CN114958829A

CN114958829A - Large fragment DNA cyclization connection method

Info

Publication number: CN114958829A
Application number: CN202210684676.1A
Authority: CN
Inventors: 任军; 高芳芳; 张鹏
Original assignee: Xukang Medical Science & Technology Suzhou Co ltd
Current assignee: Xukang Medical Science & Technology Suzhou Co ltd
Priority date: 2022-06-16
Filing date: 2022-06-16
Publication date: 2022-08-30

Abstract

The invention belongs to the field of genetic engineering, and particularly relates to a large-fragment DNA circularization connection method and application thereof in sequencing library construction, a kit for realizing the method and the application, and a circularized DNA molecule and a sequencing library formed by the method.

Description

Large fragment DNA cyclization connection method

Technical Field

Background

In the second-generation sequencing, the construction of a large fragment library is an important and basic experimental technology. Genetic mapping using next generation sequencing for species with unknown or no genomic information of the closely related species is one of the ways to rapidly understand a species. In addition, the second generation sequencing also has important use value in the screening and detecting process of the chromosome structure variation of the human genome at present.

terminal-Pair sequencing (MP sequencing) is one of the most commonly used next-generation sequencing technologies. The sequencing technology only carries out paired sequencing on two ends of a DNA fragment to obtain a sequencing result of a matched-end read (Mate-pair, MP). The sequencing result provides not only the sequence information of the reads themselves but also the distance information between the paired ends, from which the distance of the paired MP sequences on the genome can be determined.

The implementation of end-pairing sequencing relies on the construction of large-fragment end-pairing libraries (mate-pair library). In the library construction process, there are two general categories of methods: physical disruption or ordinary enzymatic cleavage; and transposase cleavage disruption.

Physical disruption or ordinary enzymatic cleavage generally involves: 1) breaking gDNA by physical method (DNA fragmenting instrument) or ordinary enzyme cutting method and recovering target size DNA fragment; 2) adding universal biotin labeling joints at two ends of the recovered DNA fragments; 3) circularizing the large DNA fragment with biotin-labeled linker; 4) digesting the non-circularized linear DNA fragments; 5) fragmenting the cyclized DNA fragment, and capturing the DNA fragment with the biotin label by using a streptavidin magnetic bead; 6) and amplifying the captured DNA fragments to build a DNA library for sequencing.

The transposase disruption method generally involves: 1) fragmenting the gDNA by transposase and attaching a biotin-labeled linker; 2) recovering DNA fragments with target sizes and filling the tail ends of the DNA fragments; 3) circularizing the large DNA fragment with biotin-labeled linker; 4) digesting the unclycled DNA fragments; 5) fragmenting the cyclized DNA fragment, and capturing the DNA fragment with the biotin label by using a streptavidin magnetic bead; 6) and amplifying the captured DNA fragments to build a DNA library for sequencing.

In both end-pairing library construction methods, the circularization and ligation of large-fragment DNA are the main part of library construction, and the ligation efficiency is the key node for limiting the large-fragment library construction. Furthermore, since the valid data of end-pairing sequencing is derived from the end-paired DNA fragments, the circularization ligation efficiency of the DNA fragments also affects the overall efficiency of end-pairing sequencing.

Currently there are two main ways based on intramolecular cyclic ligation: one is a blunt-end direct ligation scheme; one is to circularize large fragments of DNA using a recombinase system. Blunt-end direct ligation protocols rely only on collision probability of self-twisting of large-fragment DNA molecules, and therefore cyclization efficiency is low, usually less than 10%. Circularization of large DNA fragments based on recombinase systems also has problems, for example, a large proportion of linker fragments with recombination sites in different directions at both ends are generated, resulting in failure of recombination; in addition, the recombination efficiency of the recombinase system in vitro is not high, and the factors cause the cyclization efficiency of the whole system to be not more than 10%.

To improve the efficiency of DNA ligation, more common research has focused on the optimization of various functional enzymes and buffers during the library construction process. For example, researchers have proposed that high efficiency ligation can be achieved by optimizing the ligation buffer system and by adding reagents such as high polymer polyethylene glycol (PEG), small molecule propylene glycol, glycerol, and the like. Although these studies can also effectively improve the ligation efficiency, they either require complicated and high-standard equipment to produce the modified enzyme, or they cannot be used for patent protection, or they require the purchase of commercial reagents at a high price.

Therefore, it is urgently required to develop a simple method for improving the efficiency of circularization and ligation of large-fragment DNA.

Disclosure of Invention

In intermolecular ligation, cohesive-end ligation is considered to be a more efficient means of DNA ligation than blunt-end ligation. When two DNA molecules are ligated using a sticky end, it is considered that the DNA ligation efficiency is affected by the GC content of the sticky end and the length of the sticky end. A high GC abundance of the sticky ends will help to improve ligation efficiency; and for cohesive ends containing A, G, T, C nucleotides, it is more advantageous to use longer cohesive ends than shorter ones. Whereas sticky ends rich in a and T will significantly reduce ligation efficiency. See, e.g., GURNEET BOLA,2005, evaluation of the Role of G, C-nucleotides and Length of overhangins in T4 DNA 9 ase Efficiency, Vol.8:1-7,

December 2005,M&i, UBC; and Tina Gao et al, incorporated Overhang GC-Content Increases Sticky-End Ligation Efficiency，Journal of Experimental Microbiology and Immunology(JEMI),

April 2015,M&I UBC。

However, after intensive research on circularization of large-fragment DNA, the present inventors surprisingly found that introduction of only 2bp AT sticky ends is a more effective means of circularization ligation for achieving intramolecular circularization of large-fragment DNA using USER enzyme to form sticky ends. Based on this surprising finding, the present inventors have completed the present invention. As demonstrated in the examples herein, in circularization ligation of large pieces of DNA, introduction of optimized sticky ends using the linker of the present invention can improve ligation efficiency and the ratio of the ligation products of interest. In addition, the inventor also finds that on the basis of the above, the cyclization and ligation efficiency can be further improved by adding an endonuclease treatment step such as PvuI-HF after the USER enzyme treatment and before the cyclization and ligation.

Accordingly, in one aspect, the present invention provides a method of circularization of DNA, the method comprising the steps of:

(i) adding circularized linkers to both ends of the target DNA fragment to obtain a target DNA fragment with circularized linkers,

wherein the circularization linker is composed of a long 5' phosphorylated oligonucleotide chain and a short 5' phosphorylated oligonucleotide chain complementary thereto, wherein the first base at the 5' end of the long oligonucleotide chain is an A base, and the second base is a U base; and is provided with

Wherein the obtained target DNA fragment with the cyclized linker has AU in the form of inverted terminal repeats at both ends of the target DNA fragment;

(ii) (ii) treating the target DNA fragment with circularized adaptor from step (i) using uracil specific excision reagents to produce DNA fragments with 3' AT sticky ends on both ends;

(iii) (iii) treating the DNA fragment containing the 3'AT sticky ends resulting from step (ii) with a restriction enzyme to produce digested DNA fragments, wherein the restriction enzyme is capable of recognizing double-stranded DNA and producing 3' AT sticky ends;

(iv) (iv) incubating the DNA fragments resulting from step (iii) under conditions that allow circularization of the digested DNA fragments to obtain circularized DNA molecules.

In one embodiment, in step (i), the circularization adapters are ligated to both ends of the target DNA fragment by transposition reaction.

In another embodiment, in step (i), the circularized adapters are ligated to both ends of the target DNA fragment by TA-tail ligation.

In some preferred embodiments, the restriction enzyme used in step (iii) is selected from the group consisting of: PvuI, PacI, AsiSI, BstKTI, RgaI, and SfaAI, preferably the restriction enzyme is PvuI.

The target DNA fragment according to the method of the present invention is preferably a large fragment DNA of 1kbp to 200kbp, preferably 3kbp to 30kbp, more preferably 5kbp to 20kbp, more preferably 6kbp to 15kbp, and still more preferably 8kbp to 12 kbp.

In a further aspect, the invention also provides the use of the method of the invention in the construction of sequencing libraries, and circularised adaptors and kits for use in the method of the invention and for use in the construction of sequencing libraries, as well as circularised DNA molecules and sequencing libraries produced by the method of the invention.

Drawings

FIG. 1 schematically shows a large-fragment DNA circularization ligation method according to the present invention.

FIG. 2 schematically shows a cyclized linker useful in the method of the present invention.

FIG. 3 schematically shows a transposition cyclization scheme according to the present invention.

FIG. 4 schematically shows a TA tail link cyclization scheme in accordance with the present invention.

