WO2016037358A1 - 分离的寡核苷酸及其在核酸测序中的用途 - Google Patents

分离的寡核苷酸及其在核酸测序中的用途 Download PDF

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WO2016037358A1
WO2016037358A1 PCT/CN2014/086418 CN2014086418W WO2016037358A1 WO 2016037358 A1 WO2016037358 A1 WO 2016037358A1 CN 2014086418 W CN2014086418 W CN 2014086418W WO 2016037358 A1 WO2016037358 A1 WO 2016037358A1
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Prior art keywords
stranded dna
linker
strand
dna fragment
double
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PCT/CN2014/086418
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English (en)
French (fr)
Inventor
耿春雨
巴林杰丹尼斯·G.
张艳艳
傅书锦
贺玲瑜
章文蔚
蒋慧
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深圳华大基因科技有限公司
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Priority to PCT/CN2014/086418 priority Critical patent/WO2016037358A1/zh
Priority to US15/510,877 priority patent/US9890375B2/en
Priority to EP14901591.9A priority patent/EP3192869B1/en
Priority to AU2014406026A priority patent/AU2014406026B2/en
Priority to DK14901591.9T priority patent/DK3192869T3/da
Priority to ES14901591T priority patent/ES2726149T3/es
Priority to CN201480081861.5A priority patent/CN107075513B/zh
Priority to JP2017514336A priority patent/JP6483249B2/ja
Priority to ES14901739.4T priority patent/ES2682068T3/es
Priority to PCT/CN2014/087589 priority patent/WO2016037389A1/en
Priority to EP14901739.4A priority patent/EP3191630B1/en
Priority to CN201410505310.9A priority patent/CN105400864B/zh
Priority to DK14901597.6T priority patent/DK3192900T3/en
Priority to CN201480081852.6A priority patent/CN107075731B/zh
Priority to PCT/CN2014/088543 priority patent/WO2016037394A1/zh
Priority to US15/510,904 priority patent/US10023906B2/en
Priority to ES14901597.6T priority patent/ES2689353T3/es
Priority to AU2014405969A priority patent/AU2014405969B2/en
Priority to JP2017514335A priority patent/JP6438126B2/ja
Priority to EP14901597.6A priority patent/EP3192900B1/en
Priority to US15/510,890 priority patent/US10544451B2/en
Priority to DK18174793.2T priority patent/DK3388519T3/da
Priority to ES14901593T priority patent/ES2708804T3/es
Priority to CN201480081687.4A priority patent/CN106795514B/zh
Priority to PCT/CN2014/091852 priority patent/WO2016037416A1/zh
Priority to ES18174793T priority patent/ES2738480T3/es
Priority to CN201410677537.1A priority patent/CN105400776B/zh
Priority to EP14901593.5A priority patent/EP3192877B1/en
Priority to EP18174793.2A priority patent/EP3388519B1/en
Priority to AU2014405991A priority patent/AU2014405991B2/en
Priority to DK14901593.5T priority patent/DK3192877T3/en
Priority to JP2017513705A priority patent/JP6679576B2/ja
Publication of WO2016037358A1 publication Critical patent/WO2016037358A1/zh
Priority to HK16103684.7A priority patent/HK1215721A1/zh
Priority to HK16104236.8A priority patent/HK1217726A1/zh
Priority to US16/576,582 priority patent/US10995367B2/en

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Definitions

  • the invention relates to the field of biotechnology.
  • it relates to isolated oligonucleotides and their use in nucleic acid sequencing. More particularly, it relates to an isolated oligonucleotide, a kit, a method of adding a linker at both ends of a double-stranded DNA fragment, a method of constructing a sequencing library for a double-stranded DNA fragment, and a nucleic acid sequencing method.
  • High-throughput sequencing has become one of the foundations of modern molecular biology, biotechnology, and medicine.
  • the research on rapid, accurate and economical gene expression levels and nucleotide sequence determination methods has been continuously developed; the second generation high-throughput sequencing technology based on sequencing while synthesizing is becoming mature.
  • Major sequencing companies have focused on the development of new sequencing products, the shortening of sequencing processes and cost reductions.
  • sequencing products based on second-generation sequencing technologies include whole-genome resequencing, whole transcriptome sequencing, and small-molecule RNA sequencing.
  • second-generation sequencing combined with microarray technology--the target sequence capture sequencing technology can use a large number of oligonucleotide probes to complement a specific region on the genome to enrich a specific segment, and then These segments were sequenced using second generation sequencing technology to achieve human total exome sequencing (WES).
  • WES human total exome sequencing
  • the present invention aims to solve at least one of the technical problems existing in the prior art.
  • Complete Genomics (sometimes referred to herein as "CG") currently has a second-generation sequencing technology developed independently for human genome sequencing.
  • the library construction process mainly includes: genomic DNA disruption, first linker ligation, double-stranded cyclization and enzymatic cleavage, second linker ligation, single-stranded cyclization. Two of the joints are important throughout the building process.
  • a linker is a DNA sequence that is ligated to both ends of a DNA fragment by ligation and can be identified during sequencing and used as a starting site for sequencing for the instrument to read subsequent sequence information.
  • the present invention proposes a means for adding a linker at both ends of a DNA fragment.
  • the invention proposes an isolated oligonucleotide.
  • the oligonucleotide comprises: a first strand, the 5' terminal nucleotide of the first strand has a phosphate group, and the 3' terminal nucleotide of the first strand is double a deoxynucleotide; and a second strand, the 5' terminal nucleotide of the second strand does not have a phosphate group, and the 3' terminal nucleotide of the second strand 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 strand and the second strand.
  • the isolated oligonucleotide can be used as a linker for constructing a sequencing library, and when constructing a sequencing library, it is possible to simultaneously connect different junctions at both ends of the nucleic acid fragment while avoiding interconnection between the joints and improving The efficiency of the connection reduces the economic and time cost of building a sequencing library.
  • the invention proposes a kit.
  • the kit comprises: a first linker and a second linker, wherein the first linker and the second linker are both isolated oligonucleotides as described above, wherein the first linker The second joint is different.
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the kit can be used as a linker for constructing a sequencing library, and when constructing a sequencing library, simultaneous connection of different joints at both ends of the nucleic acid fragment can be realized, and interconnection between the joints is avoided, and connection efficiency is improved. Reduces the economic and time cost of building a sequencing library.
  • the invention provides a method of adding a linker at both ends of a double stranded DNA fragment.
  • 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
  • the method comprises: The double-stranded DNA fragment is ligated to the first linker and the second linker to obtain a first linker product, wherein the first linker and the second linker are different, and the first linker and the second linker are both previously described An isolated oligonucleotide; replacing a second strand of the first linker with a first single stranded DNA and replacing a second strand of the second linker with a second single stranded DNA, wherein the first single
  • the stranded DNA is capable of specifically binding to a first strand of the first linker to form a double stranded structure, the second single stranded DNA being capable of specifically binding
  • the oligonucleotide can be used as a linker to construct a sequencing library while simultaneously connecting different junctions at both ends of the nucleic acid fragment, The interconnection between the joints is avoided, the connection efficiency is improved, and the economic and time cost of constructing the sequencing library is reduced.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed.
  • the invention proposes a method of constructing a sequencing library for a double-stranded DNA fragment.
  • 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
  • the method comprises: according to the foregoing a method of ligating a linker at both ends of a double-stranded DNA fragment, ligating a linker at both ends of the double-stranded DNA fragment to obtain a DNA fragment having a linker at both ends; and separating the DNA fragment from the linker a stranded DNA fragment; and cyclizing the single-stranded DNA fragment to obtain a single-stranded DNA loop, the single-stranded DNA loop constituting the sequencing library.
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed. Further, by isolating single-stranded DNA and performing a single-strand loop-forming reaction, a sequencing library such as a sequencing library for a CG sequencing platform can be efficiently obtained.
  • the invention provides a nucleic acid sequencing method.
  • the method comprises: constructing a sequencing library according to the method of constructing a sequencing library for a double-stranded DNA fragment as described above; and sequencing the sequencing library.
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the foregoing description of the features and advantages of the isolated oligonucleotides in accordance with embodiments of the present invention is equally applicable to the method and will not be described herein.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed. Further, by isolating single-stranded DNA and performing a single-strand loop-forming reaction, a sequencing library such as a sequencing library for a CG sequencing platform can be efficiently obtained. Thereby, the efficiency of sequencing can be further improved, and the cost of sequencing can be reduced.
  • the invention also provides an apparatus for adding a linker at both ends of a double-stranded DNA fragment.
  • Root 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
  • the device comprises: a first linking unit The first connecting unit is configured to connect the DNA segment with the first joint and the second joint to obtain a first joint product, wherein the first joint and the second joint are different, and the first The linker and the second linker are both isolated oligonucleotides as described above; a substitution unit for replacing the second strand of the first linker with the first single stranded DNA, and using the second single strand DNA replacing a second strand of the second linker, wherein the first single stranded DNA is capable of specifically matching a first strand of the first linker to form a double stranded structure, the second single stranded DNA being capable of The first linking unit The first connecting unit is configured to connect the DNA segment with the
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed.
  • the invention also provides an apparatus for constructing a sequencing library for a double-stranded DNA fragment.
  • 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
  • the apparatus comprises: a device for adding a linker at both ends of a double-stranded DNA fragment for ligating a linker at both ends of the double-stranded DNA fragment to obtain a DNA fragment having a linker at both ends; a single-stranded DNA fragment separation device, the single-strand a DNA fragment separation device for separating a single-stranded DNA fragment from the DNA fragment to which the linker is ligated; and a cyclization device for cyclizing the single-stranded DNA fragment to obtain a single strand A DNA loop that constitutes the sequencing library.
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the first single stranded DNA and the second single stranded DNA can be used to replace the two joints, respectively. a second strand and a more stable double-stranded structure with the first strand, and further, by using the first single-stranded DNA and the second single-stranded DNA as primers, PCR amplification can be performed to form a stable linker at both ends DNA fragment. Further, by isolating single-stranded DNA and performing a single-strand loop-forming reaction, a sequencing library such as a sequencing library for a CG sequencing platform can be efficiently obtained.
  • the invention also proposes a nucleic acid sequencing system.
  • 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.
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the foregoing description of the features and advantages of the isolated oligonucleotides in accordance with embodiments of the present invention is equally applicable to the system and will not be described herein.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed. Further, by isolating single-stranded DNA and performing a single-strand loop-forming reaction, a sequencing library such as a sequencing library for a CG sequencing platform can be efficiently obtained. Thereby, the efficiency of sequencing can be further improved, and the cost of sequencing can be reduced.
  • the invention also proposes an apparatus for constructing a sequencing library against genomic DNA.
  • the apparatus comprises: means for fragmenting the genomic DNA to obtain a fragmented product; means for dephosphorylation of the fragmented product for obtaining a phosphorylated fragmented product; means for end-repairing the dephosphorylated fragmented product to obtain a double-stranded DNA fragment; means for using the double-stranded DNA fragment with the first linker Connecting with a second linker to obtain a first ligation product, wherein the first linker and the second linker are different, and the first linker and the second linker are both isolated oligonucleotides as described above; Means for replacing a second strand of the first linker with a first single stranded DNA and replacing a second strand of the second linker with a second single stranded DNA, wherein the first single stranded DNA is capable of The first strand of the first linker specifically matches to form a
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed. Further, by isolating single-stranded DNA and performing a single-strand loop-forming reaction, a sequencing library such as a sequencing library for a CG sequencing platform can be efficiently obtained.
  • Figure 1 shows a schematic flow diagram of the construction of a sequencing library in accordance with one embodiment of the present invention.
  • 1 Break the DNA fragment.
  • 2 Dephosphorylated, terminally repaired fragments (each end is a hydroxyl group).
  • 3 Connector A.
  • 4 Connector B.
  • 5 Single chain C.
  • 6 Single chain D.
  • 7 Sequence of tags on single-stranded C.
  • 8 The final product is a cyclic single chain.
  • Figure 2 shows an electropherogram in accordance with one embodiment of the present invention.
  • Figure 3 shows an electropherogram in accordance with one embodiment of the present invention.
  • Figure 4 shows a schematic flow diagram of a method of adding a linker at both ends of a double-stranded DNA fragment, in accordance with one embodiment of the present invention.
  • Figure 5 is a schematic view showing the structure of an apparatus for adding a linker at both ends of a double-stranded DNA fragment according to an embodiment of the present invention.
  • Figure 6 shows a schematic representation of the structure of an apparatus for constructing a sequencing library for double-stranded DNA fragments, in accordance with one embodiment of the present invention.
  • Figure 7 shows a schematic structural view of a nucleic acid sequencing system in accordance with one embodiment of the present invention.
  • the invention proposes an isolated oligonucleotide.
  • the oligonucleotide comprises: a first strand, the 5' terminal nucleotide of the first strand has a phosphate group, and the 3' terminal nucleotide of the first strand is double a deoxynucleotide; and a second strand, the 5' terminal nucleotide of the second strand does not have a phosphate group, and the 3' terminal nucleotide of the second strand 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 strand and the second strand.
  • the isolated oligonucleotide can be used as a linker for constructing a sequencing library, and when constructing a sequencing library, it is possible to simultaneously connect different junctions at both ends of the nucleic acid fragment while avoiding interconnection between the joints and improving The efficiency of the connection reduces the economic and time cost of building a sequencing library.
  • a first protruding end comprising: a first protruding end, the first protruding end is located at a 3' end of the first chain; and an optional second protruding end, the second protruding end is located at the The 5' end of the second strand.
  • the length of the first protruding end is greater than the length of the second protruding end.
  • the first protruding end has a length of about 6 to 12 nt.
  • the second protruding end has a length of 0 to 4 nt.
  • the first chain has a length of about 20 to 25 nt.
  • the second chain has a length of about 10-15 nt.
  • the sequence of the first strand is: 5'GGCTCCGTCGAAGCCCGACGC3' (SEQ ID NO: 1)
  • the sequence of the second strand is: 5'CTTCGACGGAGCC3' (SEQ ID NO: 2)
  • the sequence of the first strand is: 5'ACGTCGGGGCCAAGCGGTCGTC3' (SEQ ID NO: 3)
  • the sequence of the second strand is: 5'TTGGCCCCGGCTT3' (SEQ ID NO: 4).
  • the invention proposes a kit.
