Goose double enzyme digestion simplified genome sequencing library construction kit and library construction method and application

Abstract

This invention discloses a goose double-enzyme digestion simplified genome sequencing library construction kit, library construction method, and application. The kit includes Kit A, Kit B, and Kit C, and magnetic beads. The invention uses two conventional endonucleases to control the location and number of target fragments, and uses improved enrichment primers to significantly increase the number of library pools for GBS sequencing. This leverages the large amount of NGS data and makes goose population genetics research more feasible. In theory, this library construction strategy can exponentially increase the number of individuals to be tested simply by modifying the corresponding adapter and index sequences.

Description

Technical Field

[0001] The present invention relates to the technical field of genome sequencing, and in particular to a goose double-enzyme digestion simplified genome sequencing library construction kit, a library construction method and an application. Background Art

[0002] Next generation sequencing (NGS) is widely used in genetic research. Based on the coverage of the genome, NGS is generally divided into whole genome sequencing (WGS) and reduced representation genome sequencing (RRGS). WGS sequences almost all genomic regions. Its advantages are: (1) obtaining the most complete genetic variation information of individuals or populations; (2) reducing the interference of repetitive sequences caused by PCR amplification enrichment fragments. Its disadvantages are also very obvious: (1) high cost and difficulty in large-scale sequencing, especially for crops and livestock with many offspring; (2) high requirements for the analysis platform, requiring a high-level hardware computing platform, and a large initial investment.

[0003] Simplified genome sequencing uses a specific type of endonuclease or physical disruption to obtain a portion of the entire genome for sequencing. Its advantages are: (1) The endonuclease that disrupts the genome is cheap and easy to obtain; (2) The sequencing volume is small and the cost is low; (3) The small amount of data does not require high computing hardware, and a medium to high-end personal computer can carry out the analysis; (4) The entire experimental cycle is shortened.

[0004] Types of RRGS: divided into single enzyme digestion, double enzyme digestion, etc. (1) Single enzyme digestion RRGS: The early RAD / GBS technology belongs to this category, which only uses one endonuclease to fragment the genome, and then performs adapter ligation and sequencing. (2) Double enzyme digestion: Add an enzyme to the single enzyme digestion RRGS, or perform physical interruption in the second step to control the fragment length. The two technical routes have their own advantages and disadvantages: (1) Single enzyme digestion is simple and convenient to operate, and only one enzyme is needed for digestion, but the sequencing position is repetitive and cross-linked. (2) Double enzyme digestion has good reproducibility and can control the number of fragments, but it involves two endonucleases, which increases the cost and reduces the ease of operation.

[0005] Simplified genome sequencing technology was first published by Robert J. Elshire in 2011 and continues to this day. While various other simplified genome sequencing technologies have evolved, their essence remains unchanged, and the technical approaches share significant similarities. The single-enzyme digestion library construction protocol published by Robert J. Elshire uses ApeKI as an example for library construction and sequencing. The specific steps are as follows:

[0006] Step 1: Select an appropriate endonuclease (ApeKI) to fragment the genome into small fragments of 100-1000bp. You can simulate enzyme digestion or directly use existing endonucleases for actual operation. The fragments after enzyme digestion will leak the endonuclease-specific sticky end sequence.

[0007] Step 2: Synthesize 5' and 3' adapters that are complementary to the sticky ends. 5-18 bases (base barcodes) are added between the 5' adapter and the sticky end sequence as identification sequences to distinguish individuals. The 5' adapter is also called a barcode adapter (BA), and the 3' adapter also has a sequence complementary to the endonuclease sticky end, but without a barcode, and is called a common adapter (CA).

[0008] Step 3: Under the action of T4 ligase, the adapters with complementary sequences and the digested fragments achieve complementary pairing, forming a complete sequence to be sequenced. However, many of these fragments are self-ligated, too small, or too large, and cannot be used for sequencing.

[0009] Step 4: Obtain the ideal size fragment through magnetic beads or gel screening, generally 350bp. To increase the uniformity of sequence distribution, the screening range can be expanded to 500-800bp.

[0010] Step 5: Enrich the fragments after screening through PCR test, and then use magnetic beads or gel running to remove unnecessary fragments. The enrichment primers used in this step are a key technology that distinguishes traditional RRGS and the present invention. In the enrichment primers of traditional RRGS, one section of the sequence is complementary to the probe sequence of the Illumina sequencer for sequencing reaction, and the other half of the sequence is the same as the BA sequence. Enrichment will achieve two effects: (1) increase the complementary pairing sequence with the probe on the Illumina sequencer; (2) at the same time, the number of fragments after screening is exponentially increased. The structure after amplification is as follows Figure 1 shown.

