thus, when constructing a sequencing library, the oligonucleotides can be used as linkers to simultaneously connect different linkers to both ends of a nucleic acid fragment, while preventing linkers from ligating to each other, improving ligation efficiency, and reducing the economic and time costs of constructing the sequencing library. The above description of the features and advantages of the isolated oligonucleotides according to the embodiments of the present invention also applies to this method and will not be repeated here. In addition, when constructing the sequencing library, the first and second single-stranded DNAs can be used to replace the second strands of the two linkers, respectively, and form a more stable double-stranded structure with the first strand. Furthermore, by using the first and second single-stranded DNAs as primers for PCR amplification, DNA fragments with stable linkers at both ends can be generated. By further isolating single-stranded DNA and performing a single-strand circularization reaction, a sequencing library can be effectively obtained, such as a sequencing library for a CG sequencing platform, thereby further improving sequencing efficiency and reducing sequencing costs.

In the sixth aspect of the present invention, the present invention also provides a device for adding connectors at both ends of a double-stranded DNA fragment. According to an embodiment of the present invention, the double-stranded DNA fragment has two blunt ends, and the four terminal nucleotides of the double-stranded DNA fragment do not have a phosphate group, and the device includes: a first connection unit, the first connection unit is used to connect the DNA fragment with a first connector and a second connector to obtain a first connection product, wherein the first connector and the second connector are different, and the first connector and the second connector are both the separated oligonucleotides described above; a replacement unit, the replacement unit is used to replace the second chain of the first connector with a first single-stranded DNA, and replace the second chain of the second connector with a second single-stranded DNA, wherein the first single-stranded DNA can specifically match the first chain of the first connector to form a double-stranded structure, and the second single-stranded DNA can specifically match the first chain of the first connector to form a double-stranded structure. DNA can specifically match the first strand of the second adapter to form a double-stranded structure; a second connecting unit, the second connecting unit is used to connect the first single-stranded DNA and the second single-stranded DNA to the DNA fragment respectively to obtain a second connection product; and an amplification unit, the amplification unit is used to amplify the second connection product using a first primer and a second primer to obtain an amplified product, wherein the first primer contains the same sequence as one of the first single-stranded DNA and the second single-stranded DNA, and the second primer contains the same sequence as the other of the first single-stranded DNA and the second single-stranded DNA, and has an additional biotin at the 5' end compared to the other of the first single-stranded DNA and the second single-stranded DNA. As mentioned above, since the 3' ends of the first and second chains in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the 5' terminal nucleotide of the second chain does not have a phosphate group, these ends will not be able to connect to each other with other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Thus, the oligonucleotide can be used as a linker to connect different linkers at both ends of the nucleic acid fragment when constructing a sequencing library, while avoiding the connection between the linkers, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library. The aforementioned description of the features and advantages of the isolated oligonucleotides according to the embodiments of the present invention also applies to this apparatus and will not be repeated here. Furthermore, when constructing a sequencing library, the first and second single-stranded DNAs can be used to replace the second strands of the two linkers, respectively, and form a more stable double-stranded structure with the first strand. Furthermore, by using the first and second single-stranded DNAs as primers for PCR amplification, DNA fragments with stable linkers at both ends can be generated.

In a seventh aspect of the present invention, the present invention further proposes a device for constructing a sequencing library for double-stranded DNA fragments. According to an embodiment of the present invention, the double-stranded DNA fragment has two blunt ends, and the four terminal nucleotides of the double-stranded DNA fragment do not have a phosphate group, and the device includes: the aforementioned device for adding adapters to the two ends of the double-stranded DNA fragment, which is used to connect adapters at both ends of the double-stranded DNA fragment to obtain DNA fragments with adapters connected at both ends; a single-stranded DNA fragment separation device, which is used to separate single-stranded DNA fragments from the DNA fragments with adapters connected at both ends; and a cyclization device, which is used to cyclize the single-stranded DNA fragments to obtain single-stranded DNA rings, which constitute the sequencing library. As mentioned above, since the 3' ends of the first and second chains in the oligonucleotides according to the embodiments of the present invention are both dideoxynucleotides, and the 5' terminal nucleotide of the second chain does not have a phosphate group, these ends will not be able to connect to each other with other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Thus, the oligonucleotide can be used as a connector to connect different connectors at both ends of the nucleic acid fragment when constructing a sequencing library, while avoiding mutual connection between the connectors, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library. The above description of the features and advantages of the isolated oligonucleotide according to the embodiment of the present invention is also applicable to this device and will not be repeated here. In addition, when constructing a sequencing library, the first single-stranded DNA and the second single-stranded DNA can be used to replace the second chains of the two connectors respectively, and form a more stable double-stranded structure with the first chain. Further, by using the first single-stranded DNA and the second single-stranded DNA as primers and performing PCR amplification, a DNA fragment with stable connectors at both ends can be formed. Further, by isolating the single-stranded DNA and performing a single-stranded circularization reaction, a sequencing library can be effectively obtained, such as a sequencing library for a CG sequencing platform.

In its eighth aspect, the present invention also provides a nucleic acid sequencing system. According to an embodiment of the present invention, the system comprises: the aforementioned apparatus for constructing a sequencing library for double-stranded DNA fragments; and a sequencing device for sequencing the sequencing library. As previously described, since the 3'-termini of the first and second strands of the oligonucleotides according to the embodiments of the present invention are both dideoxynucleotides, and the 5'-terminal nucleotide of the second strand lacks a phosphate group, these termini cannot ligate with other nucleic acid fragments, thereby preventing interlinking between oligonucleotides. Thus, when constructing a sequencing library, the oligonucleotides can be used as linkers to simultaneously connect different linkers to both ends of a nucleic acid fragment, while preventing interlinking between linkers, improving linking efficiency and reducing the economic and time costs of constructing the sequencing library. The aforementioned description of the features and advantages of the isolated oligonucleotides according to the embodiments of the present invention also applies to this system and will not be repeated here. Furthermore, when constructing a sequencing library, the first and second single-stranded DNAs can be used to replace the second strands of the two linkers, respectively, and form a more stable double-stranded structure with the first strand. Furthermore, by using the first and second single-stranded DNAs as primers for PCR amplification, DNA fragments with stable linkers at both ends can be generated. By further isolating single-stranded DNA and performing a single-strand circularization reaction, a sequencing library can be effectively obtained, such as a sequencing library for a CG sequencing platform, thereby further improving sequencing efficiency and reducing sequencing costs.

In the ninth aspect of the present invention, the present invention also proposes a device for constructing a sequencing library for genomic DNA. According to an embodiment of the present invention, the device includes: means for fragmenting the genomic DNA to obtain fragmented products; means for dephosphorylating the fragmented products to obtain dephosphorylated fragmented products; means for performing end repair on the dephosphorylated fragmented products to obtain double-stranded DNA fragments; means for connecting the double-stranded DNA fragments with a first connector and a second connector to obtain a first connection product, wherein the first connector and the second connector are different, and the first connector and the second connector are both the separated oligonucleotides described above; means for replacing the second chain of the first connector with a first single-stranded DNA, and replacing the second chain of the second connector with a second single-stranded DNA, wherein the first single-stranded DNA can specifically match the first chain of the first connector to form a double-stranded structure, and the second single-stranded DNA can specifically match the first chain of the second connector to form a double-stranded structure. The invention further comprises the steps of: amplifying the second ligated product using a first primer and a second primer, wherein the amplified product is a DNA fragment with a linker connected at both ends, wherein the first primer comprises a sequence identical to that of one of the first and second single-stranded DNAs, and the second primer comprises a sequence identical to that of the other of the first and second single-stranded DNAs, and has an additional biotin at the 5' end compared to the other of the first and second single-stranded DNAs; separating the single-stranded DNA fragment from the DNA fragment with a linker connected at both ends; and circularizing the single-stranded DNA fragment to obtain a single-stranded DNA ring, wherein the single-stranded DNA ring constitutes the sequencing library. As previously described, since the 3' ends of the first and second chains in the oligonucleotides according to the embodiments of the present invention are both dideoxynucleotides, and the 5' terminal nucleotide of the second chain does not have a phosphate group, these ends cannot be connected to other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Thus, the oligonucleotide can be used as a connector to connect different connectors at both ends of the nucleic acid fragment when constructing a sequencing library, while avoiding the mutual connection between the connectors, improving the connection efficiency, and reducing the economic and time costs of constructing a sequencing library. The above description of the features and advantages of the oligonucleotides according to the separation of the embodiments of the present invention is also applicable to this device and will not be repeated here. In addition, when constructing a sequencing library, the first single-stranded DNA and the second single-stranded DNA can be used to replace the second chains of the two connectors respectively, and form a more stable double-stranded structure with the first chain. Further, by using the first single-stranded DNA and the second single-stranded DNA as primers, PCR amplification can be performed to form DNA fragments with stable connectors at both ends. Further, by isolating the single-stranded DNA and performing a single-stranded circular reaction, a sequencing library can be effectively obtained, such as a sequencing library for a CG sequencing platform.

Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

Figure 1 shows a schematic diagram of a sequencing library construction process according to one embodiment of the present invention. 1: DNA fragments after shearing. 2: Dephosphorylated and end-repaired fragments (each end is hydroxyl group). 3: Adapter A. 4: Adapter B. 5: Single strand C. 6: Single strand D. 7: Tag sequence on single strand C. 8: Final circular single strand product.

