WO2019205135A1 - 用于对单链核酸分子进行环化处理的夹板核酸分子及其应用 - Google Patents
用于对单链核酸分子进行环化处理的夹板核酸分子及其应用 Download PDFInfo
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Definitions
- the present invention relates to the field of biotechnology, and in particular to the use of splint nucleic acid molecules for the cyclization of single-stranded nucleic acid molecules and their use, and more particularly to the use of loops for single-stranded nucleic acid molecules Processed splint nucleic acid molecule, method for cyclization of single-stranded nucleic acid molecule, method for constructing sequencing library, sequencing library, DNA sequence analysis method, kit, device for cyclization of single-stranded nucleic acid molecule, and sequencing Library system, DNA sequence analysis system.
- BGISEQ-500 and MGI2000 provide a one-stop sequencing process, and the sequencing process is clear.
- the sequencing platform not only provides an optional library preparation system and sample loading system, but also supports a variety of supporting library preparation solutions. It is one of the industry's leading high-throughput sequencing platforms using optimized joint probe anchor polymerization (cPAS) and improved DNA nanosphere (DNB) core sequencing technology.
- cPAS joint probe anchor polymerization
- DNS DNA nanosphere
- the existing DNA nanosphere (DNB) sequencing technology library construction process includes: under the action of splint nucleic acid molecules and ligase, the single-stranded nucleic acid to be cyclized is self-ligated to undergo a cyclization reaction, and the cyclized single strand The nucleic acid molecule is further subjected to rolling circle replication to form a DNA nanosphere (DNB) sequencing library.
- the cyclization reaction is a very important link in the process of building a database.
- the inventors found in the process of in-depth study of the factors affecting the cyclization reaction that if the selection of the splint nucleic acid molecules is improper, the efficiency of the cyclization reaction will be caused.
- the present invention aims to solve at least one of the technical problems in the related art to some extent.
- the present invention proposes a novel splint nucleic acid molecule (splint oligo), and proposes a new method for constructing a library based on the splint nucleic acid molecule.
- splint nucleic acid molecule according to the present invention, the cyclization efficiency of a single-stranded nucleic acid molecule can be effectively improved.
- the invention proposes a splint nucleic acid molecule for use in the cyclization of a single-stranded nucleic acid molecule.
- the splint nucleic acid molecule consists of a 5' end fragment and a 3' end fragment, wherein the 5' end fragment is adapted to form a first complementary region with the 5' end of the single stranded nucleic acid molecule
- the 3' end fragment is adapted to form a second complementary region with the 3' end of the single stranded nucleic acid molecule, and the length of the first complementary region and the second complementary region are different.
- the first complementary region and the second complementary region can be annealed at different temperatures, thereby effectively avoiding the to-be-cyclized
- the linkage between single-stranded nucleic acid molecules effectively increases the self-looping efficiency of single-stranded nucleic acid molecules.
- the invention provides a method of cyclization of a single-stranded nucleic acid molecule.
- the method comprises: placing a reaction mixture containing the aforementioned splint nucleic acid molecule, the single-stranded nucleic acid molecule, and a ligase under conditions suitable for ligation to obtain the single-stranded nucleic acid molecule Cyclization product.
- the first complementary region and the second complementary region can be annealed at different temperatures, thereby being effective
- the linkage between the single-stranded nucleic acid molecules to be cyclized is avoided, and the loop-forming efficiency of the single-stranded nucleic acid molecule is effectively improved.
- the single-stranded nucleic acid molecule can be effectively prevented from forming a linear complex with the splint nucleic acid molecule or the single-stranded nucleic acid molecule can be refolded to form a double-stranded DNA structure, and the intramolecular ligation and cyclization rate of the single-stranded nucleic acid molecule is compared with the prior art. Significantly increased.
- the invention proposes a method of constructing a sequencing library.
- the method comprises: obtaining a cyclization product of the single-stranded nucleic acid molecule, and a single-stranded nucleic acid molecule based on the single-stranded nucleic acid molecule carrying the insert, according to the method described above
- the cyclized product is subjected to a digestion process to obtain a circularized sequencing library, wherein the single-stranded nucleic acid molecule carrying the insert comprises: an insert, a first linker, the first linker being ligated to the 5' end of the insert a second connector, the second connector being coupled to the 3' end of the insert.
- a method for constructing a sequencing library can effectively prevent a single-stranded nucleic acid molecule from forming a linear complex with a splint nucleic acid molecule or re-refolding to form a double-stranded DNA structure, and the single-stranded nucleic acid is compared with the prior art.
- the intramolecular ligation and cyclization rate of the molecule is significantly improved.
- the invention proposes a sequencing library.
- the sequencing library has a base separation rate of no more than 0.5%.
- the positive and negative strands are balanced, and the base separation ratios of A and T, G, and C are low, not higher than 0.5%, and the accuracy of DNA quantification and determination of SNP is high.
- the invention proposes a sequencing library.
- the sequencing library is obtained by the method described above.
- the nucleic acid molecule in the sequencing library according to an embodiment of the present invention has a high cyclization rate.
- the invention proposes a DNA sequence analysis method.
- the method comprises sequencing the sequencing library described above to obtain a sequencing result comprising a plurality of sequencing reads; comparing the sequencing result with a reference sequence to obtain DNA sequence information .
- the DNA sequence information obtained by the method according to an embodiment of the present invention has a high accuracy rate.
- the invention proposes a kit.
- the kit comprises a splint nucleic acid molecule as described above.
- the splint nucleic acid molecules contained therein are different in length due to the first complementary region and the second complementary region, the first complementary region and the second complementary region may be Annealing at different temperatures can effectively avoid the linkage between single-stranded nucleic acid molecules that require cyclization, and effectively improve the self-looping efficiency of single-stranded nucleic acid molecules.
- the invention provides a device for the cyclization of a single-stranded nucleic acid molecule.
- the device is adapted to cyclize the single-stranded nucleic acid molecule under conditions suitable for ligation of a reaction mixture comprising a splint nucleic acid molecule, the single-stranded nucleic acid molecule, and a ligase, In order to obtain a cyclized product of the single-stranded nucleic acid molecule.
- the apparatus according to an embodiment of the present invention is adapted to perform a method of cyclizing a single-stranded nucleic acid molecule according to an embodiment of the present invention, which can effectively prevent a single-stranded nucleic acid molecule from forming a linear complex with a splint nucleic acid molecule or re-refolding into a double-stranded
- the DNA structure, the intramolecular ligation and cyclization rate of single-stranded nucleic acid molecules is significantly improved.
- the invention proposes a system for constructing a sequencing library.
- the system comprises: means for cyclizing a single-stranded nucleic acid molecule, the means for cyclizing a single-stranded nucleic acid molecule, adapted to be based on a single-stranded nucleic acid molecule carrying an insert, Performing a method of cyclizing a single-stranded nucleic acid molecule as described above to obtain a cyclized product of the single-stranded nucleic acid molecule; and a digestion treatment device that loops the single-stranded nucleic acid molecule
- the ligated device is ligated to digest the cyclized product of the single-stranded nucleic acid molecule to obtain a circularized sequencing library;
- the single-stranded nucleic acid molecule carrying the insert comprises: an insert; a first linker The first linker is coupled to the 5' end of the insert; the second linker is coupled to the
- the above system according to an embodiment of the present invention is adapted to perform a method of constructing a sequencing library according to an embodiment of the present invention, which can effectively prevent a single-stranded nucleic acid molecule from forming a linear complex with a splint nucleic acid molecule or re-refolding into a double-stranded DNA.
- Structure, the intramolecular ligation and cyclization rate of single-stranded nucleic acid molecules are significantly improved relative to the prior art.
- the invention provides a DNA sequence analysis system.
- the system comprises: a sequencing device adapted to sequence a sequencing library as described above to obtain a sequencing result comprising a plurality of sequencing reads; a comparison device, said ratio The device is coupled to the sequencing device and is adapted to align the sequencing result with a reference sequence to obtain DNA sequence information.
- the above system according to an embodiment of the present invention is adapted to perform the method of DNA sequence analysis according to an embodiment of the present invention, and the obtained DNA sequence information has high accuracy.
- FIG. 1 is a schematic view showing the structure of a splint nucleic acid molecule according to an embodiment of the present invention
- FIG. 2 is a schematic diagram showing the formation of single-stranded nucleic acid molecules by bridging nucleic acid molecules having a symmetrical structure according to an embodiment of the present invention
- Figure 3 is a schematic view showing the structure of a single-stranded nucleic acid molecule according to an embodiment of the present invention
- FIG. 4 is a schematic structural diagram of a system for constructing a sequencing library according to an embodiment of the present invention.
- FIG. 5 is a schematic structural diagram of a system for constructing a sequencing library according to another embodiment of the present invention.
- FIG. 6 is a schematic structural diagram of a system for constructing a sequencing library according to another embodiment of the present invention.
- Figure 7 is a schematic structural view of a DNA sequence analysis system according to an embodiment of the present invention.
- Figure 8 is a schematic illustration of the overall construction of a DNB sequencing library in accordance with an embodiment of the present invention.
- a "splint nucleic acid molecule” as used herein refers to a nucleic acid molecule suitable for two single-stranded nucleic acids that will require cyclization through its two end regions. The ends are pulled closer to increase the efficiency of the cyclization.
- an acid single-stranded nucleic acid molecule that requires cyclization or sometimes simply referred to as a "single-stranded nucleic acid molecule”, as used herein, refers to a nucleic acid molecule containing a single-stranded region, particularly the nucleic acid molecule.
- the two ends are single-stranded regions, and preferably, the nucleic acid molecules are all single-stranded regions in their entire length.
- the "complementary region" as used herein may contain a certain number of mismatched bases, unless otherwise specified.
- the number of mismatched bases used herein means that within the range of the complementary region, the two strands forming the complementary region are determined by the chain having the largest number of bases having no double-stranded structure. .
- the 5' end fragment forms a first complementary region with the 5' end of the single-stranded nucleic acid molecule
- 3 bases in the 5' end fragment do not form a double-stranded structure
- the single-stranded nucleic acid The molecule has one base that does not form a double-stranded structure, and the complementary region contains three mismatched bases.
- heat-resistant ligase means that the ligase is not inactivated at high temperatures, such as 95 ° C, and at a low temperature, such as at 37 ° C, the activity of the ligase is at least restored to 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the highest activity.
- base separation means that, due to DNA double-strand complementation and the composition of the bases on the genome, it is theoretically possible to sequence the reads at various positions if the randomness is good enough.
- the probability of occurrence of A and T is equal, and the probability of occurrence of C and G is also equal.
- the base separation rate can be calculated by: setting the read length to L, starting from the base of the 11th position of Read to the end, and calculating the absolute value of the AT at each position to occupy all the bases at the position. The percentage is denoted as Pn(
- first”, “second”, “third” and the like are used for the purpose of convenience of description and are not intended to imply or express There are differences in order or importance, and it does not mean that the content defined by the terms “first”, “second”, “third” and the like has only one component.
- the invention proposes a splint nucleic acid molecule for use in the cyclization of a single-stranded nucleic acid molecule.
- the splint nucleic acid molecule consists of a 5' end fragment and a 3' end fragment, wherein the 5' end fragment is adapted to form a first complementary region with the 5' end of the single stranded nucleic acid molecule
- the 3' end fragment is adapted to form a second complementary region with the 3' end of the single stranded nucleic acid molecule, and the length of the first complementary region and the second complementary region are different.
- a splint nucleic acid molecule according to an embodiment of the invention is a splint oligo suitable for use in the construction of circularized sequencing libraries.