[0011] Step 6: Perform another fragment size screening and quality control before sequencing.

[0012] The above is the traditional RRGS technical process. The most common change is the fragmentation method, which achieves a more evenly distributed label by changing the type and quantity of different endonucleases.

[0013] The simplified genome sequencing of geese currently reported is 2bRAD, a RRGS technology based on type IIB endonuclease library construction and sequencing commonly used in aquatic genetics research. Although it can also be used to discover a large number of markers in geese, it has the following disadvantages: (1) Type IIB endonucleases do not have enough restriction sites in the goose genome, and single restriction enzyme library construction and sequencing can only obtain about 20K markers. (2) Type IIB endonucleases are not common, and the fragments they cut are only 33bp in length. On the one hand, special data analysis methods are required to concatenate five restriction enzyme tags for subsequent sequencing analysis, which increases the complexity. More importantly, the short sequence of 33bp cannot provide more sequence information. A single type IIB endonuclease has fewer restriction sites in the goose genome, and multiple enzymes are required to obtain a sufficient number of markers. Applying this technology to goose genetics research will increase the cost of sequencing library construction experiments. The technology described in the simplified genome sequencing technology articles or patents that have been published in chickens and ducks is not applicable to geese. The most important thing is that the endonuclease selection is species-specific, and targeted experiments need to be designed for enzyme selection and subsequent library construction. In addition, the multi-library mixing capability of the above methods needs to be improved to increase sequencing efficiency and further reduce sequencing costs. Summary of the Invention

[0014] The purpose of the present invention is to address the problems existing in the prior art and to provide a goose double-enzyme digestion simplified genome sequencing library construction kit, library construction method and application.

[0015] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: a goose double enzyme digestion simplified genome sequencing library construction kit, the kit comprising:

[0016] Kit A: Contains: 1000U / mL EcoRI, 1000U / mL CviAII, 10× Buffer, and ddH2O.

[0017] Kit B: includes: 400,000 U / mL T4 ligase, 10× buffer, 20 pm / μL BA linker, 20 pm / μL CA linker, and ddH2O;

[0018] The BA adapter is a double-stranded DNA formed by annealing the sequences shown in SEQ ID No. 1 and SEQ ID No. 2, wherein SEQ ID No. 2 is 5' phosphorylated. m and n in SEQ ID No. 1 and SEQ ID No. 2 represent any short nucleotide barcode sequence of 5-20 bp in length, and the 4 bases AATT at the front of n are the sticky end of EcoRI.

[0019] The CA linker is a double-stranded DNA formed by annealing the sequences shown in SEQ ID No. 3 and SEQ ID No. 4, wherein SEQ ID No. 3 is 5' phosphorylated;

[0020] Kit C: includes: 2 U / μL high-fidelity PCR polymerase mix, 10 μM / μL enrichment primers, and ddH2O;

[0021] The enrichment primer F is shown in SEQ ID No. 5, and R is shown in SEQ ID No. 6, wherein h in SEQ ID No. 5 represents the index sequence in the upstream primer, and y in SEQ ID No. 6 represents the index sequence in the downstream primer;

[0022] and magnetic beads.

[0023] As a preferred embodiment of the present invention, the magnetic beads are streptavidin affinity magnetic beads.

[0024] For those skilled in the art, the application of the kit of the present invention in the construction of a goose double-enzyme digestion simplified genome sequencing library also belongs to the scheme claimed for protection.

[0025] The second technical solution provided by the present invention is to provide a method for constructing a library for goose double-enzyme digestion simplified genome sequencing using the kit of the present invention, the steps comprising:

[0026] (1) Genome fragmentation: Goose genomic DNA was extracted and fragmented using Kit A with a 20 μL enzyme digestion system to obtain the genomic fragmentation product, fragDNA;

[0027] (2) Adapter ligation: The genomic fragmentation product fragDNA was ligated using Kit B at 16°C for 12 h, inactivated at 65°C for 10 min, and the ligation product LigMix was obtained;

[0028] (3) Fragment screening: Add magnetic beads to the ligation product LigMix for double-end screening;