FIG. 2 shows an electropherogram according to an embodiment of the present invention.

FIG3 shows an electropherogram according to an embodiment of the present invention.

FIG4 shows a schematic flow chart of a method for adding linkers to both ends of a double-stranded DNA fragment according to one embodiment of the present invention.

FIG5 shows a schematic structural diagram of a device for adding linkers to both ends of a double-stranded DNA fragment according to one embodiment of the present invention.

FIG6 shows a schematic structural diagram of an apparatus for constructing a sequencing library for double-stranded DNA fragments according to an embodiment of the present invention.

FIG7 shows a schematic structural diagram of a nucleic acid sequencing system according to an embodiment of the present invention.

DETAILED DESCRIPTION

The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

It should be noted that the terms "first" and "second" used herein are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

Isolated oligonucleotides, kits

In the first aspect of the present invention, the present invention provides a separated oligonucleotide. According to an embodiment of the present invention, the oligonucleotide includes: a first chain, wherein the 5' terminal nucleotide of the first chain has a phosphate group, and the 3' terminal nucleotide of the first chain is a dideoxynucleotide; and a second chain, wherein the 5' terminal nucleotide of the second chain does not have a phosphate group, and the 3' terminal nucleotide of the second chain is a dideoxynucleotide, wherein the length of the first chain is greater than the length of the second chain, and a double-stranded structure is formed between the first chain and the second chain. Since the 3' ends of the first and second chains in the oligonucleotide are both dideoxynucleotides, and the 5' terminal nucleotide of the second chain does not have a phosphate group, these ends will not be able to connect with other nucleic acid fragments, thereby preventing the oligonucleotides from connecting with each other. Thus, the separated oligonucleotide can be used as a linker for constructing a sequencing library, and when constructing a sequencing library, different linkers can be connected to both ends of the nucleic acid fragment at the same time, while avoiding the connection between the linkers, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library.

According to one embodiment of the present invention, the number of nucleotides on the second strand that do not match the first strand does not exceed 3. This can further improve the ligation efficiency when constructing the sequencing library, further reducing the economic and time costs of constructing the sequencing library.

According to one embodiment of the present invention, the method comprises: a first overhang located at the 3' end of the first strand; and an optional second overhang located at the 5' end of the second strand. This can further improve ligation efficiency during sequencing library construction, further reducing the economic and time costs of constructing a sequencing library.

According to one embodiment of the present invention, the length of the first overhang is greater than the length of the second overhang, thereby further improving the ligation efficiency when constructing a sequencing library and further reducing the economic and time costs of constructing a sequencing library.

According to one embodiment of the present invention, the length of the first overhang is about 6 to 12 nt, thereby further improving the ligation efficiency when constructing a sequencing library and further reducing the economic and time costs of constructing a sequencing library.

According to one embodiment of the present invention, the length of the second overhang is 0 to 4 nt, thereby further improving the ligation efficiency when constructing a sequencing library and further reducing the economic and time costs of constructing a sequencing library.

According to one embodiment of the present invention, the first chain and the second chain are both DNA.

According to one embodiment of the present invention, the length of the first chain is about 20 to 25 nt. This can further improve the ligation efficiency when constructing a sequencing library, further reducing the economic and time costs of constructing a sequencing library.

According to one embodiment of the present invention, the length of the second strand is about 10 to 15 nt. This can further improve the ligation efficiency when constructing a sequencing library, further reducing the economic and time costs of constructing a sequencing library.

According to one embodiment of the present invention, the sequence of the first strand is: 5'GGCTCCGTCGAAGCCCGACGC3' (SEQ ID NO: 1), and the sequence of the second strand is: 5'CTTCGACGGAGCC3' (SEQ ID NO: 2); or the sequence of the first strand is: 5'ACGTCGGGGCCAAGCGGTCGTC3' (SEQ ID NO: 3), and the sequence of the second strand is: 5'TTGGCCCCGGCTT3' (SEQ ID NO: 4). Thus, the ligation efficiency during sequencing library construction can be further improved, further reducing the economic and time costs of sequencing library construction.

According to a second aspect of the present invention, a kit is provided. According to an embodiment of the present invention, the kit comprises: a first adapter and a second adapter, wherein the first adapter and the second adapter are both the isolated oligonucleotides described above, wherein the first adapter is different from the second adapter.

As previously mentioned, since the 3' ends of the first and second chains in the oligonucleotide according to the embodiment of the present invention are dideoxynucleotides, and the 5' terminal nucleotides of the second chain do not have a phosphate group, these ends cannot be interconnected with other nucleic acid fragments, thereby preventing the mutual connection between the oligonucleotides. Thus, this test kit can be used as a joint for building a sequencing library, and when building a sequencing library, it is possible to achieve simultaneous connection of different joints at the two ends of nucleic acid fragments, while avoiding the mutual connection between the joints, improving connection efficiency, and reducing the economic and time cost of building a sequencing library. The above description of the features and advantages of the oligonucleotides separated according to the embodiment of the present invention is equally applicable to this test kit and will not be repeated here.

According to one embodiment of the present invention, the method further comprises: a first single-stranded DNA, wherein the first single-stranded DNA can match the first strand of the first adapter to form a double-stranded structure; and a second single-stranded DNA, wherein the second single-stranded DNA can match the first strand of the second adapter to form a double-stranded structure. Thus, when constructing a sequencing library, the first single-stranded DNA and the second single-stranded DNA can be used to replace the second strands of the two adapters, respectively, and form a more stable double-stranded structure with the first strand. Furthermore, by using the first single-stranded DNA and the second single-stranded DNA as primers for PCR amplification, DNA fragments with stable adapters at both ends can be formed.

According to one embodiment of the present invention, the length of the double-stranded structure formed by the first single-stranded DNA and the first strand of the first adapter is greater than the length of the double-stranded structure formed between the first strand and the second strand in the first adapter; and the length of the double-stranded structure formed by the second single-stranded DNA and the first strand of the second adapter is greater than the length of the double-stranded structure formed between the first strand and the second strand in the second adapter. Thus, the ligation efficiency during sequencing library construction can be further improved, further reducing the economic and time costs of sequencing library construction.

According to one embodiment of the present invention, the method further comprises: a first primer, the first primer being identical to one of the first single-stranded DNA and the second single-stranded DNA; and a second primer, the second primer having an additional biotin at the 5' end compared to the other of the first single-stranded DNA and the second single-stranded DNA. This can further improve the ligation efficiency during sequencing library construction, further reducing the economic and time costs of constructing the sequencing library. Furthermore, the use of reagents capable of specific biological recognition can effectively isolate single-stranded nucleic acid molecules, which can then be used to construct sequencing libraries for CG sequencing platforms.

According to one embodiment of the present invention, the sequence of the first chain of the first linker is: 5'GGCTCCGTCGAAGCCCGACGC3' (SEQ ID NO: 1); the sequence of the second chain of the first linker is: 5'CTTCGACGGAGCC3' (SEQ ID NO: 2); the sequence of the first chain of the second linker is: 5'ACGTCGGGGCCAAGCGGTCGTC3' (SEQ ID NO: 3); the sequence of the second chain of the second linker is: 5'TTGGCCCCGGCTT3' (SEQ ID NO: 4); the sequence of the first single-stranded DNA is: 5'AGACAAGCTC(N) _m GATCGGGCTTCGACGGAG3', wherein (N) _m represents a tag sequence with a length of m nucleotides, wherein m is any integer from 4 to 10, and N=A, T, G or C; and the sequence of the second single-stranded DNA is 5'TCCTAAGACCGCTTGGCCCCG3' (SEQ ID NO: 5). This can further improve the ligation efficiency during sequencing library construction, further reducing the economic and time costs of sequencing library construction. Furthermore, the use of reagents that can specifically identify organisms can effectively separate single-stranded nucleic acid molecules, which can then be used to construct sequencing libraries for CG sequencing platforms.

Use of isolated oligonucleotides in nucleic acid sequencing

In a third aspect of the present invention, a method for adding linkers to both ends of a double-stranded DNA fragment is provided. According to an embodiment of the present invention, the double-stranded DNA fragment has two blunt ends, and none of the four terminal nucleotides of the double-stranded DNA fragment has a phosphate group. Referring to FIG4 , the method comprises:

S100: Ligating the double-stranded DNA fragments with the first adapter and the second adapter

The double-stranded DNA fragment is ligated with a first adapter and a second adapter to obtain a first ligation product, wherein the first adapter and the second adapter are different and both are the isolated oligonucleotides described above.

According to one embodiment of the present invention, the step of connecting the double-stranded DNA fragment to the first adapter and the second adapter is completed in a one-step reaction.

According to one embodiment of the present invention, the DNA fragments are obtained by the following steps: fragmenting a DNA sample to obtain fragmented products; dephosphorylating the fragmented products to obtain dephosphorylated fragmented products; and end-repairing the dephosphorylated fragmented products to obtain double-stranded DNA fragments. Thus, DNA fragments suitable for constructing a sequencing library can be effectively obtained.

According to one embodiment of the present invention, the DNA sample is at least a portion of genomic DNA or a reverse transcription product of RNA, thereby effectively constructing a sequencing library for the genomic DNA or RNA.