- the first complementary region and the second complementary region can be performed at different temperatures, thereby effectively avoiding the to-be-cyclized
- the linkage between single-stranded nucleic acid molecules effectively increases the self-looping efficiency of single-stranded nucleic acid molecules.
- the complementary regions of unequal length are an asymmetric complementary structure.
- the long end is first annealed and the short end is annealed. Furthermore, it facilitates the occurrence of single-stranded intramolecular ligation and greatly increases the self-looping rate of single-stranded nucleic acid molecules.
- a bridged nucleic acid molecule having a symmetric complementary structure is used, which is symmetrically complementary to a single-stranded template, and the first complementary region and the second complementary region are formed to be equal in length, which is easier. The linkage between the single-stranded molecules occurs, forming a false linear complex structure rather than a cyclic product.
- the length of the first complementary region is the same as the length of the 5' end segment
- the length of the second complementary region is the same as the length of the 3' end segment.
- the 5' end and the 3' end of the single-stranded nucleic acid molecule are infinitely close, facilitating ligation of the 5' end and the 3' end of the single-stranded nucleic acid molecule.
- the length of the first complementary region is greater than the length of the second complementary region.
- the length of the first complementary region is at least 1.5 times the length of the second complementary region.
- the length of the first complementary region is at least 2 times the length of the second complementary region.
- the length of the first complementary region is at least 10 bp longer than the length of the second complementary region.
- the length of the first complementary region is at least 13 bp longer than the length of the second complementary region.
- the Tm values forming the first complementary region and the second complementary region differ by 10 °C.
- the inventors have found that the Tm values forming the first complementary region and the second complementary region differ by 10 ° C to form a two-stage gradient annealing, which is more advantageous for self-cyclization of single-stranded nucleic acid molecules.
- the 5' end fragment is 25 bp in length and the 3' end fragment is 11 bp in length. Further, the 5' end fragment can first anneal to the 5' end of the single-stranded nucleic acid molecule at a higher temperature, and the 3' end fragment is annealed to the 3' end of the single-stranded nucleic acid molecule at a lower temperature. More single-stranded nucleic acid molecules in the mode are easy to form intramolecular ligation, which greatly increases the self-looping rate of single-stranded nucleic acid molecules.
- the single-stranded nucleic acid molecule comprises, with reference to Figure 3: an insert; a first linker, the first linker being connected to the 5' end of the insert; a second linker, the second a linker is ligated to the 3' end of the insert; wherein the 5' end fragment of the splint nucleic acid molecule is adapted to form the first complementary region with at least a portion of the first linker, 3 of the splint nucleic acid molecule The 'end segment is adapted to form the second complementary region with at least a portion of the second linker.
- the single-stranded nucleic acid molecule can be further used for database construction and sequencing analysis after cyclization.
- the manner of providing the insert fragment described in the present application is not particularly limited, and may be obtained by breaking and denaturation treatment of genomic DNA, or directly provided by single-stranded DNA or RNA. It may also be a recombinant nucleic acid molecule obtained by inserting another target nucleic acid molecule into a nucleic acid vector.
- the insert is derived from at least a portion of a genomic fragment, in particular, the genomic fragment is obtained by disruption and denaturation of genomic DNA.
- the splint nucleic acid molecule according to an embodiment of the present invention can cyclize a single-stranded nucleic acid molecule formed of genomic DNA to obtain a genome sequencing library.
- the insert has a length of 100 to 600 bp.
- the splint nucleic acid molecule according to an embodiment of the present invention is applicable not only to the construction of a DNA library having an insertion fragment of 100 to 300 bp, but also to the construction of a larger insertion fragment such as a DNA library of 300 to 600 bp.
- the splint nucleic acid molecule according to the embodiment of the present invention is used for the construction of a DNA library having an insert length of 100 to 600 bp, the cyclization rate of the single-stranded DNA is high, and the base separation rate of the library is low.
- the single-stranded nucleic acid molecule has a first linker and a second linker, the first linker being linked to the 5' end of the insert and the second linker being ligated to the 3' end of the insert
- the single-stranded nucleic acid molecule has a length of 136 to 636 bp.
- the 5'-end fragment has the nucleotide sequence shown in SEQ ID NO: 1.
- AAGTCGGATCGTAGCCATGTCGTTC (SEQ ID NO: 1).
- the above 5'-end fragment according to an embodiment of the present invention may be ligated to a complementary sequence at the 5' end of a single-stranded nucleic acid molecule at 35 to 65 °C.
- the 3' end fragment has the nucleotide sequence set forth in SEQ ID NO: 2.
- TGTGAGCCAAG (SEQ ID NO: 2).
- the above 3'-end fragment according to an embodiment of the present invention may be ligated to the complementary sequence at the 3' end of the single-stranded nucleic acid molecule at 32 to 42 °C.
- the splint nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO:3.
- AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAG SEQ ID NO: 3
- the above-mentioned splint nucleic acid molecule according to the embodiment of the present invention can realize the sequential annealing connection of the 5′ end fragment and the 3′ end fragment at different temperatures, thereby greatly avoiding the formation of a linear complex or a single-stranded nucleic acid between the single-stranded nucleic acid molecule and the splint nucleic acid molecule.
- the phenomenon of molecular re-refolding to form a double-stranded DNA structure greatly increases the self-looping rate of single-stranded nucleic acid molecules.
- the splint nucleic acid molecule is DNA.
- the first complementary region and the second complementary region each independently contain no more than 5 mismatched bases, preferably no more than 4 mismatched bases, for example, preferably no more than
- the three mismatch bases preferably have no more than two mismatch bases, preferably no more than one mismatch base, and most preferably no mismatch bases.
- the inventors have found that the number of the first complementary region and the second complementary mismatch base is controlled within 5, the annealing success rate and the self-looping rate of the single-stranded nucleic acid molecule are high, and the first complementary region and the second The smaller the number of complementary mismatched bases, the higher the annealing success rate of the splint nucleic acid molecules and the single-stranded nucleic acid molecules, and the higher the self-forming rate of the single-stranded nucleic acid molecules.
- the invention provides a method of cyclization of a single-stranded nucleic acid molecule.
- the method comprises: placing a reaction mixture containing a splint nucleic acid molecule, the single-stranded nucleic acid molecule, and a ligase under conditions suitable for ligation to obtain cyclization of the single-stranded nucleic acid molecule product.
- the single-stranded nucleic acid molecule can be effectively prevented from forming a linear complex with the splint nucleic acid molecule or re-refolding to form a double-stranded DNA structure, and the intramolecular ligation and cyclization of the single-stranded nucleic acid molecule is compared with the prior art.
- the rate has increased significantly.
- the ligase is a thermostable ligase.
- heat-resistant ligase as used in the present application means that the ligase is not inactivated at a high temperature, such as 95 ° C, and at least at a low temperature, such as 37 ° C, the activity of the ligase can be restored to at least the highest. 50%, 60%, 70%, 80%, 90%, 95%, 99% of the activity.
- the thermotolerant ligase is capable of withstanding temperatures of at least 50 ° C, preferably at least 70 ° C, preferably at least 80 ° C, more preferably at least 90 ° C. Furthermore, the thermostable ligase can mediate the hybridization of the single-stranded nucleic acid molecule with the splint nucleic acid molecule at a higher temperature, such as 50 ° C.
- the secondary structure of the DNA strand is less and the specificity is higher at high temperature, such as Taq DNA ligase can be Annealing at 50 ° C, the original T4 ligase anneals the oligonucleotide strand to the template strand at 37 ° C, the original T4 ligase is more likely to produce a variety of secondary structures, connection mismatch.
- the thermotolerant ligase is Taq DNA ligase.
- a conventional ligase such as T4 DNA ligase
- T4 DNA ligase two DNA molecules can be ligated as long as the 5'-phosphate end and the 3'-hydroxy terminus are present;
- Taq DNA ligase is a high-fidelity ligase, It has high fidelity, it relies on a oligo oligo to connect, and if there is a base on the oligo that does not match the template, the connection cannot occur.
- the ligase according to an embodiment of the present invention uses Taq DNA ligase, which greatly reduces the mismatch rate.
- the method comprises: (1) placing the reaction mixture at a first temperature for a first predetermined time, the first temperature being suitable for a 5' end fragment of the splint nucleic acid molecule The 5' end of the single-stranded nucleic acid molecule forms a first complementary region; (2) the reaction mixture is placed at a second temperature for a second predetermined time, the second temperature being suitable for 3 of the splint nucleic acid molecule An 'end fragment forms a second complementary region with the 3' end of the single-stranded nucleic acid molecule; (3) placing the reaction mixture at a third temperature for a third predetermined time, the third temperature being adapted to At least one of the first complementary region and the second complementary region undergoes a melting reaction; (4) returns to step (1), and steps (1) and (2) are sequentially performed.
- the above method according to an embodiment of the present invention can re-anneal the product which does not form a cyclized product, such as a linear complex or a refolded double-stranded DNA, and re-anneal, greatly improving the connection efficiency and reducing the connection bias.
- a cyclized product such as a linear complex or a refolded double-stranded DNA
- the method further comprises: (5) returning to step (3), performing steps (3), (1) and (2) at least one cycle in sequence.
- the ligation reaction is a dynamic process in which the reactants collide with each other in the liquid under the action of power and enzymes to form products. The energy and power required for the reactants of different GC contents are different. The product will not increase any more.
- the unconnected molecules that reach the equilibrium state in the first round are restarted, and the connection continues in the second round. As a result, most of the molecules reach a relatively balanced state, The maximum cyclization efficiency is achieved, which significantly reduces the separation of bases.
- steps (3), (1), and (2) 2 to 18 cycles are sequentially performed in step (5).
- the inventors have found that the ligation process is carried out for 4 to 20 cycles as described above, and all molecules reach a relatively equilibrium state, and the cyclization rate is high, which significantly reduces the separation of bases.
- the single-stranded nucleic acid molecule is obtained by denaturation of a sample containing a double-stranded nucleic acid molecule.
- the denaturation treatment is carried out for 3 to 5 minutes at a temperature of 95 °C. Further, the secondary structure of the double-stranded nucleic acid molecule is opened, and the double-stranded chain is single-stranded.
- the inventors have found that the greater the difference between the temperatures formed by the first complementary region and the second complementary region, the less likely the single-stranded nucleic acid molecules are to form an intermolecular connection, but at the same time the second complementary
- the formation temperature of the zone needs to be not too different from the optimum temperature of the ligase, which poses a problem that needs to be considered.
- the first temperature is higher than the second temperature.
- the activity of the ligase at the second temperature is not less than 50%, preferably not less than 80%, and preferably not less than 90%, of the highest activity of the ligase, Not less than 91%, not less than 92%, not less than 93%, not less than 94%, not less than 95%, not less than 96%, not less than 97%, not less than 98%, not low At 99%, preferably, it is 100% of the highest activity of the ligase.
- the difference between the second temperature and the optimum temperature of the ligase is no more than 10 degrees Celsius, preferably no more than 8 degrees Celsius, preferably no more than 5 degrees Celsius, such as no more than 4 degrees Celsius, more preferably no More than 3 degrees Celsius, further preferably no more than 2 degrees Celsius.
- the optimum temperature refers to the temperature at which the ligase activity is the highest, and the optimum temperature of the Taq DNA ligase used in the specific examples of the present application is 45 °C.
- the second temperature is lower than the optimum temperature of the ligase, and the difference between the second temperature and the optimum temperature of the ligase is 2 to 8 degrees Celsius.
- first temperature and the second temperature are within the range of the above temperature difference, and are suitable for forming the first complementary region and the second complementary phase, and it is not easy to form an intermolecular connection between the single-stranded nucleic acid molecules, and the second
- the difference between the temperature and the optimum temperature is within an acceptable range and is suitable for annealing formation of the second complementary region.