[0029] (4) Fragment enrichment: using Kit C, pre-denaturation at 94°C for 2 min, followed by 13–15 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and extension at 68°C for 50 s, followed by final extension at 72°C for 5 min and storage at 4°C to obtain the primary sequencing library libMix;

[0030] (5) Final screening: LibMix performs fragment filtration according to the magnetic bead ratio and screening method in step (4) to obtain mature sequenced DNA within the target size range, i.e., the mature DNA library libMix;

[0031] (6) Quality inspection: The constructed DNA library libMix was quality inspected using the Quibt method; the qualified library had the following criteria: a. single peak type, with a clear main peak, no large broad peaks and miscellaneous peaks, and no large fragments; b. concentration above 0.2 ng / μL; c. flat baseline, and qualified qPCR concentration.

[0032] Furthermore, in step (1), the enzyme digestion system composition and enzyme digestion scheme are as follows: 20 μL of the enzyme digestion system includes 0.5 μL of CviAII endonuclease, 2 μL of 10×Buffer, 200 ng of DNA and is adjusted to 20 μL with ddH2O, digested at 25°C overnight or for 16 hours, and then 0.5 μL of EcoRI endonuclease is added to the system for a second digestion, and digested at 37°C overnight or for 16 hours.

[0033] Furthermore, in step (2), the adapter ligation system is: 2 μL of T4 ligase, 4 μL of 10× Buffer, 1 μL of adapter mixture, 20 μL of fragDNA, and ddH2O is added to 40 μL.

[0034] Furthermore, the linker mixture is prepared by mixing BA linkers and CA linkers in a volume ratio of 1:16.

[0035] Furthermore, in step (3), the ratio of magnetic beads added is 0.535-0.6 times that of the ligation product.

[0036] The beneficial technical effects of the present invention are:

[0037] (1) This invention establishes a simplified goose genome sequencing technology. Using two conventional endonucleases to control the location and number of target fragments, and using improved enrichment primers, the number of library pools for GBS sequencing is significantly increased. This leverages the large amount of NGS data, making goose population genetics research more feasible. Theoretically, this library construction strategy can exponentially increase the number of sequenced individuals simply by modifying the corresponding adapter and index sequences.

[0038] (2) The benefits of using double enzyme digestion in the present invention are: 1) improved repeatability. The dual recognition of the digestion sites by the two enzymes can improve the specificity of the digestion fragments and increase the repeatability of sequencing sites between individuals; 2) more convenient regulation of the number of selectable fragments. Due to the addition of the low-frequency enzyme EcoRI, the large number of fragments obtained by the high-frequency enzyme CviAII digestion can be reduced, saving subsequent data resources.

[0039] (3) Cost control: After the technology is established, users can build their own databases according to their needs.

[0040] (4) The ability to mix libraries is greatly increased: Due to the introduction of library index, library identification can be performed when GBS BA controls individual identification, which greatly increases the number of libraries that can be mixed in GBS experiments and the number of individuals that can be sequenced in one experiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0042] Figure 1 , is a structural diagram of the product after amplification using the existing simplified genome sequencing technology. In the figure: the orange sequence is the sequence that is complementary to the probe on the Illumina sequencing instrument; the blue ones are BA and CA sequences; N represents the genome fragment obtained by enzyme cutting, which is also the fragment to be sequenced.

[0043] Figure 2 , Agarose gel electrophoresis of genomic fragments after double digestion with CviAII and EcoRI in Example 3. Lane 1 is control DNA, undigested, lane 2 is Sangon Ladder H2 molecular weight standards (50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, and 1031 bp), and lanes 3-6 show the fragment distribution after digestion of the four individual DNAs to be tested.

[0044] Figure 3 , the structural diagram of the linker sequence in Example 3;

[0045] Figure 4 , Length distribution result diagram of Chinese library fragments in Example 3;

[0046] Figure 5 , Graph showing the length distribution of the Chinese library fragments in Example 4. DETAILED DESCRIPTION

[0047] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0048] Consumables: 96-well plate, 1.5 mL EP tube, 96-pillar magnet, and 10-200 μL pipette tips.

[0049] Equipment: PCR instrument (capable of nested PCR), electrophoresis instrument, pipette, etc.

[0050] Example 1

[0051] Goose double enzyme digestion simplified genome sequencing library construction kit, including:

[0052] Kit A: Contains: 1000U / mL EcoRI, 1000U / mL CviAII, 10× Buffer, and ddH2O.

