Patent Application
20240076657
A computer readable text file, entitled “I121-0003US_SeqList.txt” created on or about Jun. 28, 2023, with a file size of 4 KB, contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
The present disclosure belongs to the field of biotechnology. More specifically, the present disclosure provides a method for rapidly constructing cyclized library and cyclization adapters for the method.
At present, there are many well-established library construction methods based on next-generation sequencing (NGS, also known as High-Throughput Sequencing) technology. However, these library construction methods are generally based on the conventional library construction process, which comprises the following steps: the genomic nucleic acid strands are fragmented by physical or enzymatic means; the obtained fragments are subjected to end-repair by exonuclease and two ends of the fragments are filled in to ensure both ends are blunt, and then A is added to the 3′ end of the fragments by polymerase to generate a single-base sticky end; same adapters are added to both ends of the fragments with A at the 3′ end. The adapter is formed by complementary pairing of a long nucleic acid strand and a short strand, and gap is ligated by ligase after complementary pairing of A and T; the fragment ligated with adapter is used as a template and a nucleic acid single-stranded primer complementary with the adapter is added as primer for Polymerase Chain Reaction (PCR); two single strands of the template are separated by denaturation, and after primer binding to the corresponding single strand, the corresponding single strand is extended to be a completely complementary double-stranded target product, with one end being A adapter and the other end being B adapter; the ligation products are purified and recycled by magnetic beads; the purified and recycled DNA double-stranded products are denatured to obtain single-stranded DNA; the target DNA single strands are cyclized by using a nucleic acid single-stranded primer for assisting cyclization and the screening of the phosphoric acid group at the 5′ end of the DNA single strands; the unnecessary and remaining uncyclized single strands are removed by using exonuclease, and the like; the cyclized products are purified and recycled by magnetic beads; the cyclized single-stranded circular nucleic acid products are subjected to subsequent sequencing; DNA Nanoballs (DNBs) formed by rolling circle amplification are used for reading nucleic acid sequence information. This process involves several tube-changing and purification steps, which could be improved in terms of ease of operation and time of library construction.
Adapters used in NGS high-throughput sequencing should be a specially designed DNA sequence. The characteristic sequence in an adapter will function as the sequence of start site and be paired with sequencing primer during sequencing. The subsequent sequence can be determined through the extension of the primer. An adapter may be ligated to both ends of a DNA fragment by ligation and the like. The ligation of adapters with sticky ends is usually used to achieve directional ligation and avoid ligation between adapters. In the conventional library construction, it is necessary to ensure that adapters are ligated to both ends of DNA double strands and the redundant adapter products are removed by purification.
With the proceeding of national sequencing services for large-scale residents in many countries, there is a need for an easy-operating and time-saving method for constructing a cyclized library in the prior art.
To solve the problems in the prior art, the present disclosure provides a method for rapidly constructing cyclized library and cyclization adapters for the method.
Therefore, on one aspect, the present disclosure provides a method for constructing cyclized library, the method comprising:
In one embodiment, in 1), the target nucleic acid is fragmented into random fragments by ultrasound or enzymatic cleavage.
In one embodiment, the 3′ overhang of the fragments and the 3′ overhang of the cyclization adapter are 1-5 nt in length, e.g., 3 nt or 2 nt, preferably 1 nt.
In one embodiment, the 3′ overhang of the fragmented fragments is an A and the 3′ overhang of the cyclization adapter is a T.
In one embodiment, in 2), the fragment is treated with exonuclease, polymerase and T4 polynucleotide kinase to perform end repair and A tailing with 5′ end phosphorylation and extra A deoxynucleotide at the 3′ end.
In one embodiment, in 3), the incompletely complementary double strands comprise a nick in one strand or a mis-matching region between the double strands.
In one embodiment, in 3), the two strands comprise two mis-matching regions between the double strands and include a barcode sequence for distinguishing samples between the two mis-matching regions.
In one embodiment, in 3), the cyclization adapter comprises cyclization adapter (a) or cyclization adapter (b) as follows:
In one embodiment, in 3), the cyclization adapter is the cyclization adapter (a), the ligated products are digested by exonuclease and the digested products are purified in one step to obtain a cyclized library.