Detailed Description

Definition of

As used herein, a nucleotide pair refers to a pair of complementary nucleotides that are hydrogen bonded based on the Watson-Crick base-complementary pairing rules. For example, a nucleotide pair consisting of 5'-AU-3' on one strand and 3'-TA-5' complementary thereto on the other strand, as used herein, can be represented as a dinucleotide pair,

5'-A U-3'

3'-T A-5'，

or simply "AU:: TA" dinucleotide pairs.

In this context, the expression "sticky end generating region" refers to a region in the 5 'terminal part of a DNA molecule comprising the U residue and the nucleotide located 5' of this residue, e.g.the circularizing linker according to the invention and the region on the double stranded DNA molecule comprising AU dinucleotides.

In some embodiments, on circularized adaptors according to the invention, the sticky end generating region consists of the AU dinucleotide AT the 5 'extreme end of the long oligonucleotide chain of the adaptor molecule (i.e., A is the first residue AT the 5' end; U is the second residue AT the 5 'end) and forms a 5' single stranded overhang of the adaptor molecule, or in other embodiments, the sticky end generating region consists of the AU dinucleotide and an AT dinucleotide on the short oligonucleotide chain that is reverse complementary thereto.

In some embodiments of the double-stranded DNA molecules produced according to the methods of the invention, the sticky-end generating region is formed by a dinucleotide pair

5'-A U-3'

3'-T A-5'

And are present as inverted terminal repeats at both ends of the DNA molecule. Thus, the double-stranded DNA molecule has the following structure:

5'AU-(N) ₁ -AT3'

3'TA-(N) ₂ -UA5'

wherein, (N) ₁ And (N) ₂ Denotes a region of complementary nucleic acid located between two inverted terminal repeats. It will be appreciated that for the nucleic acid region in the DNA molecule, it may be of any length, sequence and/or origin, for example, different lengths of the target nucleic acid and/or other additional sequences.

Herein, reference to a DNA molecule having inverted terminal repeats means that the DNA molecule has inverted repeat sequences (IR) at both ends. For example, the DNA molecule described above having an AU:: TA dinucleotide pair inverted terminal repeats.

In this context, when a DNA molecule or oligonucleotide molecule is said to comprise a long strand of nucleotides and a short strand of nucleotides complementary thereto, it is meant that the DNA molecule has a duplex region consisting of the short strand and the long strand portion complementary thereto.

Herein, when referring to a partially double-stranded oligonucleotide, it is meant that the oligonucleotide has a double-stranded portion and a single-stranded portion at its 5 'or 3' end.

The expression "transposable element" and "transposable element" are used interchangeably herein and refer to a double-stranded oligonucleotide molecule capable of forming a functional transposable complex with a transposase in a transposition reaction. Transposases are capable of recognizing and binding to a transposable element (or a nucleic acid molecule comprising a transposable element) to form a transposable complex. In an in vitro transposition reaction, a transposable complex can transfer a transposable element (or a nucleic acid molecule comprising a transposable element) to a generated target DNA fragment while fragmenting the target DNA. The transposable element is generally composed of two oligonucleotide strands that are complementary, one of the oligonucleotide strands being referred to as the "transfer strand" and the other oligonucleotide strand being referred to as the "non-transfer strand". In an in vitro transposition reaction, the 3 'end of the transferred strand is covalently linked to the 5' end of the target DNA or transferred to the target DNA; while the non-transferred strand complementary to the transferred strand is not covalently linked to or transferred to the target DNA in an in vitro transposition reaction. The transposable element may optionally contain other sequences at the 5 'end of the transferred strand and/or at the 3' end of the non-transferred strand, in addition to the transposon end sequence (i.e., the minimal double stranded DNA fragment capable of forming a transposition complex with the transposase and initiating the transposition reaction), without affecting the transposition event. In a preferred embodiment, the transfer strand of the transposable element has the sequence: 5'AGATGTGTATAAGAGACAG 3' (SEQ ID NO: 3); the non-transferred strand has the sequence: 5'CTGTCTCTTATACACATCT 3' (SEQ ID NO: 4).

Herein, a transfer strand refers to an oligonucleotide strand in a transposable element that is transferred to a target nucleic acid molecule in a transposition reaction; a non-transferred strand refers to an oligonucleotide strand that is complementary to a transferred strand in a transposable element but is not transferred to a target nucleic acid molecule in a transposition reaction.

Herein, the transposition reaction refers to a reaction in which a transposition complex (also referred to herein as a "transposome") formed by a transposase and an at least partially double-stranded DNA molecule containing a transposable element transfers and ligates the DNA molecule containing the transposable element to the fragmented target DNA molecule while fragmenting the target DNA molecule. For more details on transposition reactions, see US2010/0120098a1, which is incorporated herein by reference in its entirety. The essential components of the transposition reaction are a transposase and a DNA nucleic acid molecule containing a transposable element. After the transposition reaction, the DNA nucleic acid strand containing the non-transferred strand that is not linked to the target nucleic acid can be removed from the product DNA molecule by displacement using a DNA polymerase having a strand displacement activity, and simultaneously the end of the product DNA molecule is filled in, as described in US2010/0120098a 1. As schematically shown in FIGS. 1 and 3, in some embodiments of the present invention, a transposition reaction may be employed to fragment a target DNA molecule while simultaneously ligating a circularizing adaptor carrying a transposable element to the resulting DNA fragment to obtain a circularized adaptor-carrying target DNA fragment that can be used in step (ii) of the method of the present invention.

In this context, TA tail ligation refers to the ligation of two molecules by base complementation of T and A between a molecule with a protruding "T" tail at the 3 'end and another molecule with a "A" tail at the 3' end. In this context, TA tail ligation is intended to refer to such a manner of ligation between two molecules, and not to the purpose of molecular ligation or the nature of the molecules being ligated. In other words, reference to two molecules being linked by a TA-tail linkage strategy only means that the two molecules have complementary T/a tails and are linked via this complementarity. As schematically shown in FIGS. 1 and 4, in some embodiments of the invention, TA tail ligation may be used to ligate a circularizing linker with a 3'-T tail to a DNA molecule to be circularized with a 3' -A tail. As a known general approach, a target nucleic acid can be treated with a DNA polymerase having terminal transferase activity (e.g., Taq DNA polymerase) to add a single template-independent A residue at the 3' end of blunt-ended double-stranded DNA.

In this context, the term "auxiliary sequence region" in connection with the circularized linker of the invention refers to an oligonucleotide of 0 to 75bp in length. When the helper sequence region is 0bp, as understood by those skilled in the art, it means that the circularized linker according to the present invention does not comprise the helper sequence region. When the auxiliary sequence region is greater than 1bp, for example 10-75bp, as understood by the person skilled in the art, it means that the circularization adapter according to the present invention comprises an auxiliary sequence region, which in this case may be single-stranded or double-stranded or partially double-stranded and consists of an auxiliary sequence located on the long oligonucleotide chain of the circularization adapter alone (single-stranded auxiliary sequence region) or with a complementary auxiliary sequence located on the short oligonucleotide chain that is fully or partially complementary thereto (double-stranded or partially double-stranded auxiliary sequence region).

I. The method of the invention

Based on the intensive research, the present inventors propose a method for achieving efficient circularization of a DNA molecule by using a specially optimized circularization adaptor containing U bases to generate a large fragment of DNA having AT cohesive ends and further combining restriction enzymes capable of recognizing double-stranded DNA and generating 3' AT cohesive ends.

Accordingly, in one aspect, the present invention provides a DNA circularization ligation method, characterized in that the method comprises the steps of:

wherein the circularization linker is composed of a long 5' phosphorylated oligonucleotide chain and a short 5' phosphorylated oligonucleotide chain complementary thereto, wherein the first base at the 5' end of the long oligonucleotide chain is an A base, and the second base is a U base; and is

(ii) (ii) treating the target DNA fragment with circularized adaptor resulting from step (i) with uracil specific excision reagents to produce DNA fragments with 3' AT sticky ends on both ends;

(iii) (iii) treating the DNA fragment resulting from step (ii) with a restriction enzyme to produce an digested DNA fragment, wherein the restriction enzyme is capable of recognizing double-stranded DNA and producing a 3' AT sticky end;

Some examples of the process of the present invention are schematically illustrated in fig. 1. Some examples of cyclized linkers of the present invention are schematically illustrated in FIG. 2.

In step i) of the invention, blunt-ended large fragment DNA molecules of AU:: TA dinucleotide pairs with inverted repeats at both ends will be generated by the use of the circularized linkers of the invention. In this step, ligation of the circularized linker can be performed using various means known in the art, but ligation using transposition reaction and ligation using T/A tail complementation are preferred. Accordingly, the portion of the circularized linker used for ligation to the target DNA fragment (also referred to herein as an "internal ligation region") can be designed based on the choice of the manner of ligation. After the circularized linker ligation reaction, the reaction product is preferably purified to remove, for example, enzymes and reagents used for the ligation reaction.