[0053] Kit B: includes: 400,000 U / mL T4 ligase, 10× buffer, 20 μM BA linker, 20 μM CA linker, and ddH2O.

[0054] The BA adapter is a double-stranded DNA formed by annealing the sequences shown in SEQ ID No. 1 and SEQ ID No. 2, wherein SEQ ID No. 2 is 5' phosphorylated. m and n in SEQ ID No. 1 and SEQ ID No. 2 represent any short nucleotide barcode sequence of 5-20 bp in length, and the 4 bases AATT at the front of n are the sticky end of EcoRI.

[0055] The CA linker is a double-stranded DNA formed by annealing the sequences shown in SEQ ID No. 3 and SEQ ID No. 4, wherein SEQ ID No. 3 is 5' phosphorylated;

[0056] Kit C: includes: 2 U / μL high-fidelity PCR polymerase mix, 10 μM / μL enrichment primers, and ddH2O;

[0057] The enrichment primer F is shown in SEQ ID No. 5, and the enrichment primer R is shown in SEQ ID No. 6, wherein h in SEQ ID No. 5 represents the index sequence in the upstream primer, and y in SEQ ID No. 6 represents the index sequence in the downstream primer;

[0058] and streptavidin affinity magnetic beads.

[0059] Example 2

[0060] A method for constructing a library for goose double-enzyme digestion simplified genome sequencing using the kit of Example 1, comprising the following steps:

[0061] (1) Genome fragmentation: Goose genomic DNA was extracted and fragmented using Kit A with a 20 μL enzyme digestion system to obtain the genomic fragmentation product, fragDNA;

[0062] Enzyme digestion system composition and digestion protocol: 20 μL of the enzyme digestion system includes 0.5 μL of CviAII endonuclease, 2 μL of 10× Buffer, and 200 ng of DNA, which is adjusted to 20 μL with ddH2O and digested at 25°C overnight or for 16 hours. Then, 0.5 μL of EcoRI endonuclease is added to the system for a second digestion, and digestion is carried out at 37°C overnight or for 16 hours.

[0063] (2) Adapter ligation: The genomic fragmentation product fragDNA was ligated using Kit B at 16°C for 12 h, inactivated at 65°C for 10 min, and ligated to obtain the ligation product ligDNA;

[0064] The adapter ligation system was as follows: T4 ligase 2 μL, 10× Buffer 4 μL, adapter mixture 1 μL, fragDNA 20 μL, and ddH2O was added to 40 μL.

[0065] The adapter mixture adaMix is ​​a mixture of BA adapters and CA adapters in a volume ratio of 1:16.

[0066] (3) Fragment screening: Add magnetic beads to the ligation product ligMix at a ratio of 0.535-0.6 times the ligation product for double-end screening;

[0067] (4) Fragment enrichment: Using Kit C, perform pre-denaturation at 94°C for 2 min, followed by 13–15 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and extension at 68°C for 50 s. Finally, perform a final extension at 72°C for 5 min and store at 4°C to obtain the primary sequencing library libMix.

[0068] (5) Final screening: libMix is ​​subjected to fragment filtration according to the magnetic bead ratio and screening method of step (4) to obtain a mature sequencing DNA library libMix within the target size range;

[0069] (6) Quality inspection: The constructed DNA library libMix was quality inspected using the Quibt method. The qualified library had the following criteria: a. Single peak type, with a clear main peak, no broad peaks or miscellaneous peaks, and no large fragments; b. Concentration above 0.2 ng / μL; c. Flat baseline, and qualified qPCR concentration.

[0070] Example 3

[0071] Preliminary experiments were conducted on 4 geese to perform simplified genome sequencing and pre-build libraries

[0072] A. Endonuclease Selection and Genome Fragmentation

[0073] (1) The classic single enzyme digestion simplified genome sequencing was changed to double enzyme digestion, and the IIB type endonuclease was no longer used; (2) Through simulated enzyme digestion, a preliminary combination of multiple enzyme digestion was determined, mainly looking at the number of 100-999bp fragments given by the simulated enzyme digestion. Through the simulation data and the actual enzyme digestion effect observation, it was confirmed that EcoRI (a low-frequency enzyme, which controls the bottom line of the number of enzyme digestion tags) and CviAII (a high-frequency enzyme that increases the upper limit of the number of enzyme digestion tags) are the two endonucleases of the present invention; (3) The composition of the enzyme digestion system and the enzyme digestion scheme: 20μL enzyme digestion system includes 0.5μL CviAII endonuclease, 2μL 10×Buffer, 200ng DNA and adjusted to 20μL with ddH2O, digested at 25℃ overnight (~16 hours), and then 0.5μL EcoRI endonuclease was added to the system for a second digestion, and digested at 37℃ overnight (~16 hours) to obtain the genomic fragmentation product fragDNA. The results are as follows Figure 2 shown.