  • the kit A first linker and a second linker are included, the first linker and the second linker being the isolated oligonucleotides described above, wherein the first linker is different from the second linker.
  • the kit can be used as a linker for constructing a sequencing library, and when constructing a sequencing library, simultaneous connection of different joints at both ends of the nucleic acid fragment can be realized, and interconnection between the joints is avoided, and connection efficiency is improved. Reduces the economic and time cost of building a sequencing library.
  • a first single-stranded DNA capable of matching a first strand of the first linker to form a double-stranded structure
  • a second single-stranded DNA is capable of matching the first strand of the second linker to form a double stranded structure.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, further, by adopting
  • the first single-stranded DNA and the second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed.
  • the length of the double-stranded structure formed by the first single-stranded DNA and the first strand of the first linker is greater than the first strand and the second strand of the first linker Forming a length of the double-stranded structure; and the length of the double-stranded structure formed by the second single-stranded DNA and the first strand of the second linker is greater than the first strand and the first The length of the double-stranded structure is formed between the two strands.
  • a first primer the first primer being identical to one of the first single stranded DNA and the second single stranded DNA
  • a second primer the second primer
  • biotin at the 5' end compared to the other of the first single stranded DNA and the second single stranded DNA.
  • the efficiency of the ligation when constructing the sequencing library can be further improved, further reducing the economic and time cost of constructing the sequencing library.
  • a reagent capable of specifically recognizing a living organism a single-stranded nucleic acid molecule can be efficiently isolated, and thus can be used to construct a sequencing library of a CG sequencing platform.
  • the invention provides a method of adding a linker at both ends of a double stranded DNA fragment.
  • 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 with reference to FIG. 4, the method includes :
  • the step of linking the double-stranded DNA fragment to the first linker and the second linker is carried out in a one-step reaction.
  • the DNA fragment is obtained by fragmenting a DNA sample to obtain a fragmented product; dephosphorating the fragmented product to obtain a dephosphorylation treatment a fragmented product; and subjecting the dephosphorylated fragmented product to a terminal repair treatment to obtain the double-stranded DNA fragment.
  • the DNA sample is at least a portion of genomic DNA or a reverse transcription product of RNA.
  • a sequencing library can be efficiently constructed for genomic DNA or RNA.
  • the length of the double-stranded structure formed by the first single-stranded DNA and the first strand of the first linker is greater than the first strand and the second strand of the first linker Forming a length of the double-stranded structure; and the length of the double-stranded structure formed by the second single-stranded DNA and the first strand of the second linker is greater than the first strand and the first The length of the double-stranded structure is formed between the two strands.
  • a reagent capable of specifically recognizing biotin a single-stranded nucleic acid molecule can be efficiently isolated, and thus can be used to construct a sequencing library of a CG sequencing platform.
  • the second strand of the first linker is replaced with a first single stranded DNA and the second strand of the second linker is replaced with a second single stranded DNA by a thermal cleavage-annealing treatment.
  • the thermal cracking is carried out at about 60 degrees Celsius.
  • the first single stranded DNA and the second single stranded DNA are ligated to the double stranded DNA fragment, respectively, to obtain a second ligation product.
  • the first single-stranded DNA and the second single-stranded DNA are respectively ligated to the double-stranded DNA fragment by a gap-filling reaction.
  • S400 amplifying the second ligation product by using a first primer and a second primer
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed.
  • the invention proposes a method of constructing a sequencing library for a double-stranded DNA fragment.
  • 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:
  • a linker is ligated to both ends of the double-stranded DNA fragment in accordance with the method of ligating a linker at both ends of the double-stranded DNA fragment as described above to obtain a DNA fragment to which a linker is ligated at both ends.
  • separating the single-stranded DNA fragment from the DNA fragment to which the linker is ligated at both ends further comprises: contacting the DNA fragment having the linker at both ends with the magnetic beads to form a magnetic bead-DNA complex a streptavidin to which the magnetic beads are attached; and contacting the magnetic bead-DNA complex with a solution having a pH higher than 7 to obtain the single-stranded DNA Fragment.
  • the single-stranded DNA fragment can be efficiently separated, thereby improving the efficiency of constructing the sequencing library and reducing the cost of constructing the sequencing library.
  • the solution having a pH above 7 is a sodium hydroxide solution.
  • the concentration of the sodium hydroxide solution is about 0.5 to 2M.
  • the concentration of the sodium hydroxide solution is about 1M.
  • the DNA fragment to which the linker is ligated is previously screened before separating the single-stranded DNA fragment from the DNA fragment to which the linker is ligated. Thereby, sequencing library construction can be performed for a predetermined region.
  • the screening is performed by contacting the DNA fragment to which the linker is ligated to the probe, wherein the probe is specific for a predetermined sequence.
  • the predetermined sequence comprises at least one exon.
  • the probes are provided in the form of a microchip array. Thereby, the single-stranded DNA fragment can be efficiently cyclized.
  • the single-stranded DNA fragment is then cyclized to obtain a single-stranded DNA loop, which constitutes the sequencing library.
  • the single-stranded DNA fragment is cyclized by using a single-stranded nucleic acid molecule, wherein the single-stranded nucleic acid molecule defines a first segment and a second segment, and the A segment is capable of matching a sequence comprising a 5' terminal nucleotide and a 3' terminal nucleotide of said single stranded DNA fragment, said second segment being capable of cooperating with a 5' terminal nucleus comprising said single stranded DNA fragment
  • the sequence of one of the nucleotides and the 3' terminal nucleotide is matched.
  • the first section and the second section are connected adjacently.
  • 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) .
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed. Further, by isolating single-stranded DNA and performing a single-strand loop-forming reaction, a sequencing library such as a sequencing library for a CG sequencing platform can be efficiently obtained.
  • the invention provides a nucleic acid sequencing method.
  • the method comprises: constructing a sequencing library according to the method of constructing a sequencing library for a double-stranded DNA fragment as described above; and sequencing the sequencing library.
  • the sequencing library is sequenced using a CG sequencing platform.
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not work with other nucleic acid sheets
  • the segments are interconnected to prevent interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed.
  • a sequencing library such as a sequencing library for a CG sequencing platform can be efficiently obtained. Thereby, the efficiency of sequencing can be further improved, and the cost of sequencing can be reduced.
  • the invention also provides an apparatus for adding a linker at both ends of a double-stranded DNA fragment.
  • 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 with reference to FIG. 5, the apparatus 100 includes The first connection unit 101, the replacement unit 102, the second connection unit 103, and the amplification unit 104. specifically:
  • the first connecting unit 101 is configured to connect the DNA segment with the first joint and the second joint to obtain a first joint product, wherein the first joint and the second joint are different, and the first joint and The second linker is the isolated oligonucleotide described above.
  • the first linking unit is configured to link the DNA fragment to the first linker and the second linker in a one-step reaction.
  • a replacement unit 102 for replacing a second strand of the first linker with a first single stranded DNA and a second strand of the second linker with a second single stranded DNA, wherein the first single stranded DNA is capable of The first strand is specifically matched to the first linker to form a double stranded structure, the second single stranded DNA being capable of specifically binding to the first strand of the second linker to form a double stranded structure.
  • the length of the double-stranded structure formed by the first single-stranded DNA and the first strand of the first linker is greater than the first strand and the second strand of the first linker Forming a length of the double-stranded structure; and the length of the double-stranded structure formed by the second single-stranded DNA and the first strand of the second linker is greater than the first strand and the first The length of the double-stranded structure is formed between the two strands.
  • connection efficiency for constructing the sequencing library can be further improved, further reducing the economic and time cost of constructing the sequencing library.
  • a reagent capable of specifically recognizing biotin a single-stranded nucleic acid molecule can be efficiently isolated, and thus can be used to construct a sequencing library of a CG sequencing platform.
  • the permutation unit 102 is configured to displace the second strand of the first linker using a first single stranded DNA and to displace the second stranded DNA using a second single stranded DNA by a thermal lysis-annealing treatment The second chain of the second joint.
  • the thermal cracking is carried out at about 60 degrees Celsius.
  • the second linking unit 103 is configured to cause the first single stranded DNA and the second single stranded DNA to be ligated to the DNA fragment, respectively, to obtain a second ligation product.
  • the second linking unit 103 is configured to connect the first single-stranded DNA and the second single-stranded DNA to the double-stranded DNA fragment by a gap-filling reaction, respectively. .
  • the amplification unit 104 is configured to amplify the second ligation product by using a first primer and a second primer to obtain an amplification product, wherein the first primer comprises the first single-stranded DNA and a sequence identical to one of the second single-stranded DNA, the second primer comprising the same sequence as the other of the first single-stranded DNA and the second single-stranded DNA, and the first single-stranded DNA There is additional biotin at the 5' end compared to the other of the second single stranded DNA.
  • the DNA fragment acquisition unit comprising: a fragmentation component for fragmenting the DNA sample, To obtain a fragmentation product; a dephosphorylation module for dephosphorylation of the fragmented product to obtain a dephosphorylated fragmented product; and an end repair component,
  • a terminal repair module is used for end-repairing the dephosphorylated fragmented product to obtain the double-stranded DNA fragment.
  • the double-stranded DNA fragment obtaining unit further comprises: a genomic DNA extraction component for extracting genomic DNA from the biological sample; and/or a reverse transcription component, the inverse A transcription component is used to perform 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 a reverse transcription product of RNA constitutes the DNA sample.
  • a sequencing library can be efficiently constructed for genomic DNA or RNA.
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed.
  • the invention also provides an apparatus for constructing a sequencing library for a double-stranded DNA fragment.
  • the double-stranded DNA fragment has two blunt ends, and the double-stranded DNA piece The four terminal nucleotides of the segment do not have a phosphate group, and referring to FIG. 6, the apparatus 1000 includes the apparatus 100 for adding a linker at both ends of the double-stranded DNA fragment, and the single-stranded DNA fragment separation device 200. And a cyclization device 300. specifically:
  • a device 100 for adding a linker at both ends of a double-stranded DNA fragment is used to ligate a linker at both ends of the double-stranded DNA fragment to obtain a DNA fragment to which a linker is ligated.
  • the single-stranded DNA fragment separation device 200 is for separating a single-stranded DNA fragment from the DNA fragment to which the linker is ligated.
  • a cyclization device 300 is used to cyclize the single-stranded DNA fragment to obtain a single-stranded DNA loop, which constitutes the sequencing library.
  • the single-stranded DNA fragment separation device 200 further includes: a magnetic bead trap unit for contacting the DNA fragment to which the linker is attached at both ends with the magnetic beads so that Forming a magnetic bead-DNA complex, wherein the magnetic bead is linked with streptavidin; an alkaline lysis unit, wherein the alkaline lysis unit is provided with a solution having a pH higher than 7, for using the magnetic
  • the bead-DNA complex is contacted with a solution having a pH lower than 7 to obtain the single-stranded DNA fragment.
  • the solution having a pH above 7 is a sodium hydroxide solution.
  • the concentration of the sodium hydroxide solution is about 0.5 to 2M.
  • the concentration of the sodium hydroxide solution is about 1M.
  • the screening device is provided with a probe, wherein the probe is specific to a predetermined sequence.
  • the predetermined sequence comprises at least one exon.
  • the probes are provided in the form of a microchip array.
  • the cyclization device 300 is provided with a single-stranded nucleic acid molecule, wherein the single-stranded nucleic acid molecule defines a first segment and a second segment, and the first segment Compatible with a sequence comprising a 5' terminal nucleotide and a 3' terminal nucleotide of the single stranded DNA fragment, the second segment being capable of interacting with a 5' terminal nucleotide comprising the single stranded DNA fragment Sequence matching of one of the 3' terminal nucleotides.
  • the first section and the second section are connected adjacently.
  • 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) .
  • the single-stranded DNA fragment can be efficiently cyclized by using a single-stranded nucleic acid molecule.
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the first stranded DNA and the second single stranded DNA can be used to replace the second strand of the two adaptors, respectively. Further, a more stable double-stranded structure is formed with the first strand, and further, by performing PCR amplification using the first single-stranded DNA and the second single-stranded DNA as primers, a DNA fragment having a stable linker at both ends can be formed. Further, by isolating single-stranded DNA and performing a single-strand loop-forming reaction, a sequencing library such as a sequencing library for a CG sequencing platform can be efficiently obtained.
  • the invention also proposes a nucleic acid sequencing system.
  • the system 10000 comprises: the apparatus 1000 and the sequencing apparatus 2000 for constructing a sequencing library for double-stranded DNA fragments as described above, the sequencing apparatus 2000 for sequencing the sequencing library .
  • the sequencing device 2000 is a CG sequencing platform.
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed.
  • a sequencing library such as a sequencing library for a CG sequencing platform can be efficiently obtained. Thereby, the efficiency of sequencing can be further improved, and the cost of sequencing can be reduced.
  • the invention also proposes an apparatus for constructing a sequencing library against genomic DNA.
  • the apparatus comprises:
  • the di-ligands are all isolated oligonucleotides as described above;
  • the amplification product is a DNA fragment having a linker attached to both ends
  • the first primer comprises the same sequence as one of the first single-stranded DNA and the second single-stranded DNA
  • the second primer comprises the same sequence as the other of the first single stranded DNA and the second single stranded DNA, and the other of the first single stranded DNA and the second single stranded DNA Extra biotin compared to the 5' end;
  • the 3' end of the first strand and the second strand in the oligonucleotide according to the embodiment of the present invention are both dideoxynucleotides, and the nucleotide at the 5' end of the second strand is not With a phosphate group, these ends will not be able to be linked to other nucleic acid fragments, thereby preventing interconnection between the oligonucleotides.
  • the oligonucleotide as a linker can realize simultaneous ligation of different linkers at both ends of the nucleic acid fragment, while avoiding interconnection between the linkers, improving the connection efficiency, and reducing the construction of the sequencing library. Economic and time costs.
  • the first single-stranded DNA and the second single-stranded DNA can be used to respectively replace the second strand of the two linkers, and form a more stable double-stranded structure with the first strand, and further, by adopting the A single-stranded DNA and a second single-stranded DNA are used as primers for PCR amplification, and a DNA fragment having a stable linker at both ends can be formed. Further, by isolating single-stranded DNA and performing a single-strand loop-forming reaction, a sequencing library such as a sequencing library for a CG sequencing platform can be efficiently obtained.