S200: Use the first single-stranded DNA to replace the second strand of the first adapter, and the second single-stranded DNA to replace the second strand of the second adapter

A first single-stranded DNA is used to replace the second strand of the first adapter, and a second single-stranded DNA is used to replace the second strand of the second adapter, wherein the first single-stranded DNA can specifically match with the first strand of the first adapter to form a double-stranded structure, and the second single-stranded DNA can specifically match with the first strand of the second adapter to form a double-stranded structure.

According to one embodiment of the present invention, the length of the double-stranded structure formed by the first single-stranded DNA and the first strand of the first adapter is greater than the length of the double-stranded structure formed between the first strand and the second strand in the first adapter; and the length of the double-stranded structure formed by the second single-stranded DNA and the first strand of the second adapter is greater than the length of the double-stranded structure formed between the first strand and the second strand in the second adapter. Thus, the ligation efficiency during sequencing library construction can be further improved, further reducing the economic and time costs of sequencing library construction.

According to one embodiment of the present invention, the sequence of the first chain of the first linker is: 5'GGCTCCGTCGAAGCCCGACGC3' (SEQ ID NO: 1); the sequence of the second chain of the first linker is: 5'CTTCGACGGAGCC3' (SEQ ID NO: 2); the sequence of the first chain of the second linker is: 5'ACGTCGGGGCCAAGCGGTCGTC3' (SEQ ID NO: 3); the sequence of the second chain of the second linker is: 5'TTGGCCCCGGCTT3' (SEQ ID NO: 4); and the sequence of the first single-stranded DNA is: 5'AGACAAGCTC(N) _m GATCGGGCTTCGACGGAG3', wherein (N) _m represents a tag sequence with a length of m nucleotides, wherein m is any integer from 4 to 10, and N=A, T, G or C; the sequence of the second single-stranded DNA is 5'TCCTAAGACCGCTTGGCCCCG3' (SEQID NO: 5). This can further improve ligation efficiency during sequencing library construction, further reducing the economic and time costs of constructing sequencing libraries. Furthermore, the use of reagents that specifically recognize biotin can effectively separate single-stranded nucleic acid molecules, which can then be used to construct sequencing libraries for CG sequencing platforms.

According to one embodiment of the present invention, the second strand of the first adapter is replaced by the first single-stranded DNA, and the second strand of the second adapter is replaced by the second single-stranded DNA through a thermal cleavage-annealing process. According to some specific examples of the present invention, the thermal cleavage is performed at approximately 60 degrees Celsius. This can further improve the ligation efficiency when constructing a sequencing library, further reducing the economic and time costs of constructing the sequencing library.

S300: connecting the first single-stranded DNA and the second single-stranded DNA to the double-stranded DNA fragments respectively

The first single-stranded DNA and the second single-stranded DNA are respectively ligated to the double-stranded DNA fragment to obtain a second ligation product.

According to one embodiment of the present invention, the first single-stranded DNA and the second single-stranded DNA are respectively connected to the double-stranded DNA fragment through a gap-filling reaction.

S400: Amplify the second ligation product using the first primer and the second primer

The second ligation product is amplified using a first primer and a second primer to obtain an amplified product, wherein the amplified product is a DNA fragment with adapters connected at both ends, wherein the first primer contains a sequence identical to that of one of the first single-stranded DNA and the second single-stranded DNA, and the second primer contains a sequence identical to that of the other of the first single-stranded DNA and the second single-stranded DNA, and has an additional biotin at the 5' end compared to the other of the first single-stranded DNA and the second single-stranded DNA.

As previously mentioned, since the 3' ends of the first and second chains in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the 5' terminal nucleotides of the second chain do not have a phosphate group, these ends will not be able to interconnect with other nucleic acid fragments, thereby preventing the mutual connection between the oligonucleotides. Thus, the oligonucleotide can be used as a joint to connect different joints at both ends of the nucleic acid fragment when constructing a sequencing library, while avoiding the mutual connection between the joints, improving the connection efficiency, and reducing the economic and time costs of constructing a sequencing library. The above description of the features and advantages of the oligonucleotides separated according to the embodiment of the present invention is equally applicable to this method and will not be repeated here. In addition, when constructing a sequencing library, the first single-stranded DNA and the second single-stranded DNA can be utilized to replace the second chain of the two joints respectively, and form a more stable double-stranded structure with the first chain. Further, by adopting the first single-stranded DNA and the second single-stranded DNA as primers, PCR amplification is performed to form DNA fragments with stable joints at both ends.

In a fourth aspect, the present invention provides a method for constructing a sequencing library for double-stranded DNA fragments. According to an embodiment of the present invention, the double-stranded DNA fragments have two blunt ends, and none of the four terminal nucleotides of the double-stranded DNA fragments have a phosphate group, and the method comprises:

First, according to the aforementioned method for ligating adapters to both ends of a double-stranded DNA fragment, adapters are ligated to both ends of the double-stranded DNA fragment to obtain a DNA fragment with adapters ligated to both ends.

Next, single-stranded DNA fragments are isolated from the DNA fragments with adapters attached to both ends. According to one embodiment of the present invention, isolating single-stranded DNA fragments from the DNA fragments with adapters attached to both ends further comprises: contacting the DNA fragments with adapters attached to both ends with magnetic beads to form a magnetic bead-DNA complex, wherein the magnetic beads are attached to streptavidin; and contacting the magnetic bead-DNA complex with a solution having a pH greater than 7 to obtain the single-stranded DNA fragments. This allows for efficient isolation of single-stranded DNA fragments, thereby improving the efficiency and reducing the cost of sequencing library construction. According to one embodiment of the present invention, the solution having a pH greater than 7 is a sodium hydroxide solution. According to one embodiment of the present invention, the concentration of the sodium hydroxide solution is approximately 0.5 to 2 M. According to another embodiment of the present invention, the concentration of the sodium hydroxide solution is approximately 1 M. According to one embodiment of the present invention, prior to isolating single-stranded DNA fragments from the DNA fragments with adapters attached to both ends, the DNA fragments with adapters attached to both ends are pre-screened. This allows for sequencing library construction targeting a predetermined region. According to one embodiment of the present invention, the screening is performed by contacting the DNA fragments with adapters at both ends with a probe, wherein the probe is specific for a predetermined sequence. According to a specific example of the present invention, the predetermined sequence includes at least one exon. According to another embodiment of the present invention, the probe is provided in the form of a microchip array. Thus, the single-stranded DNA fragments can be effectively circularized.

Then, the single-stranded DNA fragment is circularized to obtain a single-stranded DNA ring, which constitutes the sequencing library. According to one embodiment of the present invention, the single-stranded DNA fragment is circularized by using a single-stranded nucleic acid molecule, wherein a first segment and a second segment are defined on the single-stranded nucleic acid molecule, and the first segment can match a sequence comprising the 5' terminal nucleotide and the 3' terminal nucleotide of the single-stranded DNA fragment, and the second segment can match a sequence comprising one of the 5' terminal nucleotide and the 3' terminal nucleotide of the single-stranded DNA fragment. In this way, the circularization efficiency can be further improved. According to one embodiment of the present invention, the first segment and the second segment are adjacently connected. According to one embodiment of the present invention, the sequence of the first segment is 5'TCGAGCTTGTCT3' (SEQ ID NO: 6); and the sequence of the second segment is 5'TCCTAAGACCGC3' (SEQ ID NO: 7).

As mentioned above, since the 3' ends of the first and second chains in the oligonucleotides according to the embodiments of the present invention are both dideoxynucleotides, and the 5' terminal nucleotides of the second chain do not have a phosphate group, these ends will not be able to connect to each other with other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Thus, the oligonucleotides can be used as a linker to connect different linkers at both ends of the nucleic acid fragment when constructing a sequencing library, while avoiding the connection between the linkers, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library. The above description of the features and advantages of the isolated oligonucleotides according to the embodiments of the present invention is also applicable to this method and will not be repeated here. In addition, when constructing a sequencing library, the first single-stranded DNA and the second single-stranded DNA can be used to replace the second chains of the two linkers respectively, and form a more stable double-stranded structure with the first chain. Further, by using the first single-stranded DNA and the second single-stranded DNA as primers and performing PCR amplification, DNA fragments with stable linkers at both ends can be formed. Further, by isolating the single-stranded DNA and performing a single-stranded circularization reaction, a sequencing library can be effectively obtained, such as a sequencing library for a CG sequencing platform.

In its fifth aspect, the present invention provides a nucleic acid sequencing method. According to an embodiment of the present invention, the method comprises: constructing a sequencing library according to the aforementioned method for constructing a sequencing library for double-stranded DNA fragments; and sequencing the sequencing library. According to one embodiment of the present invention, the sequencing library is sequenced using a CG sequencing platform.