- the first temperature is 35 to 65 ° C
- the first predetermined time is 2 min
- the first temperature is 50 ° C.
- the 5' end fragment of the splint nucleic acid molecule is completely complementary to the 5' end of the single stranded nucleic acid molecule to form a first complementary region.
- the second temperature is 32 to 42 ° C
- the second predetermined time is 30 min
- the second temperature is 37 ° C.
- the 3' end fragment of the splint nucleic acid molecule is completely complementary to the 3' end of the single stranded nucleic acid molecule to form a second complementary region.
- the third temperature is 94-98 ° C
- the third predetermined time is 30 s
- the third temperature is 95 ° C.
- the invention proposes a method of constructing a sequencing library.
- the method comprises: obtaining a cyclized product of the single-stranded nucleic acid molecule according to the method described above based on a single-stranded nucleic acid molecule carrying the insert; and the single-stranded nucleic acid molecule The cyclized product is subjected to a digestion treatment to obtain a circularized sequencing library.
- the single-stranded nucleic acid molecule carrying the insert comprises: an insert; a first linker, the first linker being linked to the 5' end of the insert; a second linker, the second linker and the insert The 3' end of the fragment is connected.
- a method for constructing a sequencing library can effectively prevent a single-stranded nucleic acid molecule from forming a linear complex with a splint nucleic acid molecule or re-refolding to form a double-stranded DNA structure, and the single-stranded nucleic acid is compared with the prior art.
- the intramolecular ligation and cyclization rate of the molecule is significantly improved.
- the digestion treatment is carried out under the action of a DNA exonuclease. Further, the unchained single-stranded nucleic acid molecule and the renatured double-stranded DNA molecule in the cyclization process can be digested, thereby further increasing the cyclization ratio of the sequencing library.
- the exonuclease comprises digestive enzyme I and digestive enzyme III. Further, digestive enzyme I can hydrolyze uncircularized single-stranded nucleic acid DNA molecules, digestive enzyme III can hydrolyze single nucleotides in the 3'-end direction of double-stranded DNA, and digestive enzyme-treated products retain only circular DNA.
- the digestion treatment is carried out at 37 ° C for 30 min.
- the uncircularized single-stranded nucleic acid molecule and the renatured double-stranded DNA molecule can be thoroughly digested without excessive digestion to produce a non-target product.
- the digesting treatment further comprises: subjecting the digestion treatment product to a fourth purification treatment. Furthermore, the interference of the enzymatic or ion used in the cyclization treatment and the digestion treatment on subsequent construction or sequencing can be excluded, and the purity of the circularization sequencing library and the accuracy of the sequencing result can be further improved.
- the single-stranded nucleic acid molecule carrying the insert is obtained by randomly breaking the genomic sample, first purification, end repair, and addition of a joint, the joint including the first linker And the second linker; performing a second purification and a PCR amplification process on the genomic sample to which the linker is ligated; and performing a third purification and denaturation treatment on the PCR amplification process product to obtain a denatured treatment product, the denaturation
- the processed product is the single-stranded nucleic acid molecule carrying the insert.
- the denaturation treatment is carried out at a temperature of 95 ° C for 3-5 min. Further, the PCR amplification product can be completely melted to obtain a single-stranded genome sample.
- the method further comprises: subjecting the circularized sequencing library to DNB amplification treatment to obtain a DNB sequencing library.
- the DNB amplification treatment comprises the following steps: taking a circularized sequencing library, adding water and DNB (DNA nanospheres) preparation buffer, vortexing and mixing for brief centrifugation, and the PCR instrument is at 95 ° C for 1 min, 65 ° C for 1 min, The reaction was carried out at 40 ° C for 1 min and maintained at 4 ° C.
- the temperature is 4 ° C, take out the mixture of DNB polymerase mixture I and DNB polymerase II, vortex and mix for brief centrifugation, and carry out the reaction on the PCR machine at 30 ° C for 20 min.
- the temperature is 4 ° C and taken out.
- Add DNB stop buffer, pipette and wide mouth tips to mix and mix slowly, then obtain DNB sequencing library, and store at 4 °C for use.
- the invention proposes a sequencing library.
- the sequencing library has a base separation rate of no more than 0.5%.
- the positive and negative strands are balanced, and the base separation ratios of A and T, G, and C are low, not higher than 0.5%, and the accuracy of DNA quantification and determination of SNP is high.
- the invention proposes a sequencing library.
- the sequencing library is obtained by the method described above.
- the nucleic acid molecule in the sequencing library according to an embodiment of the present invention has a high cyclization rate, and the base separation rate is not higher than 0.5%.
- the invention proposes a DNA sequence analysis method.
- the method comprises sequencing the sequencing library described above to obtain a sequencing result comprising a plurality of sequencing reads; comparing the sequencing result with a reference sequence to obtain DNA sequence information .
- the DNA sequence information obtained by the method according to an embodiment of the present invention has a high accuracy rate.
- the invention proposes a kit.
- the kit comprises a splint nucleic acid molecule as described above.
- the splint nucleic acid molecules contained therein are different in length due to the first complementary region and the second complementary region, the first complementary region and the second complementary region may be Annealing at different temperatures can effectively avoid the linkage between single-stranded nucleic acid molecules that require cyclization, and effectively improve the self-looping efficiency of single-stranded nucleic acid molecules.
- the invention provides a device for the cyclization of a single-stranded nucleic acid molecule.
- the device is adapted to cyclize the single-stranded nucleic acid molecule under conditions suitable for ligation of a reaction mixture comprising a splint nucleic acid molecule, the single-stranded nucleic acid molecule, and a ligase, In order to obtain a cyclized product of the single-stranded nucleic acid molecule.
- the single-stranded nucleic acid molecule can be effectively prevented from forming a linear complex with the splint nucleic acid molecule or re-refolding to form a double-stranded DNA structure, and the intramolecular ligation and cyclization rate of the single-stranded nucleic acid molecule are remarkably improved.
- the above apparatus according to an embodiment of the present invention is adapted to perform a method of cyclizing a single-stranded nucleic acid molecule according to an embodiment of the present invention, the additional technical features, advantages and effects of which are described above with respect to single-stranded nucleic acid molecules The method is the same.
- the invention proposes a system for constructing a sequencing library.
- the system comprises: a device 100 for cyclization of a single-stranded nucleic acid molecule, the device 100 for cyclization of a single-stranded nucleic acid molecule, adapted to be based on carrying an insert a single-stranded nucleic acid molecule, performing the method of cyclizing a single-stranded nucleic acid molecule as described above to obtain a cyclized product of the single-stranded nucleic acid molecule; and a digestion treatment device 200, the digestion treatment device 200
- the apparatus 100 for cyclizing a single-stranded nucleic acid molecule is ligated, and is adapted to digest the cyclized product of the single-stranded nucleic acid molecule to obtain a circularized sequencing library; wherein the single-stranded nucleic acid carrying the insert
- the molecule comprises: an insert; a first linker,
- the system further includes: a fourth purification processing device 300 connected to the digestion device 200 for performing digestion treatment products Four purification treatments.
- the system further includes a DNB amplification processing device 400 for performing a DNB amplification process on the circularized sequencing library to obtain DNB sequencing library.
- the above system according to an embodiment of the present invention is adapted to perform a method of constructing a sequencing library according to an embodiment of the present invention, the additional technical features, advantages and effects of which are the same as those described above for constructing a sequencing library.
- the invention provides a DNA sequence analysis system.
- the system comprises: a sequencing device 1000 adapted to sequence a sequencing library as described above to obtain sequencing results comprising a plurality of sequencing reads; Comparing device 2000, the comparison device 2000 is coupled to the sequencing device 1000 and is adapted to compare the sequencing result with a reference sequence to obtain DNA sequence information.
- the above system according to an embodiment of the present invention is adapted to perform a method of DNA sequence analysis according to an embodiment of the present invention, the additional technical features, advantages and effects of which are the same as those of the DNA sequence analysis described above.
- the inventors of the present application increase the cyclization efficiency of single-stranded nucleic acid molecules in the process of building a library, so that the single-stranded DNA is sufficiently self-ligated into a loop, and on the one hand, the number of reaction cycles of the cyclization is increased, each round After the ligation reaction, the unligated linear complex or double-stranded DNA is denatured again, and is annealed to the splint nucleic acid molecule having an asymmetric structure of the present invention, and then self-ligated and cyclized, and thus reciprocated, and multi-wheel connection is performed to achieve maximum Cyclization efficiency.
- this scheme requires multiple high temperature denaturation, and the current collocation step in the prior art uses T4 DNA ligase, which cannot meet the needs of multiple high temperature denaturation, and the Taq DNA used in the present application.
- the ligase is a thermostable ligase that is active at higher temperatures.
- the present invention uses Taq DNA ligase instead of T4 DNA ligase to cyclize single-stranded DNA, which not only greatly reduces the mismatch rate, but also allows the ligation reaction. Multiple high temperature denaturation and circulation can be performed to achieve maximum cyclization efficiency.
- Cyclization of a single-stranded molecule using Taq DNA ligase can open a double-stranded structure at a high temperature and carry out multiple rounds of ligation, allowing each strand of DNA to sufficiently and uniformly generate an enzymatic reaction, thereby improving the ligation efficiency. , significantly improved the base separation of sequencing;
- the splint nucleic acid molecule (splint oligo) and the linker of the PCR product are an asymmetric complementary structure, such as the first 25 bases are completely complementary to the one end of the PCR product, and the latter 11 bases are different from the PCR product. One end of the linker is completely complementary. When annealed with single-stranded DNA, the formed structure is more stable, which is more conducive to the self-joining in the next step;
- the cyclization method of the single-stranded DNA molecule of the invention can reduce the base separation rate of the DNA library in the second-generation sequencing, and the base separation rate is less than 0.5%, thereby improving the sequencing quality;
- the present invention is applicable not only to a DNA library construction of 100 to 300 bp but also to a larger insertion fragment such as a DNA library of 300 to 600 bp.
- FIG. 1 the schematic diagram of the overall construction of the DNB sequencing library can be referred to FIG.
- the inventors performed DNA sequencing library construction and sequencing test on the human standard DNA (NA12878) by using the cyclization method of the single-stranded nucleic acid molecule of the present invention, and the specific steps are as follows:
- Duty/cycle (0%) 10; Intensity 5; Cycle/burst 200; time (s) 60; number of cycles 5.
- a linker mixture (containing at least 8 kinds of tags) and 25 ⁇ L of a ligase reaction solution were added, and the mixture was incubated at 23 ° C for 1 hour; hydrated to 100 ⁇ L, and purified using Ampure XP beads, and the amount of magnetic beads used was 0.5X (50 ⁇ L).
- the purified ligation product was added to the PCR reaction solution in a volume of 100 ⁇ L, and the following reaction was carried out on a PCR machine: 95 ° C for 3 min, (98 ° C for 20 s, 60 ° C for 15 s, 72 ° C for 30 s, for 6 cycles), 72 ° C for 5 min, Finally kept at 4 ° C to save.
- the PCR product was subjected to Qubit concentration detection, and 330 ng of PCR product was supplemented with TE to 60 ⁇ L. 10 ⁇ L of splint nucleic acid molecule (10 ⁇ M) was added (the sequence is shown in SEQ ID NO: 3), mixed, and placed in a PCR apparatus at 95 ° C for incubation. -5 min, the double-stranded DNA was denatured into single-stranded DNA, immediately transferred to ice and left for 2 min.
- AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAG SEQ ID NO: 3
- the mixture was vortexed and briefly centrifuged, and the apparatus was maintained at 95 ° C for 1 min, 65 ° C for 1 min, 40 ° C for 1 min, and 4 ° C.