  • the ligation of the double stranded DNA fragment to the first linker and the second linker is accomplished in a one step reaction.
  • the method further includes:
  • Means for extracting genomic DNA from a biological sample Means for extracting genomic DNA from a biological sample.
  • At least a portion of the genomic DNA and/or a reverse transcription product of RNA constitutes the DNA sample.
  • a means for isolating a single-stranded DNA fragment from the DNA fragment to which the linker is ligated is configured to be suitable for isolating the single-stranded DNA fragment by: a DNA fragment to which a linker is attached is contacted with a magnetic bead to form a magnetic bead-DNA complex, wherein streptavidin is attached to the magnetic bead; and the magnetic bead-DNA complex is at a pH lower than 7 The solution is contacted to obtain the single-stranded DNA fragment.
  • the single-stranded DNA fragment can be efficiently separated, thereby improving the efficiency of constructing the sequencing library and reducing the cost of constructing the sequencing library.
  • the method further comprises: means for screening the DNA fragment to which the linker is ligated in advance before separating the single-stranded DNA fragment from the DNA fragment to which the linker is ligated.
  • the screening is carried out by contacting the DNA fragment to which the linker is ligated to the probe, wherein the probe is specific for a predetermined sequence.
  • the predetermined sequence comprises at least one exon.
  • the probe is in the form of a microchip array Provided by the style.
  • the means for cyclizing the single-stranded DNA fragment is configured to cyclize the single-stranded DNA fragment using a single-stranded nucleic acid molecule, wherein the single-stranded nucleic acid molecule Defining a first segment and a second segment, and the first segment is capable of matching a sequence comprising a 5' terminal nucleotide and a 3' terminal nucleotide of the single stranded DNA fragment, the second The segment is capable of matching a sequence comprising one of the 5' terminal nucleotide and the 3' terminal nucleotide of the single stranded DNA fragment.
  • the first section and the second section are connected adjacently.
  • 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) .
  • the single-stranded DNA fragment can be efficiently cyclized by using a single-stranded nucleic acid molecule.
  • the technical solution according to an embodiment of the present invention may have at least one of the following advantages:
  • the technical solution according to the embodiment of the present invention solves the problem that there are too many joint connection steps in the construction of the Complete Genomics sequencing platform library, and the overall library construction time is too long and the cost is too high.
  • the conventional multi-step method of adding the two-end joints is discarded when the joint is connected, and a new method of adding the joints at both ends in the same reaction is adopted.
  • connection manner of the two joints at the same time also needs to solve the problems of joint self-joining, segment interconnection and the like; and the joint designed by the present invention has a unique sequence structure and is connected by the same novel joint.
  • the method solves the problems of segment interconnection, joint self-joining, low segment connection efficiency, label sequence introduction position, etc., and successfully shortens the entire joint connection process into three steps; greatly shortens the time required for joint connection, obviously Reduced costs.
  • the technical solution according to an embodiment of the present invention combines an original joint ligation method with a nucleic acid probe capture technique, and further adjusts the traditional library construction scheme of Complete Genomics by further designing; successfully reduces the joint ligation process from twice to once. Significantly shortened library construction costs and time; and successfully created a single-joint full exome sequencing product based on the Complete Genomics sequencing platform.
  • a sequencing library can be constructed in the following embodiments in accordance with the following steps:
  • genomic nucleic acid strand is interrupted into fragments
  • dephosphorylation of the target fragment is used to block the 5' end of the fragment of interest to prevent fragment self-ligation.
  • both linkers A and B are polynucleotide duplexes consisting of one long chain (first strand) and one short chain (second strand).
  • the long chain can be linked to the target nucleic acid fragment by having a phosphate group at the 5' end, and the short chain is bound to the long chain by base complementary pairing, since the short chain end is a closed sequence, and the target nucleic acid fragment is not linked;
  • nucleic acid single strand C (shown in number 5 in Figure 1) and nucleic acid single strand D (shown in Figure 1 number 6).
  • Single-stranded C has a tag sequence (shown in Figure 1 in Figure 7), and the remaining fragments are complementary to the long strand of linker A; single-stranded D can be paired with the long chain of linker B.
  • short bonds with weak joints, single chain C, D and long chain of joints Complementary pairing.
  • extension and ligation reaction the linkage of single-stranded C and single-strand D to the target fragment is achieved.
  • step 4 using the product of step 4 as a template, single-chain C, D as a primer for polymerase chain reaction, amplification and enrichment of the product with the tag sequence;
  • step 5 product is subjected to oligonucleotide probe hybridization capture; the specific steps include probe hybridization, hybridization product elution, hybridization product enrichment step; and in the hybridization product enrichment step, in a double strand of the target nucleic acid Biotin modification is introduced into the chain.
  • the single strand of the nucleic acid is cyclized and the remaining uncyclized single strand is removed.
  • step length screening of step 7 can be carried out after the other steps before the single-strand separation, depending on the specific requirements of the sequencing and the actual changes in the size of the product fragments after each step. If it is confirmed by quality control that the size of the product of each step has always met the requirements, step 8 can be removed.
  • step 7 the steps introduced by full exon sequencing can be achieved.
  • the target nucleic acid fragment is subjected to dephosphorylation end-blocking treatment, and becomes a blunt-end fragment which is closed at both ends, completely avoiding the occurrence of interaction between fragments, and making the connection
  • dephosphorylation end-blocking treatment After the treatment of steps 2 and 3, the target nucleic acid fragment is subjected to dephosphorylation end-blocking treatment, and becomes a blunt-end fragment which is closed at both ends, completely avoiding the occurrence of interaction between fragments, and making the connection
  • the utilization of the pre-fragments is extremely guaranteed.
  • the specific linker design of the present invention introduces a phosphate group at the 5' end of the long chain of the linker A, B; and introduces a blocking sequence at both the 3' end of the long chain of the linker and the double end of the short chain. . Due to the presence of the blocking sequence, the blocked end can not only be connected to the target nucleic acid fragment, but also can not be connected to other joints added at the same time; ensuring that the 5' end of the long chain of the linker can be accurately aligned when the joint is connected in step 4. Connect to the 3' end of the target fragment.
  • This design is very effective in preventing the occurrence of joint interconnections, making it possible to simultaneously connect different joints and to ensure the efficiency of the connection reaction.
  • step 5 the characteristics of the long and short chains in the linker structure are skillfully utilized; since the short-chain complementary pairing bases are few and the binding is unstable, the long-chain separation is performed at a relatively mild temperature. Then, through the slow annealing reaction, the single-stranded C and D with longer binding ability and the longer binding ability are combined with the long chain of the linker; and the extended double-stranded head is formed after the extension.
  • the single-stranded C and D with longer binding ability and the longer binding ability are combined with the long chain of the linker; and the extended double-stranded head is formed after the extension.
  • This unique design has the characteristics of mild reaction conditions; thereby, the reaction system, the reaction time, and the reaction sequence are appropriately adjusted; and the three reactions of the segment replacement, the connection and the extension are carried out in the same reaction step 5, and The operation is simple, the reaction is rapid, and the processing time is greatly reduced.
  • the joint connection is successfully shortened from the original five steps to three steps of joint connection, gap filling, and polymerase chain reaction, and the operation amount is greatly reduced, thereby eliminating the use of various reagents and saving. A lot of time and cost.
  • the present invention not only the comprehensive replacement from the specific method of the joint connection, but also the subversive change of the traditional library construction scheme of CG company, and the novel single-stranded nucleic acid library structure (Fig. 1 mark 8) is proposed.
  • the conventional two-joint ligation process is reduced to a single-joint ligation process; the introduction of the polymerase chain reaction is reduced, and the quality of sequencing is improved.
  • the streamlining of the steps shortened the library construction time by as much as 3-4 days. High cost The amount is reduced; it has a huge advantage over the traditional solution.
  • the present invention successfully develops an efficient sequencing method suitable for human whole exome sequencing by modifying and supplementing the traditional sequencing library construction scheme of Complete Genomics, and combining the novel joint ligation method previously described.
  • Library construction program A novel human all-exome sequencing product based on the Complete Genomics sequencing platform was developed to achieve a breakthrough in the sequencing of all exomes based on the Complete Genomics platform.
  • a sequencing library is constructed in accordance with the following steps in an embodiment of the invention:
  • genomic nucleic acid strand is interrupted into fragments
  • dephosphorylation of the target fragment is used to block the 5' end of the fragment of interest to prevent fragment self-ligation.
  • nucleic acid single strand C (shown in number 5 in Figure 1) and nucleic acid single strand D (shown in Figure 1 number 6).
  • Single-stranded C has a tag sequence (shown in Figure 1 in Figure 7), and the remaining fragments are complementary to the long strand of linker A; single-stranded D can be paired with the long chain of linker B.
  • the short-chain drop of the weakly bonded linker the complementary pairing of the single-chain C, D and the long chain of the linker are caused.
  • extension and ligation reaction the linkage of single-stranded C and single-strand D to the target fragment is achieved.
  • step 4 using the product of step 4 as a template, single-chain C, D as a primer for polymerase chain reaction, amplification and enrichment of the product with the tag sequence;
  • step 5 product is subjected to oligonucleotide probe hybridization capture; the specific steps include probe hybridization, hybridization product elution, hybridization product enrichment step; and in the hybridization product enrichment step, in a double strand of the target nucleic acid Biotin modification is introduced into the chain.
  • the single strand of the nucleic acid is cyclized and the remaining uncyclized single strand is removed.
  • Interrupted fragment selection magnetic bead purification or gel recovery can be used. This embodiment employs a magnetic bead purification method.
  • Enzyme free water 12.2 ⁇ l 10x NEB Buffer 2 1.8 ⁇ l 0.1M adenosine triphosphate 0.8 ⁇ l 25mM deoxyribonucleoside triphosphate 0.8 ⁇ l Bovine serum albumin 0.4 ⁇ l T4 deoxyribonucleic acid polymerase (3U/ ⁇ l) 2 ⁇ l total capacity 18 ⁇ l
  • the system was mixed and added to the product of the previous step, mixed and incubated at 12 ° C for 20 min. Purification was carried out using 80 ⁇ l of PEG 32 magnetic beads, and 40 ⁇ l of TE buffer solution was dissolved to recover the product. (There are many ways to purify the reaction product, there are magnetic beads, Column purification, gel recovery, and the like. Can be used for replacement. This embodiment is purified by a magnetic bead method unless otherwise specified. )
  • Linker A, B linkage The sequence of the linker used in this protocol is as follows (the sequence is from 5' to 3' end from left to right, the terminal modification group is in "//”, and "phos” indicates phosphorylation, “ Dd” shows dideoxy, “bio” shows biotin).
  • Short chain TTGGCCCCGGCT/-ddT/.
  • the above system was mixed with the previous system and incubated at 20 ° C for 1 h. Purification was carried out using 100 ⁇ l of Ampure XP and 40 ⁇ l of TE buffer solution.
  • This step completes the ligation of the nucleic acid fragment of interest to linker A and linker B.
  • the electrophoresis results of the products before and after the connection are shown in Fig. It can be seen from Fig. 2 that the size of the segment after the connection of the step 5 is increased significantly, indicating that the joint connection of the solution is very successful. In particular, after step 7 polymerase chain reaction, the bands are more concentrated, and the screening enrichment effect is obvious.
  • Enzyme free water 19.88 ⁇ l 10x Taq Buffer 8 ⁇ l 0.1M adenosine triphosphate 0.8 ⁇ l 25mM deoxyribonucleoside triphosphate 0.32 ⁇ l
  • reaction system was prepared at 37 ° C to prepare the following reaction system:
  • Hybridization capture Take 500 ng-1 ⁇ g of the reaction product in the above step, concentrate and evaporate and add to the following system 1 to dissolve:
  • the mixed reaction system 1 was subjected to a reaction at 95 ° C for 5 minutes, and was continuously placed at 65 ° C.
  • System 3 was added to systems 1, 2 and reacted at 65 ° C for 20-24 h.
  • binding was carried out using streptavidin-coated magnetic beads, and after the completion of the binding, the magnetic beads were dissolved in 50 ⁇ l of enzyme-free water.
  • the dissolved magnetic beads are added to the reaction system and mixed, and the reaction is carried out according to the following table:
  • the single-strand E sequence is as follows: TCGAGCTTGTCTTCCTAAGACCGC (SEQ ID NO: 8)
  • Reaction System 1 was added to the single chain product of Step 9. Mix well.
  • the reaction system 2 was added to the reaction system 1, mixed, and incubated at 37 ° C for 1.5 h.
  • Purification was carried out using 500 ⁇ l of PEG32 magnetic beads, and 40-80 ⁇ l of enzyme-free water/TE buffer was used for reconstitution.
  • the final product concentration and total amount of this example are as follows:
  • FIG. 3 is a graph showing the results of electrophoresis of the product after step 11 using 6% polyacrylamide denaturing gel electrophoresis.
  • the products 1, 3, and 5 were subjected to gel electrophoresis fragment screening after hybridization in step 8, and the products 2, 4, and 6 were subjected to the fragment size screening step.
  • the size of the product screened by the gel electrophoresis fragment is more concentrated, but the fragment which is not subjected to fragment size screening can also be subjected to normal sequencing. Prove that the program is completely successful.
  • the isolated oligonucleotide of the present invention can be effectively used as a linker for constructing a sequencing library, and when constructing a sequencing library, it is possible to simultaneously connect different junctions at both ends of the nucleic acid fragment while avoiding interconnection between the joints, Increased connection efficiency and reduced economic and time cost of building sequencing libraries.