In step ii) of the present invention, the U base in the molecule of the adapter-bearing DNA product from step (i) may be cleaved using uracil-specific cleavage reagents to form a single nucleotide nicked intermediate. The remaining nucleic acid sequence from the nick to the 5 'end (i.e., single base A) can dissociate from the original complementary strand without high temperature due to the unstable structure of the intermediate product, thereby forming complementary 3' AT cohesive end structures AT both ends of the product. In this step, the uracil-specific excision reagents used to excise the U bases can be any reagent that is capable of specifically recognizing and excising uracil bases in a double-stranded DNA molecule and leaving a single nucleotide gap, e.g., USER enzyme and UNG enzyme. The USER enzyme is a mixture of Uracil DNA Glycosylase (UDG) and DNA glycosylase-lyase Endo VIII. Preferably, step ii) of the present invention is carried out, for example, at about 20-45 deg.C, e.g., about 30-45 deg.C, e.g., about 31 deg.C, about 32 deg.C, about 33 deg.C, about 34 deg.C, about 35 deg.C, about 36 deg.C, about 37 deg.C, about 38 deg.C, about 39 deg.C, about 40 deg.C, about 41 deg.C, about 42 deg.C, about 43 deg.C, about 44 deg.C, about 45 deg.C. The reaction time of step ii) may vary depending on the uracil-specific cleavage reagent used. In one embodiment, in step ii) using the USER enzyme, the reaction is at about 30 ℃ to 45 ℃ for at least 20 minutes, e.g., 20 to 50 minutes, such as 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, or a length of time between any two. After the USER treatment, the reaction product is preferably purified, for example, using magnetic beads, to minimize interference with subsequent DNA circularization reactions.

In the process of fragmentation of genomic DNA, a mixture of DNA fragments with varying sizes is typically produced. In intensive studies, the inventors found that the intramolecular ligation efficiency of the method can be further improved by fragmenting a large part of the DNA into smaller fragments by reprocessing the DNA fragment mixture generated in step (ii) with a restriction enzyme capable of recognizing double-stranded DNA and generating a 3' AT sticky end before circularization (see examples of the present application). Therefore, the method of the present invention further comprises, after the step (ii) of the present invention, a step of treating the DNA fragment with a restriction enzyme. The restriction enzyme recognizes the double-stranded DNA of step (ii) and generates a 3'AT sticky end, thereby ensuring that the resulting cleavage fragment also has a 3' AT palindromic complementary structure AT both ends. As shown in fig. 1-4. Preferably, the restriction enzyme has a restriction recognition site of 6-8 bp. More preferably, the restriction enzyme is selected from the group consisting of: PvuI, PacI, AsiSI, BstKTI, RgaI, and SfaAI, and it is further preferred that the restriction enzyme is PvuI.

In step (iv) of the present invention, the DNA fragment molecule having the palindromic 3' AT cohesive ends may be circularized under conditions that allow for intramolecular circularization. In order to avoid the influence of intermolecular linkage, a cyclization reaction system may be prepared, and the concentration of the molecule to be cyclized may be controlled at a low level. The ligase system used for the cyclization reaction is not limited. The conditions of the cyclization reaction can be determined according to the ligase system used. For example, the reaction can be carried out using the T4 ligase system at, for example, about 20-30 deg.C (e.g., 25 deg.C) for about 10-20 hours (e.g., about 12 hours).

Optionally, after the circularization reaction of step (iv), a step of digesting the linear DNA molecules that have not undergone circularization may be included. The means for effecting digestion of the linear DNA molecule is not limited. The reaction conditions can be readily determined depending on the digestion mode chosen. For example, a combination of Exonuclease I (Exonuclease I enzyme) and ATP-dependent DNase may be used. For example, using the enzyme combination, the reaction is carried out at about 30-40 ℃ for about 30 minutes or more, and at about 70-75 ℃ for about 10-30 minutes.

In an embodiment of the method according to the invention, the target DNA fragment molecule to be circularised in step i) preferably has a larger fragment size, e.g.a fragment size of 1kbp to 200kbp, e.g.4 kbp,5kbp,6kbp,7kbp,8kbp,9kbp,10kbp,11kbp,12kbp,13kbp,14kbp,15kbp,16kbp,17kbp,18kbp,19kbp,20kbp or more, preferably 3kbp to 30kbp, more preferably 5kbp to 20kbp, more preferably 6kbp to 15kbp, still more preferably 8kbp to 12 kbp.

The circularization efficiency of the method of the present invention can be determined by quantifying the molecular weight of the circularized DNA after the circularization reaction and comparing it with the amount of DNA put before the start of the circularization reaction. Circularization efficiency can be expressed as the percentage of the circularized DNA molecular weight to the DNA input. The method for quantifying the cyclized molecular weight is not limited. For example, digestion of the residual linear DNA in the cyclization reaction can be followed by DNA quantification using a Qubit kit.

As shown in the examples of the application, compared with the introduction of the GC-containing multi-base sticky end, the invention combines the additional restriction enzyme treatment before cyclization by using the optimized U-base-containing linker to complete the cyclization and ligation of the large-fragment DNA, thereby not only being simpler and easier in operation process, but also improving the ligation efficiency and the proportion of the target cyclized DNA product, and the overall cyclization efficiency of the large-fragment DNA reaches about 25 percent on average.

Without wishing to be bound by theory, it is believed that in the method of the present invention, the 5 'single base a remaining after USER treatment has fewer hydrogen bonds formed by the complementation of 2 bases (a and T) and small intermolecular forces between the bases and the complementary strand, resulting in a very unstable structure of the intermediate molecule having a gap, and thus the residual a is sufficiently dissociated from the original complementary strand AT normal temperature without high temperature denaturation, thereby releasing the 3' AT cohesive end on the complementary strand. It is presumed that sufficient release of such cohesive ends is more favorable for intramolecular cyclization, compared to a scheme in which a U-containing linker is used to generate a multi-base cohesive end (for example, a linker with 5' -terminal ACGU as shown in comparative example 1). While the specific restriction enzyme treatment prior to circularization can further promote intramolecular circularization of the sticky end of the 3' palindromic AT with 2bp by controlling the size of the DNA fragment to some extent (as shown in examples 2 and 3).

Therefore, the large-fragment DNA circularization connection method of the present invention has at least the following advantages:

(1) the operation flow is simple and easy to implement;

(2) the ligation efficiency and the occupation ratio of the target circularized DNA product are high;

(3) the cyclized linker disclosed by the invention can be compatible with various sequencing linker adding modes, and can be applied to a transposase system and a non-transposase system;

(4) the cyclized linker of the invention can flexibly adapt to the construction needs of various sequencing libraries by adding various auxiliary sequence regions.

In combination with the aforementioned advantages, the method of the invention will be particularly useful for forming circularized large fragment DNA molecules required in sequencing library construction. Thus, in some preferred embodiments, the present invention provides methods for the production of circularized large fragment DNA molecules, particularly circularized DNA molecules for use in sequencing libraries, e.g., in end-pairing sequencing library construction,

wherein the circularization linker is composed of a long oligonucleotide chain and a complementary short oligonucleotide chain, and the linker has the following structure according to the 5 'to 3' direction of the long oligonucleotide chain:

cohesive end-forming region-auxiliary sequence region-internal joining region,

wherein said sticky end generating region consists of the 5' -most AU dinucleotide on the long oligonucleotide strand, or of this dinucleotide and a dinucleotide AT complementary to it on the short oligonucleotide strand in the reverse direction,

wherein the internal joining region is a 10-50bp double-stranded or partially double-stranded nucleic acid region determined by the joining means of the adaptor to the target nucleic acid,

wherein, in embodiments in which circularized linker ligation is achieved by a transposition reaction, the internal linking region comprises a transposable element, e.g., of Tn5 transposase; or

Wherein, in embodiments wherein circularized linker ligation is achieved by TA tail ligation, the internal linking region comprises 3 'from the long oligonucleotide chain'

A 3'T overhang formed by the endmost T residue (e.g., a single residue 3' T overhang);

wherein the auxiliary sequence region is 0-75bp in length, i.e. may be present or absent, wherein, in the presence of the auxiliary sequence region, the auxiliary sequence region is a single-stranded or double-stranded or partially double-stranded oligonucleotide selected on the basis of the application of the molecule to be circularised, e.g. an oligonucleotide which may be used for identifying, detecting (e.g. fluorescence detection, biotin detection) or sorting the products of the method of the invention; for example, helper sequences for sequencing pooling, e.g., index (index) or barcode (barcode) nucleic acids or sequencing primer binding sites.