- the temperature was 4 ° C, take 40 ⁇ L of DNB polymerase mixture I and 4 ⁇ L of DNB polymerase mixture II, vortex and mix for brief centrifugation, and centrifuge on a PCR machine at 30 ° C for 20 min. At a temperature of 4 ° C, take it out and place it on an ice box. Add 20 ⁇ L of DNB Stop Buffer, pipette and wide-mouth tips to mix slowly, do not shake and violently blow, and store at 4 °C for later use.
- the sequencing method is PE100+10+100.
- Duty/cycle (0%) 10; Intensity 5; Cycle/burst 200; time (s) 60; number of cycles 5.
- a linker mixture (containing at least 8 kinds of tags) and 25 ⁇ l of a ligase reaction solution were added, and the mixture was incubated at 23 ° C for 1 hour; hydrated to 100 ⁇ L, and purified using Ampure XP beads, and the amount of magnetic beads used was 0.5X (50 ⁇ L).
- the purified ligation product was added to the PCR reaction solution in a volume of 100 ⁇ L, and the following reaction was carried out on a PCR machine: 95 ° C for 3 min, (98 ° C for 20 s, 60 ° C for 15 s, 72 ° C for 30 s, for 6 cycles), 72 ° C for 5 min, Finally kept at 4 ° C to save.
- the PCR product was subjected to Qubit concentration detection, 330 ng of PCR product was supplemented with TE to 60 ⁇ L, and 10 ⁇ L of the original BGISEQ bridging nucleic acid molecule (equal length of the first complementary region and the second complementary region formed with the single-stranded nucleic acid molecule) (10 ⁇ M) was added. ), mix and incubate in a PCR machine at 95 ° C for 3-5 min, the double-stranded DNA is denatured into single-stranded DNA, immediately transferred to ice, placed for 2 min.
- the sequencing method is PE100+10+100.
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Abstract
本发明提出了用于对单链核酸分子进行环化处理的夹板核酸分子及其应用。该夹板核酸分子由5'端片段和3'端片段组成，其中，所述5'端片段适于与所述单链核酸分子的5'末端形成第一互补区，所述3'端片段适于与所述单链核酸分子的3'末端形成第二互补区，以及所述第一互补区与所述第二互补区的长度不同。
Description
优先权信息
无。
本发明涉及生物技术领域，具体地，本发明涉及用于用于对单链核酸分子进行环化处理的夹板核酸分子及其应用，更具体地，本发明涉及用于对单链核酸分子进行环化处理的夹板核酸分子、对单链核酸分子进行环化处理的方法、构建测序文库的方法、测序文库、DNA序列分析方法、试剂盒、对单链核酸分子进行环化处理的装置、构建测序文库的系统、DNA序列分析系统。
BGISEQ-500和MGI2000作为一个开放性平台，提供了一站式测序操作流程，测序流程清晰明确，该测序平台不仅提供了可选的文库制备系统和样本加载系统，而且支持多种配套文库制备方案，其采用优化的联合探针锚定聚合技术(cPAS)和改进的DNA纳米球(DNB)核心测序技术，是行业领先的高通量测序平台之一。
然而，现有的建库技术仍需要进一步开发和改进。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的：
现有的DNA纳米球(DNB)测序技术的建库流程包括：在夹板核酸分子和连接酶的作用下，使得待环化的单链核酸发生自我连接发生环化反应，环化后的单链核酸分子进一步进行滚环复制形成DNA纳米球(DNB)测序文库。显然，环化反应是建库过程中非常重要的环节，然而，发明人在对环化反应的影响因素进行深入研究的过程中发现，如果夹板核酸分子的选择不当，会造成环化反应的效率较低，具体地，并非全部待环化的单链核酸都能自我连接成环，而是绝大多数产物为待环化的单链核酸与夹板核酸分子形成的线性复合物，或是重新复性的双链DNA结构，从而会造成测序时正负链的不平衡，导致A和T、G和C碱基分离。这种分离会影响DNA定量和SNP确定的准确性。
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。为此，本发明提出了一种新型的夹板核酸分子(splint oligo)，并基于该夹板核酸分子提出了新的构建文库的方法。利用根据本发明的夹板核酸分子，可以有效地提高单链核酸分子的环化效率。
在本发明的第一方面，本发明提出了一种用于对单链核酸分子进行环化处理的夹板核酸分子。根据本发明的实施例，所述夹板核酸分子由5’端片段和3’端片段组成，其中，所 述5’端片段适于与所述单链核酸分子的5’末端形成第一互补区，所述3’端片段适于与所述单链核酸分子的3’末端形成第二互补区，以及所述第一互补区与所述第二互补区的长度不同。根据本发明的实施例，由于第一互补区和第二互补区的长度不相同，所以第一互补区和第二互补区可以在不同的温度下退火形成，从而能够有效地避免了待环化的单链核酸分子间的连接，有效地提高单链核酸分子的自身成环效率。
在本发明的第二方面，本发明提出了一种对单链核酸分子进行环化处理的方法。根据本发明的实施例，所述方法包括：将含有前面所述夹板核酸分子、所述单链核酸分子、连接酶的反应混合物置于适于连接的条件下，以便获得所述单链核酸分子的环化产物。如前所述，根据本发明的实施例，由于第一互补区和第二互补区的长度不相同，所以第一互补区和第二互补区可以在不同的温度下退火形成，从而能够有效地避免待环化的单链核酸分子间的连接，有效地提高单链核酸分子的成环效率。具体地，可有效避免单链核酸分子与夹板核酸分子形成线性复合物或单链核酸分子重新复性形成双链DNA结构，相对于现有技术，单链核酸分子的分子内连接和环化率显著提高。
在本发明的第三方面，本发明提出了一种构建测序文库的方法。根据本发明的实施例，所述方法包括：基于携带插入片段的单链核酸分子，根据前面所述的方法，获得所述单链核酸分子的环化产物，以及对所述单链核酸分子的环化产物进行消化处理，以便获得环化测序文库，其中，所述携带插入片段的单链核酸分子包括：插入片段，第一接头，所述第一接头与所述插入片段的5’端相连，第二接头，所述第二接头与所述插入片段的3’端相连。根据本发明实施例的构建测序文库的方法，环化过程中可有效避免单链核酸分子与夹板核酸分子形成线性复合物或重新复性形成双链DNA结构，相对于现有技术，单链核酸分子的分子内连接和环化率显著提高。
在本发明的第四方面，本发明提出了一种测序文库。根据本发明的实施例，所述测序文库的碱基分离率不高于0.5％。根据本发明实施例的测序文库用于测序时，正负链平衡，A和T、G和C的碱基分离率低，不高于0.5％，DNA定量和确定SNP的准确性高。
在本发明的第五方面，本发明提出了一种测序文库。根据本发明的实施例，所述测序文库是通过前面所述的方法获得。根据本发明实施例的测序文库中的核酸分子的环化率高。
在本发明的第六方面，本发明提出了一种DNA序列分析方法。根据本发明的实施例，所述方法包括对前面所述的测序文库进行测序，以便获得包含多个测序读段的测序结果；将所述测序结果与参考序列进行比对，以便获得DNA序列信息。根据本发明实施例的方法获得的DNA序列信息准确率高。
在本发明的第七方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括前面所述的夹板核酸分子。如前所述，利用根据本发明实施例的试剂盒，其中所含有 的夹板核酸分子，由于第一互补区和第二互补区的长度不相同，所以第一互补区和第二互补区可以在不同的温度下退火形成，从而能够有效地避免了需要环化的单链核酸分子间的连接，有效地提高了单链核酸分子的自身成环效率。
在本发明的第八方面，本发明提出了一种对单链核酸分子进行环化处理的装置。根据本发明的实施例，所述装置适于将含有夹板核酸分子、所述单链核酸分子、连接酶的反应混合物在适于连接的条件下，将所述单链核酸分子进行环化处理，以便获得所述单链核酸分子的环化产物。根据本发明实施例的装置适于执行根据本发明实施例的对单链核酸分子进行环化处理的方法，可有效避免单链核酸分子与夹板核酸分子形成线性复合物或重新复性形成双链DNA结构，单链核酸分子的分子内连接和环化率显著提高。
在本发明的第九方面，本发明提出了一种构建测序文库的系统。根据本发明的实施例，所述系统包括：对单链核酸分子进行环化处理的装置，所述对单链核酸分子进行环化处理的装置，适于基于携带插入片段的单链核酸分子，执行前面所述的对单链核酸分子进行环化处理的方法，以获得所述单链核酸分子的环化产物；以及消化处理装置，所述消化处理装置与所述对单链核酸分子进行环化处理的装置相连，适于对所述单链核酸分子的环化产物进行消化处理，以便获得环化测序文库；其中，所述携带插入片段的单链核酸分子包括：插入片段；第一接头，所述第一接头与所述插入片段的5’端相连；第二接头，所述第二接头与所述插入片段的3’端相连。根据本发明实施例的上述系统适于执行根据本发明实施例的构建测序文库的方法，环化过程中可有效避免单链核酸分子与夹板核酸分子形成线性复合物或重新复性形成双链DNA结构，相对于现有技术，单链核酸分子的分子内连接和环化率显著提高。
在本发明的第十方面，本发明提出了一种DNA序列分析系统。根据本发明的实施例，所述系统包括：测序装置，所述测序装置适于对权前面所述的测序文库进行测序，以便获得包含多个测序读段的测序结果；对比装置，所述比对装置与所述测序装置相连，适于将所述测序结果与参考序列进行比对，以便获得DNA序列信息。根据本发明实施例的上述系统适于执行根据本发明实施例的DNA序列分析的方法，获得的DNA序列信息准确率高。
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。
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是根据本发明实施例的夹板核酸分子的结构示意图；