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Abstract

提供了分离的寡核苷酸及其在核酸测序中的用途，该分离的寡核苷酸包括：第一链，所述第一链的5'末端核苷酸具有磷酸基团，并且所述第一链的3'末端核苷酸为双脱氧核苷酸；以及第二链，所述第二链的5'末端核苷酸不具有磷酸基团，并且所述第二链的3'末端核苷酸为双脱氧核苷酸，其中，所述第一链的长度大于所述第二链的长度，并且所述第一链和所述第二链之间形成双链结构。

Description

分离的寡核苷酸及其在核酸测序中的用途 技术领域
本发明涉及生物技术领域。具体而言，涉及分离的寡核苷酸及其在核酸测序中的用途。更具体的，涉及一种分离的寡核苷酸、一种试剂盒、一种在双链DNA片段两端添加接头的方法、一种针对双链DNA片段构建测序文库的方法以及一种核酸测序方法。
背景技术
高通量测序已经成为了现代分子生物学、生物技术、医学等多领域的基础之一。在近几年，对迅速、精确、经济的基因表达水平和核苷酸序列的测定方法的研究不断推陈出新；以边合成边测序为基本原理的第二代高通量测序技术已趋于成熟，各大测序公司纷纷将重点放在了新测序产品的开发、测序流程的缩短和成本降低上。目前已有的基于第二代测序技术的测序产品有全基因组重测序、全转录组测序、小分子RNA测序等。特别的，第二代测序结合微阵列技术而衍生出来的应用--目标序列捕获测序技术能够使用大量寡核苷酸探针与基因组上的特定区域互补结合，从而富集到特定区段，然后用第二代测序技术对这些区段进行测序，以实现人全外显子组测序(WES)。这种测序方式数据分析压力小，较之全基因组测序有明显优势。
然而，目前关于核酸测序的相关技术仍有待改进。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。
首先，需要说明的是，本发明是基于发明人的下列发现而完成的：
Complete Genomics公司(在本文中有时简称为“CG”)目前已有一套独立自主开发的第二代测序技术，适用于人全基因组测序。其文库构建流程主要包括：基因组DNA打断、第一次接头连接、双链环化并酶切、第二次接头连接、单链分离环化。其中两次接头连接在整个建库流程非常重要。接头是一段DNA序列，通过连接固定在DNA片段两端后，在测序时能被识别并作为测序的起始位点，供仪器读取其后的序列信息。为保证读取的序列信息易于分析，在一个DNA片段的两端(5’端和3’端)需要加上两种不同的接头；为了实现这种特定的方向性连接，同时避免接头间的相互连接，可以采用粘性末端接头连接的方式；但这种方式要求具有粘性末端的片段，难以避免片段间相互连接的问题。而Complete Genomics公司测序文库构建则采用了分多步骤分别添加两端接头的方式。为获得两端均连接上接头的片段，需要经过DNA片段一端连接接头、变性退火延伸、在DNA片段另一端连接接头、缺口补平、聚合酶链式反应在内五个步骤。其中多次的延伸反应所需试剂费用高昂，多个步骤间需要进行多次纯化回收，总体成本高且缺乏效率。并且，在目前的文库构建方案中，这样的接头连接过程要进行两次。
为此，本发明提出了一种适用于在DNA片段两端添加接头的手段。
在本发明的第一方面，本发明提出了一种分离的寡核苷酸。根据本发明的实施例，该寡核苷酸包括：第一链，所述第一链的5’末端核苷酸具有磷酸基团，并且所述第一链的3’末端核苷酸为双脱氧核苷酸；以及第二链，所述第二链的5’末端核苷酸不具有磷酸基团，并且所述第二链的3’末端核苷酸为双脱氧核苷酸，其中，所述第一链的长度大于所述第二链的长度，并且所述第一链和所述第二链之间形成双链结构。由于该寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该分离的寡核苷酸可以作为接头用于构建测序文库，并且在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。
根据本发明的第二方面，本发明提出了一种试剂盒。根据本发明的实施例，该试剂盒包括：第一接头和第二接头，所述第一接头和第二接头均为前面所述的分离的寡核苷酸，其中，所述第一接头与所述第二接头不同。如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该试剂盒可以作为接头用于构建测序文库，并且在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该试剂盒，在此不再赘述。
在本发明的第三方面，本发明提供了一种在双链DNA片段两端添加接头的方法。根据本发明的实施例，所述双链DNA片段具有两个平端末端，并且所述双链DNA片段的四个末端核苷酸均不具有磷酸基团，并且所述方法包括：将所述双链DNA片段与第一接头和第二接头进行连接，以便获得第一连接产物，其中，所述第一接头和第二接头不同，并且所述第一接头和第二接头均为前面所述的分离的寡核苷酸；使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链，其中，所述第一单链DNA能够与所述第一接头的第一链特异性匹配形成双链结构，所述第二单链DNA能够与所述第二接头的第一链特异性匹配形成双链结构；使所述第一单链DNA和所述第二单链DNA分别与所述双链DNA片段发生连接，以便获得第二连接产物；以及利用第一引物和第二引物，对所述第二连接产物进行扩增，以便获得扩增产物，其中，所述扩增产物为两端连接有接头的DNA片段，其中，所述第一引物包含与所述第一单链DNA和所述第二单链DNA之一相同的序列，所述第二引物包含与所述第一单链DNA和所述第二单链DNA的另一个相同的序列，并且与所述第一单链DNA和所述第二单链DNA的所述另一个相比在5’末端具有额外的生物素。如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同 时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该方法，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。
在本发明的第四方面，本发明提出了一种针对双链DNA片段构建测序文库的方法。根据本发明的实施例，所述双链DNA片段具有两个平端末端，并且所述双链DNA片段的四个末端核苷酸均不具有磷酸基团，并且该方法包括：根据前面所述的在双链DNA片段两端连接接头的方法，在所述双链DNA片段的两端连接接头，以便获得两端连接有接头的DNA片段；从所述两端连接有接头的DNA片段分离单链DNA片段；以及将所述单链DNA片段进行环化，以便获得单链DNA环，所述单链DNA环构成所述测序文库。如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该方法，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。进一步通过分离单链DNA，并且进行单链成环反应，可以有效地获得测序文库，例如用于CG测序平台的测序文库。
在本发明的第五方面，本发明提供了一种核酸测序方法。根据本发明的实施例，该方法包括：根据前面所述的针对双链DNA片段构建测序文库的方法，构建测序文库；以及对所述测序文库进行测序。如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该方法，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。进一步通过分离单链DNA，并且进行单链成环反应，可以有效地获得测序文库，例如用于CG测序平台的测序文库。从而可以进一步提高测序的效率，降低测序的成本。
在本发明的第六方面，本发明还提供了一种在双链DNA片段两端添加接头的装置。根 据本发明的实施例，所述双链DNA片段具有两个平端末端，并且所述双链DNA片段的四个末端核苷酸均不具有磷酸基团，并且该装置包括：第一连接单元，所述第一连接单元用于将所述DNA片段与第一接头和第二接头进行连接，以便获得第一连接产物，其中，所述第一接头和第二接头不同，并且所述第一接头和第二接头均为前面所述的分离的寡核苷酸；置换单元，所述置换单元用于使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链，其中，所述第一单链DNA能够与所述第一接头的第一链特异性匹配形成双链结构，所述第二单链DNA能够与所述第二接头的第一链特异性匹配形成双链结构；第二连接单元，所述第二连接单元用于使所述第一单链DNA和所述第二单链DNA分别与所述DNA片段发生连接，以便获得第二连接产物；以及扩增单元，所述扩增单元用于利用第一引物和第二引物，对所述第二连接产物进行扩增，以便获得扩增产物，其中，所述第一引物包含与所述第一单链DNA和所述第二单链DNA之一相同的序列，所述第二引物包含与所述第一单链DNA和所述第二单链DNA的另一个相同的序列，并且与所述第一单链DNA和所述第二单链DNA的所述另一个相比在5’末端具有额外的生物素。如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该装置，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。
在本发明的第七方面，本发明还提出了一种针对双链DNA片段构建测序文库的设备。根据本发明的实施例，所述双链DNA片段具有两个平端末端，并且所述双链DNA片段的四个末端核苷酸均不具有磷酸基团，并且所述设备包括：前面所述的在双链DNA片段两端添加接头的装置，用于在所述双链DNA片段的两端连接接头，以便获得两端连接有接头的DNA片段；单链DNA片段分离装置，所述单链DNA片段分离装置用于从所述两端连接有接头的DNA片段分离单链DNA片段；以及环化装置，所述环化装置用于将所述单链DNA片段进行环化，以便获得单链DNA环，所述单链DNA环构成所述测序文库。如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该设备，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头 的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。进一步通过分离单链DNA，并且进行单链成环反应，可以有效地获得测序文库，例如用于CG测序平台的测序文库。
在本发明的第八方面，本发明还提出了一种核酸测序***。根据本发明的实施例，该***包括：前面所述的针对双链DNA片段构建测序文库的设备；以及测序设备，所述测序设备用于对所述测序文库进行测序。如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该***，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。进一步通过分离单链DNA，并且进行单链成环反应，可以有效地获得测序文库，例如用于CG测序平台的测序文库。从而可以进一步提高测序的效率，降低测序的成本。
在本发明的第九方面，本发明还提出了一种用于针对基因组DNA构建测序文库的装置。根据本发明的实施例，该装置包括：手段，用于对所述基因组DNA进行片段化，以便获得片段化产物；手段，用于对所述片段化产物进行去磷酸化处理，以便获得经过去磷酸化处理的片段化产物；手段，用于对所述经过去磷酸化处理的片段化产物进行末端修复，以便获得双链DNA片段；手段，用于将所述双链DNA片段与第一接头和第二接头进行连接，以便获得第一连接产物，其中，所述第一接头和第二接头不同，并且所述第一接头和第二接头均为前面所述的分离的寡核苷酸；手段，用于使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链，其中，所述第一单链DNA能够与所述第一接头的第一链特异性匹配形成双链结构，所述第二单链DNA能够与所述第二接头的第一链特异性匹配形成双链结构；手段，用于使所述第一单链DNA和所述第二单链DNA分别与所述DNA片段发生连接，以便获得第二连接产物；手段，利用第一引物和第二引物，对所述第二连接产物进行扩增，以便获得扩增产物，其中，所述扩增产物为两端连接有接头的DNA片段，其中，所述第一引物包含与所述第一单链DNA和所述第二单链DNA之一相同的序列，所述第二引物包含与所述第一单链DNA和所述第二单链DNA的另一个相同的序列，并且与所述第一单链DNA和所述第二单链DNA的所述另一个相比在5’末端具有额外的生物素；手段，用于从所述两端连接有接头的DNA片段分离单链DNA片段；以及手段，用于将所述单链DNA片段进行环化，以便获得单链DNA环，所述单链DNA环构成所述测序文库。 如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该装置，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。进一步通过分离单链DNA，并且进行单链成环反应，可以有效地获得测序文库，例如用于CG测序平台的测序文库。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例的构建测序文库的流程示意图。1：打断后DNA片段。2：经过去磷酸化、末端修复后的片段(每个末端均为羟基)。3：接头A。4：接头B。5：单链C。6：单链D。7:单链C上的标签序列。8：最终产物环状单链。
图2显示了根据本发明一个实施例的电泳图。
图3显示了根据本发明一个实施例的电泳图。
图4显示了根据本发明一个实施例的在双链DNA片段两端添加接头的方法的流程示意图。
图5显示了根据本发明一个实施例的在双链DNA片段两端添加接头的装置的结构示意图。
图6显示了根据本发明一个实施例的针对双链DNA片段构建测序文库的设备的结构示意图。
图7显示了根据本发明一个实施例的核酸测序***的结构示意图。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，在本文中所采用的术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
分离的寡核苷酸、试剂盒
在本发明的第一方面，本发明提出了一种分离的寡核苷酸。根据本发明的实施例，该寡核苷酸包括：第一链，所述第一链的5’末端核苷酸具有磷酸基团，并且所述第一链的3’末端核苷酸为双脱氧核苷酸；以及第二链，所述第二链的5’末端核苷酸不具有磷酸基团，并且所述第二链的3’末端核苷酸为双脱氧核苷酸，其中，所述第一链的长度大于所述第二链的长度，并且所述第一链和所述第二链之间形成双链结构。由于该寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该分离的寡核苷酸可以作为接头用于构建测序文库，并且在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。
根据本发明的一个实施例，所述第二链上与所述第一链不匹配的核苷酸数目不超过3个。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
根据本发明的一个实施例，包括：第一突出端，所述第一突出端位于所述第一链的3’端；以及任选的第二突出端，所述第二突出端位于所述第二链的5’端。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
根据本发明的一个实施例，所述第一突出端的长度大于所述第二突出端的长度。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
根据本发明的一个实施例，所述第一突出端的长度为大约6～12nt。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
