WO2023155847A1

WO2023155847A1 - Method and kit for constructing sequencing library for detecting chromosome copy number variation

Info

Publication number: WO2023155847A1
Application number: PCT/CN2023/076541
Authority: WO
Inventors: 侯丽敏; 陈迪; 张建光; 张丽
Original assignee: 北京贝瑞和康生物技术有限公司
Priority date: 2022-02-17
Filing date: 2023-02-16
Publication date: 2023-08-24
Also published as: CN114197061A

Abstract

Provided are a method and kit for constructing a sequencing library for detecting chromosome copy number variation. The method comprises the following steps: 1) providing a genome DNA or RNA/DNA hybrid double-strand sample; 2) adding endonuclease and DNA polymerase into the sample, randomly shearing DNA double strands or RNA/DNA hybrid double strands by means of the nick translation principle to obtain a DNA fragment, and adding A at the tail end of the fragment to obtain an A-added DNA fragment; 3) performing ligation on the A-added DNA fragment and a sequencing adapter to obtain a ligation product; and 4) purifying the ligation product to obtain a sequencing library. The kit comprises: 1) a reagent for performing random nicking and performing nick translation and A-addition at a tail end; 2) a reagent for ligation of the A-added DNA fragment and a sequencing adapter; and 3) a reagent for purifying the ligation product.

Description

Methods and kits for constructing sequencing libraries for detection of chromosomal copy number variation

technical field

The present invention relates to a method for rapidly constructing a high-throughput next-generation sequencing library, in particular, a method for constructing a sequencing library for detecting chromosome copy number variation.

Background technique

Chromosomal copy number variation (CNV) is an important form of human genetic variation. It is widely distributed in the human genome. It covers a wide range of chromosomes and has a high mutation frequency. It can lead to severe changes in the phenotype of mutant individuals and even the death of mutant individuals.

Currently, techniques for detecting CNV include traditional karyotyping combined with fluorescence in situ hybridization (FISH), Chromosome Microarray Analysis (CMA, such as aCGH and SNP-array), fluorescent quantitative polymerase chain PCR (qPCR), multiplex ligation probe amplification (MLPA), high-throughput sequencing (NGS), etc. (Nord, et al, 2015; Manning and Hudgins, 2010; Trask, 1991;). In recent years, the increasingly mature NGS has been widely used in clinical research due to its outstanding advantages such as high throughput, high accuracy, high sensitivity, high degree of automation and low operating cost (Xuan, et al, 2013 ).

In the existing library construction technology of NGS, the first step of library construction is gDNA fragmentation, and the most common implementation methods are physical fragmentation and enzymatic fragmentation. The physical fragmentation method mainly includes ultrasound or confocal acoustic wave (Covaris), which requires special instruments, and has problems such as long time consumption, large amount of gDNA required, and low product recovery efficiency. Enzyme fragmentation, most commonly NEBNext DNA double-strand fragmentation enzyme and Tn5 from NEB. The T7 endonuclease in NEBNext DNA double-strand fragmentation enzyme has patent protection, and the fragmented DNA fragments need end repair and A process; the cost of Tn5 library construction is relatively high, and the amount of DNA input and Tn5 must be strictly limited proportion.

Therefore, it is of great significance to find new and effective fragmentation techniques and rapid library construction methods for detecting chromosomal copy number variation.

Contents of the invention

The above problems can be effectively solved by providing two rapid library construction methods for randomly interrupting gDNA based on different forms of polymerase in the nick translation method for detecting chromosomal copy number variation.

According to a first aspect of the present invention, a method for constructing a high-throughput sequencing library is provided, comprising the following steps:

1) Provide genomic DNA or RNA/DNA hybrid double-stranded samples;

2) Add endonuclease and DNA polymerase to the above sample, use the principle of nick translation to randomly interrupt the DNA double strand or RNA/DNA hybrid double strand to obtain DNA fragments, and add A to the end of the fragment to obtain the A-added DNA fragments;

3) Ligate the A-added DNA fragments with a sequencing adapter to obtain a ligation product;

4) purifying the ligation product to obtain a sequencing library.

In a preferred embodiment, the endonuclease is a Vibrio-derived endonuclease, preferably a Vvn endonuclease derived from Vibrio vulnificus (Vvn).

In a preferred embodiment, the DNA polymerase is a polymerase single enzyme or a mixed enzyme of the same type of polymerase, which is characterized in that the enzyme has 5'-3' polymerization activity and 5'-3' excision active but does not have 3'-5'exo-cutting activity; preferably, the DNA polymerase is Taq DNA Polymerase; preferably, the reaction temperature in step 2) is fixed, and the temperature range is preferably 40-65°C, more preferably 50- 60°C.