Other means of joining the linkers known in the art are also contemplated by the present invention. For example, the linker can be attached to the target nucleic acid by other sticky ends. In such embodiments, the internal linking region will comprise a single stranded complementary overhang that is complementary to a single stranded overhang on the target nucleic acid.

The cyclized linker used in the methods of the invention can carry a conjugate, e.g., an affinity tag. The affinity tag may be one of a pair of binding pair members. Examples of binding pairs that may be mentioned include, but are not limited to: biotin-avidin, biotin-streptavidin, ligand-receptor, antigen-antibody. For example, the circularized linker may be formed by including one member of a binding pair on the circularized linker; and the other member of the binding pair is included on, for example, a capture reagent immobilized on a solid phase, thereby facilitating isolation of the DNA molecule with the circularized linker.

Transposition cycle

In a further preferred embodiment of the present invention, the process of the present invention comprises the steps of:

1) fragmenting a target DNA (preferably genomic DNA) by transposase and simultaneously adding circularizing adapters according to the present invention to both ends of the generated target DNA fragment;

2) the target DNA fragment with the linker is subjected to a strand displacement reaction (for example, using an enzyme having a strand displacement activity such as Klenow large fragment) to fill in the ends;

3) digesting with uracil specific excision reagent (preferably USER enzyme) to obtain DNA fragments with two ends containing 3' AT sticky ends;

4) (iii) treating the DNA fragment resulting from step (ii) with a restriction enzyme, wherein the restriction enzyme is capable of recognizing double-stranded DNA and generating a 3' AT sticky end;

5) performing cyclization and connection on the obtained DNA fragment containing the cohesive end to obtain a cyclization DNA molecule;

6) optionally digesting the non-circularized linear DNA molecule to obtain said circularized DNA molecule.

Transposases useful in the present invention are not particularly limited, and include, but are not limited to: mu, Mu E392Q, Tn5, superactive Tn5(Goryshin and Reznikoff, J.biol.chem.,273:7367(1998)), EZ-Tn5 ^TM Transposases (Epicentre Biotechnologies, Madison, Wisconsin), Tn5 variants, RAG, Tn7, Tn10, Vibhar transposases, and Tn 552. Variants of Tn5 transposase are disclosed in U.S. patents 5,925,545; 5,965,443, respectively; 7,083,980, respectively; 7,608,434, respectively; and U.S. patent application 14/686,961. These patents and patent applications are incorporated herein by reference. Preferably, the transposase used in the method of the invention is selected from the group consisting of: tn5, superactive Tn5, and EZ-Tn5 ^TM A transposase; in particular EZ-Tn5 ^TM A transposase.

Transposable elements useful in the present invention can comprise any nucleic acid suitable for forming a functional complex with a transposase in an in vitro transposition reaction. Examples of transposable elements include, but are not limited to, transposon end sequences that can be recognized by wild type or mutant Tn5 transposases, e.g., 19bp Outside End (OE) transposon ends, Inside End (IE) transposon ends, or chimeric end (ME) transposon ends, or R1 and R2 transposon ends. (see, e.g., US2010/0120098, which is hereby incorporated by reference).

The circularized linker according to the present invention used in the above method preferably consists of a long oligonucleotide chain phosphorylated at the 5 'end and a short complementary oligonucleotide chain phosphorylated at the 5' end,

wherein, according to the 5 'to 3' direction of the long oligonucleotide chain, the linker has the following structure:

cohesive end-forming region-auxiliary sequence region-internal joining region,

wherein the sticky end forming region consists of AU dinucleotides at the 5 '-most end of the long oligonucleotide chain and forms a 5' -single-stranded overhang of the linker,

wherein the internal joining region has or consists of a transposable element double-stranded oligonucleotide at its 3' end,

wherein the auxiliary sequence region is 0-75bp in length, i.e., may be present or absent.

In some embodiments, the linker comprises an auxiliary sequence region, e.g., an oligonucleotide comprising a barcode sequence, located between the sticky end generating region and the internal linking region.

A preferred illustrative example of this embodiment is shown schematically in fig. 3.

In a preferred embodiment, the circularized adapter molecule is a double stranded oligonucleotide molecule having a 5 'overhang and a 3' blunt end, in the 5 'to 3' direction of the long oligonucleotide chain, and wherein

(i) The long oligonucleotide chain consists of a transfer chain of AU dinucleotides, auxiliary sequences and transposable elements from the 5 'to 3' direction; the short strand of oligonucleotide chain consists of non-transferred strand of transposable element and complementary auxiliary sequence from 5 'to 3' direction, wherein the auxiliary sequence and complementary auxiliary sequence form double-stranded or partially double-stranded auxiliary sequence region;

(ii) the long oligonucleotide chain consists of a transfer chain of AU dinucleotides, auxiliary sequences and transposable elements from the 5 'to 3' direction; the short strand of the oligonucleotide chain consists of the non-transferred strand of the transposable element in the 5 'to 3' direction, wherein the auxiliary sequence forms a single-stranded auxiliary sequence region; or

(iii) Preferably, the long oligonucleotide chain consists of a transferred strand of AU dinucleotides and transposable elements in the 5 'to 3' direction; the short strand of oligonucleotide chain consists of the non-transferred strand of the transposable element.

In a more preferred embodiment, the transposable element is 19bp of the transposon end sequence of Tn5 transposase, and preferably:

the transfer strand of the transposable element has the sequence: 5'P-AGATGTGTATAAGAGACAG-3' (SEQ ID NO:3), and

the non-transferred strand of the transposable element has the sequence: 5'P-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 4).

In some embodiments, circularized linkers according to the present invention may comprise an auxiliary sequence region located between the single-stranded AU dinucleotide cohesive end generating region and the transposable element double-stranded oligonucleotide. The length of the auxiliary sequence region can be between 10 and 30 bp. The sequence of the desired auxiliary sequence region can be selected by the person skilled in the art depending on the specific application of the linker molecule. For example, in sequencing applications, barcode or index sequences of, for example, 10-15bp can be provided by the helper sequence region for identification and grouping of target DNA fragments.

In a preferred embodiment, the circularizing adaptor used in the above method therefore consists of a long oligonucleotide chain phosphorylated at the 5 'end and a short complementary oligonucleotide chain phosphorylated at the 5' end, wherein,

the long oligonucleotide chain has the sequence: 5'P-AU (N) AGATGTGTATAAGAGACAG-3'; and the short oligonucleotide strand has the sequence: 5' P-CTGTCTCTTATACACATCT (N ') -3',

wherein N is a nucleotide chain of 0-75bp, for example 0-50bp,

n 'is the complement of N, either wholly or partially, or N' may be absent,

preferably, both N and N' are absent, and the long oligonucleotide chain has the sequence of SEQ ID NO 1; and the short oligonucleotide chain has the sequence of SEQ ID NO 2.

In a further preferred embodiment, the circularizing linker therefore consists of a long oligonucleotide chain phosphorylated at the 5 'end and a short complementary oligonucleotide chain phosphorylated at the 5' end, wherein,

the long oligonucleotide chain has the sequence (SEQ ID NO: 1): 5 'P-AUAGATGTGTATAAGAGACAG-3';

the oligonucleotide short strand has the sequence (SEQ ID NO: 2): 5 'P-CTGTCTCTTATACACATCT-3'.

In some embodiments according to the invention, the circularized linker may carry a label, e.g., an affinity label, such as a biotin label, that facilitates identification, detection and/or purification of the product molecule (e.g., circularized molecule). However, it will be appreciated by those skilled in the art that the presence of such a label is not essential for circularisation of the molecule, although in some cases the inclusion of such a label may be advantageous for subsequent processing of the circularised molecule.

In some embodiments, the label may be coupled or conjugated to a nucleotide of an internal linking region (e.g., a transfer strand of a transposable element) or an auxiliary sequence region of the circularized linker, and preferably, the label is conjugated to a long oligonucleotide chain. In a preferred embodiment, the circularizing linker comprises a transposable element transfer strand carrying a biotin tag, such as the sequence: AGATGTGTA(Biotin) TAAGAGACAG are provided.

In a more preferred embodiment, the circularizing linker is composed of a long oligonucleotide chain and a short complementary oligonucleotide chain, wherein,

the long oligonucleotide chain has the nucleotide sequence shown in SEQ ID NO: biotinylation sequence of 5: 5'P-AUAGATGTGTA(Biotin) TAAGAGACAG-3'; and the short oligonucleotide strand has the sequence of SEQ ID NO: 2: 5 'P-CTGTCTCTTATACACATCT-3'.