根据本发明的一个实施例，所述第二突出端的长度为0～4nt。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
根据本发明的一个实施例，所述第一链和第二链均为DNA。
根据本发明的一个实施例，所述第一链的长度为大约20～25nt。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
根据本发明的一个实施例，所述第二链的长度为大约10～15nt。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
根据本发明的一个实施例，所述第一链的序列为：5’GGCTCCGTCGAAGCCCGACGC3’(SEQ ID NO:1)，以及所述第二链的序列为：5’CTTCGACGGAGCC3’(SEQ ID NO:2)；或者所述第一链的序列为：5’ACGTCGGGGCCAAGCGGTCGTC3’(SEQ ID NO:3)，以及所述第二链的序列为：5’TTGGCCCCGGCTT3’(SEQ ID NO:4)。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
根据本发明的第二方面，本发明提出了一种试剂盒。根据本发明的实施例，该试剂盒 包括：第一接头和第二接头，所述第一接头和第二接头均为前面所述的分离的寡核苷酸，其中，所述第一接头与所述第二接头不同。
如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该试剂盒可以作为接头用于构建测序文库，并且在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该试剂盒，在此不再赘述。
根据本发明的一个实施例，进一步包括：第一单链DNA，所述第一单链DNA能够与所述第一接头的第一链匹配形成双链结构；以及第二单链DNA，所述第二单链DNA能够与所述第二接头的第一链匹配形成双链结构。由此，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。
根据本发明的一个实施例，所述第一单链DNA与所述第一接头的第一链形成的双链结构的长度大于所述第一接头中所述第一链和所述第二链之间形成双链结构的长度；以及所述第二单链DNA与所述第二接头的第一链形成的双链结构的长度大于所述第二接头中所述第一链和所述第二链之间形成双链结构的长度。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
根据本发明的一个实施例，进一步包括：第一引物，所述第一引物与所述第一单链DNA和所述第二单链DNA之一相同；以及第二引物，所述第二引物与所述第一单链DNA和所述第二单链DNA的另一个相比在5’末端具有额外的生物素。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。并且，采用能够特异性识别生物的试剂，可以有效地分离单链核酸分子，进而可以用于构建CG测序平台的测序文库。
根据本发明的一个实施例，所述第一接头的第一链的序列为：5’GGCTCCGTCGAAGCCCGACGC3’(SEQ ID NO:1)；所述第一接头的第二链的序列为：5’CTTCGACGGAGCC3’(SEQ ID NO:2)；所述第二接头的第一链的序列为：5’ACGTCGGGGCCAAGCGGTCGTC3’(SEQ ID NO:3)；所述第二接头的第二链的序列为：5’TTGGCCCCGGCTT3’(SEQ ID NO:4)；所述第一单链DNA的序列为：5’AGACAAGCTC(N)mGATCGGGCTTCGACGGAG3’，其中，(N)m表示长度为m个核苷酸的标签序列，其中，m为4～10中的任意整数，N＝A、T、G或者C；以及所述第二单链DNA的序列为5’TCCTAAGACCGCTTGGCCCCG3’(SEQ ID NO:5)。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。并且，采用能够特异性识别生物的试剂，可以有效地分离单链核酸分子，进而可以用于构建CG测序平台的测序文 库。
分离的寡核苷酸在核酸测序中的用途
在本发明的第三方面，本发明提供了一种在双链DNA片段两端添加接头的方法。根据本发明的实施例，所述双链DNA片段具有两个平端末端，并且所述双链DNA片段的四个末端核苷酸均不具有磷酸基团，并且参照图4，所述方法包括：
S100：将双链DNA片段与第一接头和第二接头进行连接
将所述双链DNA片段与第一接头和第二接头进行连接，以便获得第一连接产物，其中，所述第一接头和第二接头不同，并且所述第一接头和第二接头均为前面所述的分离的寡核苷酸。
根据本发明的一个实施例，将所述双链DNA片段与第一接头和第二接头进行连接的步骤是在一步反应中完成的。
根据本发明的一个实施例，所述DNA片段是通过下列步骤获得的：对DNA样本进行片段化，以便获得片段化产物；对所述片段化产物进行去磷酸化处理，以便获得去磷酸化处理的片段化产物；以及对所述经过去磷酸化处理的片段化产物进行末端修复处理，以便获得所述双链DNA片段。由此，可以有效地获得适于构建测序文库的DNA片段。
根据本发明的一个实施例，所述DNA样本为基因组DNA的至少一部分或者RNA的反转录产物。由此，可以有效地针对基因组DNA或者RNA构建测序文库。
S200：使用第一单链DNA置换第一接头的第二链，第二单链DNA置换第二接头的第二链
使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链，其中，所述第一单链DNA能够与所述第一接头的第一链特异性匹配形成双链结构，所述第二单链DNA能够与所述第二接头的第一链特异性匹配形成双链结构。
根据本发明的一个实施例，所述第一单链DNA与所述第一接头的第一链形成的双链结构的长度大于所述第一接头中所述第一链和所述第二链之间形成双链结构的长度；以及所述第二单链DNA与所述第二接头的第一链形成的双链结构的长度大于所述第二接头中所述第一链和所述第二链之间形成双链结构的长度。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
根据本发明的一个实施例，所述第一接头的第一链的序列为：5’GGCTCCGTCGAAGCCCGACGC3’(SEQ ID NO:1)；所述第一接头的第二链的序列为：5’CTTCGACGGAGCC3’(SEQ ID NO:2)；所述第二接头的第一链的序列为：5’ACGTCGGGGCCAAGCGGTCGTC3’(SEQ ID NO:3)；所述第二接头的第二链的序列为：5’TTGGCCCCGGCTT3’(SEQ ID NO:4)；以及所述第一单链DNA的序列为：5’AGACAAGCTC(N)mGATCGGGCTTCGACGGAG3’，其中，(N)m表示长度为m个核苷酸的标签序列，其中，m为4～10中的任意整数，N＝A、T、G或者C；所述第二单链DNA的序列为5’TCCTAAGACCGCTTGGCCCCG3’(SEQ ID NO:5)。由此，可以进一步提高在构 建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。并且，采用能够特异性识别生物素的试剂，可以有效地分离单链核酸分子，进而可以用于构建CG测序平台的测序文库。
根据本发明的一个实施例，通过热裂解-退火处理，使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链。根据本发明的一些具体示例，所述热裂解是在大约60摄氏度下进行的。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
S300：使第一单链DNA和第二单链DNA分别与双链DNA片段发生连接
使所述第一单链DNA和所述第二单链DNA分别与所述双链DNA片段发生连接，以便获得第二连接产物。
根据本发明的一个实施例，通过缺口补平反应，使所述第一单链DNA和所述第二单链DNA分别与所述双链DNA片段发生连接。
S400：利用第一引物和第二引物，对所述第二连接产物进行扩增
利用第一引物和第二引物，对所述第二连接产物进行扩增，以便获得扩增产物，其中，所述扩增产物为两端连接有接头的DNA片段，其中，所述第一引物包含与所述第一单链DNA和所述第二单链DNA之一相同的序列，所述第二引物包含与所述第一单链DNA和所述第二单链DNA的另一个相同的序列，并且与所述第一单链DNA和所述第二单链DNA的所述另一个相比在5’末端具有额外的生物素。
如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该方法，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。
在本发明的第四方面，本发明提出了一种针对双链DNA片段构建测序文库的方法。根据本发明的实施例，所述双链DNA片段具有两个平端末端，并且所述双链DNA片段的四个末端核苷酸均不具有磷酸基团，并且该方法包括：
首先，根据前面所述的在双链DNA片段两端连接接头的方法，在所述双链DNA片段的两端连接接头，以便获得两端连接有接头的DNA片段。
其次，从所述两端连接有接头的DNA片段分离单链DNA片段。根据本发明的一个实施例，从所述两端连接有接头的DNA片段分离单链DNA片段进一步包括：使所述两端连接有接头的DNA片段与磁珠接触，以便形成磁珠-DNA复合物，其中，所述磁珠上连接有链霉亲和素；以及将所述磁珠-DNA复合物与pH高于7的溶液接触，以便获得所述单链DNA 片段。由此，可以有效地分离单链DNA片段，从而提高构建测序文库的效率，降低构建测序文库的成本。根据本发明的一个实施例，所述pH高于7的溶液为氢氧化钠溶液。根据本发明的一个实施例，所述氢氧化钠溶液的浓度为大约0.5～2M。根据本发明的另一个实施例，所述氢氧化钠溶液的浓度为大约1M。根据本发明的一个实施例，在从所述两端连接有接头的DNA片段分离单链DNA片段之前，预先对所述两端连接有接头的DNA片段进行筛选。由此，可以针对预定的区域进行测序文库构建。其中，根据本发明的一个实施例，所述筛选是通过所述两端连接有接头的DNA片段与探针接触进行的，其中，所述探针对于预定序列是特异性的。根据本发明的一个具体示例，所述预定序列包括至少一个外显子。根据本发明的另一个实施例，所述探针是以微芯片阵列的形式提供的。由此，能够有效地将单链DNA片段进行环化。
然后，将所述单链DNA片段进行环化，以便获得单链DNA环，所述单链DNA环构成所述测序文库。根据本发明的一个实施例，通过采用单链核酸分子将所述单链DNA片段进行环化，其中，所述单链核酸分子上限定出第一区段和第二区段，并且所述第一区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的序列匹配，所述第二区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的之一的序列匹配。由此，可以进一步提高成环效率。根据本发明的一个实施例，所述第一区段和所述第二区段是毗邻连接的。根据本发明的一个实施例，所述第一区段的序列为5’TCGAGCTTGTCT3’(SEQ ID NO:6)；以及所述第二区段的序列为5’TCCTAAGACCGC3’(SEQ ID NO:7)。
如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该方法，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。进一步通过分离单链DNA，并且进行单链成环反应，可以有效地获得测序文库，例如用于CG测序平台的测序文库。
在本发明的第五方面，本发明提供了一种核酸测序方法。根据本发明的实施例，该方法包括：根据前面所述的针对双链DNA片段构建测序文库的方法，构建测序文库；以及对所述测序文库进行测序。根据本发明的一个实施例，采用CG测序平台，对所述测序文库进行测序。
如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片 段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该方法，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。进一步通过分离单链DNA，并且进行单链成环反应，可以有效地获得测序文库，例如用于CG测序平台的测序文库。从而可以进一步提高测序的效率，降低测序的成本。
在本发明的第六方面，本发明还提供了一种在双链DNA片段两端添加接头的装置。根据本发明的实施例，所述双链DNA片段具有两个平端末端，并且所述双链DNA片段的四个末端核苷酸均不具有磷酸基团，并且参照图5，该装置100包括：第一连接单元101、置换单元102、第二连接单元103和扩增单元104。具体地：
第一连接单元101用于将所述DNA片段与第一接头和第二接头进行连接，以便获得第一连接产物，其中，所述第一接头和第二接头不同，并且所述第一接头和第二接头均为前面所述的分离的寡核苷酸。根据本发明的一个实施例，所述第一连接单元被配置为在一步反应中将所述DNA片段与第一接头和第二接头进行连接。
置换单元102用于使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链，其中，所述第一单链DNA能够与所述第一接头的第一链特异性匹配形成双链结构，所述第二单链DNA能够与所述第二接头的第一链特异性匹配形成双链结构。根据本发明的一个实施例，所述第一单链DNA与所述第一接头的第一链形成的双链结构的长度大于所述第一接头中所述第一链和所述第二链之间形成双链结构的长度；以及所述第二单链DNA与所述第二接头的第一链形成的双链结构的长度大于所述第二接头中所述第一链和所述第二链之间形成双链结构的长度。由此，可以进一步提高在后续构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。根据本发明的一个实施例，所述第一接头的第一链的序列为：5’GGCTCCGTCGAAGCCCGACGC3’(SEQ ID NO:1)；所述第一接头的第二链的序列为：5’CTTCGACGGAGCC3’(SEQ ID NO:2)；所述第二接头的第一链的序列为：5’ACGTCGGGGCCAAGCGGTCGTC3’(SEQ ID NO:3)；所述第二接头的第二链的序列为：5’TTGGCCCCGGCTT3’(SEQ ID NO:4)；所述第一单链DNA的序列为：5’AGACAAGCTC(N)mGATCGGGCTTCGACGGAG3’，其中，(N)m表示长度为m个核苷酸的标签序列，其中，m为4～10中的任意整数，N＝A、T、G或者C；以及所述第二单链DNA的序列为5’TCCTAAGACCGCTTGGCCCCG3’(SEQ ID NO:5)。由此，可以进一步提高用于构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。并且，采用能够特异性识别生物素的试剂，可以有效地分离单链核酸分子，进而可以用于构建CG测序平台的测序文库。
根据本发明的一个实施例，所述置换单元102被配置为通过热裂解-退火处理，使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链。根据本发明的一个实施例，所述热裂解是在大约60摄氏度下进行的。由此，可以进一步提高在构建测序文库时的连接效率，进一步降低了构建测序文库的经济和时间成本。
第二连接单元103用于使所述第一单链DNA和所述第二单链DNA分别与所述DNA片段发生连接，以便获得第二连接产物。根据本发明的一个实施例，所述第二连接单元103被配置为通过缺口补平反应，使所述第一单链DNA和所述第二单链DNA分别与所述双链DNA片段发生连接。