In a preferred embodiment, the DNA polymerase is a mixture of two types of polymerases, wherein one type of polymerase has 5'-3' polymerization activity and 3'-5' and 5'-3 ' exonucleolytic activity; another polymerase is a polymerase having 5'-3' polymerization activity and 5'-3' exonucleolytic activity but not having 3'-5' exonucleolytic activity; preferably, the Described DNA polymerase is the mixed enzyme of DNA Polymerase I and Taq DNA Polymerase; Preferably, wherein the reaction temperature of step 2) is variable, first reacts with lower temperature, then reacts with higher reaction temperature, wherein lower The reaction temperature is preferably 30-50°C, more preferably 32-37°C; the higher reaction temperature is preferably 60-75°C, more preferably 68-72°C.

In a preferred embodiment, wherein steps 2) and 3) are completed in a single reaction tube , no intermediate DNA purification steps are required.

In a preferred embodiment, the method for constructing a high-throughput sequencing library does not comprise a PCR amplification step.

In a preferred embodiment, wherein said library is used to detect chromosomal copy number variation.

According to a second aspect of the present invention, a kit for constructing a high-throughput sequencing library is provided, including:

1) Make random cuts and perform cut translation and add A reagent at the end;

2) a reagent for ligating the A-added DNA fragment with a sequencing adapter; and

3) Reagents for purifying the ligated product.

In a preferred embodiment, the reagents for randomly making nicks and performing nick translation and adding A to the end include endonuclease, DNA polymerase and dNTP.

In a preferred embodiment, the DNA polymerase is a polymerase single enzyme or a mixed enzyme of the same type of polymerase, which is characterized in that the enzyme has 5'-3' polymerization activity and 5'-3' excision Activity but no 3'-5' exoactivity; preferably, the DNA polymerase is Taq DNA Polymerase.

In a preferred embodiment, the DNA polymerase is a mixture of two types of polymerases, wherein one type of polymerase has 5'-3' polymerization activity and 3'-5' and 5'-3 ' exonucleolytic activity; another polymerase is a polymerase having 5'-3' polymerization activity and 5'-3' exonucleolytic activity but not having 3'-5' exonucleolytic activity; preferably, the The DNA polymerase is a mixed enzyme of DNA Polymerase I and Taq DNA Polymerase.

In a preferred embodiment, the reagent for ligating the A-added DNA fragments to the sequencing adapters includes ligase.

In a preferred embodiment, the ligase is T4 DNA ligase or T7 DNA ligase.

In a preferred embodiment, the reagents for purifying the ligated product are selected from purification columns, Qiagen columns, purification magnetic beads or Beckman Ampure XP beads

It should be understood that the content described in this section is not intended to identify key or important features of the embodiments of the present invention, nor is it intended to limit the scope of the present invention. Other features of the present invention will be easily understood from the following description.

Description of drawings

Figure 1: Schematic diagram of the principle of the method of the present invention.

Figure 2: Schematic diagram of the library construction method of the present invention.

Figure 3: Gel diagram for determining the amount of Vvn endonuclease, where gDNA: genome control, no interruption reaction; VVn-negative: negative control, except for VVn, the rest of the reactants were added to the reaction system for interruption reaction; VVn -10 ⁰ , 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^- ⁴ : represent adding 1.0uL of VVn stock solution, 10-fold dilution, 100-fold dilution, 1000-fold dilution, 10000-fold dilution to the system respectively, and the rest The reaction product of the reactants. M: Marker.

Figure 4: Gel map for determining the amount of Taq DNA Polymerase, where 0.25, 0.5, 1.0, 2.0: represent the volume of Taq DNA Polymerase added in the reaction system is 0.25uL, 0.5uL, 1.0uL, 2.0uL, and the rest of the reactants are added Reaction product; M: Marker.

Figures 5a-5k: results of sequencing the aneuploid samples of different chromosomes after constructing the library using the method of the present invention.

Figure 6: The results of sequencing different chromosomal microdeletion syndrome samples after library construction using the method of the present invention, where the ordinate refers to the copy number of the detection region, and the abscissa represents the entire chromosome region. The entire chromosome is evenly divided into several regions, and each point represents the copy number of a region, and then a trend line (blue solid line) is obtained according to the distribution of all points. The red box in the figure lists the region of microdeletion in each sample.

Detailed ways

Embodiments of the present invention will be described in more detail below. It should be understood, however, that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for a more thorough and complete understanding of this invention. It should also be understood that the drawings and embodiments of the present invention are for exemplary purposes only, and are not intended to limit the protection scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the definitions in this specification shall prevail.

As used herein, the terms "comprises," "including," "has," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion.

When amounts, concentrations, or other values or parameters are expressed in terms of ranges, preferred ranges, or ranges bounded by a series of upper preferred values and lower preferred values, it is to be understood that any range upper or preferred value combined with any lower range limit All ranges formed by any pairing of values or preferred values, whether or not such ranges are individually disclosed. For example, when the range "1 to 5" is disclosed, the recited range should be construed to include the ranges "1 to 4," "1 to 3," "1-2," "1-2, and 4-5" , "1-3 and 5", etc. When a numerical range is described herein, unless otherwise stated, that range is intended to include its endpoints and all integers and fractions within the range.