TA Tail ligation cyclization

The method for fragmenting genome DNA can be realized by physical disruption (a DNA fragmenting instrument and the like) and ordinary DNA fragmentation enzyme cutting besides transposition reaction and joint. DNA fragmented by this method can be used for circularization linker ligation and subsequent uracil nucleic acid treatment and circularization steps after DNA fragment end repair and A addition.

In a preferred embodiment of the present invention, the process of the present invention therefore comprises the steps of:

1) obtaining blunt-end target DNA fragments (e.g., large fragments of genomic DNA obtained by physical or enzymatic cleavage and end repair), adding a 3' A tail by terminal transferase;

2) connecting the cyclic adapters to two ends of the target DNA fragment by using TA tail connection;

3) digesting the DNA fragment with the linker by using uracil specific excision reagent (preferably USER enzyme) to obtain DNA fragments with 3' AT cohesive ends AT two ends;

5) performing circularization connection on the obtained DNA fragment containing the cohesive end to obtain a circularized DNA molecule;

6) optionally, digesting the non-circularized linear DNA molecule to obtain said circularized DNA molecule.

The circularization linker according to the present invention for use in the above method preferably consists of a long oligonucleotide chain phosphorylated at the 5 'end and a short complementary oligonucleotide chain phosphorylated at the 5' end, wherein the linker has the following structure in the 5 'to 3' direction of the long oligonucleotide chain:

cohesive end-forming region-auxiliary sequence region-internal joining region,

wherein the cohesive end forming region is composed of a dinucleotide pair formed by complementing a dinucleotide at the 5 '-most end of the long oligonucleotide chain with a dinucleotide at the 3' -most end of the short oligonucleotide chain,

5'-A U-3'

3'-T A-5'

wherein the internal linking region has a 3' T overhang (i.e., a 3' T tail) formed by the 3' endmost T residue of the long oligonucleotide chain, and

wherein the auxiliary sequence region is 0-75bp in length, i.e., can be present or absent.

In some embodiments, the internal linking region can have internal linker sequences for an MP sequencing library, e.g., a circularized linker (CA) sequence included in the Roche454 system and an internal linker (IA) sequence included in the SOLiD system.

In some embodiments, the linker further comprises an auxiliary sequence region, e.g., an oligonucleotide comprising a barcode sequence, located between the sticky end generating region and the internal circularizing attachment region.

A preferred illustrative example of this embodiment is shown schematically in fig. 4.

In a preferred embodiment, the circularizing linker is composed of a long oligonucleotide chain and a short complementary oligonucleotide chain, wherein,

the long oligonucleotide chain has the sequence: 5'P-AU- (N) -T-3';

the oligonucleotide short strand has the sequence: 5' P- (N ') -AT-3 ';

wherein N and N ' are complementary oligonucleotides of 10-50bp, e.g.about 15-30bp, wherein the 3' -most T of the long oligonucleotide chain forms a single base 3' -overhang of the adaptor.

Cyclized linker

In a further aspect, the present invention provides a circularization linker useful in the DNA circularization method of the present invention, which is a double-stranded oligonucleotide molecule composed of a long oligonucleotide chain and a short oligonucleotide chain complementary thereto, wherein the first base at the 5' end of the long oligonucleotide chain is an A base and the second base is a U base. Preferably, the linker molecule comprises a duplex region consisting of the short oligonucleotide strand and the long oligonucleotide strand portion complementary thereto and one or two overhangs. Preferably, the linker molecule has an overhang consisting of long-chain nucleotides of the oligonucleotide and a blunt end.

In one embodiment, the adaptor molecule is a double-stranded oligonucleotide molecule having a 5 'overhang of at least 2bp and a 3' blunt end, in the 5 'to 3' direction of the long oligonucleotide chain; in yet another embodiment, the adaptor molecule is a double stranded oligonucleotide molecule having a 5 'blunt end and a 3' overhang of at least 1 bp. In still other embodiments, the linker molecule comprises a duplex region of at least 15bp, such as 19-30bp, formed by the complementarity of the short oligonucleotide strand to at least a portion of the long oligonucleotide strand.

Preferably, the circularized linker of the present invention is composed of a long oligonucleotide chain and a short oligonucleotide chain complementary thereto, wherein the linker has the following structure in the 5 'to 3' direction of the long oligonucleotide chain:

cohesive end-forming region-auxiliary sequence region-internal joining region,

wherein the internal linking region is a 10-30bp double-stranded or at least partially double-stranded nucleic acid region for ligation of the adaptor to the target nucleic acid,

wherein the helper sequence region is 0-75bp long, i.e., may be present or absent, in some embodiments the helper sequence region is 0bp, and the linker does not contain a helper sequence region; in another embodiment, the adaptor comprises an auxiliary sequence region, and the auxiliary sequence region is a single-stranded or double-stranded or partially double-stranded oligonucleotide of 10-75 bp.

Preferably, the cyclized linker according to the invention has at least one, preferably two, phosphorylated 5' ends.

In one embodiment, the internal linking region comprises a double-stranded oligonucleotide of a transposable element, e.g., a double-stranded oligonucleotide of a transposable element of Tn5 transposase. Thus, the circularized adapters can be used to ligate to target molecules in a transposition reaction to produce target nucleic acid molecules with circularized adapters at both ends.

In another embodiment, the internal linking region has a 3'T overhang formed by the 3' endmost T residue of the long oligonucleotide chain. Thus, the circularized adapters can be used to ligate to target molecules in a TA tail ligation strategy to produce target nucleic acid molecules with circularized adapters at both ends.

In some embodiments, the circularized linker according to the present invention further comprises a helper sequence region. The sequence of the auxiliary sequence region may be determined based on the intended use of the molecule to be circularized. In order to facilitate the in vitro transposition reaction on the target DNA, the length of the helper sequence region located at the 5' end of the transferred strand is preferably less than 75bp, or 50bp, or 25 bp.

In some preferred embodiments, the circularized adapters according to the present invention are used for generation of circularized molecules in sequencing library construction.

The circularised linker according to the present invention may have a length of 10-100bp, e.g. 15bp,20bp,25bp,30bp,35bp,40bp,45bp,50bp,55bp,60bp,65bp,70bp,75bp,80bp,85bp,90bp,95bp,100bp, or a length between any two of said lengths, preferably 20-40bp long, e.g. 21bp long. However, it will be apparent to those skilled in the art that the circularized linkers of the present invention may also be longer, e.g., greater than 120bp, greater than 150bp, as desired.

In some preferred embodiments, circularized adapters according to the present invention are double stranded oligonucleotide adapter molecules with a 5 'overhang of at least 2bp, in the 5' to 3 'direction of the long oligonucleotide chain, wherein the first base of the 5' overhang is an a base and the second base is a U base. More preferably, the adaptor molecule is a double stranded DNA molecule having a 5 'overhang and a 3' blunt end, wherein the duplex region of the adaptor molecule is at least 19bp long. Still preferably, the adaptor molecule consists of an AU dinucleotide 5' overhang and a 19bp duplex region.

In some further preferred embodiments, the linker molecule consists of a long oligonucleotide chain and a short oligonucleotide chain, wherein, in the 5 'to 3' direction,

the long oligonucleotide chain has the sequence: AU dinucleotide + optional helper sequence + transfer strand of transposable element;

the oligonucleotide short strand has the sequence: a transposable element complementary to the transferred strand, a non-transferred strand + optionally a complementary helper sequence complementary to all or part of the helper sequence;

preferably, both the long oligonucleotide chain and the short oligonucleotide chain have a phosphorylated 5' end.

In some embodiments, the transfer strand of the transposable element has the nucleotide sequence set forth in SEQ ID NO 3; the non-transferred strand of the transposable element has a nucleotide sequence shown in SEQ ID NO. 4.

In a preferred embodiment, the circularizing linker therefore consists of a long oligonucleotide chain phosphorylated at the 5 'end and a short complementary oligonucleotide chain phosphorylated at the 5' end, wherein,

wherein N is a nucleotide chain of 0-75bp, for example 0-50bp,

n 'is the complement of N, either wholly or partially, or N' may be absent,

In other embodiments, the linker molecule is conjugated to a label, e.g., an affinity label, such as a biotin label. In a preferred embodiment, the long oligonucleotide chain constituting the linker has the sequence of SEQ ID NO: biotinylation sequence of 5: 5'P-AUAGATGTGTA(Biotin) TAAGAGACAG-3'; and the short oligonucleotide strand has the sequence of SEQ ID NO: 2: 5 'P-CTGTCTCTTATACACATCT-3'.