As previously mentioned, since the 3' ends of the first and second chains in the oligonucleotides according to the embodiments of the present invention are both dideoxynucleotides, and the 5' terminal nucleotide of the second chain does not have a phosphate group, these ends will not be able to connect to other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Thus, when constructing a sequencing library, the oligonucleotide can be used as a linker to connect different linkers at both ends of the nucleic acid fragment, while avoiding the connection between the linkers, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library. The previous description of the features and advantages of the isolated oligonucleotides according to the embodiments of the present invention also applies to this method and will not be repeated here. In addition, when constructing a sequencing library, the first single-stranded DNA and the second single-stranded DNA can be used to replace the second chains of the two linkers respectively, and form a more stable double-stranded structure with the first chain. Furthermore, by using the first single-stranded DNA and the second single-stranded DNA as primers and performing PCR amplification, DNA fragments with stable linkers at both ends can be formed. Further, by isolating the single-stranded DNA and performing a single-stranded circularization reaction, a sequencing library can be effectively obtained, such as a sequencing library for a CG sequencing platform. This can further improve the efficiency of sequencing and reduce the cost of sequencing.

In a sixth aspect of the present invention, the present invention further provides a device for adding linkers to both ends of a double-stranded DNA fragment. According to an embodiment of the present invention, the double-stranded DNA fragment has two blunt ends, and none of the four terminal nucleotides of the double-stranded DNA fragment has a phosphate group. Referring to FIG5 , the device 100 includes: a first linking unit 101, a displacement unit 102, a second linking unit 103, and an amplification unit 104. Specifically:

The first ligation unit 101 is configured to ligate the DNA fragment to a first adapter and a second adapter to obtain a first ligation product, wherein the first adapter and the second adapter are different and both are the isolated oligonucleotides described above. According to one embodiment of the present invention, the first ligation unit is configured to ligate the DNA fragment to the first adapter and the second adapter in a one-step reaction.

The replacement unit 102 is used to replace the second chain of the first adapter with a first single-stranded DNA, and to replace the second chain of the second adapter with a second single-stranded DNA, wherein the first single-stranded DNA can specifically match the first chain of the first adapter to form a double-stranded structure, and the second single-stranded DNA can specifically match the first chain of the second adapter to form a double-stranded structure. According to one embodiment of the present invention, the length of the double-stranded structure formed by the first single-stranded DNA and the first chain of the first adapter is greater than the length of the double-stranded structure formed between the first chain and the second chain in the first adapter; and the length of the double-stranded structure formed by the second single-stranded DNA and the first chain of the second adapter is greater than the length of the double-stranded structure formed between the first chain and the second chain in the second adapter. Thus, the connection efficiency during the subsequent construction of the sequencing library can be further improved, further reducing the economic and time costs of constructing the sequencing library. According to one embodiment of the present invention, the sequence of the first chain of the first linker is: 5'GGCTCCGTCGAAGCCCGACGC3' (SEQ ID NO: 1); the sequence of the second chain of the first linker is: 5'CTTCGACGGAGCC3' (SEQ ID NO: 2); the sequence of the first chain of the second linker is: 5'ACGTCGGGGCCAAGCGGTCGTC3' (SEQ ID NO: 3); the sequence of the second chain of the second linker is: 5'TTGGCCCCGGCTT3' (SEQ ID NO: 4); the sequence of the first single-stranded DNA is: 5'AGACAAGCTC(N) _m GATCGGGCTTCGACGGAG3', wherein (N) _m represents a tag sequence with a length of m nucleotides, wherein m is any integer from 4 to 10, and N=A, T, G or C; and the sequence of the second single-stranded DNA is 5'TCCTAAGACCGCTTGGCCCCG3' (SEQ ID NO: 5). This can further improve the ligation efficiency when constructing sequencing libraries, further reducing the economic and time costs of constructing sequencing libraries. Furthermore, the use of reagents that specifically recognize biotin can effectively separate single-stranded nucleic acid molecules, which can then be used to construct sequencing libraries for CG sequencing platforms.

According to one embodiment of the present invention, the replacement unit 102 is configured to replace the second strand of the first adapter with the first single-stranded DNA, and replace the second strand of the second adapter with the second single-stranded DNA, through a thermal cleavage-annealing process. According to one embodiment of the present invention, the thermal cleavage is performed at approximately 60 degrees Celsius. This can further improve the ligation efficiency during sequencing library construction, further reducing the economic and time costs of sequencing library construction.

The second ligation unit 103 is configured to ligate the first single-stranded DNA and the second single-stranded DNA to the DNA fragment, respectively, to obtain a second ligation product. According to one embodiment of the present invention, the second ligation unit 103 is configured to ligate the first single-stranded DNA and the second single-stranded DNA to the double-stranded DNA fragment, respectively, through a gap-filling reaction.

The amplification unit 104 is configured to amplify the second ligation product using a first primer and a second primer to obtain an amplified product, wherein the first primer comprises a sequence identical to that of one of the first single-stranded DNA and the second single-stranded DNA, and the second primer comprises a sequence identical to that of the other of the first single-stranded DNA and the second single-stranded DNA, and has an additional biotin at the 5' end compared to the other of the first single-stranded DNA and the second single-stranded DNA.

According to one embodiment of the present invention, a double-stranded DNA fragment acquisition unit (not shown) is further included. The DNA fragment acquisition unit includes: a fragmentation component for fragmenting the DNA sample to obtain fragmented products; a dephosphorylation component for dephosphorylating the fragmented products to obtain dephosphorylated fragmented products; and an end-repair component for end-repairing the dephosphorylated fragmented products to obtain the double-stranded DNA fragments. In this way, DNA fragments suitable for constructing a sequencing library can be effectively obtained.

According to one embodiment of the present invention, the double-stranded DNA fragment acquisition unit further includes: a genomic DNA extraction component for extracting genomic DNA from a biological sample; and/or a reverse transcription component for performing a reverse transcription reaction on an RNA sample to obtain a reverse transcription product, wherein at least a portion of the genomic DNA and/or the reverse transcription product of the RNA constitutes the DNA sample. Thus, a sequencing library can be efficiently constructed for the genomic DNA or RNA.

As previously mentioned, since the 3' ends of the first and second chains in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the 5' terminal nucleotides of the second chain do not have a phosphate group, these ends will not be able to interconnect with other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Thus, the oligonucleotide can be used as a joint to connect different joints at both ends of the nucleic acid fragment when constructing a sequencing library, while avoiding the mutual connection between the joints, improving the connection efficiency, and reducing the economic and time costs of constructing a sequencing library. The above description of the features and advantages of the oligonucleotides separated according to the embodiment of the present invention is also applicable to this device and will not be repeated here. In addition, when constructing a sequencing library, the first single-stranded DNA and the second single-stranded DNA can be utilized to replace the second chain of the two joints respectively, and form a more stable double-stranded structure with the first chain. Further, by adopting the first single-stranded DNA and the second single-stranded DNA as primers, PCR amplification is performed to form DNA fragments with stable joints at both ends.

In a seventh aspect, the present invention further provides an apparatus for constructing a sequencing library for double-stranded DNA fragments. According to an embodiment of the present invention, the double-stranded DNA fragments have two blunt ends, and none of the four terminal nucleotides of the double-stranded DNA fragments have a phosphate group. Referring to FIG6 , the apparatus 1000 includes: the aforementioned apparatus 100 for adding linkers to both ends of the double-stranded DNA fragments, the single-stranded DNA fragment separation apparatus 200, and the cyclization apparatus 300. Specifically:

The device 100 for adding adapters to the ends of double-stranded DNA fragments is used to attach adapters to the ends of the double-stranded DNA fragments to obtain DNA fragments with adapters attached to both ends. The single-stranded DNA fragment separation device 200 is used to separate single-stranded DNA fragments from the DNA fragments with adapters attached to both ends. The circularization device 300 is used to circularize the single-stranded DNA fragments to obtain single-stranded DNA rings, which constitute the sequencing library.

According to one embodiment of the present invention, the single-stranded DNA fragment separation device 200 further includes: a magnetic bead capture unit, configured to contact the DNA fragments, each end of which has a linker attached, with magnetic beads to form a magnetic bead-DNA complex, wherein the magnetic beads are attached to streptavidin; and an alkaline lysis unit, configured to contain a solution having a pH greater than 7 and to contact the magnetic bead-DNA complex with a solution having a pH less than 7 to obtain the single-stranded DNA fragments. This allows for efficient separation of single-stranded DNA fragments, thereby improving the efficiency and reducing the cost of sequencing library construction. According to one embodiment of the present invention, the solution having a pH greater than 7 is a sodium hydroxide solution. According to one embodiment of the present invention, the concentration of the sodium hydroxide solution is approximately 0.5 to 2 M. According to another embodiment of the present invention, the concentration of the sodium hydroxide solution is approximately 1 M.

According to one embodiment of the present invention, the method further comprises: a screening device (not shown in the figure), wherein the screening device is used to screen the DNA fragments with connectors connected to both ends before separating the single-stranded DNA fragments from the DNA fragments with connectors connected to both ends. According to one embodiment of the present invention, the screening device is provided with a probe, wherein the probe is specific for a predetermined sequence. According to one embodiment of the present invention, the predetermined sequence includes at least one exon. According to one embodiment of the present invention, the probe is provided in the form of a microchip array.