[0074] B. Synthesis and Use of Linker Sequences

[0075] 1Connector structure

[0076] aThe linker sequence structure is as follows Figure 3 As shown, the orange sequence is the sequence that is complementary to the probe on the Illumina sequencing instrument; b green is the Illumina index sequence, and users can also develop new index sequences for synthesis and use; c blue is the BA and CA sequences; d N represents the genomic fragment obtained by enzyme digestion, which is also the fragment to be sequenced.

[0077] 2. Synthesis and application of BA linker and CA linker:

[0078] (1) Synthesize the BA sequence according to this sequence. P represents the need to add a phosphorylated linker. A total of 4 pairs of sequences are shown in Table 1. The library can be sequenced for 4 individuals. The underlined sequence (AATT) represents the EcoRI enzyme-digested sticky end; HPLC purification is required; after synthesis, the dilution is 100 μM.

[0079] Table 1 SEQ ID No. 1 and SEQ ID No. 2 corresponding to 4 pairs of BA linkers

[0080]

[0081]

[0082] (2) Synthesize CA according to the following sequence (Table 2). P indicates the addition of a phosphorylated linker. The italicized AT sequence represents the sticky end sequence of CviAII. The black base sequence is the library construction sequence used for Illumina TRUE Seq library construction. Purify by HPLC; dilute to 100 μM after synthesis for later use.

[0083] Table 2 CA linker composed of SEQ ID No. 3 and SEQ ID No. 4

[0084]

[0085] (3) BA and CA linkers need to be annealed to form double strands for ligation experiments. A 50 μL reaction system contains: 5 μL of each paired primer single strand, 5× annealing buffer, and ddH2O to make up to 50 μL. Mix well and heat to 94°C, then cool to 25°C at a rate of 1°C / min and store at 4°C. Prepare adaMix by mixing BA and CA at a ratio of 1:16.

[0086] C. Connector connection

[0087] 1. React the digested fragments obtained in step A with the adapter mixture adaMix prepared in step B using the following system configuration: 2 μL T4 ligase, 4 μL T4 ligase buffer (10×), 1 μL adaMix, and 20 μL fragDNA. Finally, make up to 40 μL with ddH2O. Ligate at 16°C for 12 hours and inactivate at 65°C for 10 minutes to obtain the ligation product, LigMix.

[0088] D. Fragment screening

[0089] Use Beckman's SPRISelect magnetic beads for double-end screening with a LigMix ratio of 0.535-0.6. The screening process can be carried out according to the instructions. Fresh 85% anhydrous ethanol should be prepared in advance, and selMix should be obtained after screening.

[0090] E: Fragment enrichment

[0091] 1. Enrichment Primer Synthesis and Use: The following primer sequences were used for synthesis and PCR amplification of enriched sequencing fragments. The bold sequences in the sequence are the UDI0066-I5 and I7 sequences used in the library construction kit published by Illumina:

[0092] GBS-P5: 5'-AATGATACGGCGACCACCGAGATCTACACGCTTGCGCACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'

[0093] GBS-P7: 5'-CAAGCAGAAGACGGCATACGAGATTCCGACACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3'

[0094] 2. PCR enrichment: Dilute the above primers to a 10 μM concentration before use. The system is: Platinum PCR SuperMix High Fidelity PCR mix 45 μL, GBS-P5 / P7 0.7 μL each, and LigMix 3.6 μL. Make up to 40 μL and set up the PCR instrument according to the following program: pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 68°C for 50 s, repeat these 3 steps for 13-15 cycles, final extension at 72°C for 5 min, and store at 4°C. The obtained libMix cannot be sequenced directly.

[0095] F. Final screening

[0096] The libMix obtained in E was fragment filtered according to the magnetic bead ratio and screening method in D to obtain library DNA within the target size range.