In other embodiments, the circularized adapters according to the present invention are double stranded oligonucleotide adapter molecules with 3' overhangs of at least 1bp, in the 5' to 3' direction of the long oligonucleotide chain. More preferably, the linker molecule is a double-stranded DNA molecule having a 3 'mutated end and a 5' blunt end, in the 5 'to 3' direction of the long oligonucleotide chain, wherein the blunt end has a pair of dinucleotides formed by the complementation of the 5 'endmost dinucleotide of the long oligonucleotide chain with the 3' endmost dinucleotide of the short oligonucleotide chain,

5'-A U-3'

3'-T A-5'

and wherein, preferably, the 3 'mutant end is formed by the 3' endmost T residue of the long oligonucleotide chain, i.e., the 3 'mutant end is a single base 3' T mutant end.

In other embodiments, the circularizing adaptor has a duplex region between the 5 'blunt end and the 3' overhang of 10-50bp long. Still preferably, the adaptor molecule has a duplex region of 20-30bp, e.g. 21 bp. This duplex of the circularized linker may comprise an auxiliary sequence region suitable for sequencing library construction or sequencing requirements. For example, in one embodiment, the circularized linker comprises an internal linker sequence for an MP sequencing library, e.g., a circularized linker (CA) sequence included in the Roche454 system and an internal linker (IA) sequence included in the SOLiD system; and/or include barcode sequences or index sequences.

Sequencing library construction

In a further aspect of the invention, the invention provides the use of a method and circularised linker molecule according to the invention in the construction of a sequencing library involving the generation of circularised molecules.

In one embodiment, therefore, the present invention provides a method of constructing a sequencing library (e.g., a Mate-pair sequencing library), comprising:

(a) obtaining a circularized DNA molecule with a circularized linker by the method of the present invention;

(b) fragmenting the circularized DNA fragments and capturing the DNA fragments with the (biotin-labeled) circularized adaptors (e.g.using streptavidin magnetic beads);

(c) and amplifying the captured DNA fragment to establish a DNA library for sequencing.

In some embodiments, a circularised linker according to the invention with a helper sequence region may be employed in step (a) for e.g. sequencing purposes. The helper sequence region may comprise an oligonucleotide selected from: molecule index sequence, anchor site, reporter molecule label, bar code sequence and primer combining site. The primer binding site may be a sequencing primer site, which may include a nucleic acid sequence for annealing a sequencing primer to a sequencing library nucleic acid molecule in a sequencing reaction. The anchor site may comprise a sequence complementary or substantially complementary to the capture probe; or may comprise a member of a binding pair, for example one of biotin/streptavidin, or one of a ligand/receptor, conjugated/coupled to a nucleotide of the anchor site. Useful reporter labels may be reporter labels capable of signaling, e.g., fluorescence, chemiluminescence, bioluminescence, etc., e.g., nucleotides and/or oligonucleotides conjugated or conjugated to fluorophores, chromophores, radioisotopes, etc.

In a preferred embodiment, the helper sequence region comprises a barcode sequence to identify the target nucleic acid molecule.

In still further aspects, the invention also provides circularized DNA molecules and sequencing library compositions that can be produced by the methods of the invention.

In further aspects, the invention provides a kit comprising a circularised linker molecule of the invention and a restriction enzyme of the invention, and the use of said kit for preparing circularised DNA molecules and/or for constructing a sequencing library, preferably a Mate-pair sequencing library.

Examples

The present invention will be described in further detail with reference to the following embodiments. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present invention have not been shown or described in detail to avoid obscuring the present invention from too much description, and a detailed description of such related operations may not be necessary for a person of ordinary skill in the art because such related operations are fully understood from the description of the present application and from the knowledge of one of ordinary skill in the art.

Example 1:

cyclized linker preparation

Providing a circularized linker comprising a sense strand (SEQ ID NO: 5) and an antisense strand (SEQ ID NO: 2), wherein both the sense strand and the antisense strand are phosphorylated at 5 'end, and the second base at 5' end of the sense strand is U base, and the linker sequence is shown in the following Table 1:

TABLE 1

Step 1: genomic DNA extraction

1ml of whole blood of a healthy person is taken, and genome DNA is extracted by adopting a genome DNA extraction kit (manufacturer: Kangji century goods number: CW 2087). No significant degradation of DNA was detected by 0.8% agarose gel electrophoresis.

And 2, step: annealing of joints

The reaction solution was prepared as follows:

and (3) fully and uniformly mixing the prepared reaction liquid, and applying a reaction program: at 98 ℃ for 2 min; 15 cycles (95 ℃, 2min, (-5 ℃/cycle)); keeping at 4 ℃; to obtain an annealed joint.

And step 3: transposome assembly

The reaction solution for transposome assembly was prepared as follows:

and (3) fully and uniformly mixing the prepared reaction liquid, wherein the reaction procedure is as follows: 35 ℃ for 1 h; keeping at 4 ℃.

And 4, step 4: transposition reaction

The reaction system shown in the following table was prepared:

and (3) fully and uniformly mixing the prepared reaction liquid, wherein the reaction procedure is as follows: incubate at 55 ℃ for 15 min. After completion, the cells were purified using a Zymo Genomic DNA & Concentrator kit (cat # D4011), and eluted with 30ul of an elution buffer. After this transposition reaction, the genomic DNA is fragmented and the linker is added. The fragmented DNA was detected by 0.8% agarose gel electrophoresis to determine the DNA size.

And 5: strand displacement reaction

The reaction system shown in the following table was prepared:

and (3) fully and uniformly mixing the prepared reaction liquid, wherein the reaction procedure is as follows: incubate at 25 ℃ for 15min and at 75 ℃ for 20 min.

Step 6: sticky end generation

The reaction system shown in the following table was prepared:

and (3) fully and uniformly mixing the prepared reaction liquid, wherein the reaction procedure is as follows: incubate at 37 ℃ for 30 min. During the reaction, the product can form a sticky end without high temperature denaturation. After the reaction, 40. mu.l of water was added, 40. mu.l (0.4X) of magnetic beads (Beckman Agencour AMPure XP beads, cat. No. A63880) were added for purification, wherein the reaction products were allowed to bind to the magnetic beads at room temperature for 15min, and mixed uniformly every 2min, after the incubation was completed, the magnetic beads were washed with 70% alcohol 2 times, the products bound to the magnetic beads were eluted with 30. mu.l of nuclease-free water, and the amount of Qubit was determined (Kit: dsDNA HS Assay Kit for Qubit, Q32851).

And 7: DNA cyclization

The reaction system shown in the following table was prepared:

and (3) fully and uniformly mixing the prepared reaction liquid, wherein the reaction procedure is as follows: incubate at 25 ℃ for 12 h.

And 8: linear DNA digestion

The reaction system shown in the following table was prepared:

adding the prepared reaction liquid into the reaction product obtained in the step (7), and fully and uniformly mixing, wherein the reaction procedure is as follows: incubation was carried out at 37 ℃ for 30min and at 75 ℃ for 10 min.

And step 9: quantitive of Qubit

Quantification was performed using a Kit (dsDNA HS Assay Kit for Qubit, Q32851), and the quantitative statistics are shown in Table 2;

TABLE 2

As is clear from Table 2, the circularization efficiency of the large fragment DNA completed in example 1 was about 20%.

Comparative example 1:

the assay uses a GC-containing 4-base sticky-end linker for control, which comprises a sense strand (SEQ ID NO: 7) and an antisense strand (SEQ ID NO:8), both of which are phosphorylated at 5 'end, and the fourth base at 5' end of the sense strand is U base, and the linker sequence is shown in Table 3 below:

TABLE 3

Genomic DNA extraction, transposome assembly, transposition reaction, strand displacement reaction, sticky end generation, DNA circularization, linear DNA digestion, and Qubit quantification were performed in the same manner as in example 1; however, in the sticky end formation step, a high temperature treatment which is more favorable for the detachment thereof is used in consideration of the longer length of 5' -ACG left after the USER enzyme treatment. As shown by the results below, even under this more favorable processing condition, the cyclization ligation efficiency of the control linker is still significantly lower than that of the linker of the present invention.

Specifically, 1ml of whole blood was taken from a healthy person and three sample experimental replicates were performed. The extraction of genomic DNA, linker annealing, transposome assembly, transposition reaction, and strand displacement reaction of steps 1 to 5 were carried out according to the same procedure as in example 1. Then, the following steps were performed as follows: sticky end formation, circularization and quantification.