扩增单元104用于利用第一引物和第二引物，对所述第二连接产物进行扩增，以便获得扩增产物，其中，所述第一引物包含与所述第一单链DNA和所述第二单链DNA之一相同的序列，所述第二引物包含与所述第一单链DNA和所述第二单链DNA的另一个相同的序列，并且与所述第一单链DNA和所述第二单链DNA的所述另一个相比在5’末端具有额外的生物素。
根据本发明的一个实施例，进一步包括双链DNA片段获取单元(图中未示出)，所述DNA片段获取单元包括：片段化组件，所述片断化组件用于对DNA样本进行片段化，以便获得片段化产物；去磷酸化组件，所述去磷酸化组件用于对所述片段化产物进行去磷酸化处理，以便获得经过去磷酸化处理的片段化产物；以及末端修复组件，所述末端修复组件用于对所述经过去磷酸化处理的片段化产物进行末端修复，以便获得所述双链DNA片段。由此，可以有效地获得适于构建测序文库的DNA片段。
根据本发明的一个实施例，所述双链DNA片段获取单元进一步包括：基因组DNA提取组件，所述基因组DNA提取组件用于从生物样本提取基因组DNA；和/或反转录组件，所述反转录组件用于对RNA样本进行反转录反应，以便获得反转录产物，其中，所述基因组DNA的至少一部分和/或RNA的反转录产物构成所述DNA样本。由此，可以有效地针对基因组DNA或者RNA构建测序文库。
如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该装置，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。
在本发明的第七方面，本发明还提出了一种针对双链DNA片段构建测序文库的设备。根据本发明的实施例，所述双链DNA片段具有两个平端末端，并且所述双链DNA片 段的四个末端核苷酸均不具有磷酸基团，并且参照图6，所述设备1000包括：前面所述的在双链DNA片段两端添加接头的装置100、单链DNA片段分离装置200和环化装置300。具体地：
在双链DNA片段两端添加接头的装置100用于在所述双链DNA片段的两端连接接头，以便获得两端连接有接头的DNA片段。单链DNA片段分离装置200用于从所述两端连接有接头的DNA片段分离单链DNA片段。环化装置300用于将所述单链DNA片段进行环化，以便获得单链DNA环，所述单链DNA环构成所述测序文库。
根据本发明的一个实施例，所述单链DNA片段分离装置200进一步包括：磁珠捕获单元，所述磁珠捕获单元用于使所述两端连接有接头的DNA片段与磁珠接触，以便形成磁珠-DNA复合物，其中，所述磁珠上连接有链霉亲和素；碱性裂解单元，所述碱性裂解单元中设置有pH高于7的溶液，用于将所述磁珠-DNA复合物与pH低于7的溶液接触，以便获得所述单链DNA片段。由此，可以有效地分离单链DNA片段，从而提高构建测序文库的效率，降低构建测序文库的成本。根据本发明的一个实施例，所述pH高于7的溶液为氢氧化钠溶液。根据本发明的一个实施例，所述氢氧化钠溶液的浓度为大约0.5～2M。根据本发明的另一个实施例，所述氢氧化钠溶液的浓度为大约1M。
根据本发明的一个实施例，进一步包括：筛选装置(图中未示出)，所述筛选装置用于在从所述两端连接有接头的DNA片段分离单链DNA片段之前，预先对所述两端连接有接头的DNA片段进行筛选。根据本发明的一个实施例，所述筛选装置中设置有探针，其中，所述探针对于预定序列是特异性的。根据本发明的一个实施例，所述预定序列包括至少一个外显子。根据本发明的一个实施例，所述探针是以微芯片阵列的形式提供的。
根据本发明的一个实施例，所述环化装置300中设置有单链核酸分子，其中，所述单链核酸分子上限定出第一区段和第二区段，并且所述第一区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的序列匹配，所述第二区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的之一的序列匹配。根据本发明的一个实施例，所述第一区段和所述第二区段是毗邻连接的。根据本发明的一个实施例，所述第一区段的序列为5’TCGAGCTTGTCT3’(SEQ ID NO:6)；以及所述第二区段的序列为5’TCCTAAGACCGC3’(SEQ ID NO:7)。由此，能够有效地通过采用单链核酸分子将单链DNA片段进行环化。
如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该设备，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链， 并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。进一步通过分离单链DNA，并且进行单链成环反应，可以有效地获得测序文库，例如用于CG测序平台的测序文库。
在本发明的第八方面，本发明还提出了一种核酸测序***。根据本发明的实施例，参照图7，该***10000包括：前面所述的针对双链DNA片段构建测序文库的设备1000和测序设备2000，所述测序设备2000用于对所述测序文库进行测序。根据本发明的一个实施例，所述测序设备2000为CG测序平台。
如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该***，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。进一步通过分离单链DNA，并且进行单链成环反应，可以有效地获得测序文库，例如用于CG测序平台的测序文库。从而可以进一步提高测序的效率，降低测序的成本。
在本发明的第九方面，本发明还提出了一种用于针对基因组DNA构建测序文库的装置。根据本发明的实施例，该装置包括：
手段，用于对所述基因组DNA进行片段化，以便获得片段化产物；
手段，用于对所述片段化产物进行去磷酸化处理，以便获得经过去磷酸化处理的片段化产物；
手段，用于对所述经过去磷酸化处理的片段化产物进行末端修复，以便获得双链DNA片段；
手段，用于将所述双链DNA片段与第一接头和第二接头进行连接，以便获得第一连接产物，其中，所述第一接头和第二接头不同，并且所述第一接头和第二接头均为前面所述的分离的寡核苷酸；
手段，用于使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链，其中，所述第一单链DNA能够与所述第一接头的第一链特异性匹配形成双链结构，所述第二单链DNA能够与所述第二接头的第一链特异性匹配形成双链结构；
手段，用于使所述第一单链DNA和所述第二单链DNA分别与所述DNA片段发生连接，以便获得第二连接产物；
手段，利用第一引物和第二引物，对所述第二连接产物进行扩增，以便获得扩增产 物，其中，所述扩增产物为两端连接有接头的DNA片段，其中，所述第一引物包含与所述第一单链DNA和所述第二单链DNA之一相同的序列，所述第二引物包含与所述第一单链DNA和所述第二单链DNA的另一个相同的序列，并且与所述第一单链DNA和所述第二单链DNA的所述另一个相比在5’末端具有额外的生物素；
手段，用于从所述两端连接有接头的DNA片段分离单链DNA片段；以及
手段，用于将所述单链DNA片段进行环化，以便获得单链DNA环，所述单链DNA环构成所述测序文库。
如前所述，由于根据本发明实施例的寡核苷酸中的第一链和第二链的3’末端均为双脱氧核苷酸，并且在第二链的5’末端核苷酸不具有磷酸基团，这些末端将无法与其他核酸片段相互连接，从而可以防止寡核苷酸之间的互相连接。由此，该寡核苷酸作为接头在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。前面关于根据本发明实施例的分离的寡核苷酸的特征和优点的描述同样适用该装置，在此不再赘述。另外，在构建测序文库时，可以利用该第一单链DNA和第二单链DNA分别置换两个接头的第二链，并且与第一链形成更稳定的双链结构，进一步，通过采用第一单链DNA和第二单链DNA作为引物，进行PCR扩增，可以形成在两端具有稳定接头的DNA片段。进一步通过分离单链DNA，并且进行单链成环反应，可以有效地获得测序文库，例如用于CG测序平台的测序文库。
根据本发明的一个实施例，将所述双链DNA片段与第一接头和第二接头进行连接是在一步反应中完成的。
根据本发明的一个实施例，进一步包括：
手段，用于从生物样本提取基因组DNA；和/或
手段，用于从对RNA样本进行反转录反应，
其中，
所述基因组DNA的至少一部分和/或RNA的反转录产物构成所述DNA样本。
根据本发明的一个实施例，用于从所述两端连接有接头的DNA片段分离单链DNA片段的手段，被配置为适于通过下列步骤分离所述单链DNA片段：使所述两端连接有接头的DNA片段与磁珠接触，以便形成磁珠-DNA复合物，其中，所述磁珠上连接有链霉亲和素；以及将所述磁珠-DNA复合物与pH低于7的溶液接触，以便获得所述单链DNA片段。由此，可以有效地分离单链DNA片段，从而提高构建测序文库的效率，降低构建测序文库的成本。
根据本发明的一个实施例，进一步包括：手段，用于在从所述两端连接有接头的DNA片段分离单链DNA片段之前，预先对所述两端连接有接头的DNA片段进行筛选。根据本发明的一个实施例，所述筛选是通过所述两端连接有接头的DNA片段与探针接触进行的，其中，所述探针对于预定序列是特异性的。根据本发明的一个实施例，所述预定序列包括至少一个外显子。根据本发明的一个实施例，所述探针是以微芯片阵列的形 式提供的。
根据本发明的一个实施例，用于将所述单链DNA片段进行环化的手段被配置为采用单链核酸分子将所述单链DNA片段进行环化，其中，所述单链核酸分子上限定出第一区段和第二区段，并且所述第一区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的序列匹配，所述第二区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的之一的序列匹配。根据本发明的一个实施例，所述第一区段和所述第二区段是毗邻连接的。根据本发明的一个实施例，所述第一区段的序列为5’TCGAGCTTGTCT3’(SEQ ID NO:6)；以及所述第二区段的序列为5’TCCTAAGACCGC3’(SEQ ID NO:7)。由此，能够有效地通过采用单链核酸分子将单链DNA片段进行环化。
综上所述，根据本发明的实施例的技术方案可以具有下列优点的至少之一：
根据本发明的实施例的技术方案解决了Complete Genomics公司测序平台文库构建中存在的接头连接步骤过多，整体文库构建时间过长，成本过高的问题。
根据本发明的实施例的技术方案，在接头连接时抛弃了传统的多步骤分别添加两端接头的方式，转而采用了在同一次反应中加入两端接头的新型方法。
根据本发明的实施例的技术方案，同时加入两种接头的连接方式同样需要解决接头自连、片段互连等问题；而本发明设计的连接接头有着独特的序列构造，通过同样新颖的接头连接方法；同时解决了片段互连、接头自连、片段连接效率低、标签序列引入位置等问题；并成功地将整个接头连接过程缩短为三个步骤；大大缩短了接头连接所需时间，明显地降低了成本。
根据本发明的实施例的技术方案将独创的接头连接方法结合于核酸探针捕获技术，通过进一步设计调整Complete Genomics公司传统文库构建方案；成功将接头连接过程从两次减少为一次。显著缩短文库构建成本和时间；且成功创立了基于Complete Genomics公司测序平台的单接头的全外显子组测序产品。
由此，根据本发明的实施例，参考图1，在本发明的实施例中可以按照下列步骤构建测序文库：
1.基因组核酸链被打断成片段；
2.对目标片段进行去磷酸化；去磷酸化用于封闭目的片段5’端，防止片段自连。
3.补平片段两端，使两端均为平末端(图1中编号2所示)。
4.在目标片段的两端加上接头A(图1中编号3所示)。和接头B(图1中编号4所示)接头A和B均为为多聚核苷酸双链，由一条长链(第一链)和一条短链(第二链)组成。长链由于5’端具有磷酸基团，能与目标核酸片段进行连接，短链通过碱基互补配对结合在长链上，由于短链末端为封闭序列，不会目标核酸片段连接；
5.加入核酸单链C(图1中编号5所示)和核酸单链D(图1编号6所示)。单链C具有标签序列(图1编号7所示)，其余部分片段与接头A长链互补配对；单链D则能与接头B长链互补配对。通过退火过程，导致结合不牢固的接头短链掉落、单链C、D与接头长链 的互补配对。再通过延伸、连接反应，实现了单链C和单链D与目的片段的连接。
6.以步骤4产物为模板，单链C、D作为引物进行聚合酶链式反应，扩增富集带有标签序列的产物；
7.取步骤5产物进行寡核苷酸探针杂交捕获；具体步骤包括探针杂交、杂交产物洗脱、杂交产物富集步骤；并在杂交产物富集步骤中，在目的核酸双链的一条链上引入生物素修饰。
8.对杂交捕获后的核酸双链进行长度筛选(可选)；
9.通过核酸双链中一条链上的生物素标记，将筛选后的核酸双链分离为两条核酸单链；
10.将该核酸单链环化，并去掉剩余的未环化单链。
需要说明的是步骤7片段长度筛选可以选在单链分离前的其他步骤后进行，具体情况视乎测序具体需求和各步骤后产物片段大小的实际变化而定。如果通过质量控制确认各步骤产物的大小一直符合要求，可以去掉步骤8。
采用步骤7，可以实现全外显子测序而引入的步骤。
根据本发明的实施例，通过步骤2、3的处理；目的核酸片段经过去磷酸化的末端封闭处理后，成为了两端封闭的平末端片段，完全避免了片段间相互作用的发生，使连接前片段的利用率得到了极高的保证。
根据本发明的实施例，本发明的特殊接头设计在接头A、B的长链的5’端引入了磷酸基团；且在接头长链3’端和短链的双末端都引入了封闭序列。由于封闭序列的存在，被封闭的末端不但无法与目标核酸片段进行连接，更无法与同时加入的其他接头进行连接；确保了在步骤4进行接头连接时，接头长链的5’末端能够准确地连接至目的片段3’末端。这种设计非常有效地防止了接头互连的发生，使不同接头的连接同时进行成为了可能，且保证了连接反应的效率。
根据本发明的实施例，在步骤5里，巧妙地运用了接头结构中长短链的特性；由于短链互补配对碱基较少、结合不稳定，在相对较温和的温度就会与长链分离；再通过缓慢退火反应，简单地使具有较长碱基互补配对序列，结合能力更占优势的单链C、D与接头长链结合；延伸连接后形成了完整地双链接头。通过在单链C上引入标签序列，还能同时为接头提供识别标签。这种独特的设计有反应条件温和的特点；借此，通过对反应体系、反应时间、反应顺序的适当调整；更使片段置换、连接、延伸三个反应在同一个反应步骤5中进行，且操作简单，反应迅速，极大地降低了处理时间。
根据本发明的实施例，成功地将接头连接从原来的五步缩短为接头连接、缺口补平、聚合酶链式反应三个步骤，操作量大大减少，省去了多种试剂的使用，节约了大量的时间和成本。
根据本发明的实施例，不但从接头连接的具体方法上进行全面的更换，更颠覆性地改变了了CG公司传统的文库构建方案，提出了新颖的单链核酸文库结构(图1标记8)；将传统的两次的接头连接过程精简为仅一次接头连接过程；减少了聚合酶链式反应的引入，提升了测序的质量。更主要的是，步骤的精简将文库构建的时间缩短了3-4天之多。成本大 量降低；较于传统方案有巨大优势。
根据本发明的实施例，本发明通过对Complete Genomics公司传统的测序文库构建方案进行修改和补充，结合之前阐述的新颖接头连接方法，成功研发出了适合于人全外显子组测序的高效的文库构建方案。开发出了基于Complete Genomics测序平台的新颖的人全外显子组测序产品，实现了基于Complete Genomics平台的全外显子组测序从无到有的突破。
本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
一般方法
参考图1，在本发明的实施例中按照下列步骤构建测序文库：
1.基因组核酸链被打断成片段；
2.对目标片段进行去磷酸化；去磷酸化用于封闭目的片段5’端，防止片段自连。