Furthermore, the indefinite articles "a" and "an" preceding an element or component herein place no limitation on the quantity requirement (ie, number of occurrences) of the element or component. Thus "a" or "an" should be read to include one or at least one, and elements or components in the singular include the plural unless it is obvious that the number stated is intended to limit the singular.

Further, in the following specification, a large number of expressions will be mentioned, which are defined to have the following meanings.

"Sequencing" refers to determining the order of nucleotides (base sequence) in a nucleic acid sample such as DNA or RNA.

"High-throughput sequencing" is also called "next generation sequencing", or NGS for short. It refers to the sequence determination of hundreds of thousands to millions of DNA molecules at a time.

"Nick translation" refers to the 5'-3' exonuclease activity of a DNA molecule with a cut under the action of DNA polymerase (with 5'-3' exonuclease activity and 5'-3' polymerase activity). It can hydrolyze the DNA strand from the 5' end of the nick, and at the same time, its polymerization activity extends the DNA strand at the nick to the 3' end, resulting in the translation of the nick position from the 5' end to the 3' end. This method is commonly used to introduce radiolabeled nucleotides into DNA molecules.

As described above, the prior art lacks new and effective fragmentation techniques and rapid library construction methods that can be used to detect chromosomal copy number variations.

Methods for Construction of High-Throughput Sequencing Libraries

In order to at least partially address one or more of the above-mentioned problems and other potential problems, referring to Fig. A method for a library comprising the steps of:

1) Provide genomic DNA or RNA/DNA hybrid double-stranded samples;

4) purifying the ligation product to obtain a sequencing library.

There is no particular limitation on the content of the starting DNA in the sample, and it can be those routinely used in the art, for example, it can be 3.5ng-1000ng.

As for the endonuclease, there is no particular limitation, and those conventionally used in the art may be used. However, preferably, the endonuclease is a Vibrio-derived endonuclease, more preferably a Vvn endonuclease derived from halophilic Vibrio (Vibrio vulnificus) (Vvn). Further, the endonuclease can also be a wild-type Vvn endonuclease or a mutant thereof.

As for the DNA polymerase, there is no particular limitation, and those conventionally used in the art may be used. However, preferably, the DNA polymerase can be used in one of the following two ways.

In the first mode, the DNA polymerase is a single polymerase or the same type of polymerase Mixed enzymes of synthases, characterized in that the enzymes have 5'-3' polymerization activity and 5'-3' exo-cleavage activity but no 3'-5' exo-cleavage activity. Preferably, the DNA polymerase used in this first approach is Taq DNA Polymerase. Preferably, in the first mode, the reaction temperature in step 2) is fixed, and the temperature range is preferably 40-65°C, more preferably 50-60°C. For example, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C or any value between these values. Further preferably, a combination of the above-mentioned Vvn endonuclease and Taq DNA Polymerase is used to carry out the above step 2), so that the Vvn endonuclease randomly generates a single-stranded nick on the double-stranded DNA, and Taq DNA Polymerase uses this nick from The 5' end is translated to the 3' end to achieve random fragmentation of DNA and add A. When using this combination, typically, the amount of Vvn used in the step (2) is 0.11ug-1.1ug, preferably 0.33ug-1.1ug, more preferably 0.88ug. For example, 0.11ug, 0.12ug, 0.13ug, 0.14ug, 0.15ug, 0.16ug, 0.17ug, 0.18ug, 0.19ug, 0.20ug, 0.21ug, 0.22ug, 0.23ug, 0.24ug, 0.25ug, 0.26ug, 0.27ug, 0.28ug, 0.29ug, 0.30ug, 0.31ug, 0.32ug, 0.33ug, 0.34ug, 0.35ug, 0.36ug, 0.37ug, 0.38ug, 0.39ug, 0.40ug, 0.41ug, 0.42ug , 0.43ug, 0.44ug, 0.45ug, 0.46ug, 0.47ug, 0.48ug, 0.49ug, 0.50ug, 0.51ug, 0.52ug, 0.53ug, 0.54ug, 0.55ug, 0.56ug, 0.57ug, 0.58ug, 0.59 ug, 0.60ug, 0.61ug, 0.62ug, 0.63ug, 0.64ug, 0.65ug, 0.66ug, 0.67ug, 0.68ug, 0.69ug, 0.70ug, 0.71ug, 0.72ug, 0.73ug, 0.74ug, 0.75ug, 0.76ug, 0.77ug, 0.78ug, 0.79ug, 0.80ug, 0.81ug, 0.82ug, 0.83ug, 0.84ug, 0.85ug, 0.86ug, 0.87ug, 0.88ug, 0.89ug, 0.90ug, 0.91ug, 0.92ug , 0.93ug, 0.94ug, 0.95ug, 0.96ug, 0.97ug, 0.98ug, 0.99ug, 1.00ug, 1.01ug, 1.02ug, 1.03ug, 1.04ug, 1.05ug, 1.06ug, 1.07ug, 1.08ug, 1.09 ug, 1.1ug, or any value in between. When using this combination, typically, the usage activity unit amount of Taq DNA Polymerase in the step (2) is 1.25U～5U, preferably 1.25U～2.5U, More preferably 1.25U. For example, 1.25U, 1.30U, 1.35U, 1.40U, 1.45U, 1.50U, 1.55U, 1.60U, 1.65U, 1.70U, 1.75U, 1.80U, 1.85U, 1.90U, 1.95U, 2.0U, 2.05U, 2.10U, 2.15U, 2.20U, 2.25U, 2.30U, 2.35U, 2.40U, 2.45U, 2.50U, 2.55U, 2.60U, 2.65U, 2.70U, 2.75U, 2.80U , 2.85U, 2.90U, 2.95U, 3.0U, 3.05U, 3.10U, 3.15U, 3.20U, 3.25U, 3.30U, 3.35U, 3.40U, 3.45U, 3.50U, 3.55U, 3.60U, 3.65 U, 3.70U, 3.75U, 3.80U, 3.85U, 3.90U, 3.95U, 4.0U, 4.05U, 4.10U, 4.15U, 4.20U, 4.25U, 4.30U, 4.35U, 4.40U, 4.45U, 4.50U, 4.55U, 4.60U, 4.65U, 4.70U, 4.75U, 4.80U, 4.85U, 4.90U, 4.95U, 5.0U, or any value in between. When using this combination, typically, the reaction conditions of the step (1) can be: 45°C-68°C, 20 minutes, preferably the reaction temperature is 50°C-62°C, more preferably 50°C or 60°C. For example, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C or any value in between.