图2是根据本发明实施例的具有对称结构的桥连核酸分子形成单链核酸分子间连 接的示意图；
图3是根据本发明实施例的单链核酸分子的结构示意图；
图4是根据本发明实施例的构建测序文库的系统的结构示意图；
图5是根据本发明另一实施例的构建测序文库的系统的结构示意图；
图6是根据本发明另一实施例的构建测序文库的系统的结构示意图；
图7是根据本发明实施例的DNA序列分析系统的结构示意图；以及
图8是根据本发明实施例的整体的构建DNB测序文库的流程示意图。
发明详细描述
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
术语解释
如无特别说明，参考图1，在本文中所使用的“夹板核酸分子”是指这样一种核酸分子，该核酸分子适于通过其两端区域将需要进行环化处理的单链核酸两个末端进行拉近从而提高环化效率。
如无特别说明，在本文中所使用的“需要进行环化处理的酸单链核酸分子”或者有时简单称为“单链核酸分子”是指含有单链区域的核酸分子，尤其是该核酸分子的两端为单链区域，优选地，该核酸分子全长均为单链区域。
如无特别说明，在本文中所使用的“5’端片段”是指在夹板核酸分子上携带5’-磷酸基团的区域，并不能理解为该“5’端片段”是一个游离的区域。
如无特别说明，在本文中所使用的“3’端片段”是指在夹板核酸分子上携带3’-羟基的区域，并不能理解为该“3’端片段”是一个游离的区域。
如无特别说明，在本文中所使用的“互补区”是指通过碱基之间的互补关系形成的含有双链结构的区域。
如无特别说明，在本文中所使用的“互补区”可以含有一定数目的错配碱基。
如无特别说明，在本文中所使用的错配碱基的数目是指在该互补区的范围内，形成该互补区的两条链中未形成双链结构的碱基数目最多的链决定的。例如，5’端片段与单链核酸分子的5’末端形成第一互补区时，在该第一互补区内，5’端片段内有3个碱基未形成双链结构，而单链核酸分子则有1个碱基未形成双链结构，则该互补区含有3个错配碱基。
如无特别说明，在本文中所使用的“耐热连接酶”是指在高温下，如95℃下连接酶不失活，并在低温下，如37℃下连接酶的活性能至少恢复至最高活性的50％、60％、70％、80％、90％、95％、99％或100％。
如无特别说明，在本文中所使用的“碱基分离”是指，由于DNA双链互补以及基因组 上碱基的构成决定了理论上如果随机性足够好的话，测序的读段在各个位置上出现A和T的概率相等，出现C和G的概率也相等，而当测序存在偏向性时，则读段上各个位置的碱基分布出现A的含量不等于T的含量，C的含量不等于G的含量，即出现所谓的碱基分离。碱基分离率可通过如下方式计算获得：设读段(read)长度为L，从Read第11个位置的碱基开始到结束，计算每个位置的A-T的绝对值占该位置所有碱基的百分比，记为Pn(|A-T|)，其中n为read上的碱基位置(取值范围为11～L)，然后取平均值，作为碱基分离率。
如无特别说明，在本文中所使用的“第一”、“第二”、“第三”等类似术语均为用于描述方便而进行区分的目的，并不以任何目的暗示或者明示互相之间存在顺序或者重要性等差异，同时并不意味着由“第一”、“第二”、“第三”等类似术语所限定的内容仅有一种成分构成。
用于对单链核酸分子进行环化处理的夹板核酸分子
在本发明的第一方面，本发明提出了一种用于对单链核酸分子进行环化处理的夹板核酸分子。根据本发明的实施例，所述夹板核酸分子由5’端片段和3’端片段组成，其中，所述5’端片段适于与所述单链核酸分子的5’末端形成第一互补区，所述3’端片段适于与所述单链核酸分子的3’末端形成第二互补区，以及所述第一互补区与所述第二互补区的长度不同。根据本发明实施例的夹板核酸分子是一种寡义核苷酸链(splint oligo)，适于应用于构建环化测序文库。根据本发明的实施例，由于第一互补区和第二互补区的长度不相同，所以第一互补区和第二互补区可以在不同的温度下进行，从而能够有效地避免了待环化的单链核酸分子间的连接，有效地提高单链核酸分子的自我成环效率。换句话说，参考图1，其长度不等的互补区是一种非对称的互补结构，当该夹板核酸分子与单链核酸分子退火时，长的一端首先发生退火，短的一端再退火，进而有利于单链分子内连接的发生，大大提高单链核酸分子自我成环率。而现有技术中，参考图2，采用的是具有对称的互补结构的桥连核酸分子，其与单链模板是对称互补的，形成的第一互补区和第二互补区长度相等，更容易发生单链分子间的连接，形成一种错误的线性复合体结构，而不是环状产物。
根据本发明的实施例，所述第一互补区的长度与所述5’端片段的长度相同，所述第二互补区的长度与所述3’端片段的长度相同。进而单链核酸分子的5’端和3’端无限接近，有利于单链核酸分子的5’端和3’端进行连接。
根据本发明的实施例，所述第一互补区的长度大于所述第二互补区的长度。
根据本发明的实施例，所述第一互补区的长度为所述第二互补区的长度的至少1.5倍。
根据本发明的实施例，所述第一互补区的长度为所述第二互补区的长度的至少2倍。
根据本发明的实施例，所述第一互补区的长度比所述第二互补区的长度长至少10bp。
根据本发明的实施例，所述第一互补区的长度比所述第二互补区的长度长至少13bp。
根据本发明的实施例，形成所述第一互补区和第二互补区的Tm值相差10℃。发明人发现，形成所述第一互补区和第二互补区的Tm值相差10℃更容易形成双阶段的梯度退火，进而更有利于单链核酸分子的自我环化。
根据本发明的实施例，所述5’端片段长25bp，所述3’端片段长11bp。进而5’端片段在较高温度下可首先与单链核酸分子的5’末端进行退火，降到较低温度下，3’端片段与单链核酸分子的3’末端进行退火，这种退火模式下更单链核酸分子易于形成分子内连接，大大提高了单链核酸分子的自我成环率。
根据本发明的实施例，所述单链核酸分子包括，参考图3：插入片段；第一接头，所述第一接头与所述插入片段的5’端相连；第二接头，所述第二接头与所述插入片段的3’端相连；其中，所述夹板核酸分子的5’端片段适于与所述第一接头的至少一部分形成所述第一互补区，所述夹板核酸分子的3’端片段适于与所述第二接头的至少一部分形成所述第二互补区。进而单链核酸分子环化后可进一步用于建库和测序分析。
需要说明的是，本申请所述的插入片段的提供方式不受特别限制，它可以是基因组DNA进行打断和变性处理后获得的，也可以是以单链DNA或RNA的形式直接提供的，也可以是其它靶标核酸分子插入核酸载体后获得的重组核酸分子。根据本发明的具体实施例，所述插入片段来源于基因组片段的至少一部分，具体地，所述基因组片段是由基因组DNA进行打断和变性处理后获得。进而根据本发明实施例的夹板核酸分子可对由基因组DNA形成的单链核酸分子进行环化处理，而获得基因组测序文库。
根据本发明的实施例，所述插入片段的长度为100～600bp。根据本发明实施例的夹板核酸分子不仅适用于插入片段为100～300bp的DNA文库的构建，也适用于更大插入片段如300～600bp的DNA文库的构建。根据本发明实施例的夹板核酸分子用于插入片段的长度为100～600bp的DNA文库的构建，单链DNA的环化率高，文库的碱基分离率低。
根据本发明的具体实施例，所述单链核酸分子具有第一接头和第二接头，所述第一接头与插入片段的5’端相连，所述第二接头与插入片段的3’端相连，所述单链核酸分子的长度为136～636bp。
根据本发明的实施例，所述5’端片段具有SEQ ID NO：1所示的核苷酸序列。
AAGTCGGATCGTAGCCATGTCGTTC(SEQ ID NO：1)。
根据本发明实施例的上述5’端片段可在35～65℃与单链核酸分子的5’末端的互补序列配对连接。
根据本发明的实施例，所述3’端片段具有SEQ ID NO：2所示的核苷酸序列。
TGTGAGCCAAG(SEQ ID NO：2)。
根据本发明实施例的上述3’端片段可在32～42℃与单链核酸分子的3’末端的互补序 列配对连接。
根据本发明的实施例，所述夹板核酸分子具有SEQ ID NO：3所示的核苷酸序列。
AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAG(SEQ ID NO：3)。
根据本发明实施例的上述夹板核酸分子可在不同温度下实现5’端片段和3’端片段先后退火连接，进而极大地避免了单链核酸分子与夹板核酸分子形成线性复合物或单链核酸分子重新复性形成双链DNA结构的现象的发生，大大提高了单链核酸分子的自我成环率。
根据本发明的实施例，所述夹板核酸分子为DNA。
根据本发明的实施例，其特征在于，所述第一互补区和所述第二互补区分别独立地含有不超过5个错配碱基，优选不超过4错配碱基，例如优选不超过3个错配碱基，优选不超过2个错配碱基，优选不超过1个错配碱基，最优选不含有错配碱基。发明人发现，第一互补区和所述第二互补错配碱基数控制在5个以内，退火成功率和单链核酸分子的自我成环率高，并且第一互补区和所述第二互补错配碱基数越少，夹板核酸分子与单链核酸分子的退火成功率越高，单链核酸分子自我成环率越高。
对单链核酸分子进行环化处理的方法
在本发明的第二方面，本发明提出了一种对单链核酸分子进行环化处理的方法。根据本发明的实施例，所述方法包括：将含有夹板核酸分子、所述单链核酸分子、连接酶的反应混合物置于适于连接的条件下，以便获得所述单链核酸分子的环化产物。根据本发明实施例的方法，可有效避免单链核酸分子与夹板核酸分子形成线性复合物或重新复性形成双链DNA结构，相对于现有技术，单链核酸分子的分子内连接和环化率显著提高。
根据本发明的实施例，所述连接酶为耐热连接酶。需要说明的是，本申请所述的“耐热连接酶”是指在高温下，如95℃下连接酶不失活，并在低温下，如37℃下连接酶的活性能至少恢复至最高活性的50％、60％、70％、80％、90％、95％、99％。
根据本发明的具体实施例，所述耐热连接酶能够耐受至少50℃，优选至少70℃，优选至少80℃，更优选至少90℃的温度。进而耐热连接酶可在较高温，如50℃下介导单链核酸分子与夹板核酸分子的杂交，高温下DNA链的二级结构比较少，特异性更高，如Taq DNA连接酶可在50℃下退火，原始的T4连接酶是在37℃下让寡核苷酸链与模板链发生退火，原始的T4连接酶更容易产生多种二级结构，发生连接错配。
根据本发明的实施例，所述耐热连接酶为Taq DNA连接酶。常规的连接酶如T4DNA连接酶进行DNA连接的时候，只要存在5’-磷酸末端和3’-羟基末端就可让两条DNA分子发生连接；而Taq DNA连接酶是一种高保真连接酶，具有高保真性，它依赖于一条寡义核苷酸链(oligo)进行连接，这条oligo上只要有一个碱基与模板不匹配，就不能发生连接。根据本发明实施例的连接酶采用Taq DNA连接酶，极大程度降低了错配率。
根据本发明的实施例，所述方法包括：(1)将所述反应混合物置于第一温度下持续第一预定时间，所述第一温度适于所述夹板核酸分子的5’端片段与所述单链核酸分子的5’末端形成第一互补区；(2)将所述反应混合物置于第二温度下持续第二预定时间，所述第二温度适于所述夹板核酸分子的3’端片段与所述单链核酸分子的3’末端形成第二互补区；(3)将所述反应混合物置于第三温度下持续第三预定时间，所述第三温度适于使所述第一互补区和所述第二互补区的至少之一发生解链反应；(4)返回至步骤(1)，依次实施步骤(1)和(2)。根据本发明实施例的上述方法可将没有形成环化的产物，如线性复合物或重新复性形成的双链DNA的再次打开，重新进行退火，大大提高了连接效率，降低了连接偏向性。
根据本发明的实施例，所述方法进一步包括：(5)返回至步骤(3)，依次实施步骤(3)、(1)和(2)至少一个循环。发明人发现，连接反应是一个动态过程，反应物在动力、酶的作用下，在液体中相互碰撞形成产物，不同GC含量的反应物需要的能量和动力是不一样的，达到某个平衡时，产物就不会再增加了。通过多轮循环的上述反应，会让在第一轮达到平衡态的没有连接的分子，重新启动，在第二轮继续发生连接，以此下去，多数分子都达到了一个相对平衡的状态，以达到最大的环化效率，明显降低了碱基的分离现象。