According to one embodiment of the present invention, the circularization device 300 is provided with a single-stranded nucleic acid molecule, wherein a first segment and a second segment are defined on the single-stranded nucleic acid molecule, and the first segment can match a sequence comprising the 5' terminal nucleotide and the 3' terminal nucleotide of the single-stranded DNA fragment, and the second segment can match a sequence comprising one of the 5' terminal nucleotide and the 3' terminal nucleotide of the single-stranded DNA fragment. According to one embodiment of the present invention, the first segment and the second segment are adjacently connected. According to one embodiment of the present invention, the sequence of the first segment is 5'TCGAGCTTGTCT3' (SEQ ID NO: 6); and the sequence of the second segment is 5'TCCTAAGACCGC3' (SEQ ID NO: 7). Thus, the single-stranded DNA fragment can be effectively circularized by using the single-stranded nucleic acid molecule.

As mentioned above, since the 3' ends of the first and second chains in the oligonucleotides according to the embodiments of the present invention are both dideoxynucleotides, and the 5' terminal nucleotides of the second chain do not have phosphate groups, these ends will not be able to connect to each other with other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Thus, when constructing a sequencing library, the oligonucleotide can be used as a linker to connect different linkers at both ends of the nucleic acid fragment, while avoiding the connection between the linkers, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library. The above description of the features and advantages of the isolated oligonucleotides according to the embodiments of the present invention is also applicable to this device and will not be repeated here. In addition, when constructing a sequencing library, the first single-stranded DNA and the second single-stranded DNA can be used to replace the second chains of the two linkers respectively, and form a more stable double-stranded structure with the first chain. Further, by using the first single-stranded DNA and the second single-stranded DNA as primers and performing PCR amplification, DNA fragments with stable linkers at both ends can be formed. Further, by isolating the single-stranded DNA and performing a single-stranded circularization reaction, a sequencing library can be effectively obtained, such as a sequencing library for a CG sequencing platform.

In its eighth aspect, the present invention also provides a nucleic acid sequencing system. According to an embodiment of the present invention, referring to FIG7 , system 10000 comprises: the aforementioned apparatus 1000 for constructing a sequencing library for double-stranded DNA fragments; and a sequencing apparatus 2000 for sequencing the sequencing library. According to one embodiment of the present invention, sequencing apparatus 2000 is a CG sequencing platform.

As previously described, since the 3' ends of the first and second strands in the oligonucleotides according to the embodiments of the present invention are both dideoxynucleotides, and the 5' terminal nucleotide of the second strand does not have a phosphate group, these ends will not be able to connect to other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Therefore, when constructing a sequencing library, the oligonucleotide can be used as a linker to connect different linkers to both ends of the nucleic acid fragment, while avoiding the connection between the linkers, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library. The previous description of the features and advantages of the isolated oligonucleotides according to the embodiments of the present invention also applies to this system and will not be repeated here. In addition, when constructing a sequencing library, the first and second single-stranded DNAs can be used to replace the second strands of the two linkers respectively, and form a more stable double-stranded structure with the first strand. Furthermore, by using the first and second single-stranded DNAs as primers and performing PCR amplification, DNA fragments with stable linkers at both ends can be formed. Further, by isolating the single-stranded DNA and performing a single-stranded circularization reaction, a sequencing library can be effectively obtained, such as a sequencing library for a CG sequencing platform. This can further improve the efficiency of sequencing and reduce the cost of sequencing.

In a ninth aspect of the present invention, the present invention further provides a device for constructing a sequencing library for genomic DNA. According to an embodiment of the present invention, the device comprises:

means for fragmenting the genomic DNA to obtain fragmentation products;

means for dephosphorylating the fragmented product to obtain a dephosphorylated fragmented product;

Means for performing end repair on the dephosphorylated fragmented products to obtain double-stranded DNA fragments;

means for ligating the double-stranded DNA fragment with a first adapter and a second adapter to obtain a first ligation product, wherein the first adapter and the second adapter are different and both the first adapter and the second adapter are the isolated oligonucleotides described above;

Means for replacing the second strand of the first adapter with a first single-stranded DNA, and replacing the second strand of the second adapter with a second single-stranded DNA, wherein the first single-stranded DNA can specifically match with the first strand of the first adapter to form a double-stranded structure, and the second single-stranded DNA can specifically match with the first strand of the second adapter to form a double-stranded structure;

means for ligating the first single-stranded DNA and the second single-stranded DNA to the DNA fragment, respectively, to obtain a second ligation product;

means, amplifying the second ligation product using a first primer and a second primer to obtain an amplified product, wherein the amplified product is a DNA fragment with adapters connected to both ends, wherein the first primer comprises a sequence identical to that of one of the first single-stranded DNA and the second single-stranded DNA, and the second primer comprises a sequence identical to that of the other of the first single-stranded DNA and the second single-stranded DNA, and has an additional biotin at the 5' end compared to the other of the first single-stranded DNA and the second single-stranded DNA;

Means for separating single-stranded DNA fragments from the DNA fragments with linkers connected to both ends; and

Means for circularizing the single-stranded DNA fragments to obtain single-stranded DNA circles, which constitute the sequencing library.

As mentioned above, since the 3' ends of the first and second chains in the oligonucleotides according to the embodiments of the present invention are both dideoxynucleotides, and the 5' terminal nucleotides of the second chain do not have a phosphate group, these ends will not be able to connect to each other with other nucleic acid fragments, thereby preventing the oligonucleotides from connecting to each other. Thus, the oligonucleotides can be used as connectors to connect different connectors at both ends of the nucleic acid fragment when constructing a sequencing library, while avoiding the connection between the connectors, improving the connection efficiency, and reducing the economic and time costs of constructing the sequencing library. The above description of the features and advantages of the isolated oligonucleotides according to the embodiments of the present invention is also applicable to this device and will not be repeated here. In addition, when constructing a sequencing library, the first single-stranded DNA and the second single-stranded DNA can be used to replace the second chains of the two connectors respectively, and form a more stable double-stranded structure with the first chain. Further, by using the first single-stranded DNA and the second single-stranded DNA as primers and performing PCR amplification, DNA fragments with stable connectors at both ends can be formed. Further, by isolating the single-stranded DNA and performing a single-stranded circularization reaction, a sequencing library can be effectively obtained, such as a sequencing library for a CG sequencing platform.

According to one embodiment of the present invention, ligating the double-stranded DNA fragment to the first adapter and the second adapter is completed in a one-step reaction.

According to one embodiment of the present invention, the method further comprises:

Means for extracting genomic DNA from a biological sample; and/or

Means for performing reverse transcription reaction on RNA samples,

in,

At least a portion of the genomic DNA and/or the reverse transcription product of RNA constitutes the DNA sample.

According to one embodiment of the present invention, the means for separating single-stranded DNA fragments from DNA fragments having adapters attached to both ends is configured to separate the single-stranded DNA fragments by: contacting the DNA fragments having adapters attached to both ends with magnetic beads to form a magnetic bead-DNA complex, wherein the magnetic beads are attached to streptavidin; and contacting the magnetic bead-DNA complex with a solution having a pH lower than 7 to obtain the single-stranded DNA fragments. Thus, the single-stranded DNA fragments can be effectively separated, thereby improving the efficiency of sequencing library construction and reducing the cost of sequencing library construction.

According to one embodiment of the present invention, the method further comprises: a means for pre-screening the DNA fragments connected to the adapters before separating the single-stranded DNA fragments from the DNA fragments connected to the adapters. According to one embodiment of the present invention, the screening is performed by contacting the DNA fragments connected to the adapters with a probe, wherein the probe is specific for a predetermined sequence. According to one embodiment of the present invention, the predetermined sequence includes at least one exon. According to one embodiment of the present invention, the probe is provided in the form of a microchip array.

According to one embodiment of the present invention, the means for circularizing the single-stranded DNA fragment is configured to circularize the single-stranded DNA fragment using a single-stranded nucleic acid molecule, wherein a first segment and a second segment are defined on the single-stranded nucleic acid molecule, and the first segment can match a sequence comprising the 5' terminal nucleotide and the 3' terminal nucleotide of the single-stranded DNA fragment, and the second segment can match a sequence comprising one of the 5' terminal nucleotide and the 3' terminal nucleotide of the single-stranded DNA fragment. According to one embodiment of the present invention, the first segment and the second segment are adjacently connected. According to one embodiment of the present invention, the sequence of the first segment is 5'TCGAGCTTGTCT3' (SEQ ID NO: 6); and the sequence of the second segment is 5'TCCTAAGACCGC3' (SEQ ID NO: 7). Thus, the single-stranded DNA fragment can be effectively circularized by using a single-stranded nucleic acid molecule.

In summary, the technical solution according to the embodiment of the present invention can have at least one of the following advantages:

The technical solution according to the embodiments of the present invention solves the problems of excessive number of adapter connection steps, long overall library construction time, and high cost in Complete Genomics' sequencing platform library construction.

According to the technical solution of the embodiment of the present invention, the traditional method of adding two end connectors separately in multiple steps is abandoned during connector connection, and a new method of adding two end connectors in the same reaction is adopted instead.

According to the technical solution of the embodiments of the present invention, the connection method of adding two connectors at the same time also needs to solve problems such as connector self-ligation and fragment interconnection. The connector designed by the present invention has a unique sequence structure and uses an equally novel connector connection method to solve problems such as fragment interconnection, connector self-ligation, low fragment connection efficiency, and label sequence introduction position. It also successfully shortens the entire connector connection process to three steps, greatly shortens the time required for connector connection, and significantly reduces costs.