In the second mode, the DNA polymerase is a mixture of two types of polymerases, one of which has 5'-3' polymerization activity and 3'-5' and 5'-3' Exonucleolytic activity; another polymerase is a polymerase that has 5'-3' polymerization activity and 5'-3' exonucleolytic activity but does not have 3'-5' exonucleolytic activity. Preferably, the DNA polymerase used in the second mode is a mixed enzyme of DNA Polymerase I and Taq DNA Polymerase. Preferably, in the second mode, the reaction temperature of step 2) is variable, first react at a lower temperature, and then react at a higher reaction temperature, wherein the lower reaction temperature is preferably 30-50°C, more preferably Preferably 32-37°C, such as 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C , 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C or any value between these values; the higher reaction temperature is preferably 60-75°C, more preferably 68-72°C, for example can be 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C or any value between these values. Typically, the reaction conditions of step 2) can be: 37°C for 5min-20min; 68°C for 10min, preferably 37°C for 5min-10min; 68°C for 10min, more preferably 37°C for 10min; 68°C for 10min. Further, use the combination of the above-mentioned Vvn endonuclease+DNA Polymerase I+Taq DNA Polymerase to carry out the above step 2), the Vvn randomly generates a single-stranded nick on the double-stranded DNA, and DNA Polymerase I uses this nick from the 5' The end is translated to the 3' end to realize random fragmentation of DNA, and Taq DNA Polymerase realizes the addition of A to fragmented DNA. When using this combination, typically, the amount of Vvn used in the step (2) is 0.011ug-0.11ug, preferably 0.033ug-0.11ug, more preferably 0.088ug. For example, 0.011ug, 0.012ug, 0.013ug, 0.014ug, 0.015ug, 0.016ug, 0.017ug, 0.018ug, 0.019ug, 0.020ug, 0.021ug, 0.022ug, 0.023ug, 0.024ug, 0.025ug, 0.026ug, 0.027ug, 0.028ug, 0.029ug, 0.030ug, 0.031ug, 0.032ug, 0.033ug, 0.034ug, 0.035ug, 0.036ug, 0.037ug, 0.038ug, 0.039ug, 0.040ug, 0.041ug, 0.042ug , 0.043ug, 0.044ug, 0.045ug, 0.046ug, 0.047ug, 0.048ug, 0.049ug, 0.050ug, 0.051ug, 0.052ug, 0.053ug, 0.054ug, 0.055ug, 0.056ug, 0.057ug, 0.058ug, 0.059 ug, 0.060ug, 0.061ug, 0.062ug, 0.063ug, 0.064ug, 0.065ug, 0.066ug, 