根据本发明的实施例，在步骤(5)中依次实施步骤(3)、(1)和(2)2～18个循环。发明人发现，所述连接处理进行上述的4～20个循环，所有分子都达到一个相对平衡的状态，环化率高、明显降低了碱基的分离现象。
根据本发明的实施例，所述单链核酸分子是通过对含有双链核酸分子的样本进行变性处理而获得的。
根据本发明的具体实施例，所述变性处理是在温度为95℃的条件下进行3～5min。进而双链核酸分子的二级结构被打开，由双链解链为单链。
另外，发明人发现，第一互补区，第二互补区形成的温度之间的差异越大，单链核酸分子之间就越不容易形成分子间的连接，但同时还要照顾到第二互补区的形成温度需要满足与连接酶的最适温度不要差别太大，这就造成了一个需要兼顾的问题。
根据本发明的实施例，所述第一温度高于所述第二温度。
根据本发明的具体实施例，在所述第二温度下，所述连接酶的活性不低于所述连接酶最高活性的50％，优选不低于80％，例如优选不低于90％、不低于91％、不低于92％、不低于93％、不低于94％、不低于95％、不低于96％、不低于97％、不低于98％、不低于99％，优选地，为所述连接酶最高活性的100％。
根据本发明的具体实施例，所述第二温度与所述连接酶的最适温度的差异不超过10摄氏度，优选不超过8摄氏度，优选不超过5摄氏度，例如不超过4摄氏度，更优选不超过3 摄氏度，进一步优选不超过2摄氏度。其中，最适温度是指连接酶活性最高时的时温度，如本申请具体实施例中所用的Taq DNA连接酶的最适温度是45℃。
根据本发明的具体实施例，所述第二温度低于所述连接酶的最适温度，并且所述第二温度与所述连接酶的最适温度的差异为2～8摄氏度。
发明人发现，第一温度和第二温度在上述温度差的范围内，适于第一互补区和第二互补区分阶段形成，单链核酸分子之间不容易形成分子间的连接，且第二温度与最适温度的差异在可接受的范围内，适于第二互补区的退火形成。
根据本发明的实施例，所述第一温度为35～65℃，所述第一预定时间为2min，优选地，所述第一温度为50℃。进而所述夹板核酸分子的5’端片段与所述单链核酸分子的5’末端完全互补形成第一互补区。
根据本发明的实施例，所述第二温度为32～42℃，所述第二预定时间为30min，优选地，所述第二温度为37℃。进而所述夹板核酸分子的3’端片段与所述单链核酸分子的3’末端完全互补形成第二互补区。
根据本发明的实施例，所述第三温度为94-98℃，所述第三预定时间为30s，优选地，所述第三温度为95℃。进而可将反应混合物中所形成的双链区进行彻底解链，解链产物再次进入新一轮的退火连接，如此循环，以达到最大的环化效率，明显降低碱基的分离现象。
构建测序文库的方法
在本发明的第三方面，本发明提出了一种构建测序文库的方法。根据本发明的实施例，所述方法包括：基于携带插入片段的单链核酸分子，根据前面所述的方法，获得所述单链核酸分子的环化产物；以及对所述单链核酸分子的环化产物进行消化处理，以便获得环化测序文库。其中，所述携带插入片段的单链核酸分子包括：插入片段；第一接头，所述第一接头与所述插入片段的5’端相连；第二接头，所述第二接头与所述插入片段的3’端相连。根据本发明实施例的构建测序文库的方法，环化过程中可有效避免单链核酸分子与夹板核酸分子形成线性复合物或重新复性形成双链DNA结构，相对于现有技术，单链核酸分子的分子内连接和环化率显著提高。
根据本发明的实施例，所述消化处理是在DNA外切酶的作用下进行的。进而可将环化过程中未环化的单链核酸分子、复性形成的双链DNA分子进行消化，进一步提高测序文库的环化比例。
根据本发明的实施例，所述DNA外切酶包括消化酶I和消化酶III。进而消化酶I可以水解未环化的单链核酸DNA分子，消化酶III可以水解双链DNA3’端方向的单核苷酸，消化酶处理后的产物只保留了环状DNA。
根据本发明的实施例，所述消化处理是在37℃条件下进行30min。进行可将未环化的 单链核酸分子、复性形成的双链DNA分子进行彻底消化，而不产生过度消化而产生非目标产物。
根据本发明的实施例，所述消化处理后进一步包括：将消化处理产物进行第四纯化处理。进而可排除环化处理以及消化处理中所使用的酶或离子对后续建库或测序的干扰，进一步提高环化测序文库的纯度以及测序结果的准确性。
根据本发明的实施例，所述携带插入片段的单链核酸分子是通过如下方式获得的：将基因组样本进行随机打断、第一纯化、末端修复以及加接头处理，接头包括所述第一接头和所述第二接头；将连接有接头的待测基因组样本进行第二纯化以及PCR扩增处理；以及将PCR扩增处理产物进行第三纯化以及变性处理，以便获得变性处理产物，所述变性处理产物即为所述携带插入片段的单链核酸分子。
根据本发明的具体实施例，所述变性处理是在温度为95℃条件下进行3-5min。进而可将PCR扩增产物进行彻底解链，获得单链基因组样本。
根据本发明的实施例，进一步包括：将所述环化测序文库进行DNB扩增处理，以便获得DNB测序文库。具体地，所述DNB扩增处理包括如下步骤：取环化测序文库，加水和DNB(DNA纳米球)制备缓冲液，涡旋混匀短暂离心，PCR仪上以95℃1min，65℃1min，40℃1min的条件进行反应，4℃保持。温度到4℃时取出，加入DNB聚合酶混合液I和DNB聚合酶混合液II，涡旋混匀短暂离心，PCR仪上以30℃20min的条件进行反应，温度达4℃时取出，置于冰盒上，加入DNB终止缓冲液，移液器和阔口吸头缓慢吹打混匀，即得DNB测序文库，4℃保存备用。
测序文库
在本发明的第四方面，本发明提出了一种测序文库。根据本发明的实施例，所述测序文库的碱基分离率不高于0.5％。根据本发明实施例的测序文库用于测序时，正负链平衡，A和T、G和C的碱基分离率低，不高于0.5％，DNA定量和确定SNP的准确性高。
在本发明的第五方面，本发明提出了一种测序文库。根据本发明的实施例，所述测序文库是通过前面所述的方法获得。根据本发明实施例的测序文库中的核酸分子的环化率高，碱基分离率不高于0.5％。
DNA序列分析方法
在本发明的第六方面，本发明提出了一种DNA序列分析方法。根据本发明的实施例，所述方法包括对前面所述的测序文库进行测序，以便获得包含多个测序读段的测序结果；将所述测序结果与参考序列进行比对，以便获得DNA序列信息。根据本发明实施例的方法获得的DNA序列信息准确率高。
试剂盒
在本发明的第七方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括前面所述的夹板核酸分子。如前所述，利用根据本发明实施例的试剂盒，其中所含有的夹板核酸分子，由于第一互补区和第二互补区的长度不相同，所以第一互补区和第二互补区可以在不同的温度下退火形成，从而能够有效地避免了需要环化的单链核酸分子间的连接，有效地提高了单链核酸分子的自身成环效率。
对单链核酸分子进行环化处理的装置
在本发明的第八方面，本发明提出了一种对单链核酸分子进行环化处理的装置。根据本发明的实施例，所述装置适于将含有夹板核酸分子、所述单链核酸分子、连接酶的反应混合物在适于连接的条件下，将所述单链核酸分子进行环化处理，以便获得所述单链核酸分子的环化产物。根据本发明实施例的装置，可有效避免单链核酸分子与夹板核酸分子形成线性复合物或重新复性形成双链DNA结构，单链核酸分子的分子内连接和环化率显著提高。
根据本发明实施例的上述装置适于执行根据本发明实施例的对单链核酸分子进行环化处理的方法，其附加技术特征、优势和效果与前面所述描述的对单链核酸分子进行环化的方法相同。
构建测序文库的系统
在本发明的第九方面，本发明提出了一种构建测序文库的系统。根据本发明的实施例，参考图4，所述系统包括：对单链核酸分子进行环化处理的装置100，所述对单链核酸分子进行环化处理的装置100，适于基于携带插入片段的单链核酸分子，执行前面所述的对单链核酸分子进行环化处理的方法，以获得所述单链核酸分子的环化产物；以及消化处理装置200，所述消化处理装置200与所述对单链核酸分子进行环化处理的装置100相连，适于对所述单链核酸分子的环化产物进行消化处理，以便获得环化测序文库；其中，所述携带插入片段的单链核酸分子包括：插入片段；第一接头，所述第一接头与所述插入片段的5’端相连；第二接头，所述第二接头与所述插入片段的3’端相连。
根据本发明的再一实施例，参考图5，所述系统进一步包括：第四纯化处理装置300，所述第四纯化处理装置300与所述消化装置200相连，用于将消化处理产物进行第四纯化处理。
根据本发明的再一实施例，参考图6，所述系统进一步包括DNB扩增处理装置400，所述DNB扩增处理装置400用于将所述环化测序文库进行DNB扩增处理，以便获得DNB测序文库。
根据本发明实施例的上述系统适于执行根据本发明实施例的构建测序文库的方法，其附加技术特征、优势和效果与前面所述描述的构建测序文库的方法相同。
DNA序列分析系统
在本发明的第十方面，本发明提出了一种DNA序列分析系统。根据本发明的实施例，参考图7，所述系统包括：测序装置1000，所述测序装置1000适于对权前面所述的测序文库进行测序，以便获得包含多个测序读段的测序结果；对比装置2000，所述比对装置2000与所述测序装置1000相连，适于将所述测序结果与参考序列进行比对，以便获得DNA序列信息。根据本发明实施例的上述系统适于执行根据本发明实施例的DNA序列分析的方法，其附加技术特征、优势和效果与前面所述描述的DNA序列分析的方法相同。
综上所述，本申请的发明人为了提高建库过程中单链核酸分子的环化效率，使单链DNA充分自我连接成环，本发明一方面增加环化的反应循环数，每一轮连接反应后将未连接成功的线性复合物或双链DNA再次变性，与本发明的具有非对称结构的夹板核酸分子退火，再自我连接环化，如此循环往复，进行多轮连接以达到最大的环化效率。另一方面，该方案需要多次高温变性，而目前现有技术中的建库环化步骤使用的是T4DNA连接酶，该酶无法满足多次高温变性的需求，而本申请中采用的Taq DNA连接酶是一种耐高温连接酶，在较高温下也具有活性，本发明使用Taq DNA连接酶替代T4DNA连接酶对单链DNA进行环化，不仅大大降低了错配率，还可使连接反应可进行多次高温变性、循环，来达到最大的环化效率。
因此，本发明所提出的技术方案的有益效果可概括为以下几点：
(1)使用Taq DNA连接酶进行单链分子的环化，可在高温下打开双链结构，进行多轮连接，让DNA的每一条链都能充分地均一地发生酶反应，提高了连接效率，明显改善了测序的碱基分离情况；
(2)夹板核酸分子(splint oligo)与PCR产物带的接头是一种非对称的互补结构，如前25个碱基与PCR产物的一端接头完全互补，后11个碱基与PCR产物的另一端接头完全互补，当与单链DNA退火时，形成的结构更稳定，更有利于下一步分子内的自我连接；
(3)利用本发明单链DNA分子的环化方法可以降低二代测序中DNA文库的碱基分离率，使碱基分离率在0.5％以下，提高了测序质量；
(4)本发明不仅适用于100～300bp的DNA文库构建，也适用于更大的插入片段如300～600bp的DNA文库。
下面将结合实施例对本发明的方案进行解释。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，例如可以采购自Illumina公司。
以下实施例中，整体的构建DNB测序文库的流程示意图可参考图8。
在本实施例中，发明人利用本发明的单链核酸分子的环化方法，对人源标准品DNA(NA12878)进行DNA测序文库构建与测序测试，具体步骤如下所述：
1)样本打断
取1μg基因组DNA进行Covaris E210打断，将DNA打断成100-600bp DNA碎片，主带≈200bp，打断反应体积为80μL，打断参数为：
Duty/cycle(0％)10；Intensity 5；Cycle/burst 200；时间(s)60；循环数5。