The technical solution according to the embodiments of the present invention combines an innovative adapter ligation method with nucleic acid probe capture technology. By further designing and adjusting Complete Genomics' traditional library construction protocol, the adapter ligation process was successfully reduced from two steps to one. This significantly shortened the cost and time of library construction and successfully created a single-adapter whole-exome sequencing product based on the Complete Genomics sequencing platform.

Therefore, according to an embodiment of the present invention, with reference to FIG1 , a sequencing library can be constructed according to the following steps:

1. The genomic nucleic acid chain is broken into fragments;

2. Dephosphorylate the target fragment; dephosphorylation is used to block the 5’ end of the target fragment to prevent self-ligation of the fragment.

3. Fill in both ends of the fragment to make both ends blunt (as shown by number 2 in Figure 1).

4. Add adapter A (shown as number 3 in Figure 1) and adapter B (shown as number 4 in Figure 1) to both ends of the target fragment. Adapters A and B are both double-stranded polynucleotides, consisting of a long chain (first chain) and a short chain (second chain). The long chain has a phosphate group at its 5' end, which can connect to the target nucleic acid fragment. The short chain binds to the long chain through complementary base pairing. Since the short chain ends in a closed sequence, it will not connect to the target nucleic acid fragment.

5. Add single-stranded nucleic acid C (shown as 5 in Figure 1) and single-stranded nucleic acid D (shown as 6 in Figure 1). Single-stranded nucleic acid C contains the tag sequence (shown as 7 in Figure 1), and the remaining fragments complementarily pair with the long-stranded adapter A; single-stranded nucleic acid D complements the long-stranded adapter B. Annealing causes the loosely bound short adapter strands to fall off, allowing single-stranded nucleic acid C and D to complement the long-stranded adapter. Extension and ligation reactions then connect single-stranded nucleic acid C and D to the target fragment.

6. Using the product from step 4 as a template and single-stranded C and D as primers, perform polymerase chain reaction to amplify and enrich the product with the tag sequence;

7. The product of step 5 is subjected to oligonucleotide probe hybridization capture; the specific steps include probe hybridization, hybridization product elution, and hybridization product enrichment steps; and in the hybridization product enrichment step, biotin modification is introduced on one strand of the double-stranded target nucleic acid.

8. Length screening of the nucleic acid double strands after hybridization capture (optional);

9. Separate the screened nucleic acid double strands into two single nucleic acid strands by biotin labeling one of the nucleic acid double strands;

10. Circularize the nucleic acid single strand and remove the remaining uncircularized single strand.

It should be noted that fragment length screening in step 7 can be performed after other steps before single-strand separation, depending on the specific sequencing requirements and the actual changes in product fragment size after each step. If quality control confirms that the product size of each step consistently meets the requirements, step 8 can be omitted.

By adopting step 7, the step introduced by whole exome sequencing can be achieved.

According to an embodiment of the present invention, through the processing of steps 2 and 3, the target nucleic acid fragments are subjected to the dephosphorylation and end-blocking treatment to become blunt-ended fragments with blocked ends, which completely avoids the occurrence of interactions between fragments and ensures a very high utilization rate of the fragments before connection.

According to an embodiment of the present invention, the special adapter design of the present invention introduces a phosphate group at the 5' end of the long chain of adapters A and B; and a blocking sequence is introduced at the 3' end of the long chain of the adapter and at both ends of the short chain. Due to the presence of the blocking sequence, the blocked end is not only unable to connect with the target nucleic acid fragment, but also unable to connect with other adapters added simultaneously; ensuring that when the adapter is connected in step 4, the 5' end of the long chain of the adapter can be accurately connected to the 3' end of the target fragment. This design very effectively prevents the occurrence of adapter interconnection, makes it possible to connect different adapters simultaneously, and ensures the efficiency of the ligation reaction.

According to an embodiment of the present invention, in step 5, the characteristics of the long and short chains in the connector structure are cleverly utilized. Since the short chain has fewer complementary bases and the binding is unstable, it will separate from the long chain at a relatively mild temperature. Then, through a slow annealing reaction, the single chains C and D with longer base complementary pairing sequences and more advantageous binding ability are simply combined with the long chain of the connector. After extension and connection, a complete double-stranded connector is formed. By introducing a tag sequence on the single chain C, an identification tag can also be provided for the connector at the same time. This unique design has the characteristics of mild reaction conditions. By appropriately adjusting the reaction system, reaction time, and reaction sequence, the three reactions of fragment replacement, connection, and extension can be carried out in the same reaction step 5. The operation is simple, the reaction is rapid, and the processing time is greatly reduced.

According to the embodiments of the present invention, the original five steps of linker ligation are successfully shortened to three steps: linker ligation, gap filling, and polymerase chain reaction. The operation volume is greatly reduced, the use of multiple reagents is eliminated, and a large amount of time and cost are saved.

According to the embodiments of the present invention, not only has the specific method of linker ligation been completely changed, but the traditional library construction scheme of CG has also been radically changed, and a novel single-stranded nucleic acid library structure (marked 8 in Figure 1) has been proposed. The traditional two-step linker ligation process has been streamlined to a single step, reducing the introduction of polymerase chain reaction and improving the quality of sequencing. More importantly, the streamlining of the steps has shortened the library construction time by as much as 3-4 days. This has significantly reduced costs and has huge advantages over traditional schemes.

According to the embodiments of the present invention, by modifying and supplementing Complete Genomics' traditional sequencing library construction protocol and combining it with the previously described novel adapter ligation method, an efficient library construction solution suitable for human whole-exome sequencing has been successfully developed. This has resulted in the development of a novel human whole-exome sequencing product based on the Complete Genomics sequencing platform, achieving a breakthrough in whole-exome sequencing based on the Complete Genomics platform.

Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area (e.g., reference to "Molecular Cloning Laboratory Manual" by J. Sambrook et al., translated by Huang Peitang et al., 3rd edition, Science Press) or the product instructions are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

General approach

Referring to FIG1 , in an embodiment of the present invention, a sequencing library is constructed according to the following steps:

1. The genomic nucleic acid chain is broken into fragments;

2. Dephosphorylate the target fragment; dephosphorylation is used to block the 5’ end of the target fragment to prevent self-ligation of the fragment.

3. Fill in both ends of the fragment to make both ends blunt (as shown by number 2 in Figure 1).

4. Add adapter A (shown as number 3 in Figure 1) and adapter B (shown as number 4 in Figure 1) to both ends of the target fragment. Adapters A and B are both double-stranded polynucleotides, consisting of a long chain (first chain) and a short chain (second chain). The long chain has a phosphate group at its 5' end, which can connect to the target nucleic acid fragment. The short chain binds to the long chain through complementary base pairing. Since the short chain ends in a closed sequence, it will not connect to the target nucleic acid fragment.

5. Add single-stranded nucleic acid C (shown as 5 in Figure 1) and single-stranded nucleic acid D (shown as 6 in Figure 1). Single-stranded nucleic acid C contains the tag sequence (shown as 7 in Figure 1), and the remaining fragments complementarily pair with the long-stranded adapter A; single-stranded nucleic acid D complements the long-stranded adapter B. Annealing causes the loosely bound short adapter strands to fall off, allowing single-stranded nucleic acid C and D to complement the long-stranded adapter. Extension and ligation reactions then connect single-stranded nucleic acid C and D to the target fragment.

6. Using the product from step 4 as a template and single-stranded C and D as primers, perform polymerase chain reaction to amplify and enrich the product with the tag sequence;

7. The product of step 5 is subjected to oligonucleotide probe hybridization capture; the specific steps include probe hybridization, hybridization product elution, and hybridization product enrichment steps; and in the hybridization product enrichment step, biotin modification is introduced on one strand of the double-stranded target nucleic acid.

8. Length screening of the nucleic acid double strands after hybridization capture (optional);

9. Separate the screened nucleic acid double strands into two single nucleic acid strands by biotin labeling one of the nucleic acid double strands;

10. Circularize the nucleic acid single strand and remove the remaining uncircularized single strand.

It should be noted that fragment length screening in step 7 can be performed after other steps before single-strand separation, depending on the specific sequencing requirements and the actual changes in product fragment size after each step. If quality control confirms that the product size of each step consistently meets the requirements, step 8 can be omitted.

By adopting step 7, the step introduced by whole exome sequencing can be achieved.

Example 1:

1. Genomic DNA shearing: There are many ways to shear genomic DNA, whether it is physical ultrasound or enzyme reaction, and there are very mature solutions on the market. This example uses the physical ultrasound shearing method.

Take a 96-well PCR plate, add a piece of polytetrafluoroethylene thread, add 1 μg of genomic DNA, and add TE buffer or enzyme-free water to make up to 80 μl. Seal the plate and place it on an E220 ultrasonic disruptor for ultrasonic disruption. Set the disruption conditions as follows:

DutyCycle 20% Intensity 5 CyclesperBurst 200 Interruption time 60 seconds, 5 times

2. Fragment selection: Magnetic bead purification or gel recovery can be used. This example uses magnetic bead purification.

Take the sheared DNA, add 80μl Ampure XP magnetic beads, mix well and let it stand for 7-15 minutes; place it on a magnetic rack, collect the supernatant, add 40μl Ampure XP magnetic beads to the supernatant, mix well and let it stand for 7-15 minutes; place it on a magnetic rack, aspirate the supernatant, and wash the magnetic beads twice with 75% ethanol; after drying, add 50μl TE buffer solution or enzyme-free water, mix well and let it stand for 7-15 minutes to dissolve and recover the product.