[0097] G. Library quality control

[0098] The library quality was tested using the Quibt method. The quality test results when screening the ~800bp library were as follows: Figure 4 As shown in the figure, (1) the peak type is single, with a clear main peak, no large broad peaks or miscellaneous peaks, and no large fragments; (2) the concentration is above 0.2 ng / μL; (3) the baseline is flat, and the qPCR concentration is qualified. The concentration is 3.42 ng / μL, which meets the sequencing standard.

[0099] Example 4

[0100] Pre-built library for simplified genome sequencing of 96 geese

[0101] A method for constructing a library for goose double-enzyme digestion simplified genome sequencing using the kit of the present invention comprises the following steps:

[0102] (1) Genome fragmentation: Extract goose genomic DNA (conventional DNA extraction or kit extraction can be used, DNA quality requirement: 0260 / 280 (1.7-2.0, TE dissolution), fragment the goose DNA sequence using kit A according to 20 μL enzyme digestion system to obtain genomic fragmentation product fragDNA;

[0103] Enzyme digestion system composition and digestion protocol: 20 μL of the enzyme digestion system includes 0.5 μL of CviAII endonuclease, 2 μL of 10× Buffer, and 200 ng of DNA, which is adjusted to 20 μL with ddH2O and digested at 25°C overnight or for 16 hours. Then, 0.5 μL of EcoRI endonuclease is added to the system for a second digestion, and digestion is carried out at 37°C overnight or for 16 hours.

[0104] Kit A includes: 1000U of EcoRI, 1000U of CviAII, 10× Buffer, and ddH2O;

[0105] (2) Adapter ligation: The fragmented product fragDNA was ligated using Kit B at 16°C for 12 h, inactivated at 65°C for 10 min, and the ligation product LigMix was obtained;

[0106] The adapter ligation system was as follows: T4 ligase 2 μL, 10× Buffer 4 μL, adapter mixture 1 μL, fragDNA 20 μL, and ddH2O was added to 40 μL.

[0107] The adapter mixture adaMix is ​​prepared by mixing BA adapters and CA adapters in a volume ratio of 1:16;

[0108] Kit B: includes: T4 ligase, 10× Buffer, BA linker, CA linker, and ddH2O;

[0109] In the BA linker, the m barcode sequence in SEQ ID No. 1 is shown in Table 3 below, and the corresponding complete sequences of the 12 pairs of linkers are shown in Table 4.

[0110] Table 3 Barcode sequences represented by m in SEQ ID No. 1 in 12 pairs of BA linkers corresponding to 96 individuals

[0111]

[0112] Table 4 12 pairs of BA linkers consisting of SEQ ID No. 1 and SEQ ID No. 2

[0113]

[0114]

[0115] The CA linker is a double-stranded DNA formed by annealing the sequences shown in SEQ ID No. 3 and SEQ ID No. 4, wherein SEQ ID No. 3 is 5' phosphorylated (as shown in Table 5);

[0116] Table 5 CA linker composed of SEQ ID No. 3 and SEQ ID No. 4

[0117]

[0118] (3) Fragment screening: Add magnetic beads at 0.535-0.6 times the amount of the ligation product for double-end screening;

[0119] (4) Fragment enrichment: Using Kit C, pre-denaturation at 94°C for 2 min, followed by 14 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and extension at 68°C for 50 s, followed by a final extension at 72°C for 5 min and storage at 4°C to obtain libMix;

[0120] Kit C includes: high-fidelity PCR polymerase Mix, enrichment primers, and ddH2O;

[0121] The enrichment primer F is shown in SEQ ID No. 5, and the enrichment primer R is shown in SEQ ID No. 6, wherein the index sequence in the h upstream primer in SEQ ID No. 5 is shown in Table 6, and the index sequence in the y downstream primer in SEQ ID No. 6 is shown in Table 6.

[0122] Table 6 Index sequences in SEQ ID No. 5 and SEQ ID No. 6

[0123]

[0124] (5) Final screening: libMix performs fragment filtration according to the magnetic bead ratio and screening method in step (4) to obtain library DNA within the target size range;

[0125] (6) Quality inspection: The constructed DNA library was quality inspected by the Quibt method; the qualified library had the following standards: a. Single peak type, with obvious main peak (such as Figure 5 (as shown), no large broad peaks and miscellaneous peaks, no large fragments; b, the concentration is above 0.2 ng / μL, c, the baseline is flat, and the qPCR concentration is qualified.