In one embodiment, in 3), the cyclization adapter is the cyclization adapter (b) and the ligated products are denatured to obtain a cyclized library.
In one embodiment, the cyclized single-stranded library is subjected to subsequent sequencing, i.e., DNA Nanoballs (DNBs) formed by rolling circle amplification are used for reading nucleic acid sequence information.
On the other hand, the present disclosure provides a cyclization adapter for constructing cyclized library, the cyclization adapter is formed by incompletely complementary double strands and has a 3′ overhang at both ends, and the 3′ overhang of the cyclization adapter is complementary with the 3′ overhang of the fragment to be cyclized.
In one embodiment, the 3′ overhang of the fragment to be cyclized and the 3′ overhang of the cyclization adapter are 1-5 nt in length, e.g., 3 nt or 2 nt, preferably 1 nt.
In one embodiment, the 3′ overhang of the fragment to be cyclized is an A and the 3′ overhang of the cyclization adapter is a T.
In one embodiment, the incompletely complementary double strands comprise a nick in one strand or a mis-matching region between the double strands.
In one embodiment, the two strands comprise two mis-matching regions between the double strands and include a barcode sequence for distinguishing samples between the two mis-matching regions.
In one embodiment, the cyclization adapter comprises cyclization adapter (a) or cyclization adapter (b) as follows:
The present disclosure realizes one-step cyclization of a cyclized library by using specially designed adapters. In the present disclosure, an A-sticky end is formed by end-repair and addition of an A in the fragmented DNA fragment, which is complementary with the T-sticky end of the specially designed adapter to form cyclized structure, and then the ligation at the gap is completed by ligase.
To illustrate the embodiments of the present disclosure or the technical solutions in the prior art more clearly, the drawings are briefly described as below. It is obvious that the drawings described below only involve some embodiments of the present disclosure.
The present disclosure is described clearly and completely below. It is obvious that the embodiments described are only a part of the embodiments of the present disclosure, but not all embodiments. Based on the embodiments of the present disclosure, all other embodiments obtained by those skilled in the art are within the scope of protection of the present disclosure. Unless otherwise defined, all technical and scientific terms herein have the same meanings as commonly understood by those skilled in the art to which the present disclosure belongs. The terms in the specification of the present disclosure are for the purpose of describing specific embodiments only and are not intended to limit the present disclosure.
The present disclosure provides a solution to the problems of long time-consuming of library construction, complicated operation and loss in purification in the conventional library construction process based on MGI, which includes the steps of fragmenting genomic DNA, end repair and addition of A to the end of the DNB fragments, adapter ligation, PCR amplification, separation and cyclization of a single strand, etc. Given the problems in the prior art, the inventors fundamentally improved the conventional method of adding adapters, amplification and cyclization, and designed ligation adapters with unique sequence structure, which combines the adapter ligation and product cyclization in a one-step reaction, optimizes the reaction system and purification step and significantly reduces the time of constructing a cyclized library.
As shown in
As shown in
The innovation of the present disclosure lies in the design and construction of cyclization adapters, which are used to construct a library for double-stranded DNA by using AT ligation. The sequence design may be adapted to the MGI sequencing platform. Compared with the sequences in the conventional library construction, which have two adapters ligated to both ends of the fragment, the ligation adapter in the present disclosure can be ligated to both ends of the fragment simultaneously to realize one-step cyclization by ligation. By using the cyclization adapter in the present disclosure, the PCR and cyclization reaction in the conventional library construction can be skipped, and the purification step can be omitted, which greatly reduces the operation time of the whole library construction process. Meanwhile, the reactions of each step are ensured to proceed continuously in same tube, which avoids the operation of changing tubes and the loss in the process. Finally, a cyclized library is obtained after one-step purification.
The following examples are provided for a better understanding of the present disclosure. The experimental methods in the following examples are conventional methods, unless otherwise specified. The experimental materials in the following examples are all purchased from the regular reagent store unless otherwise specified. It should be noted that the above contents of the disclosure and the detailed description below are only for the purpose of specifically illustrating the present disclosure and are not intended to limit the present disclosure in any way. Without departing from the spirit and theme of the present disclosure, the scope of the present disclosure is determined by the appended claims.