0.067ug, 0.068ug, 0.069ug, 0.070ug, 0.071ug, 0.072ug, 0.073ug, 0.074ug, 0.075ug, 0.076ug, 0.077ug, 0.078ug, 0.079ug, 0.080ug, 0.081ug, 0.082ug, 0.083ug, 0.084ug, 0.085ug, 0.086ug, 0.087ug, 0.088ug, 0.089ug, 0.090ug, 0.091ug, 0.092ug , 0.093ug, 0.094ug, 0.095ug, 0.096ug, 0.097ug, 0.098ug, 0.099ug, 0.100ug, 0.101ug, 0.102ug, 0.103ug, 0.104ug, 0.105ug, 0.106ug, 0.107ug, 0.108ug, 0.109 ug, 0.11ug, or any value in between. When using this combination, typically, the amount of DNA Polymerase I used in the step (2) is 3.2U to 10U, preferably 3.2U to 10U, more preferably 3.2U, for example, 3.20U, 3.25U, 3.25U, 3.30U, 3.35U, 3.40U, 3.45U, 3.50U, 3.55U, 3.60U, 3.65U, 3.70U, 3.75U, 3.80U, 3.85U, 3.90U, 3.95U, 4.0U, 4.05U, 4.10U, 4.15U, 4.20U , 4.25U, 4.30U, 4.35U, 4.40U, 4.45U, 4.50U, 4.55U, 4.60U, 4.65U, 4.70U, 4.75U, 4.80U, 4.85U, 4.90U, 4.95U, 5.0U, 5.20 U, 5.25U, 5.30U, 5.35U, 5.40U, 5.45U, 5.50U, 5.55U, 5.60U, 5.65U, 5.70U, 5.75U, 5.80U, 5.85U, 5.90U, 5.95U, 6.0U, 6.05U, 6.10U, 6.15U, 6.20U, 6.25U, 6.30U, 6.35U, 6.40U, 6.45U, 6.50U, 6.55U, 6.60U, 6.65U, 6.70U, 6.75U, 6.80U, 6.85U , 6.90U, 6.95U, 7.0U, 7.05U, 7.10U, 7.15U, 7.20U, 7.25U, 7.30U, 7.35U, 7.40U, 7.45U, 7.50U, 7.55U, 7.60U, 7.65U, 7.70 U, 7.75U, 7.80U, 7.85U, 7.90U, 7.95U, 8.0U, 8.05U, 8.10U, 8.15U, 8.20U, 8.25U, 8.30U, 8.35U, 8.40U, 8.45U, 8.50U, 8.55U, 8.60U, 8.65U, 8.70U, 8.75U, 8.80U, 8.85U, 8.90U, 8.95U, 9.0U, 9.05U, 9.10U, 9.15U, 9.20U, 9.25U, 9.30U, 9.35U , 9.40U, 9.45U, 9.50U, 9.55U, 9.60U, 9.65U, 9.70U, 9.75U, 9.80U, 9.85U, 9.90U, 9.95U, 10.0U, or any value in between. When using this combination, typically, the use activity unit amount of Taq DNA Polymerase in the step (2) is 1.25U～5U, preferably 1.25U～2.5U, more preferably 1.25U, for example, 1.25U can be specifically selected , 1.30U, 1.35U, 1.40U, 1.45U, 1.50U, 1.55U, 1.60U, 1.65U, 1.70U, 1.75U, 1.80U, 1.85U, 1.90U, 1.95U, 2.0U, 2.05U, 2.10 U, 2.15U, 2.20U, 2.25U, 2.30U, 2.35U, 2.40U, 2.45U, 2.50U, 2.55U, 2.60U, 2.65U, 2.70U, 2.75U, 2.80U, 2.85U, 2.90U, 2.95U, 3.0U, 3.05U, 3.10U, 3.15U, 3.20U, 3.25U, 3.30U, 3.35U, 3.40U, 3.45U, 3.50U, 3.55U, 3.60U, 3.65U, 3.70U, 3.75U , 3.80U, 3.85U, 3.90U, 3.95U, 4.0U, 4.05U, 4.10U, 4.15U, 4.20U, 4.25U, 4.30U, 4.35U, 4.40U, 4.45U, 4.50U, 4.55U, 4.60 U, 4.65U, 4.70U, 4.75U, 4.80U, 4.85U, 4.90U, 4.95U, 5.0U, or any value in between.