3. Dephosphorylation reaction: Take the product recovered in the previous step and prepare the system according to the following table:

10x NEB Buffer 2 6μl Shrimp alkaline phosphatase (1U/μl) 6 μl Total volume 12 μl

Add 12 μl of the reaction solution to the recovered product from the previous step, mix thoroughly, and proceed to the reaction according to the conditions listed below. The reaction product should be used directly in the next step. (The "cooling to 4°C at 0.1°C/s" step is optional, and the reaction time does not need to be precisely controlled. The same applies to the following steps.)

4. Fragment end repair: Prepare the system according to the table below:

Mix the system thoroughly and add it to the product from the previous step. Incubate at 12°C for 20 minutes. Purify the product using 80 μl of PEG32 magnetic beads and dissolve and recover the product in 40 μl of TE buffer. (Reaction products can be purified using a variety of methods, including magnetic bead purification, column purification, and gel recovery. These methods can be used interchangeably. In this example, magnetic bead purification is used unless otherwise specified.)

5. Connectors A and B: The linker sequences used in this protocol are as follows (the sequence from left to right is from 5' to 3' end, "//" indicates the terminal modification group, "phos" indicates phosphorylation, "dd" indicates dideoxy, and "bio" indicates biotin).

Connector A:

Long chain/Phos/GGCTCCGTCGAAGCCCGACG/ddC/

Short chain GCTTCGACGGAGC/ddC/

Connector B:

Long chain: /phos/ACGTCGGGGCCAAGCGGTCGT/ddC/

Short chain: TTGGCCCCGGCT/-ddT/.

Prepare the system according to the table below:

Enzyme-free water 11.1μl 5 μM Linker A 1.85 μl 5 μM Linker B 1.85 μl Total volume 14.8μl

Mix the above system and add it to the purified product from the previous step. After mixing, prepare the following system:

The above system was mixed with the previous system and incubated at 20°C for 1 hour. The product was purified using 100 μl of Ampure XP and dissolved in 40 μl of TE buffer solution to recover the product.

This step completes the ligation of the target nucleic acid fragment with adapters A and B. Electrophoresis results of the products before and after ligation are shown in Figure 2. As shown in Figure 2, the fragment size increases significantly after adapter ligation in step 5, demonstrating the success of this adapter ligation protocol. In particular, after the polymerase chain reaction in step 7, the bands become more concentrated, demonstrating a significant screening and enrichment effect.

6. Single chain C, D connection:

Single-strand C: /phos/AGACAAGCTCxxxxxxxxxxGATCGGGCTTCGACGGAG (the middle "x" is the variable tag sequence region)

Single-stranded D: /bio/TCCTAAGACCGCTTGGCCCCGA.

Prepare the system according to the table below:

Enzyme-free water 19.88μl 10x Taq buffer 8μl 0.1M ATP 0.8 μl 25 mM deoxyribonucleoside triphosphates 0.32μl 20 μM single-chain D 0.5 μl Total volume 30 μul

First, add 1 μl of 10 μM single-stranded C to the product recovered in the previous step, mix well, then add the above system and mix well, react at 65°C for 5 minutes, and cool to 37°C at 0.1°C/s.

Keep the above reaction system at 37°C and prepare the following reaction system:

Enzyme-free water 0.4 μl 10x Taq buffer 0.4 μl T4 DNA ligase (600 U/μl) 4.8 μl Taq polymerase (5 U/μl) 2.4 μl Total volume 8μl

Add 8 μl of the above reaction mixture to the previous reaction system at 37°C. Mix well and react at 37°C for 20 min.

The product was purified using 96 μl of Ampure XP magnetic beads and dissolved in 25 μl of TE buffer solution.

7. Polymerase chain reaction: Prepare the system according to the following table:

Take 30-40 ng of the product recovered in the previous step, make up to 25 μl with enzyme-free water or TE, add it to the above system, mix well and react according to the conditions in the table below:

After the reaction was completed, 120 μl of Ampure XP magnetic beads were used for purification, and 25 μl of enzyme-free water was used to dissolve and recover the product.

8. Hybridization capture: Take 500ng-1μg of the reaction product from the previous step, concentrate and evaporate to dryness, and then add it to the following system 1 to dissolve:

The mixed reaction system 1 was placed at 95°C for 5 minutes and then kept at 65°C.

Preparation system 2:

Add system 2 to system 1 and keep it at 65°C.

Preparation system 3:

Add system 3 to systems 1 and 2 and react at 65°C for 20-24 hours.

After the reaction is completed, streptavidin-coated magnetic beads are used for binding. After the binding is completed, the magnetic beads are dissolved in 50 μl of enzyme-free water.

Prepare the following reaction system:

Add the dissolved magnetic beads to the reaction system and mix well, then react according to the following table:

After the reaction was completed, 240 μl of Ampure XP magnetic beads were used for purification.

9. Single-stranded fragment isolation: Use streptavidin-coated magnetic beads to bind the biotinylated target fragment obtained in step 8. Use 78 μl of 0.1 M sodium hydroxide to separate the single-stranded fragments that are not bound to the magnetic beads. Add acidic buffer to neutralize the separated product. The total volume of the product after neutralization is 112 μl.

10. Single-stranded circularization: Prepare the following reaction system 1: wherein the nucleic acid single strand E has a corresponding complementary sequence for connecting the two ends of the single strand.

The single-stranded E sequence is as follows: TCGAGCTTGTCTTCCTAAGACCGC (SEQ ID NO: 8)

Enzyme-free water 43 μl Single-stranded nucleic acid E 20 μl Total volume 63 μl

Add reaction system 1 to the single-stranded product from step 9. Mix well.

Prepare reaction system 2:

Add reaction system 2 to reaction system 1, mix well, and incubate at 37°C for 1.5 h.

11. Exonuclease 1 and Exonuclease 3 treatment:

Prepare the following reaction buffer:

Add 23.7 μl of the reaction buffer prepared above to 350 μl of the reaction product from step 10. Mix well and incubate at 37°C for 30 min.

Add 15.4 μl of 500 mM EDTA and mix well.

Use 500 μl PEG32 magnetic beads for purification and recovery, and re-dissolve in 40-80 μl enzyme-free water/TE buffer.

The final product concentration and total amount of this embodiment are as follows:

The electrophoresis results are shown in Figure 3. Figure 3 shows the electrophoresis results of the products after step 11 on a 6% denaturing polyacrylamide gel. As shown in Figure 3, products 1, 3, and 5 were generated after hybridization in step 8 and then subjected to gel electrophoresis fragment selection, while products 2, 4, and 6 were not subjected to the fragment size selection step. As Figure 3 shows, the products that underwent gel electrophoresis fragment selection were more uniform in size, but the fragments that were not subjected to fragment size selection were also sequenced normally, demonstrating the complete success of this protocol.

Industrial Applicability

The isolated oligonucleotides of the present invention can be effectively used as linkers for constructing sequencing libraries. When constructing sequencing libraries, different linkers can be simultaneously connected to both ends of a nucleic acid fragment, while avoiding mutual connection between linkers, thereby improving the connection efficiency and reducing the economic and time costs of constructing a sequencing library.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in a suitable manner in any one or more embodiments or examples. In addition, it should be noted that those skilled in the art will understand that the order of the steps included in the scheme proposed in the present invention can be adjusted by those skilled in the art, which will also be included in the scope of the present invention.

While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents.

SEQUENCE LISTING

<110> Shenzhen BGI Genomics Co., Ltd.

<120> Isolated oligonucleotides and their use in nucleic acid sequencing

<130> PIOC145502PCN

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 21

<212> DNA

<213> Artificial

<220>

<223> Connector first chain

<400> 1

ggctccgtcg aagcccgacg c 21

<210> 2

<211> 13

<212> DNA

<213> Artificial

<220>

<223> Adapter second chain

<400> 2

cttcgacgga gcc 13

<210> 3

<211> 22

<212> DNA

<213> Artificial

<220>

<223> Connector first chain

<400> 3

acgtcggggc caagcggtcg tc 22

<210> 4

<211> 13

<212> DNA

<213> Artificial

<220>

<223> Adapter second chain

<400> 4

ttggccccgg ctt 13

<210> 5

<211> 21

<212> DNA

<213> Artificial

<220>

<223> Second single-stranded DNA

<400> 5

tcctaagacc gcttggcccc g 21

<210> 6

<211> 12

<212> DNA

<213> Artificial

<220>

<223> First segment of single-stranded nucleic acid molecule

<400> 6

tcgagcttgt ct 12

<210> 7

<211> 12

<212> DNA

<213> Artificial

<220>

<223> Second segment of single-stranded nucleic acid molecule

<400> 7

tcctaagacc gc 12

<210> 8

<211> 24

<212> DNA

<213> Artificial

<220>

<223> Single-stranded nucleic acid E

<400> 8

tcgagcttgt cttcctaaga ccgc 24

Claims (32)