3补平片段两端，使两端均为平末端(图1中编号2所示)。
4.在目标片段的两端加上接头A(图1中编号3所示)。和接头B(图1中编号4所示)接头A和B均为为多聚核苷酸双链，由一条长链(第一链)和一条短链(第二链)组成。长链由于5’端具有磷酸基团，能与目标核酸片段进行连接，短链通过碱基互补配对结合在长链上，由于短链末端为封闭序列，不会目标核酸片段连接；
5.加入核酸单链C(图1中编号5所示)和核酸单链D(图1编号6所示)。单链C具有标签序列(图1编号7所示)，其余部分片段与接头A长链互补配对；单链D则能与接头B长链互补配对。通过退火过程，导致结合不牢固的接头短链掉落、单链C、D与接头长链的互补配对。再通过延伸、连接反应，实现了单链C和单链D与目的片段的连接。
6.以步骤4产物为模板，单链C、D作为引物进行聚合酶链式反应，扩增富集带有标签序列的产物；
7.取步骤5产物进行寡核苷酸探针杂交捕获；具体步骤包括探针杂交、杂交产物洗脱、杂交产物富集步骤；并在杂交产物富集步骤中，在目的核酸双链的一条链上引入生物素修饰。
8.对杂交捕获后的核酸双链进行长度筛选(可选)；
9.通过核酸双链中一条链上的生物素标记，将筛选后的核酸双链分离为两条核酸单链；
10.将该核酸单链环化，并去掉剩余的未环化单链。
需要说明的是步骤7片段长度筛选可以选在单链分离前的其他步骤后进行，具体情况视乎测序具体需求和各步骤后产物片段大小的实际变化而定。如果通过质量控制确认各步骤产物的大小一直符合要求，可以去掉步骤8。
采用步骤7，可以实现全外显子测序而引入的步骤。
实施例1：
1.基因组DNA打断：基因组DNA打断有多种方式，无论是物理超声法还是酶反应法，市场上有非常成熟的方案。本实施例采用的是物理超声打断法。
取96孔PCR板一块，加入一根聚四氟乙烯线，加入基因组DNA 1μg，加入TE缓冲溶液或无酶水补齐80μl。将板封膜后至于E220超声打断仪上超声打断。打断条件设置：
工作周期(Duty Cycle) 20％
强度(Intensity) 5
循环/脉冲(Cycles per Burst) 200
打断时间 60s，5次
2.打断片段选择：可以采用磁珠纯化法或凝胶回收法。本实施例采用磁珠纯化法。
取打断后的DNA，加入80μl Ampure XP磁珠，混匀后放置7-15min；置入磁力架后收集上清，在上清中加入40μl Ampure XP磁珠，混匀后放置7-15min；置入磁力架吸去上清，用75％乙醇洗磁珠两次；晾干后加入50μl TE缓冲溶液或无酶水，混匀后放置7-15min溶解回收产物。
3.去磷酸化反应：取上步骤回收产物，按下表配制体系：
10x NEB缓冲液2 6μl
虾碱性磷酸酶(1U/μl) 6μl
总体积 12μl
将12μl反应液加入前一步的回收产物中，混匀，按下表条件进行反应。反应产物直接用于进行下一步骤。(其中“以0.1℃/s降温至4℃”步骤并非必须，反应时间也不需过于精确的控制。后同。)
Figure PCTCN2014086418-appb-000001
4.片段末端修复：按下表配制体系：
无酶水 12.2μl
10x NEB缓冲液2 1.8μl
0.1M三磷酸腺苷 0.8μl
25mM脱氧核糖核苷三磷酸 0.8μl
牛血清白蛋白 0.4μl
T4脱氧核糖核酸聚合酶(3U/μl) 2μl
总体积 18μl
将体系混匀后加入上一步骤产物中，混匀后置于12℃孵育20min。使用80μl PEG 32磁珠进行纯化，40μl TE缓冲溶液溶解回收产物。(反应产物的纯化有多种方式，有磁珠法、 柱纯化法、凝胶回收法等等。均可用于替换。本实施例如不做特殊说明，均采用磁珠法纯化。)
5.接头A、B连接：本方案中使用的接头序列如下(序列从左到右为5’端至3’端，“//”中为末端修饰基团，“phos”示磷酸化，“dd”示双脱氧，“bio”示生物素)。
接头A：
长链/Phos/GGCTCCGTCGAAGCCCGACG/ddC/
短链GCTTCGACGGAGC/ddC/
接头B：
长链：/phos/ACGTCGGGGCCAAGCGGTCGT/ddC/
短链：TTGGCCCCGGCT/-ddT/。
按下表配制体系：
无酶水 11.1μl
5μM接头A 1.85μl
5μM接头B 1.85μl
总体积 14.8μl
将以上体系混匀后加入到纯化后的上一步产物中。混匀后，配制以下体系：
Figure PCTCN2014086418-appb-000002
将以上体系与之前的体系混匀，置于20℃孵育1h。使用100μl Ampure XP纯化，40μl TE缓冲溶液溶解回收产物。
此步骤完成了目的核酸片段与接头A、接头B的连接。连接前后产物电泳结果如图2所示。由图2可知，步骤5接头连接后片段大小增大明显，说明本方案接头连接是非常成功的。而特别是通过步骤7聚合酶链式反应后，条带更为集中，筛选富集效果明显。
6.单链C、D连接：
单链C：/phos/AGACAAGCTCxxxxxxxxxxGATCGGGCTTCGACGGAG(中间“x”处为可变的标签序列区域)
单链D：/bio/TCCTAAGACCGCTTGGCCCCGA。
按下表配制体系：
无酶水 19.88μl
10x Taq缓冲液 8μl
0.1M三磷酸腺苷 0.8μl
25mM脱氧核糖核苷三磷酸 0.32μl
20μM单链D 0.5μl
总体积 30μul
先在上步骤回收产物中加入1μl的10μM的单链C，混匀后加入上述体系混匀，65℃反应5min，以0.1℃/s降温至37℃。
保持以上反应体系为37℃，配制以下反应体系：
无酶水 0.4μl
10x Taq缓冲液 0.4μl
T4DNA连接酶(600U/μl) 4.8μl
Taq聚合酶(5U/μl) 2.4μl
总体积 8μl
将以上8μl反应混合物加入之前37℃的反应体系中。混匀后37℃反应20min。
使用96μl Ampure XP磁珠进行纯化，25μl TE缓冲溶液溶解回收产物。
7.聚合酶链式反应：按下表配制体系：
Figure PCTCN2014086418-appb-000003
取30-40ng上步骤回收产物，用无酶水或TE补足25μl，加入到以上体系中，混匀后按下表条件进行反应：
Figure PCTCN2014086418-appb-000004
反应完成后使用120μl Ampure XP磁珠进行纯化，25μl无酶水溶解回收产物。
8.杂交捕获：取500ng-1μg上步骤反应产物，浓缩蒸干后加入以下体系1中溶解：
Figure PCTCN2014086418-appb-000005
Figure PCTCN2014086418-appb-000006
将混合后的反应体系1置于95℃反应5min，持续放置于65℃。
配制体系2：
Figure PCTCN2014086418-appb-000007
将体系2加入体系1中，持续放置于65℃。
配制体系3：
Figure PCTCN2014086418-appb-000008
将体系3加入体系1、2中，65℃反应20-24h。
反应完成后使用链霉亲和素包裹的磁珠进行结合，结合完成后将磁珠溶于50μl无酶水中。
配制以下反应体系：
Figure PCTCN2014086418-appb-000009
将溶解的磁珠加入反应体系中混匀，按下表进行反应：
Figure PCTCN2014086418-appb-000010
反应完成后使用240μl Ampure XP磁珠进行纯化。
9.单链分离：使用链霉亲和素包裹的磁珠结合步骤8中获得的带生物素目的片段。使用78μl 0.1M氢氧化钠将未结合磁珠的单链分离下来，加入酸性缓冲液中和获得的分离产物，中和后产物总体积112μl。
10.单链环化：配制以下反应体系1：其中核酸单链E具有相应互补序列用于连接单链两端。
单链E序列如下：TCGAGCTTGTCTTCCTAAGACCGC(SEQ ID NO:8)
无酶水 43μl
核酸单链E 20μl
总体积 63μl
将反应体系1加入步骤9单链产物中。混匀。
配制反应体系2：
Figure PCTCN2014086418-appb-000011
将反应体系2加入反应体系1中，混匀，37℃孵育1.5h。
11.外切酶1、外切酶3处理：
配置以下反应缓冲液：
Figure PCTCN2014086418-appb-000012
将23.7μl上述配置的反应缓冲液加入步骤10的350μl反应产物中。混匀后置于37℃孵育30min。
加入15.4μl 500mM乙二胺四乙酸，混匀。
使用500μl PEG32磁珠纯化回收，40-80μl无酶水/TE缓冲液回溶。
本实施例最终产物浓度和总量情况如下：
  浓度(ng/μl) 总量(ng)
产物1 2.72 108.8
产物2 2.12 84.8
产物3 4.26 170.4
产物4 1.46 58.4
产物5 3.06 122.4
产物6 1.73 69.2
电泳结果见图3。图3为步骤11后产物使用6％聚丙烯酰胺变性凝胶电泳的电泳结果图。如图3所示，产物1、3、5为步骤8杂交后进行了凝胶电泳片段筛选的，而产物2、4、6则是没有经过片段大小筛选步骤的。由图3可知，经过凝胶电泳片段筛选的产物大小更为集中，但不进行片段大小筛选的片段也能进行正常测序。证明本方案是完全成功的。
工业实用性
本发明的分离的寡核苷酸能够有效地作为接头用于构建测序文库，并且在构建测序文库时可以实现同时在核酸片段的两端连接不同的接头，同时避免了接头之间的互相连接，提高了连接效率，降低了构建测序文库的经济和时间成本。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示意性实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。另外，需要说明的是，本领域技术人员能够理解，在本发明所提出的方案中所包含的步骤顺序，本领域技术人员可以进行调整，这也将包括在本发明的范围内。
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。

Claims (71)

  1. 一种分离的寡核苷酸，其特征在于，包括：
    第一链，所述第一链的5’末端核苷酸具有磷酸基团，并且所述第一链的3’末端核苷酸为双脱氧核苷酸；以及
    第二链，所述第二链的5’末端核苷酸不具有磷酸基团，并且所述第二链的3’末端核苷酸为双脱氧核苷酸，
    其中，
    所述第一链的长度大于所述第二链的长度，并且所述第一链和所述第二链之间形成双链结构。
  2. 根据权利要求1所述的分离的寡核苷酸，其特征在于，所述第二链上与所述第一链不匹配的核苷酸数目不超过3个。
  3. 根据权利要求1所述的分离的寡核苷酸，其特征在于，包括：
    第一突出端，所述第一突出端位于所述第一链的3’端；以及
    任选的第二突出端，所述第二突出端位于所述第二链的5’端。
  4. 根据权利要求3所述的分离的寡核苷酸，其特征在于，所述第一突出端的长度大于所述第二突出端的长度。
  5. 根据权利要求4所述的分离的寡核苷酸，其特征在于，所述第一突出端的长度为大约6～12nt。
  6. 根据权利要求4所述的分离的寡核苷酸，其特征在于，所述第二突出端的长度为0～4nt。
  7. 根据权利要求1所述的分离的寡核苷酸，其特征在于，所述第一链和第二链均为DNA。
  8. 根据权利要求1所述的分离的寡核苷酸，其特征在于，所述第一链的长度为大约20～25nt。
  9. 根据权利要求1所述的分离的寡核苷酸，其特征在于，所述第二链的长度为大约10～15nt。
  10. 根据权利要求1所述的分离的寡核苷酸，其特征在于，
    所述第一链的序列为：5’GGCTCCGTCGAAGCCCGACGC3’，以及
    所述第二链的序列为：5’CTTCGACGGAGCC3’；
    或者
    所述第一链的序列为：5’ACGTCGGGGCCAAGCGGTCGTC3’，以及
    所述第二链的序列为：5’TTGGCCCCGGCTT3’。
  11. 一种试剂盒，其特征在于，包括：
    第一接头和第二接头，所述第一接头和第二接头均为权利要求1～10任一项所述的分离的寡核苷酸，
    其中，所述第一接头与所述第二接头不同。
  12. 根据权利要求11所述的试剂盒，其特征在于，进一步包括：
    第一单链DNA，所述第一单链DNA能够与所述第一接头的第一链匹配形成双链结构；以及
    第二单链DNA，所述第二单链DNA能够与所述第二接头的第一链匹配形成双链结构。
  13. 根据权利要求12所述的试剂盒，其特征在于，所述第一单链DNA与所述第一接头的第一链形成的双链结构的长度大于所述第一接头中所述第一链和所述第二链之间形成双链结构的长度；以及
    所述第二单链DNA与所述第二接头的第一链形成的双链结构的长度大于所述第二接头中所述第一链和所述第二链之间形成双链结构的长度。
  14. 根据权利要求12所述的试剂盒，其特征在于，进一步包括：
    第一引物，所述第一引物与所述第一单链DNA和所述第二单链DNA之一相同；以及
    第二引物，所述第二引物与所述第一单链DNA和所述第二单链DNA的另一个相比在5’末端具有额外的生物素。
  15. 根据权利要求14所述的试剂盒，其特征在于，
    所述第一接头的第一链的序列为：5’GGCTCCGTCGAAGCCCGACGC3’；
    所述第一接头的第二链的序列为：5’CTTCGACGGAGCC3’；
    所述第二接头的第一链的序列为：5’ACGTCGGGGCCAAGCGGTCGTC3’；
    所述第二接头的第二链的序列为：5’TTGGCCCCGGCTT3’；
    所述第一单链DNA的序列为：5’AGACAAGCTC(N)mGATCGGGCTTCGACGGAG3’，其中，(N)m表示长度为m个核苷酸的标签序列，其中，m为4～10中的任意整数，N＝A、T、G或者C；以及
    所述第二单链DNA的序列为5’TCCTAAGACCGCTTGGCCCCG3’。
  16. 一种在双链DNA片段两端添加接头的方法，所述双链DNA片段具有两个平端末端，并且所述双链DNA片段的四个末端核苷酸均不具有磷酸基团，其特征在于，所述方法包括：
    将所述双链DNA片段与第一接头和第二接头进行连接，以便获得第一连接产物，其中，所述第一接头和第二接头不同，并且所述第一接头和第二接头均为权利要求1～10任一项所述的分离的寡核苷酸；
    使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链，其中，所述第一单链DNA能够与所述第一接头的第一链特异性匹配形成双链结构，所述第二单链DNA能够与所述第二接头的第一链特异性匹配形成双链结构；
    使所述第一单链DNA和所述第二单链DNA分别与所述双链DNA片段发生连接，以便获得第二连接产物；以及
    利用第一引物和第二引物，对所述第二连接产物进行扩增，以便获得扩增产物，其中，所述扩增产物为两端连接有接头的DNA片段，其中，所述第一引物包含与所述第一单链 DNA和所述第二单链DNA之一相同的序列，所述第二引物包含与所述第一单链DNA和所述第二单链DNA的另一个相同的序列，并且与所述第一单链DNA和所述第二单链DNA的所述另一个相比在5’末端具有额外的生物素。
  17. 根据权利要求16所述的方法，其特征在于，将所述双链DNA片段与第一接头和第二接头进行连接的步骤是在一步反应中完成的。
  18. 根据权利要求16所述的方法，其特征在于，所述双链DNA片段是通过下列步骤获得的：
    对DNA样本进行片段化，以便获得片段化产物；
    对所述片段化产物进行去磷酸化处理，以便获得去磷酸化处理的片段化产物；以及
    对所述经过去磷酸化处理的片段化产物进行末端修复处理，以便获得所述双链DNA片段。
  19. 根据权利要求16所述的方法，其特征在于，
    所述第一单链DNA与所述第一接头的第一链形成的双链结构的长度大于所述第一接头中所述第一链和所述第二链之间形成双链结构的长度；以及