2)打断纯化
提前30分钟取出AMPure XP磁珠置于室温，使用前充分震荡混匀；吸取80μLAMPure XP磁珠至80μL打断产物中，用移液器轻轻吹打10次充分混匀，室温孵育5分钟；瞬时离心，将不粘管置于磁力架，静置2分钟至液体澄清，移液器吸取上清至新的不粘管中；吸取40μL AMPure XP磁珠至160μL上清中，用移液器轻轻吹打10次充分混匀，室温孵育5分钟；瞬时离心，将不粘管置于磁力架，静置2分钟至液体澄清，移液器吸取并弃掉上清；保持不粘管固定于磁力架上，加入500μL新鲜配制的80％乙醇，室温静置1分钟后小心弃去上清；重复上一步骤一次，尽量吸干管底液体；打开不粘管管盖，室温干燥3分钟；将不粘管从磁力架上取下，加入42μL TE缓冲液进行DNA洗脱，移液器吹打混匀并室温下溶解5分钟；瞬时离心，将不粘管置于磁力架上，静置2分钟至液体澄清，将40μL上清液转移到新PCR管中。
3)末端修复
根据打断纯化后的浓度取50ng样本到PCR管中，补充无核酸酶(NF)水至总体积40μL，加入10μL的BGI平台(BGISEQ500，MGI2000)DNA文库末端修复反应液，置于37℃温度下，反应30min，65℃温度下15min，使酶失活，后逐步以(0.1℃/s)速度降温至4℃。
4)接头连接
向上述反应液中加入5μL接头混合物(含至少8种标签)和连接酶反应液25μL，23℃孵育1h；补水至100μL，使用Ampure XP beads进行纯化，磁珠使用量为0.5X(50μL)。
5)连接产物纯化
使用Ampure XP beads进行纯化，磁珠使用量为0.5X(50μL)。
6)PCR反应
纯化后的连接产物加入PCR反应液，体积共100μL，在PCR仪上进行如下反应：95℃3min，(98℃20s，60℃15s，72℃30s，进行6个循环反应)，72℃5min，最后维持在 4℃保存。
7)PCR产物纯化
反应完成后使用Ampure XP beads进行纯化，磁珠使用量为1X(100μL)。
8)PCR产物变性
对PCR产物进行Qubit浓度检测，取330ng的PCR产物补充TE至60μL加入10μL的夹板核酸分子(10μM)(序列如SEQ ID NO：3所示)，混匀后放入PCR仪中95℃孵育3-5min，使双链DNA变性为单链DNA，立即转移到冰上，放置2min。
AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAG(SEQ ID NO：3)。
9)环化
配制环化反应混合液：12μL 10×Taq DNA连接缓冲液，2μL Taq DNA连接酶(NEB，40U/μL)，36μL NF水，加入到70μL的变性后的DNA溶液中；PCR仪上进行如下环化反应：95℃变性30s，50℃退火2min，37℃连接30min，一共进行4个循环反应，最后维持在4℃保存。
10)消化
配制消化反应混合液：0.8μL 10×消化缓冲液，3.9μL消化酶I，1.3μL消化酶III,2μL NF水，加入到120μL的环化反应溶液中；PCR仪上进行如下消化反应：37℃消化30min，维持在4℃保存；加入15μL终止缓冲液终止消化反应。
11)消化产物纯化
使用纯化beads进行纯化，磁珠使用量为1.2X(170μL)；使用Qubit ssDNA Kit检测消化后的单链环状产物，浓度达到0.78ng/μL以上，-20℃保存备用。
12)DNB制备
取6ng环化DNA文库，加水补至20μL，加20μL DNB(DNA纳米球)制备缓冲液，
涡旋混匀短暂离心，PCR仪上95℃1min，65℃1min，40℃1min，4℃保持。温度到4℃时取出，加入40μL DNB聚合酶混合液I和4μL DNB聚合酶混合液II，涡旋混匀短暂离心，PCR仪上30℃20min，温度达4℃时取出，置于冰盒上，加入20μL DNB终止缓冲液，移液器和阔口吸头缓慢吹打混匀，切勿震荡及剧烈吹打，4℃保存备用。
13)上机测序
测序方式为PE100+10+100。
发明人依据上述步骤，对人源标准品DNA(NA12878)建立了两个测序文库，并对该测序文库在PE100+10+100测序平台上进行测试，测序结果如表1所示。
表1：
| Read1AT分离率 | Read1GC分离率 | Read2AT分离率 | Read2GC分离率 |
| 文库1 | 0.41 | 0.35 | 0.35 | 0.36 |
| 文库2 | 0.38 | 0.35 | 0.37 | 0.41 |
由表1结果可知，两个测序文库的碱基分离率低，都在0.5％以下。
对比例
在本对比例中，发明人利用现有技术的单链核酸分子的环化方法，对人源标准品DNA(NA12878)进行DNA测序文库构建与测序测试，具体步骤如下所述：
1)样本打断
取1μg基因组DNA进行Covaris E210打断，将DNA打断成100-600bp DNA碎片，主带≈200bp，打断反应体积为80μL，打断参数为：
Duty/cycle(0％)10；Intensity 5；Cycle/burst 200；时间(s)60；循环数5。
2)打断纯化
吸取80μL AMPure XP磁珠至80μL打断产物中，用移液器轻轻吹打10次充分混匀，室温孵育5分钟；将不粘管置于磁力架，静置2分钟至液体澄清，移液器吸取上清至新的不粘管中；吸取40μL AMPure XP磁珠至160μL上清中，用移液器轻轻吹打10次充分混匀，室温孵育5分钟；将不粘管置于磁力架，静置2分钟至液体澄清，移液器吸取并弃掉上清；保持不粘管固定于磁力架上，加入500μL新鲜配制的80％乙醇洗涤两次，最后加入42μL TE缓冲液进行DNA洗脱。
3)末端修复
根据打断纯化后的浓度取50ng样本到PCR管中，补充无核酸酶(NF)水至总体积40μL，加入10μL的BGI平台(BGISEQ500，MGI2000)DNA文库末端修复反应液，置于37℃温度下，反应30min，65℃温度下15min，使酶失活，后逐步以(0.1℃/s)速度降温至4℃。
4)接头连接
向上述反应液中加入5μL接头混合物(含至少8种标签)和连接酶反应液25μl，23℃孵育1h；补水至100μL，使用Ampure XP beads进行纯化，磁珠使用量为0.5X(50μL)。
5)连接产物纯化
使用Ampure XP beads进行纯化，磁珠使用量为0.5X(50μL)。
6)PCR反应
纯化后的连接产物加入PCR反应液，体积共100μL，在PCR仪上进行如下反应：95℃3min，(98℃20s，60℃15s，72℃30s，进行6个循环反应)，72℃5min，最后维持在4℃保存。
7)PCR产物纯化
反应完成后使用Ampure XP beads进行纯化，磁珠使用量为1X(100μL)。
8)PCR产物变性
对PCR产物进行Qubit浓度检测，取330ng的PCR产物补充TE至60μL，加入10μL的原始的BGISEQ桥连核酸分子(与单链核酸分子形成的第一互补区和第二互补区长度相等)(10μM)，混匀后放入PCR仪中95℃孵育3-5min，使双链DNA变性为单链DNA，立即转移到冰上，放置2min。
9)环化
配制环化反应混合液：12μL连接缓冲液，1.2μL连接Enhancer，0.4μL T4DNA连接酶(NEB，400U/μL)，36.4μL NF水，加入到70μL的变性后的DNA溶液中；PCR仪上进行如下环化反应：37℃连接60min，最后维持在4℃保存。
10)消化
配制消化反应混合液：0.8μL 10×消化缓冲液，3.9μL消化酶I，1.3μL消化酶III，2μL NF水，加入到120μL的环化反应溶液中；PCR仪上进行如下消化反应：37℃消化30min，维持在4℃保存；加入15μL终止缓冲液终止消化反应。
11)消化产物纯化
使用纯化beads进行纯化，磁珠使用量为1.2X(170μL)；使用Qubit ssDNA Kit检测消化后的单链环状产物，浓度达到0.78ng/μL以上，-20℃保存备用。
12)DNB制备
取6ng环化DNA文库，加水补至20μL，加20μL DNB(DNA纳米球)制备缓冲液，涡旋混匀短暂离心，PCR仪上95℃1min，65℃1min，40℃1min，4℃保持。温度到4℃时取出，加入40μL DNB聚合酶混合液I和4μL DNB聚合酶混合液II，涡旋混匀短暂离心，PCR仪上30℃20min，温度达4℃时取出，置于冰盒上，加入20μL DNB终止缓冲液，移液器和阔口吸头缓慢吹打混匀，切勿震荡及剧烈吹打，4℃保存备用。
13)上机测序
测序方式为PE100+10+100。
发明人依据上述步骤，对人源标准品DNA(NA12878)建立了两个测序文库，并对该测序文库在PE100+10+100测序平台上进行测试，测序结果如表2所示。
表2：
| Read1AT分离率 | Read1GC分离率 | Read2AT分离率 | Read2GC分离率 | |
| 文库3 | 1.15 | 1.6 | 1.23 | 1.01 |
| 文库4 | 1 | 1.49 | 1.19 | 1.03 |
由表2结果可知，两个测序文库的碱基分离率都在1％以上。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“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必须针对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一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (52)
- 一种用于对单链核酸分子进行环化处理的夹板核酸分子，其特征在于，所述夹板核酸分子由5’端片段和3’端片段组成，其中，所述5’端片段适于与所述单链核酸分子的5’末端形成第一互补区，所述3’端片段适于与所述单链核酸分子的3’末端形成第二互补区，以及所述第一互补区与所述第二互补区的长度不同。
- 根据权利要求1所述的夹板核酸分子，其特征在于，所述第一互补区的长度与所述5’端片段的长度相同，所述第二互补区的长度与所述3’端片段的长度相同。
- 根据权利要求1所述的夹板核酸分子，其特征在于，所述第一互补区的长度大于所述第二互补区的长度。
- 根据权利要求1所述的夹板核酸分子，其特征在于，所述第一互补区的长度为所述第二互补区的长度的至少1.5倍；优选地，所述第一互补区的长度为所述第二互补区的长度的至少2倍；优选地，所述第一互补区的长度比所述第二互补区的长度长至少10bp；优选地，所述第一互补区的长度比所述第二互补区的长度长至少13bp。
- 根据权利要求1所述的夹板核酸分子，其特征在于，形成所述第一互补区和第二互补区的Tm值相差10℃。
- 根据权利要求1所述的夹板核酸分子，其特征在于，所述5’端片段的长度为25bp，所述3’端片段的长度为11bp。
- 根据权利要求1所述的夹板核酸分子，其特征在于，所述单链核酸分子包括：插入片段；第一接头，所述第一接头与所述插入片段的5’端相连；第二接头，所述第二接头与所述插入片段的3’端相连；其中，所述夹板核酸分子的5’端片段适于与所述第一接头的至少一部分形成所述第一互补区；所述夹板核酸分子的3’端片段适于与所述第二接头的至少一部分形成所述第二互补区。
- 根据权利要求7所述的夹板核酸分子，其特征在于，所述插入片段来源于基因组片段的至少一部分。
- 根据权利要求8所述的夹板核酸分子，其特征在于，所述基因组片段是由基因组 DNA进行打断和变性处理后获得。
- 根据权利要求7所述的夹板核酸分子，其特征在于，所述插入片段的长度为100～600bp。
- 根据权利要求7所述的夹板核酸分子，其特征在于，所述单链核酸分子的长度为136～636bp。
- 根据权利要求1所述的夹板核酸分子，其特征在于，所述5’端片段具有SEQ ID NO：1所示的核苷酸序列。