The sequences of the cyclization adapters in the examples are as follows:
wherein xxxxxxxxxx is a barcode sequence and YYYYYYYYYY is the sequence complementary with the barcode.
The two strands of the cyclization adapter (b) are both long strand b, that is, the matching region and the mis-matching region at both ends of the cyclization adapter (b) are reverse to each other, and the matching region in the middle is a palindromic sequence.
Adapter Annealing:
Purchased adapters were redissolved to a concentration of 100 μM by using TE, and
For cyclization adapter (a), it was diluted according to the following formulation and allowed to stand still at room temperature for 30 minutes, to form 10 μM cyclization adapter (a):
For cyclization adapter (b), it was diluted according to the following formulation and allowed to stand still at room temperature for 30 minutes, to form 10 μM cyclization adapter (b):
Source:
1. Genomic DNA fragmenting: there are various methods for genomic DNA fragmenting, including physical ultrasound methods and enzyme reaction methods. There are very mature solutions for genomic DNA fragmenting in the market. A physical ultrasound fragmentation method was adopted in the example.
A 96-well PCR plate was added with a polytetrafluoroethylene thread, 1 μg of extracted genomic DNA, and TE buffer or enzyme-free water to 80 μL. The plate was sealed, and then ultrasonic fragmentation was performed on the E210 ultrasonicator.
The fragmentation condition was set as follows:
2. Fragments selecting: magnetic bead purification method or gel recycling method can be used. Magnetic bead purification method was adopted in the example. 80 μL of Ampure XP magnetic beads were added into the fragmented DNA and mixed thoroughly, and the mixture was placed for 7-15 min. The mixture was placed on a magnetic rack to collect the supernatant. 40 μL of Ampure XP magnetic beads were added into the supernatant and mixed thoroughly, and the mixture was placed for 7-15 min. The mixture was placed on a magnetic rack to remove the supernatant. The magnetic beads were washed twice with 75% ethanol, dried, added with 50 μL of TE buffer or enzyme-free water and mixed thoroughly, and the mixture was placed for 7-15 min to dissolve and recycle product.
3. End repairing and adding A: 100 ng of the product recycled in the previous step was added with TE to 40 μL and used to prepare the reaction system according to the following table. The reaction mixture was prepared as shown in the following table:
10 μL of end repair reaction liquid was immediately added to 40 μL of fragmented product to carry out the following reactions. The reaction conditions are shown in the following table:
4. Adapter ligating: after the reaction, 5 μL of cyclization adapter (a) or cyclization adapter (b) at a concentration of 10 μM was immediately added to the end-repaired product. The adapter ligation mixture was prepared as shown in the following table:
After adding an appropriate amount of adapters into the end-repaired product, 25 μL adapter ligation mixture was added into the mixture to carry out the following reactions. The reaction conditions are shown in the following table:
5. After the reaction, the product was purified by using 2× magnetic beads and finally dissolved in 20 μL TE solution.
6. DNB preparing and sequencing: specific steps can refer to the instruction of MGI sequencing kit. DNBs were prepared by using a double-stranded library. The preliminary results of sequencing are as follows: GC coverage information of the experiment conducted with cyclization adapter (a) is shown in A of
Results analysis: given the problems in the prior art, all cyclization adapters would change the conventional method of adding adapters, amplification and cyclization, which combines the adapter ligation and product cyclization in a one-step reaction and significantly reduces the time of constructing a cyclized library. The products of library construction are cyclized, which can be directly sequenced after DNB preparation. The analysis of sequencing results shows that all adapters can be used to construct a library for sequencing successfully. Although the sequencing results are not optimal, the purpose of this experiment is to verify the feasibility of adapter design in the experiment and provide certain exemplary examples. Therefore, the results well prove that the adapter design of the present disclosure meets the requirements of the disclosure and shows certain implementability.
The specific examples are provided to describe the present disclosure, which are only to assist those skilled in the art to understand the present disclosure, but not to limit the present disclosure. Several simple deductions, variations or replacements can be made by those skilled in the art to which the present disclosure belongs according to the concept of the present disclosure.
This application is to commence under 35 U.S.C. § 371, the U.S. National Phase of PCT/CN2020/140877, which was filed Dec. 29, 2020.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/140877 | 12/29/2020 | WO |