1. An isolated oligonucleotide, characterized in that it comprises: The first chain has a phosphate group at its 5' end and a dideoxynucleotide at its 3' end. The first chain is 20–25 nt in length and has a first overhang located at its 3' end. The first overhang is 6–12 nt in length. The second chain has a 5' terminal nucleotide that does not have a phosphate group and a 3' terminal nucleotide that is a dideoxynucleotide. The length of the second chain is 10 to 15 nt. Optionally, the second chain has a second overhang located at the 5' end of the second chain. The length of the second overhang is 0 to 4 nt. The first chain and the second chain form a double chain structure. 2. The isolated oligonucleotide according to claim 1, characterized in that the number of nucleotides on the second strand that do not match the first strand does not exceed 3. 3. The isolated oligonucleotide according to claim 1, wherein the first strand and the second strand are both DNA. 4. The isolated oligonucleotide according to claim 1, characterized in that, The sequence of the first chain is: 5’GGCTCCGTCGAAGCCCGACGC3’, and The sequence of the second strand is: 5’CTTCGACGGAGCC3’; or The sequence of the first chain is: 5’ACGTCGGGGCCAAGCGGTCGTC3’, and The sequence of the second chain is: 5’TTGGCCCCGGCTT3’. 5. A reagent kit, characterized in that it comprises: The first connector and the second connector are both isolated oligonucleotides as described in any one of claims 1 to 4. The first connector is different from the second connector. 6. The kit according to claim 5, further comprising: A first single-stranded DNA, wherein the first single-stranded DNA is capable of matching with the first strand of the first adapter to form a double-stranded structure; and The second single-stranded DNA can match the first strand of the second adapter to form a double-stranded structure. 7. The kit according to claim 6, wherein the length of the double-stranded structure formed by the first single-stranded DNA and the first strand of the first adapter is greater than the length of the double-stranded structure formed between the first strand and the second strand in the first adapter; and The length of the double-stranded structure formed by the second single-stranded DNA and the first strand of the second adapter is greater than the length of the double-stranded structure formed between the first strand and the second strand in the second adapter. 8. The kit according to claim 6, further comprising: The first primer is identical to one of the first single-stranded DNA and the second single-stranded DNA; and The second primer has an additional biotin at the 5' end compared to the first single-stranded DNA and the other of the second single-stranded DNA. 9. The reagent kit according to claim 8, characterized in that, The sequence of the first chain of the first connector is: 5’GGCTCCGTCGAAGCCCGACGC3’; The sequence of the second chain of the first connector is: 5’CTTCGACGGAGCC3’; The sequence of the first chain of the second connector is: 5’ACGTCGGGGCCAAGCGGTCGTC3’; The sequence of the second chain of the second connector is: 5’TTGGCCCCGGCTT3’; The sequence of the first single-stranded DNA is: 5'AGACAAGCTC(N) _m GATCGGGCTTCGACGGAG3', where (N) _m represents a tag sequence of length m nucleotides, where m is any integer from 4 to 10, and N = A, T, G, or C; and The sequence of the second single-stranded DNA is 5’TCCTAAGACCGCTTGGCCCCG3’. 10. A method for adding adapters to both ends of a double-stranded DNA fragment, said double-stranded DNA fragment having two blunt ends, and all four terminal nucleotides of said double-stranded DNA fragment being free of phosphate groups, characterized in that the method comprises: The double-stranded DNA fragment is ligated with a first adapter and a second adapter to obtain a first ligation product, wherein the first adapter and the second adapter are different, and both the first adapter and the second adapter are isolated oligonucleotides as described in any one of claims 1 to 4. The first single-stranded DNA replaces the second strand of the first adapter, and the second single-stranded DNA replaces the second strand of the second adapter, wherein the first single-stranded DNA is specifically matched with the first strand of the first adapter to form a double-stranded structure, and the second single-stranded DNA is specifically matched with the first strand of the second adapter to form a double-stranded structure. The first single-stranded DNA and the second single-stranded DNA are respectively ligated to the double-stranded DNA fragment to obtain a second ligation product; and The second ligation product is amplified using a first primer and a second primer to obtain an amplification product, wherein the amplification product is a DNA fragment with adapters at both ends, wherein the first primer contains the same sequence as one of the first single-stranded DNA and the second single-stranded DNA, and the second primer contains the same sequence as the other of the first single-stranded DNA and the second single-stranded DNA, and has an additional biotin at the 5' end compared to the other of the first single-stranded DNA and the second single-stranded DNA. 11. The method according to claim 10, wherein the step of ligating the double-stranded DNA fragment to the first adapter and the second adapter is performed in a one-step reaction. 12. The method according to claim 10, wherein the double-stranded DNA fragment is obtained by the following steps: The DNA sample is fragmented to obtain fragmented products; The fragmented product is dephosphorylated to obtain a dephosphorylated fragmented product; and The dephosphorylated fragmented product is subjected to end repair treatment to obtain the double-stranded DNA fragment. 13. The method according to claim 10, characterized in that, The length of the double-stranded structure formed by the first single-stranded DNA and the first strand of the first adapter is greater than the length of the double-stranded structure formed between the first and second strands in the first adapter; and The length of the double-stranded structure formed by the second single-stranded DNA and the first strand of the second adapter is greater than the length of the double-stranded structure formed between the first strand and the second strand in the second adapter. 14. The method according to claim 10, characterized in that, The sequence of the first chain of the first connector is: 5’GGCTCCGTCGAAGCCCGACGC3’; The sequence of the second chain of the first connector is: 5’CTTCGACGGAGCC3’; The sequence of the first chain of the second connector is: 5’ACGTCGGGGCCAAGCGGTCGTC3’; The sequence of the second chain of the second connector is: 5’TTGGCCCCGGCTT3’; The sequence of the first single-stranded DNA is: 5'AGACAAGCTC(N) _m GATCGGGCTTCGACGGAG3', where (N) _m represents a tag sequence of length m nucleotides, where m is any integer from 4 to 10, and N = A, T, G, or C; and The sequence of the second single-stranded DNA is 5’TCCTAAGACCGCTTGGCCCCG3’. 15. The method according to claim 10, characterized in that, by thermal pyrolysis-annealing, the second strand of the first adapter is replaced with the first single-stranded DNA, and the second strand of the second adapter is replaced with the second single-stranded DNA. 16. The method according to claim 15, wherein the thermal decomposition is carried out at 60 degrees Celsius. 17. The method according to claim 10, characterized in that the first single-stranded DNA and the second single-stranded DNA are respectively linked to the double-stranded DNA fragment by a gap-filling reaction. 18. The method according to claim 12, wherein the DNA sample is at least a portion of genomic DNA or a reverse transcription product of RNA. 19. A method for constructing a sequencing library from a double-stranded DNA fragment, wherein the double-stranded DNA fragment has two blunt ends, and all four terminal nucleotides of the double-stranded DNA fragment are free of phosphate groups, characterized in that the method comprises: The method according to any one of claims 10 to 18, wherein adapters are ligated to both ends of the double-stranded DNA fragment to obtain a DNA fragment with adapters ligated to both ends; Separate single-stranded DNA fragments from the DNA fragments with adapters at both ends; and The single-stranded DNA fragment is circularized to obtain single-stranded DNA loops, which constitute the sequencing library. 20. The method according to claim 19, characterized in that separating the single-stranded DNA fragment from the DNA fragment with adapters at both ends further comprises: The DNA fragment with adapters at both ends is brought into contact with a magnetic bead to form a magnetic bead-DNA complex, wherein the magnetic bead is attached with streptavidin; and The magnetic bead-DNA complex is contacted with a solution at a pH higher than 7 to obtain the single-stranded DNA fragment. 21. The method according to claim 20, wherein the solution with a pH higher than 7 is a sodium hydroxide solution. 22. The method according to claim 21, wherein the concentration of the sodium hydroxide solution is 0.5–2 M. 23. The method according to claim 22, wherein the concentration of the sodium hydroxide solution is 1M. 24. The method according to claim 19, characterized in that, before isolating single-stranded DNA fragments from the DNA fragments with adapters at both ends, the DNA fragments with adapters at both ends are pre-screened. 25. The method according to claim 24, wherein the screening is performed by contacting the DNA fragment with adapters at both ends with a probe, wherein the probe is specific for a predetermined sequence. 26. The method according to claim 25, wherein the predetermined sequence comprises at least one exon. 27. The method according to claim 25, wherein the probe is provided in the form of a microchip array. 28. The method according to claim 19, characterized in that the single-stranded DNA fragment is circularized using a single-stranded nucleic acid molecule. in, The single-stranded nucleic acid molecule defines a first segment and a second segment, wherein the first segment is capable of matching the sequence of the 5' terminal nucleotide and the 3' terminal nucleotide of the single-stranded DNA fragment, and the second segment is capable of matching the sequence of one of the 5' terminal nucleotide and the 3' terminal nucleotide of the single-stranded DNA fragment. 29. The method according to claim 28, wherein the first segment and the second segment are adjacent to each other. 30. The method according to claim 29, characterized in that, The sequence of the first segment is 5’TCGAGCTTGTCT3’; and The sequence of the second segment is 5’TCCTAAGACCGC3’. 31. A nucleic acid sequencing method, characterized in that it comprises: The method according to any one of claims 19-30, constructing a sequencing library; and The sequencing library was sequenced. 32. The sequencing method according to claim 31, characterized in that the sequencing library is sequenced using a CG sequencing platform.