Step 6: sticky end generation

The reaction system shown in the following table was prepared:

and (3) fully and uniformly mixing the prepared reaction liquid, wherein the reaction procedure is as follows: the mixture was incubated at 37 ℃ for 30min and at 75 ℃ for 15min and immediately placed on ice to break off the remaining short single strand at the 3' end, thereby forming a large DNA fragment with a sticky end. Adding water 40ul, purifying with magnetic bead 40ul (0.4X), mixing at room temperature for 15min, mixing every 2min, washing with 70% alcohol for 2 times, and eluting with 30ul nuclease-free water.

And 7: DNA cyclization

The same reaction system as in example 1, step 7 was prepared:

And 8: linear DNA digestion

The same reaction system as in step 8 of example 1 was prepared:

And step 9:quantitive of Qubit

Quantitative statistics were performed in the same manner as in step 9 of example 1. The results are shown in table 4 below.

TABLE 4

As is clear from Table 4, the circularization efficiency of the large fragment DNA completed in the control example was about 15%.

Example 2

Genomic DNA extraction, transposome assembly, transposition reaction, strand displacement reaction, sticky end generation, DNA circularization, linear DNA digestion, and Qubit quantification were performed in the same manner as in example 1; but a restriction enzyme treatment step is added before the DNA cyclization step. As shown in the following results, the restriction enzyme treatment further improved the intramolecular cyclization ligation efficiency of the method of the present invention.

Specifically, 1ml of whole blood of a healthy person was taken and two experimental replicates were performed. The genomic DNA extraction, adaptor annealing, transposome assembly, transposition reaction, strand displacement reaction, and sticky end generation of steps 1 to 6 were performed according to the same procedure as in example 1. Thereafter, the subsequent steps, enzyme digestion, cyclization and quantification were performed as follows.

And 7: enzyme digestion-enzyme linked DNA cyclization

The reaction system shown in the following table was prepared:

and (3) fully and uniformly mixing the prepared reaction liquid, wherein the reaction procedure is as follows: incubate at 37 ℃ for 5 min. After completion, 10ul of T4 DNA ligase and 3ul of ATP (100mM) were added and mixed well, and the reaction sequence: incubate at 37 ℃ for 12 h.

And 8: linear DNA digestion

The same linear DNA digestion reaction system as in step 8 of example 1 was prepared:

adding the prepared reaction liquid into the reaction product obtained in the step 7, and fully and uniformly mixing, wherein the reaction procedure is as follows: incubation was carried out at 37 ℃ for 30min and at 75 ℃ for 10 min.

And step 9: quantitive of Qubit

Quantitative statistics were performed in the same manner as in step 9 of example 1. The results are shown in table 5 below;

TABLE 5

As is clear from Table 5, the circularization efficiency of the large fragment DNA completed in example 2 was about 25%.

Example 3

This example controls the circularization efficiency of DNA fragments of different sizes of interest. After strand displacement reaction, test panel tapping recovered fragments of 8-12kbp in size; meanwhile, considering the influence of PvuI enzyme digestion on the sizes of the fragments of the test group, the control group is set to cut the rubber and recover the fragments of 5-8 kbp. Each of the test and control groups was set up with 3 biological replicates.

Specifically, linker sequences shown in table 1 are provided. The extraction of genomic DNA, linker annealing, transposome assembly, transposition reaction, and strand displacement reaction of steps 1 to 5 were carried out according to the same procedure as described in example 1. The strand displacement reaction product of step 5 was subjected to DNA fragment size screening, and the subsequent test group procedure and control group procedure as follows.

DNA fragment size selection

(1) Preparing 0.6% agarose gel;

(2) adding 50ul of DNA product after strand displacement into a sample buffer solution, loading for electrophoresis, and performing electrophoresis at 100V for 120 minutes;

(3) carefully cutting the agarose gel containing the target DNA fragment, cutting the sample with the size of 8-12kbp for the test group, and cutting the sample with the size of 5-8kbp for the control group;

(4) using the Zymoclean large fragment DNA recovery kit (D4046), the DNA fragment was recovered from the agarose gel and eluted with 30ul of elution buffer.

Test group flow:

step 6: sticky end generation

Preparing a reaction system:

and (3) fully and uniformly mixing the prepared reaction liquid, wherein the reaction procedure is as follows: incubate 30min at 37 ℃. After the end, 40ul of water is supplemented, 40ul (0.4X) of magnetic beads are added for purification, the mixture is combined for 15min at room temperature, the mixture is uniformly mixed every 2min, the mixture is washed with 70% alcohol for 2 times, 30ul of nuclease-free water is eluted, and the quantity of Qubit is quantified.

And 7: enzyme digestion-enzyme linked DNA cyclization

Preparing an enzyme digestion reaction system which is the same as the step 7 of the example 2:

And 8: linear DNA digestion

The same reaction system as in step 8 of example 2 was prepared:

adding the prepared reaction liquid into the reaction product obtained in the step (5), and fully and uniformly mixing, wherein the reaction procedure is as follows: incubation was carried out at 37 ℃ for 30min and at 75 ℃ for 10 min.

And step 9: quantitive of Qubit

Quantitative statistics were performed in the same manner as in step 9 of example 2. The results are shown in table 6 below.

TABLE 6

Control group procedure:

step 6: sticky end generation

Preparing a reaction system:

and (3) fully and uniformly mixing the prepared reaction liquid, wherein the reaction procedure is as follows: incubate at 37 ℃ for 30 min. After the end, 40ul of water is supplemented, 40ul (0.4X) of magnetic beads are added for purification, the mixture is combined for 15min at room temperature, the mixture is uniformly mixed every 2min, the mixture is washed with 70% alcohol for 2 times, 30ul of nuclease-free water is eluted, and the quantity of Qubit is quantified.

And 7: DNA cyclization

The following ligation reaction system was prepared:

And 8: linear DNA digestion

The same reaction system as in step 8 of example 2 was prepared:

And step 9: qubit quantification

Quantitative statistics were performed in the same manner as in step 9 of example 2. The results are shown in table 7 below.

TABLE 7

And (3) comparing results:

group of	Efficiency of cyclization
		Test set	24.3％
Control group	17.7％

As can be seen from the above table, the average cyclization efficiency of the large-fragment DNA cyclization in the control group is about 17.7%, which is lower than 24.3% of that in the test group.

Some embodiments of the invention

1. A DNA circularization ligation method, comprising the steps of:

(iv) (iv) incubating the DNA fragments resulting from step (iii) under conditions allowing circularization of said digested DNA fragments to obtain circularized DNA molecules,

optionally, after the circularization reaction of step (iv), the linear DNA molecules that have not undergone circularization are digested.

2. The method of embodiment 1, wherein said circularized linker, in the 5 'to 3' direction of said long oligonucleotide chain, has the following structure:

cohesive end-forming region-auxiliary sequence region-internal joining region,

wherein the sticky end forming region consists of the 5 '-most AU dinucleotide of the long oligonucleotide chain or the dinucleotide AT complementary to the 5' -most AU dinucleotide of the short oligonucleotide chain,

wherein the internal linking region is a 10-30bp double-stranded or at least partially double-stranded oligonucleotide for ligation of the circularized adaptor to the target nucleic acid,

wherein the auxiliary sequence region is a single-stranded or double-stranded or partially double-stranded oligonucleotide of 0bp, or 10-75 bp.

3. The method of embodiment 2, wherein said internal joining region comprises a double-stranded oligonucleotide of a transposable element.

4. The method of embodiment 2, wherein the internal linking region comprises a 3'T overhang formed by the 3' endmost T residue of the long oligonucleotide chain.

5. The method of embodiment 2, wherein said circularized linker further comprises a helper sequence region.

6. The method of embodiment 1, wherein said circularized linker comprises a duplex region of at least 19bp formed by complementarity of said short oligonucleotide strand to a portion of said long oligonucleotide strand, and said circularized linker has a length of 21bp-50 bp.

7. The method of embodiment 6, wherein the circularized adapter molecule is a double-stranded oligonucleotide molecule having a 5 'overhang of at least 2bp and a 3' blunt end, in the 5 'to 3' direction of the long oligonucleotide chain,

preferably, wherein the 5 'overhang comprises the endmost AU dinucleotide and the molecule has a transposable element double stranded oligonucleotide at its 3' endmost.

8. The method of embodiment 6, wherein the circularized adapter molecule is a double-stranded oligonucleotide molecule having a 5 'blunt end and a 3' overhang of at least 1bp in the 5 'to 3' direction of the long oligonucleotide chain,

preferably, wherein the 5' blunt end has a dinucleotide pair formed by the complementarity of the 5' endmost dinucleotide of the long oligonucleotide chain and the 3' endmost dinucleotide of the short oligonucleotide chain,

5'-A U-3'

3'-T A-5'

and wherein the 3 'overhang comprises the 3' endmost T residue of the long oligonucleotide chain, preferably the 3 'overhang is a 1bp 3' overhang consisting of a single T residue.