Regarding steps 2) and 3) in the method of the first exemplary embodiment of the present invention, it can be done in a single reaction tube without the need for a DNA purification step in between.

Regarding the method of the first exemplary embodiment of the present invention, it may not comprise a PCR amplification step.

Regarding the method of the first exemplary embodiment of the present invention, wherein said library is used to detect chromosomal copy number variation.

Kits for Construction of High-Throughput Sequencing Libraries

In order to at least partially address one or more of the above-mentioned problems and other potential problems, a second exemplary embodiment of the present invention proposes a kit for constructing a high-throughput sequencing library, comprising:

1) Make random cuts and perform cut translation and add A reagent at the end;

3) Reagents for purifying the ligated product.

There are no particular limitations on the reagents for randomly nicking, nick translation and end-adding, and may be those routinely used in the art, but preferably, they include endonucleases, DNA polymerases and dNTPs.

In the first mode, the DNA polymerase is a polymerase single enzyme or a mixed enzyme of the same type of polymerase, which is characterized in that the enzyme has 5'-3' polymerization activity and 5'-3' excision activity But it does not have 3'-5'exo-cutting activity. Preferably, the DNA polymerase used in this first approach is Taq DNA Polymerase.

In the second mode, the DNA polymerase is a mixture of two types of polymerases, one of which has 5'-3' polymerization activity and 3'-5' and 5'-3' Exonucleolytic activity; another polymerase is a polymerase that has 5'-3' polymerization activity and 5'-3' exonucleolytic activity but does not have 3'-5' exonucleolytic activity. Preferably, the DNA polymerase used in the second mode is a mixed enzyme of DNA Polymerase I and Taq DNA Polymerase.

The reagents for ligating the A-added DNA fragments to the sequencing adapters are not particularly limited, and may be those routinely used in the art. However, preferably, the reagent for ligating the A-added DNA fragments to the sequencing adapters includes ligase.

As for the ligase, there is no particular limitation, and those conventionally used in the art may be used. However, preferably, the ligase is T4 DNA ligase or T7 DNA ligase.

There are no particular limitations on the reagents used to purify the ligation product, and may be those routinely used in the art. However, preferably, the reagents for purifying the ligated product are selected from purification columns, Qiagen columns, purification magnetic beads or Beckman Ampure XP beads.

preferred embodiment

The preferred embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings. The following descriptions are exemplary and not limiting to the present invention. Any other similar situations also fall within the protection scope of the present invention.

Embodiment 1: Determining the input amount of VVn endonuclease.

(1) Dilute VVn by 10 ^-1 /10 ^-2 /10 ^-3 /10 ^-4 respectively for use.

(2) Take 100ng of cell line genomic DNA as the initial sample, add 43uL ddH ₂ O, 5uL 10×ds HL Reaction buffer, 1uL DNA, 1uL VVn to 50uL in sequence according to Table 1. Among them, VVn includes no addition (negative control), stock solution (10 ⁰ ), 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 6 gradients.

(3) The reaction program of the PCR instrument was set at 37°C for 20 minutes; 4°C forever.

(4) Add loading buffer after the reaction is completed, and use 1.2% agarose gel electrophoresis to detect the size of the interrupted fragments. The electrophoresis instrument is set at a voltage of 120V for 40 minutes. Figure 3 shows the above electrophoresis results, and the input amount of VVn in different swimming lanes is given at the top of Figure 3. According to the results shown in Figure 3, it can be known that different input amounts of VVn have different effects on DNA interruption. When VVn is diluted to 10 When ^-1 , the DNA fragmentation size of 100-200bp is a better choice.

Table 1:

Embodiment 2: Determine the input amount of Taq DNA Polymerase.

(1) In order to determine the input amount of Taq DNA Polymerase, add 0.25, 0.5, 1.0, 2.0uL of different Taq DNA Polymerase into the reaction system respectively. According to Table 2, add ddH ₂ O, 5uL 10x PCR Reaction Buffer, 1.0uL 10mM dNTPs, 1.0uL DNA, 1.0uL VVn (diluted 10 times), 0.25/0.5/1/2.0uL Taq DNA Polymerase to 50uL.

(2) The reaction program of the PCR instrument was set as 37°C for 20 minutes; 68°C for 20 minutes; 4°C forever.

(3) Add 14.0uL ligation buffer, 5.0uL ligase, and 4.0uL Adapter to the reaction product in sequence according to Table 3 for second-generation library construction to obtain the ligation product.

(4) 1x XP magnetic bead purification to remove unligated fragments and redundant adapters to obtain a sequencing library.

(5) qPCR amplification to determine the concentration of the library. At the same time, add the qPCR product to the loading buffer, use 1.2% agarose gel electrophoresis to detect the size of the library, and set the electrophoresis The voltage is 120V, and the time is 30min. Figure 4 shows the above electrophoresis results, and the input amount of Taq DNA Polymerase in different swimming lanes is given at the top of Figure 4. According to the results shown in Figure 4, it can be known that the input amount of Taq DNA Polymerase is 0.25uL (ie 1.25U) and 0.5 When uL (ie 2.5U), the size of the library is 300bp, and the scatter is small; when the input amount of Taq DNA Polymerase is 1.0uL and 2.0uL, the library scatter is serious, and there are large fragments that are not interrupted.

Table 2:

table 3:

Embodiment 3: Determine the reaction temperature of VVn endonuclease and Taq DNA Polymerase.

(1) Add the reaction reagents sequentially according to Table 4, wherein VVn is diluted 10 times, and the input amount of Taq DNA Polymerase is 0.25uL.

(2) The reaction program of the PCR instrument was set at 45°C ˉ68°C for 20 minutes; 4°C forever with a total of 9 temperature gradients.

(3) The fragmented products were sequentially added to the ligation reagents according to Table 3 for second-generation library construction, the ligated products were obtained and purified, and the concentration of the library was determined by qPCR amplification.

(4) On-machine sequencing: use the NextSeq CN500 (National Machinery Note 20153400460) sequencer to perform on-machine sequencing according to the manufacturer's instructions.

(5) Analysis of sequencing data: compare the sequencing data with the reference sequence of the human genome, and the analysis results are shown in Table 5: the results show the concentration of the library at each reaction temperature/uniq mapped reads/uniq mapped ratio/uniq GC ratio, etc. Information, from the results of various data, it can be known that the reaction conditions can be 45°C to 68°C, and the preferred reaction temperature is 50°C to 62°C.