    所述第二单链DNA与所述第二接头的第一链形成的双链结构的长度大于所述第二接头中所述第一链和所述第二链之间形成双链结构的长度。
  20. 根据权利要求16所述的方法，其特征在于，
    所述第一接头的第一链的序列为：5’GGCTCCGTCGAAGCCCGACGC3’；
    所述第一接头的第二链的序列为：5’CTTCGACGGAGCC3’；
    所述第二接头的第一链的序列为：5’ACGTCGGGGCCAAGCGGTCGTC3’；
    所述第二接头的第二链的序列为：5’TTGGCCCCGGCTT3’；
    所述第一单链DNA的序列为：5’AGACAAGCTC(N)mGATCGGGCTTCGACGGAG3’，其中，(N)m表示长度为m个核苷酸的标签序列，其中，m为4～10中的任意整数，N＝A、T、G或者C；以及
    所述第二单链DNA的序列为5’TCCTAAGACCGCTTGGCCCCG3’。
  21. 根据权利要求16所述的方法，其特征在于，通过热裂解-退火处理，使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链。
  22. 根据权利要求21所述的方法，其特征在于，所述热裂解是在大约60摄氏度下进行的。
  23. 根据权利要求16所述的方法，其特征在于，通过缺口补平反应，使所述第一单链DNA和所述第二单链DNA分别与所述双链DNA片段发生连接。
  24. 根据权利要求18所述的方法，其特征在于，所述DNA样本为基因组DNA的至少一部分或者RNA的反转录产物。
  25. 一种针对双链DNA片段构建测序文库的方法，所述双链DNA片段具有两个平端末端，并且所述双链DNA片段的四个末端核苷酸均不具有磷酸基团，其特征在于，所述方法包括：
    根据权利要求16～24任一项所述的方法，在所述双链DNA片段的两端连接接头，以便获得两端连接有接头的DNA片段；
    从所述两端连接有接头的DNA片段分离单链DNA片段；以及
    将所述单链DNA片段进行环化，以便获得单链DNA环，所述单链DNA环构成所述测序文库。
  26. 根据权利要求25所述的方法，其特征在于，从所述两端连接有接头的DNA片段分离单链DNA片段进一步包括：
    使所述两端连接有接头的DNA片段与磁珠接触，以便形成磁珠-DNA复合物，其中，所述磁珠上连接有链霉亲和素；以及
    将所述磁珠-DNA复合物与pH高于7的溶液接触，以便获得所述单链DNA片段。
  27. 根据权利要求26所述的方法，其特征在于，所述pH高于7的溶液为氢氧化钠溶液。
  28. 根据权利要求27所述的方法，其特征在于，所述氢氧化钠溶液的浓度为大约0.5～2M。
  29. 根据权利要求28所述的方法，其特征在于，所述氢氧化钠溶液的浓度为大约1M。
  30. 根据权利要求25所述的方法，其特征在于，在从所述两端连接有接头的DNA片段分离单链DNA片段之前，预先对所述两端连接有接头的DNA片段进行筛选。
  31. 根据权利要求30所述的方法，其特征在于，所述筛选是通过所述两端连接有接头的DNA片段与探针接触进行的，其中，所述探针对于预定序列是特异性的。
  32. 根据权利要求31所述的方法，其特征在于，所述预定序列包括至少一个外显子。
  33. 根据权利要求31所述的方法，其特征在于，所述探针是以微芯片阵列的形式提供的。
  34. 根据权利要求25所述的方法，其特征在于，通过采用单链核酸分子将所述单链DNA片段进行环化，
    其中，
    所述单链核酸分子上限定出第一区段和第二区段，并且所述第一区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的序列匹配，所述第二区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的之一的序列匹配。
  35. 根据权利要求34所述的方法，其特征在于，所述第一区段和所述第二区段是毗邻连接的。
  36. 根据权利要求35所述的方法，其特征在于，
    所述第一区段的序列为5’TCGAGCTTGTCT3’；以及
    所述第二区段的序列为5’TCCTAAGACCGC3’。
  37. 一种核酸测序方法，其特征在于，包括：
    根据权利要求25～36任一项所述的方法，构建测序文库；以及
    对所述测序文库进行测序。
  38. 根据权利要求37所述的测序方法，其特征在于，采用CG测序平台，对所述测序文库进行测序。
  39. 一种在双链DNA片段两端添加接头的装置，所述双链DNA片段具有两个平端末端，并且所述双链DNA片段的四个末端核苷酸均不具有磷酸基团，其特征在于，所述装置包括：
    第一连接单元，所述第一连接单元用于将所述DNA片段与第一接头和第二接头进行连接，以便获得第一连接产物，其中，所述第一接头和第二接头不同，并且所述第一接头和第二接头均为权利要求1～10任一项所述的分离的寡核苷酸；
    置换单元，所述置换单元用于使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链，其中，所述第一单链DNA能够与所述第一接头的第一链特异性匹配形成双链结构，所述第二单链DNA能够与所述第二接头的第一链特异性匹配形成双链结构；
    第二连接单元，所述第二连接单元用于使所述第一单链DNA和所述第二单链DNA分别与所述DNA片段发生连接，以便获得第二连接产物；以及
    扩增单元，所述扩增单元用于利用第一引物和第二引物，对所述第二连接产物进行扩增，以便获得扩增产物，其中，所述第一引物包含与所述第一单链DNA和所述第二单链DNA之一相同的序列，所述第二引物包含与所述第一单链DNA和所述第二单链DNA的另一个相同的序列，并且与所述第一单链DNA和所述第二单链DNA的所述另一个相比在5’末端具有额外的生物素。
  40. 根据权利要求39所述的装置，其特征在于，所述第一连接单元被配置为在一步反应中将所述DNA片段与第一接头和第二接头进行连接。
  41. 根据权利要求39所述的装置，其特征在于，进一步包括双链DNA片段获取单元，所述DNA片段获取单元包括：
    片段化组件，所述片断化组件用于对DNA样本进行片段化，以便获得片段化产物；
    去磷酸化组件，所述去磷酸化组件用于对所述片段化产物进行去磷酸化处理，以便获得经过去磷酸化处理的片段化产物；以及
    末端修复组件，所述末端修复组件用于对所述经过去磷酸化处理的片段化产物进行末端修复，以便获得所述双链DNA片段。
  42. 根据权利要求39所述的装置，其特征在于，
    所述第一单链DNA与所述第一接头的第一链形成的双链结构的长度大于所述第一接头中所述第一链和所述第二链之间形成双链结构的长度；以及
    所述第二单链DNA与所述第二接头的第一链形成的双链结构的长度大于所述第二接头中所述第一链和所述第二链之间形成双链结构的长度。
  43. 根据权利要求39所述的装置，其特征在于，
    所述第一接头的第一链的序列为：5’GGCTCCGTCGAAGCCCGACGC3’；
    所述第一接头的第二链的序列为：5’CTTCGACGGAGCC3’；
    所述第二接头的第一链的序列为：5’ACGTCGGGGCCAAGCGGTCGTC3’；
    所述第二接头的第二链的序列为：5’TTGGCCCCGGCTT3’；
    所述第一单链DNA的序列为：5’AGACAAGCTC(N)mGATCGGGCTTCGACGGAG3’，其中，(N)m表示长度为m个核苷酸的标签序列，其中，m为4～10中的任意整数，N＝A、T、G或者C；以及
    所述第二单链DNA的序列为5’TCCTAAGACCGCTTGGCCCCG3’。
  44. 根据权利要求39所述的装置，其特征在于，所述置换单元被配置为通过热裂解-退火处理，使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链。
  45. 根据权利要求39所述的装置，其特征在于，所述第二连接单元被配置为通过缺口补平反应，使所述第一单链DNA和所述第二单链DNA分别与所述双链DNA片段发生连接。
  46. 根据权利要求41所述的装置，其特征在于，双链DNA片段获取单元进一步包括：
    基因组DNA提取组件，所述基因组DNA提取组件用于从生物样本提取基因组DNA；和/或
    反转录组件，所述反转录组件用于对RNA样本进行反转录反应，以便获得反转录产物，
    其中，所述基因组DNA的至少一部分和/或RNA的反转录产物构成所述DNA样本。
  47. 一种针对双链DNA片段构建测序文库的设备，所述双链DNA片段具有两个平端末端，并且所述双链DNA片段的四个末端核苷酸均不具有磷酸基团，其特征在于，所述设备包括：
    权利要求39～46任一项所述的在双链DNA片段两端添加接头的装置，用于在所述双链DNA片段的两端连接接头，以便获得两端连接有接头的DNA片段；
    单链DNA片段分离装置，所述单链DNA片段分离装置用于从所述两端连接有接头的DNA片段分离单链DNA片段；以及
    环化装置，所述环化装置用于将所述单链DNA片段进行环化，以便获得单链DNA环，所述单链DNA环构成所述测序文库。
  48. 根据权利要求47所述的设备，其特征在于，所述单链DNA片段分离装置进一步包括：
    磁珠捕获单元，所述磁珠捕获单元用于使所述两端连接有接头的DNA片段与磁珠接触，以便形成磁珠-DNA复合物，其中，所述磁珠上连接有链霉亲和素；以及
    碱性裂解单元，所述碱性裂解单元中设置有pH高于7的溶液，用于将所述磁珠-DNA复合物与pH低于7的溶液接触，以便获得所述单链DNA片段。
  49. 根据权利要求48所述的设备，其特征在于，所述pH高于7的溶液为氢氧化钠溶液。
  50. 根据权利要求49所述的设备，其特征在于，所述氢氧化钠溶液的浓度为大约0.5～2M。
  51. 根据权利要求50所述的设备，其特征在于，所述氢氧化钠溶液的浓度为大约1M。
  52. 根据权利要求47所述的设备，其特征在于，进一步包括：
    筛选装置，所述筛选装置用于在从所述两端连接有接头的DNA片段分离单链DNA片段之前，预先对所述两端连接有接头的DNA片段进行筛选。
  53. 根据权利要求52所述的设备，其特征在于，所述筛选装置中设置有探针，其中，所述探针对于预定序列是特异性的。
  54. 根据权利要求53所述的设备，其特征在于，所述预定序列包括至少一个外显子。
  55. 根据权利要求53所述的设备，其特征在于，所述探针是以微芯片阵列的形式提供的。
  56. 根据权利要求47所述的设备，其特征在于，所述环化装置中设置有单链核酸分子，
    其中，
    所述单链核酸分子上限定出第一区段和第二区段，并且所述第一区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的序列匹配，所述第二区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的之一的序列匹配。
  57. 根据权利要求56所述的设备，其特征在于，所述第一区段和所述第二区段是毗邻连接的。
  58. 根据权利要求56所述的设备，其特征在于，所述第一区段的序列为5’TCGAGCTTGTCT3’，所述第二区段的序列为5’TCCTAAGACCGC3’。
  59. 一种核酸测序***，其特征在于，包括：
    根据权利要求47～58任一项所述的针对双链DNA片段构建测序文库的设备；以及
    测序设备，所述测序设备用于对所述测序文库进行测序。
  60. 根据权利要求59所述的核酸测序***，其特征在于，所述测序设备为CG测序平台。
  61. 一种用于针对基因组DNA构建测序文库的装置，其特征在于，包括：
    手段，用于对所述基因组DNA进行片段化，以便获得片段化产物；
    手段，用于对所述片段化产物进行去磷酸化处理，以便获得经过去磷酸化处理的片段化产物；
    手段，用于对所述经过去磷酸化处理的片段化产物进行末端修复，以便获得双链DNA片段；
    手段，用于将所述双链DNA片段与第一接头和第二接头进行连接，以便获得第一连接产物，其中，所述第一接头和第二接头不同，并且所述第一接头和第二接头均为权利要求1～10任一项所述的分离的寡核苷酸；
    手段，用于使用第一单链DNA置换所述第一接头的第二链，并且使用第二单链DNA置换所述第二接头的第二链，其中，所述第一单链DNA能够与所述第一接头的第一链特异性匹配形成双链结构，所述第二单链DNA能够与所述第二接头的第一链特异性匹配形成双链结构；
    手段，用于使所述第一单链DNA和所述第二单链DNA分别与所述DNA片段发生连接，以便获得第二连接产物；
    手段，利用第一引物和第二引物，对所述第二连接产物进行扩增，以便获得扩增产物，其中，所述扩增产物为两端连接有接头的DNA片段，其中，所述第一引物包含与所述第一单链DNA和所述第二单链DNA之一相同的序列，所述第二引物包含与所述第一单链DNA和所述第二单链DNA的另一个相同的序列，并且与所述第一单链DNA和所述第二单链DNA的所述另一个相比在5’末端具有额外的生物素；
    手段，用于从所述两端连接有接头的DNA片段分离单链DNA片段；以及
    手段，用于将所述单链DNA片段进行环化，以便获得单链DNA环，所述单链DNA环构成所述测序文库。
  62. 根据权利要求61所述的装置，其特征在于，将所述双链DNA片段与第一接头和第二接头进行连接是在一步反应中完成的。
  63. 根据权利要求61所述的装置，其特征在于，进一步包括：
    手段，用于从生物样本提取基因组DNA；和/或
    手段，用于从对RNA样本进行反转录反应，
    其中，
    所述基因组DNA的至少一部分和/或RNA的反转录产物构成所述DNA样本。
  64. 根据权利要求61所述的装置，其特征在于，用于从所述两端连接有接头的DNA片段分离单链DNA片段的手段，被配置为适于通过下列步骤分离所述单链DNA片段：
    使所述两端连接有接头的DNA片段与磁珠接触，以便形成磁珠-DNA复合物，其中，所述磁珠上连接有链霉亲和素；以及
    将所述磁珠-DNA复合物与pH低于7的溶液接触，以便获得所述单链DNA片段。
  65. 根据权利要求61所述的装置，其特征在于，进一步包括：
    手段，用于在从所述两端连接有接头的DNA片段分离单链DNA片段之前，预先对所述两端连接有接头的DNA片段进行筛选。
  66. 根据权利要求65所述的装置，其特征在于，所述筛选是通过所述两端连接有接头的DNA片段与探针接触进行的，其中，所述探针对于预定序列是特异性的。
  67. 根据权利要求66所述的装置，其特征在于，所述预定序列包括至少一个外显子。
  68. 根据权利要求66所述的装置，其特征在于，所述探针是以微芯片阵列的形式提供的。
  69. 根据权利要求61所述的装置，其特征在于，用于将所述单链DNA片段进行环化的手段被配置为采用单链核酸分子将所述单链DNA片段进行环化，
    其中，
    所述单链核酸分子上限定出第一区段和第二区段，并且所述第一区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的序列匹配，所述第二区段能够与包含所述单链DNA片段的5’末端核苷酸和3’末端核苷酸的之一的序列匹配。
  70. 根据权利要求69所述的装置，其特征在于，所述第一区段和所述第二区段是毗邻连接的。
  71. 根据权利要求69所述的装置，其特征在于，
    所述第一区段的序列为5’TCGAGCTTGTCT3’；以及
    所述第二区段的序列为5’TCCTAAGACCGC3’。
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