[0126] Table 7 Quality inspection results of the purified test library

[0127] Library type Qubit concentration (ng / μL) Average library length (bp) qPCR concentration nM volume Qualified GBS 1.62 419 4.89 25 yes

[0128] It will be understood by those skilled in the art that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art in the art to which the present invention pertains. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with their meanings in the context of the prior art and, unless defined as such herein, will not be interpreted in an idealized or overly formal sense.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A goose double enzyme digestion simplified genome sequencing library construction kit, characterized in that: The kit comprises: Kit A: Contains: 1000U / mL EcoRI, 1000U / mL CviAII, 10× Buffer, and ddH2O. Kit B: includes: 400,000 U / mL T4 ligase, 10× buffer, 20 pm / μL BA linker, 20 pm / μL CA linker, and ddH2O; The BA adapter is a double-stranded DNA formed by annealing the sequences shown in SEQ ID No. 1 and SEQ ID No. 2, wherein SEQ ID No. 2 is 5' phosphorylated, and m and n in SEQ ID No. 1 and SEQ ID No. 2 represent any short nucleotide barcode sequence with a length of 5-20 bp, and the four bases AATT at the front of n are the sticky end of EcoRI; The CA linker is a double-stranded DNA formed by annealing the sequences shown in SEQ ID No. 3 and SEQ ID No. 4, wherein SEQ ID No. 3 is 5' phosphorylated; Kit C: includes: 2 U / μL high-fidelity PCR polymerase mix, 10 μM / μL enrichment primers, and ddH2O; The enrichment primer F is shown in SEQ ID No. 5, and R is shown in SEQ ID No. 6, wherein h in SEQ ID No. 5 represents the index sequence in the upstream primer, and y in SEQ ID No. 6 represents the index sequence in the downstream primer.

2. The goose double-enzyme digestion simplified genome sequencing library construction kit according to claim 1, characterized in that: Also includes magnetic beads.

3. The goose double-enzyme digestion simplified genome sequencing library construction kit according to claim 2, characterized in that: The magnetic beads are streptavidin affinity magnetic beads.

4. Use of the kit according to any one of claims 1 to 3 in constructing a goose double-enzyme digestion simplified genome sequencing library.

5. The use of the kit according to claim 4 in constructing a goose double enzyme digestion simplified genome sequencing library, characterized in that the steps include: (1) Genome fragmentation: Extract goose genomic DNA and fragment the goose DNA sequence using 20 μL enzyme digestion system of kit A to obtain the genomic fragmentation product fragDNA; (2) Adapter ligation: The genomic fragmentation product fragDNA was ligated using Kit B, ligated at 16°C for 12 h, inactivated at 65°C for 10 min, and the ligation product LigMix was obtained; (3) Fragment screening: Add magnetic beads to the ligation product LigMix for double-end screening; (4) Fragment enrichment: using kit C, pre-denaturation at 94°C for 2 min, denaturation at 94°C for 30 s, annealing at 62°C for 30 s, extension at 68°C for 50 s, 13-15 cycles, final extension at 72°C for 5 min, and storage at 4°C to obtain the primary sequencing library libMix; (5) Final screening: LibMix performs fragment filtering according to the magnetic bead ratio and screening method in step (3) to obtain a mature sequencing DNA library libMix within the target size range; (6) Quality control: The constructed DNA library libMix was quality checked using the Quibt method.

6. The application according to claim 5, characterized in that In step (1), the composition and digestion scheme of the enzyme digestion system are as follows: 20 μL of the enzyme digestion system includes 0.5 μL of CviAII endonuclease, 2 μL of 10× Buffer, and 200 ng of DNA, which is adjusted to 20 μL with ddH2O and digested at 25°C overnight or for 16 hours. Then, 0.5 μL of EcoRI endonuclease is added to the system for a second digestion, and digested at 37°C overnight or for 16 hours.

7. The use according to claim 5, characterized in that In step (2), the adapter ligation system is: T4 ligase 2μL, 10× Buffer 4μL, adapter mixture 1μL, fragDNA 20μL, and ddH2O is added to 40μL.

8. The application according to claim 7, characterized in that: The linker mixture is prepared by mixing the BA linker and the CA linker in a volume ratio of 1:

16.

9. The use according to claim 5, characterized in that: In step (3), the ratio of magnetic beads added is 0.535-0.6 times that of the ligation product.

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