Table 4:

table 5:

Example 4: Effect of DNA starting content on sequencing library concentration.

According to the experimental conditions determined in the above examples (VVn diluted 10 times into 8uL, Taq DNA Polymerase into 0.25uL, reaction temperature 60 ℃) to construct the library, use DNA samples with different initial content to prepare the sequencing library, calculate according to the concentration and volume of the library The total amount of the library obtained is shown in Table 6 below. It can be seen from the table that when the initial DNA content is 3.5ngˉ1000ng, the total amount of the obtained library is greater than 0.2fmol, which can meet the sequencing requirements. Therefore, the starting DNA content range for preparing the library is 3.5-1000ng, but not limited to this range.

Table 6:

Example 5: Detection verification of different aneuploid samples.

According to the experimental conditions determined in the above examples (10-fold dilution of VVn into 8uL, Taq DNA Polymerase into 0.25uL, reaction temperature of 60°C), different aneuploid DNA samples were used to construct libraries for the detection of chromosome copy number variation. The sequencing results were compared with the reference sequence of the human genome. The results of different aneuploid DNA samples detected by this method are shown in Table 7 and Figures 5a-5k below. Chromosome copy number variations can be detected correctly.

Table 7:

Example 6: A mixture of VVn and two types of polymerases achieves random fragmentation of DNA.

(1) Add reaction reagents in sequence according to Table 8, where VVn sets the gradient by adding 10 times of dilution (1uL)/100 times of dilution (3/5/8uL), the input amount of DNA Polymerase I is 0.32uL, and the input of Taq DNA Polymerase The volume is 0.25 uL.

(2) The reaction program of the PCR instrument was set as 37°C for 10 minutes (5 minutes); 68°C for 10 minutes; 4°C forever, a total of 2 temperature gradients.

(5) Analysis of sequencing data: compare the sequencing data with the reference sequence of the human genome, and the analysis results are shown in Table 9: the results show the concentration of the library under each reaction condition/uniq mapped reads/uniq mapped ratio/uniq GC ratio, etc. Information, from the results of various data, it can be known that VVn diluted 100 times and added to 5.0uL or 8uL has obvious beneficial technical effects. The reaction conditions are 37°C for 10min; 68°C for 10min; 4°C forever is the preferred reaction temperature.

Table 8:

Table 9:

Example 7: The mixed enzyme of VVn and two types of polymerases realizes the random fragmentation of DNA and detects chromosome microdeletion syndrome.

According to the experimental conditions determined in Example 6, three DNA samples of chromosome microdeletion syndrome were used to construct a library for detection of DNA copy number variation of chromosome microdeletion. The sequencing results were compared with the reference sequence of the human genome. The detection results of different DNA samples using this method are shown in Table 10 and Figure 6 below. Abnormalities in the DNA copy number in the corresponding regions can be detected correctly.

Table 10:

Example 8: VVn endonuclease+Taq DNA Polymerase realizes random fragmentation of RNA/DNA hybrid double strands.

(1) Take total RNA and use oligo(dT) primers or random primers to synthesize RNA/DNA hybrid double strands.

(2) The library was constructed according to the experimental conditions determined in the above-mentioned Example 3 (VVn was diluted 10 times into 8uL, Taq DNA Polymerase into 0.25uL, and the reaction temperature was 60°C).

(3) qPCR amplification determines the concentration of the library, and the concentration results are as shown in Table 11. As can be seen from the table, after the random fragmentation of RNA/DNA hybrid double strands is used to build a library by the inventive method, the total amount of the library obtained is Greater than 0.2 fmol, all can meet the sequencing requirements, indicating that this method can achieve random fragmentation of RNA/DNA hybrid double strands.

Table 11:

In summary, the method for constructing a high-throughput sequencing library of the present invention provides a new and effective fragmentation technology and a rapid library construction method, which can more efficiently construct a sequencing library for detecting chromosome copy number variation, and obtain Obvious beneficial technical effects have been achieved.

The foregoing examples are illustrative only, serving to explain some of the characteristic features of the invention. The appended claims are intended to claim the broadest scope conceivable and the embodiments presented herein are merely illustrations of selected implementations according to all possible combinations of embodiments. Accordingly, it is the Applicant's intent that the appended claims not be limited by the selection of examples which characterize the application. As used in the claims, the term "comprises" and its semantic variants also logically include different and varied terms, such as but not limited to "consists essentially of" or "consists of". Where necessary, numerical ranges are provided and these ranges include subranges therebetween. Variations within these ranges will also be self-evident to those skilled in the art and should not be considered as a contribution to the public, but should also be construed, where possible, to be covered by the appended claims. Moreover, advances in science and technology will create possible equivalents or sub-replacements not presently considered due to inaccuracies of language, and these changes should also be construed to be covered by the appended claims where possible .

references

1.Nord A, Salipante SJ, Pritchard C.Chapter 11–Copy Number Variant Detection Using Next-Generation Sequencing.Clinical Genomics,2015,8:165-187.