- 根据权利要求1所述的夹板核酸分子，其特征在于，所述3’端片段具有SEQ ID NO：2所示的核苷酸序列。
- 根据权利要求1所述的夹板核酸分子，其特征在于，所述夹板核酸分子具有SEQ ID NO：3所示的核苷酸序列。
- 根据权利要求1所述的夹板核酸分子，其特征在于，所述夹板核酸分子为DNA。
- 根据权利要求1所述的夹板核酸分子，其特征在于，所述第一互补区和所述第二互补区分别独立地含有不超过5个错配碱基，优选不超过4错配碱基，优选不超过3个错配碱基，优选不超过2个错配碱基，优选不超过1个错配碱基，最优选不含有错配碱基。
- 一种对单链核酸分子进行环化处理的方法，其特征在于，包括：将含有夹板核酸分子、所述单链核酸分子、连接酶的反应混合物置于适于连接的条件下，以便获得所述单链核酸分子的环化产物。
- 根据权利要求17所述的方法，其特征在于，所述连接酶为耐热连接酶。
- 根据权利要求18所述的方法，其特征在于，所述耐热连接酶能够耐受至少50℃，优选至少70℃，优选至少80℃，更优选至少90℃的温度。
- 根据权利要求18所述的方法，其特征在于，所述耐热连接酶为Taq DNA连接酶。
- 根据权利要求18所述的方法，其特征在于，所述方法包括：(1)将所述反应混合物置于第一温度下持续第一预定时间，所述第一温度适于所述夹板核酸分子的5’端片段与所述单链核酸分子的5’末端形成第一互补区；(2)将所述反应混合物置于第二温度下持续第二预定时间，所述第二温度适于所述夹板核酸分子的3’端片段与所述单链核酸分子的3’末端形成第二互补区；(3)将所述反应混合物置于第三温度下持续第三预定时间，所述第三温度适于使所述第一互补区和所述第二互补区的至少之一发生解链反应；(4)返回至步骤(1)，依次实施步骤(1)和(2)。
- 根据权利要求21所述的方法，其特征在于，进一步包括：(5)返回至步骤(3)，依次实施步骤(3)、(1)和(2)至少一个循环；优选地，在步骤(5)中依次实施步骤(3)、(1)和(2)2～18个循环。
- 根据权利要求17所述的方法，其特征在于，所述单链核酸分子是通过对含有双链核酸分子的样本进行变性处理而获得的。
- 根据权利要求23所述的方法，其特征在于，所述变性处理是在温度为95℃的条件下进行3～5min。
- 根据权利要求21所述的方法，其特征在于，所述第一温度高于所述第二温度。
- 根据权利要求21所述的方法，其特征在于，在所述第二温度下，所述连接酶的活性不低于所述连接酶最高活性的50％，优选不低于80％，优选不低于90％，优选不低于91％，优选不低于92％，优选不低于93％，优选不低于94％，优选不低于95％，优选不低于96％，优选不低于97％，优选不低于98％，优选不低于99％，优选地，为所述连接酶最高活性的100％。
- 根据权利要求21所述的方法，其特征在于，所述第二温度与所述连接酶的最适温度的差异不超过10摄氏度，优选不超过8摄氏度，优选不超过5摄氏度，优选不超过4摄氏度，更优选不超过3摄氏度，进一步优选不超过2摄氏度；优选地，所述第二温度低于所述连接酶的最适温度，并且所述第二温度与所述连接酶的最适温度的差异为2～8摄氏度。
- 根据权利要求21所述的方法，其特征在于，所述第一温度为35～65℃，所述第一预定时间为2min，优选地，所述第一温度为50℃。
- 根据权利要求21所述的方法，其特征在于，所述第二温度为32～42℃，所述第二预定时间为30min，优选地，所述第二温度为37℃。
- 根据权利要求21所述的方法，其特征在于，所述第三温度为94-98℃，所述第三预定时间为30s，优选地，所述第三温度为95℃。
- 一种构建测序文库的方法，其特征在于，包括：基于携带插入片段的单链核酸分子，根据权利要求17～30所述的方法，获得所述单链核酸分子的环化产物；以及对所述单链核酸分子的环化产物进行消化处理，以便获得环化测序文库；其中，所述携带插入片段的单链核酸分子包括：插入片段，第一接头，所述第一接头与所述插入片段的5’端相连，第二接头，所述第二接头与所述插入片段的3’端相连。
- 根据权利要求31所述的方法，其特征在于，所述消化处理是在DNA外切酶的作用下进行的。
- 根据权利要求32所述的方法，其特征在于，所述DNA外切酶包括消化酶I和消化酶III。
- 根据权利要求32所述的方法，其特征在于，所述消化处理是在37℃条件下进行30min。
- 根据权利要求31所述的方法，其特征在于，所述消化处理后进一步包括：将消化处理产物进行第四纯化处理。
- 根据权利要求31所述的方法，其特征在于，所述携带插入片段的单链核酸分子是通过如下方式获得的：将基因组样本进行随机打断、第一纯化、末端修复以及加接头处理，接头包括所述第一接头和所述第二接头；将连接有接头的待测基因组样本进行第二纯化以及PCR扩增处理；以及将PCR扩增处理产物进行第三纯化以及变性处理，以便获得变性处理产物，所述变性处理产物即为所述携带插入片段的单链核酸分子。
- 根据权利要求36所述的方法，其特征在于，所述变性处理是在温度为95℃条件下进行3-5min。
- 根据权利要求31所述的方法，其特征在于，进一步包括：将所述环化测序文库进行DNB扩增处理，以便获得DNB测序文库。
- 一种测序文库，其特征在于，所述测序文库的碱基分离率不高于0.5％。
- 一种测序文库，其特征在于，所述测序文库是通过权利要求31～38任一项所述的方法获得。
- 一种DNA序列分析方法，其特征在于，对权利要求39或40所述的测序文库进行测序，以便获得包含多个测序读段的测序结果；将所述测序结果与参考序列进行比对，以便获得DNA序列信息。
- 一种试剂盒，其特征在于，包括权利要求1～16任一项所述的夹板核酸分子。
- 一种对单链核酸分子进行环化处理的装置，其特征在于，所述装置适于将含有夹板核酸分子、所述单链核酸分子、连接酶的反应混合物在适于连接的条件下，将所述单链核酸分子进行环化处理，以便获得所述单链核酸分子的环化产物。
- 根据权利要求43所述的装置，其特征在于，所述连接酶为耐热连接酶；任选地，所述耐热连接酶能够耐受至少50℃的温度，优选至少70℃的温度，优选至少80℃，更优选至少90℃的温度；任选地，所述耐热连接酶为Taq DNA连接酶。
- 根据权利要求43所述的装置，其特征在于，所述装置适于执行以下操作：(1)将所述反应混合物置于第一温度下持续第一预定时间，所述第一温度适于所述夹板核酸分子的5’端片段与所述单链核酸分子的5’末端形成第一互补区；(2)将所述反应混合物置于第二温度下持续第二预定时间，所述第二温度适于所述夹板核酸分子的3’端片段与所述单链核酸分子的3’末端形成第二互补区；(3)将所述反应混合物置于第三温度下持续第三预定时间，所述第三温度适于使所述第一互补区和所述第二互补区的至少之一发生解链反应；(4)返回至步骤(1)，依次实施步骤(1)和(2)；任选地，进一步包括：(5)返回至步骤(3)，依次实施步骤(3)、(1)和(2)至少一个循环；优选地，在步骤(5)中依次实施步骤(3)、(1)和(2)2～18个循环。
- 根据权利要求43所述的装置，其特征在于，所述单链核酸分子是通过对含有双链核酸分子的样本进行变性处理而获得的；任选地，所述变性处理是在温度为95℃的条件下进行3～5min；任选地，所述第一温度高于所述第二温度；任选地，在所述第二温度下，所述连接酶的活性不低于所述连接酶最高活性的50％，优选不低于80％，优选不低于90％，优选不低于91％，优选不低于92％，优选不低于93％，优选不低于94％，优选不低于95％，优选不低于96％，优选不低于97％，优选不低于98％，优选不低于99％，优选地，为所述连接酶最高活性的100％；任选地，所述第二温度与所述连接酶的最适温度的差异不超过10摄氏度，优选不超过8摄氏度，优选不超过5摄氏度，优选不超过4摄氏度，更优选不超过3摄氏度，进一步优选不超过2摄氏度；优选地，所述第二温度与所述连接酶的最适温度的差异不超过10摄氏度，优选不超过8摄氏度，优选不超过5摄氏度，优选不超过4摄氏度，更优选不超过3摄氏度，进一步优选不超过2摄氏度；优选地，所述第二温度低于所述连接酶的最适温度，并且所述第二温度与所述连接酶的最适温度的差异为2～8摄氏度；任选地，所述第一温度为35～65℃，所述第一预定时间为2min，优选地，所述第一温度为50℃；任选地，所述第二温度为32～42℃，所述第二预定时间为30min，优选地，所述第二温度为37℃；任选地，所述第三温度为94-98℃，所述第三预定时间为30s，优选地，所述第三温度为95℃。
- 一种构建测序文库的系统，其特征在于，包括：对单链核酸分子进行环化处理的装置，所述对单链核酸分子进行环化处理的装置如权利要求43～46任一项所限定的，适于基于携带插入片段的单链核酸分子，执行权利要求17～30任一项所述的方法，获得所述单链核酸分子的环化产物；以及消化处理装置，所述消化处理装置与所述对单链核酸分子进行环化处理的装置相连，适于对所述单链核酸分子的环化产物进行消化处理，以便获得环化测序文库；其中，所述携带插入片段的单链核酸分子包括：插入片段；第一接头，所述第一接头与所述插入片段的5’端相连；第二接头，所述第二接头与所述插入片段的3’端相连。
- 根据权利要求47所述的系统，其特征在于，所述消化处理是在DNA外切酶的作用下进行的；任选地，所述DNA外切酶包括消化酶I和消化酶III；任选地，所述消化处理是在37℃条件下进行30min。
- 根据权利要求47所述的系统，其特征在于，所述系统进一步包括：第四纯化处理装置，所述第四纯化处理装置与所述消化装置相连，用于将消化处理产物进行第四纯化处理。
- 根据权利要求47所述的系统，其特征在于，所述携带插入片段的单链核酸分子是通过如下方式获得的：将基因组样本进行随机打断、第一纯化、末端修复以及加接头处理，接头包括所述第一接头和所述第二接头；将连接有接头的待测基因组样本进行第二纯化以及PCR扩增处理；以及将PCR扩增处理产物进行第三纯化以及变性处理，以便获得变性处理产物，所述变性处理产物即为所述携带插入片段的单链核酸分子；任选地，所述变性处理是在温度为95℃条件下进行3-5min。
- 根据权利要求47所述的系统，其特征在于，进一步包括DNB扩增处理装置，所述DNB扩增处理装置用于将所述环化测序文库进行DNB扩增处理，以便获得DNB测序文库。
- 一种DNA序列分析系统，其特征在于，测序装置，所述测序装置适于对权利要求39或40所述的测序文库进行测序，以便获得包含多个测序读段的测序结果；对比装置，所述比对装置与所述测序装置相连，适于将所述测序结果与参考序列进行 比对，以便获得DNA序列信息。
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| CN111455469A (zh) * | 2020-04-07 | 2020-07-28 | 深圳易倍科华生物科技有限公司 | 一种单链快速建库方法及建库仪器 |
| CN112111544A (zh) * | 2020-09-23 | 2020-12-22 | 复旦大学附属肿瘤医院 | 提高单链dna连接效率的方法 |
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| WO2024036445A1 (zh) * | 2022-08-15 | 2024-02-22 | 深圳华大智造科技股份有限公司 | 一种测序文库的制备方法及用于制备测序文库的试剂盒 |
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| CN116287124A (zh) * | 2023-05-24 | 2023-06-23 | 中国农业科学院农业基因组研究所 | 单链接头预连接方法、高通量测序文库的建库方法及试剂盒 |
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