9. The method of any of embodiments 1-8, wherein the circularized linker has a conjugated label, e.g. an affinity label, especially a biotin label, preferably conjugated to a long oligonucleotide chain.

10. The method of any one of embodiments 1-9, wherein the long oligonucleotide chain has the sequence: 5'P-AU (N) AGATGTGTATAAGAGACAG-3'; and the short oligonucleotide strand has the sequence: 5' P-CTGTCTCTTATACACATCT (N ') -3',

wherein N is a nucleotide chain of 0-75bp, for example 0-50bp,

n ' is the complete or partial complement of N, or N ' may be absent, preferably, both N and N ' are absent,

still preferably, the long oligonucleotide chain is biotinylated at the 11 th A residue from the 3' end,

preferably, the long oligonucleotide chain has a sequence of SEQ ID NO 1 or 5; and the short oligonucleotide chain has the sequence of SEQ ID NO 2.

11. The method of any of embodiments 1-10, wherein the circularized linker comprises a long oligonucleotide chain and a short oligonucleotide chain, wherein the long oligonucleotide chain has the sequence: 5'P-AU- (N) -T-3'; and the short oligonucleotide strand has the sequence: 5' P- (N ') -AT-3 '; wherein N and N ' are complementary oligonucleotides of 10-50bp, e.g.about 15-30bp, wherein the 3' -most T of the long oligonucleotide chain forms a single base 3' -overhang of the circularized linker.

12. The method according to any one of embodiments 1 to 11, wherein the restriction enzyme used in step (iii) is selected from the group consisting of: PvuI, PacI, AsiSI, BstKTI, RgaI, and SfaAI, preferably the restriction enzyme is PvuI.

13. The method of embodiments 1-12, wherein step ii) is performed at about 20-45 ℃, such as 30 ℃ -45 ℃ or about 35 ℃ -40 ℃.

14. The method of embodiments 1-13, wherein the uracil-specific cleaving agent is a USER enzyme, preferably wherein the reaction of step ii) lasts 20-50 minutes.

15. The method according to any one of embodiments 1 to 14, wherein the circularized ligation of step (iv) is performed using T4 ligase, preferably at 20-30 ℃ for 10-20 hours.

16. The method according to any one of embodiments 1 to 15, wherein said target DNA fragment of step i) has a length of 1K-200Kbp, preferably 2K-30Kbp, more preferably 3K-20Kbp, more preferably 5K-20Kbp, more preferably 8Kbp-12Kbp, and preferably said DNA fragment is a genomic DNA or cDNA fragment.

17. The method of any one of embodiments 1-16, wherein said step (i) comprises:

(a1) fragmenting the target DNA by transposase and simultaneously adding the circularization adapters to both ends of the generated target DNA fragment;

(b2) filling ends of the target DNA fragment produced in the step (a1) by strand displacement reaction to obtain the target DNA fragment with the circularized linker,

wherein preferably said cyclic linker is a cyclic linker molecule according to embodiments 10-11,

more preferably, the circularized linker molecule is a double stranded oligonucleotide having a 5 'overhang and a 3' blunt end in the 5 'to 3' direction of the long oligonucleotide chain, and wherein

The long oligonucleotide chain consists of a transfer chain of AU dinucleotides and transposable elements in the 5 'to 3' direction; and is

The short oligonucleotide strand consists of the non-transferred strand of the transposable element in the 5 'to 3' direction,

preferably, the transferred strand of the transposable element has the sequence shown by SEQ ID NO. 3, and the non-transferred strand of the transposable element has the sequence shown by SEQ ID NO. 4.

18. The method of any one of embodiments 1-16, wherein the method comprises the steps of:

the process of embodiment 1, characterized in that said step (i) comprises:

(a2) obtaining a target DNA fragment with a flat tail end, and adding a 3' A tail through terminal transferase;

(b2) connecting the circularized adapters to both ends of the target DNA fragment by using TA tail ligation to obtain the target DNA fragment with the circularized adapters;

wherein preferably said cyclic linker is a cyclic linker molecule according to embodiments 8-9,

more preferably, the circularized adapter is a double stranded oligonucleotide having a 5' blunt end and a 3' overhang in the 5' to 3' direction of the long oligonucleotide chain, and wherein the 5' blunt end has an AU:: TA dinucleotide pair, and wherein the 3' overhang is a single base 3' T overhang.

Preferably, the blunt-ended target DNA fragment of step i) is a genomic DNA fragment obtained after physical or enzymatic cleavage and end repair.

19. A circularized DNA molecule obtained by the method of any one of embodiments 1-18.

20. A sequencing library construction method comprising:

(a) obtaining a circularized DNA molecule by the method of any one of embodiments 1-18;

(b) fragmenting the circularized DNA molecules and capturing the DNA fragments with biotin labels (e.g.using streptavidin magnetic beads);

(c) the captured DNA is amplified to build a DNA library for sequencing.

21. The method of embodiment 20, wherein in step (a) a circularized DNA molecule is obtained using a circularized linker molecule having an auxiliary sequence region, wherein said auxiliary sequence region comprises an oligonucleotide selected from the group consisting of: molecule index, anchor site, reporter tag, barcode, primer binding site, preferably the helper sequence region comprises barcode or sequencing primer binding site.

22. A sequencing library obtained by the method of any one of embodiments 19-20.

23. A kit for circularizing DNA molecules or for sequencing library construction comprising a circularizing adaptor molecule as defined in any one of embodiments 1 to 11 and a restriction enzyme, wherein the restriction enzyme is capable of recognizing double stranded DNA and generating a 3' AT sticky end,

preferably, the restriction enzyme is selected from the group consisting of: PvuI, PacI, AsiSI, BstKTI, RgaI, and SfaAI, preferably the restriction enzyme is PvuI.

24. Use of a kit according to embodiment 23 for the preparation of circularised DNA molecules and/or for the construction of sequencing libraries.

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Claims

1. A DNA circularization ligation method, comprising the steps of:

Wherein the obtained target DNA fragment with the circularization adaptor has AU:: TA dinucleotide pair existing at two ends of the target DNA fragment in the form of inverted terminal repeats;

preferably, wherein the restriction enzyme used in step (iii) is selected from the group consisting of: PvuI, PacI, AsiSI, BstKTI, RgaI, and SfaAI.

2. The method of claim 1, characterized in that linear DNA molecules which are not circularized are digested after said step (iv).

3. The process of claim 1, characterized in that step (ii) is carried out at 20 ℃ to 45 ℃.

4. The method of claim 1, characterized in that the uracil-specific cleaving agent is a USER enzyme.

5. The method of claim 1, characterized in that said target DNA fragment of step (i) has a length of 1kbp to 200kbp, such as 5kbp to 20 kbp.

6. The method of claim 1, wherein step (i) comprises:

(a) fragmenting the target DNA by transposase and simultaneously adding the circularization adapters at both ends of the generated target DNA fragments;

(b) filling the ends of the target DNA fragment generated in the step (a) by strand displacement reaction to obtain the target DNA fragment with the cyclized linker.

7. The method of claim 6, wherein said circularized adaptor molecule is a double stranded oligonucleotide having a 5 'overhang and a 3' blunt end in the 5 'to 3' direction of the long oligonucleotide chain, and wherein

The short oligonucleotide strand consists of the non-transferred strand of the transposable element in the 5 'to 3' direction.

Preferably, the transfer strand of the transposable element has a sequence shown by SEQ ID NO. 3, and the non-transfer strand of the transposable element has a sequence shown by SEQ ID NO. 4.

8. The method of claim 1, wherein step (i) comprises:

(a) obtaining a target DNA fragment with a flat tail end, and adding a 3' A tail through terminal transferase;

(b) and connecting the circularized adapters to the two ends of the target DNA fragment by using TA tail connection to obtain the target DNA fragment with the circularized adapters.

9. The method of claim 8, wherein the circularized adapter is a double stranded oligonucleotide having a 5' blunt end and a 3' overhang in the 5' to 3' direction of the long oligonucleotide chain, and wherein the 5' blunt end has an AU:: TA dinucleotide pair, and wherein the 3' overhang is a single base 3' T overhang.

10. A sequencing library construction method comprising:

(a) obtaining a circularized DNA molecule with a circularized linker by the method of any one of claims 1-9;

(b) fragmenting the circularized DNA molecules and capturing DNA fragments with circularized adaptors;

(c) and amplifying the captured DNA fragments to build a DNA library for sequencing.