2. Trask BJ. Fluoresence in situ hybridization: application in cytogenetics 15 and gene mapping. Trends Gnent. 1991, 7:149-154.

3. Manning M, and Hudgins L. Array-based technology and recommendations for utilization in medical genetics practice for detection of chromosomal abnormalities. Genet. Med. 2010,12(11):742-745.

4. Xuan J, Yu Y, Qing T, Guo L, Shi L. Next-generation sequencing in 25 the clinic: promises and challenges. Cancer Lett. 2013, 340(2): 284-295.

Claims

A method for constructing a high-throughput sequencing library, comprising the following steps:

1) Provide genomic DNA or RNA/DNA hybrid double-stranded samples;

2) Add endonuclease and DNA polymerase to the above sample, use the principle of nick translation to randomly interrupt the DNA double strand or RNA/DNA hybrid double strand to obtain DNA fragments, and add A to the end of the fragment to obtain the A-added DNA fragments;

3) Ligate the A-added DNA fragments with a sequencing adapter to obtain a ligation product;

4) purifying the ligation product to obtain a sequencing library.
The method of claim 1, wherein the endonuclease is an endonuclease derived from Vibrio.
The method of claim 1, wherein the endonuclease is a Vvn endonuclease derived from Vibrio halophilus.
The method of claim 1, wherein the DNA polymerase is a polymerase single enzyme or a mixed enzyme of the same type of polymerase, which is characterized in that the enzyme has 5'-3' polymerization activity and 5'-3' outer cleavage activity but no 3'-5' exocleavage activity.
The method of claim 4, wherein the DNA polymerase is Taq DNA Polymerase.
The method of claim 4, wherein the reaction temperature of step 2) is fixed.
The method according to claim 4, wherein the reaction temperature of step 2) is 40-65°C.
The method of claim 4, wherein the reaction temperature of step 2) is 50-60°C.
The method of claim 1, wherein the DNA polymerase is a mixed enzyme of two types of polymerases, wherein one type of polymerase has 5'-3' polymerization activity and 3'-5' and 5'- 3' exonucleolytic activity; another polymerase is a polymerase that has 5'-3' polymerization activity and 5'-3' exonucleolytic activity but does not have 3'-5' exonucleolytic activity.
The method of claim 9, wherein the DNA polymerase is a mixed enzyme of DNA Polymerase I and Taq DNA Polymerase.
The described method of claim 9, wherein the reaction temperature of step 2) is variable, first react with lower temperature, and then react with higher reaction temperature.
The method of claim 11, wherein the lower reaction temperature is 30-50°C.
The method of claim 11, wherein the lower reaction temperature is 32-37°C.
The method of claim 11, wherein the higher reaction temperature is 60-75°C.
The method of claim 11, wherein the higher reaction temperature is 68-72°C.
The method according to claim 1, wherein steps 2) and 3) are completed in a single reaction tube without a DNA purification step in the middle.
The method of claim 1, which does not comprise a PCR amplification step.
The method according to any one of claims 1-17, wherein said library is used to detect chromosomal copy number variation.
Kits for constructing high-throughput sequencing libraries, including:

1) Make random cuts and perform cut translation and add A reagent at the end;

2) a reagent for ligating the A-added DNA fragment with a sequencing adapter; and

3) Reagents for purifying the ligated product.
The kit according to claim 19, wherein the reagents for randomly making nicks and performing nick translation and adding A to the end include endonuclease, DNA polymerase and dNTP.
The kit of claim 20, wherein the endonuclease is an endonuclease derived from Vibrio.
The kit of claim 20, wherein the endonuclease is a Vvn endonuclease derived from Vibrio halophilus.
The kit according to claim 20, wherein the DNA polymerase is a polymerase single enzyme or a mixed enzyme of the same type of polymerase, which is characterized in that the enzyme has 5'-3' polymerization activity and 5'-3' Exo-active but not 3'-5'-exo-active.
The kit of claim 23, wherein the DNA polymerase is Taq DNA Polymerase.
The described test kit of claim 20, wherein said DNA polymerase is two kinds A mixed enzyme of type polymerase, wherein one type of polymerase has 5'-3' polymerization activity and 3'-5' and 5'-3' exonuclease activity; the other polymerase has 5'- A polymerase with 3' polymerization activity and 5'-3' exonucleolytic activity but no 3'-5' exonucleolytic activity.
The test kit according to claim 25, wherein said DNA polymerase is a mixed enzyme of DNA Polymerase I and Taq DNA Polymerase.
The kit according to claim 19, wherein the reagents for ligating the A-added DNA fragments with sequencing adapters include ligase.
The test kit of claim 27, wherein the ligase is T4 DNA ligase or T7 DNA ligase.
The kit of claim 19, wherein the reagents for purifying the ligated product are selected from purification columns, Qiagen columns, purification magnetic beads or Beckman Ampure XP beads.