CN111378718A - Construction method of gene sequencing library - Google Patents
Construction method of gene sequencing library Download PDFInfo
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- CN111378718A CN111378718A CN201811627007.0A CN201811627007A CN111378718A CN 111378718 A CN111378718 A CN 111378718A CN 201811627007 A CN201811627007 A CN 201811627007A CN 111378718 A CN111378718 A CN 111378718A
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- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Abstract
The invention provides a construction method of a gene sequencing library, and particularly relates to the field of gene sequencing.
Description
Technical Field
The invention relates to the technical field of sequencing, in particular to a method for constructing a gene sequencing library.
Background
Next Generation Sequencing (NGS) has become popular since its emergence with the advantages of high throughput and low cost. With the development of the technology, a new generation of sequencing technology has applications in many aspects of scientific research and clinical detection.
At present, many scientific researches and clinical applications need to rapidly sequence the whole genome of a target or deeply sequence a target region of interest; using RNA-seq to find new variation at the transcriptome level or accurately quantifying the expression amount of mRNA; analyzing epigenetic factors such as various methylation of DNA, interactions between DNA and protein; the cancer is accurately sequenced, and the mutation site is searched, so that the method can be used for precise medical treatment and individual treatment of the cancer.
In the aspect of Sequencing technology, Sequencing by Synthesis Sequencing (SBS) technology is adopted in Sequencing instruments such as Miseq, Nextseq and Hiseq, which are developed by Illumina, so that large-scale parallel Sequencing is supported, and the Sequencing instruments are widely popular due to the advantages of high throughput, low cost and short period.
In the process of actually using sequencing, the requirement on timeliness is quite high in many times, and the time needs to be shortened as much as possible in each link of gene detection.
Sequencing library construction technology based on transposase interruption can simultaneously realize DNA fragmentation and addition of a linker, and such methods have been reported, for example, Chinese patent CN105525357B discloses a method for constructing a library by using a transposase embedding complex, which can greatly reduce the time for sample processing. However, since DNA fragmentation by transposase is related to the initial amount of target DNA, a larger initial amount of target DNA will result in a larger library fragment obtained by transposase after DNA fragmentation is achieved, and cannot meet the requirement of subsequent sequencing on the size range of the library fragment; meanwhile, different initial amounts of target DNA will yield different amounts of DNA libraries after transposase-based library construction. Thus, current transposase-interrupted library construction requires a certain amount of sample to be performed, and the resulting library is accurately quantified for downstream sequencing.
The conventional homogenization method is used for absorbing samples with equal quantity or proportion by estimating the quantity of DNA through the light absorption value, so that the homogenization purpose is realized, however, the method for quantifying the light absorption value or fluorescence is influenced by other similar absorption specific spectrums such as protein, other types of nucleic acid or substances, and the fluorescence quantification has the defects of high cost, complex operation and time consumption; the existing homogenization process can be defined as three steps of quantification-calculation-suction. The operation time for quantifying 96 samples varies from several minutes to 3 hours due to different instrument platforms; a calculation link, namely recording the concentration of each sample and calculating the specific sample absorption amount, wherein the time is about 1 hour; and adjusting a pipettor, independently sucking samples with corresponding calculated amount from each sample, and performing a downstream library construction process after homogenization between the samples, wherein the process needs 1 hour. Thus, according to the prior art process, the entire homogenization process takes 5 hours. This step is time consuming and cumbersome when performing large sample library construction, and although now aided by automated instrumentation, the costs associated therewith will increase further.
Disclosure of Invention
The invention provides a method for constructing a gene sequencing library, which comprises the following steps:
(1) contacting the magnetic particles with the transposase-embedded complex such that the magnetic particles and the transposase-embedded complex form a complex; wherein each transposase-embedding complex comprises a transposase and further comprises a first linker sequence and/or a second linker sequence; the first linker sequence comprises a first sequencing linker sequence and a transposase recognition sequence, and the second linker sequence comprises a second sequencing linker sequence and a transposase recognition sequence;
wherein, the magnetic particles in the complex are combined with the transposase through nickel ion (Ni2+) -histidine interaction;
(2) incubating the complex in (1) with a target DNA sample to generate a DNA library with linkers at both ends.
According to the invention, the construction method of the gene sequencing library comprises the following steps:
(1) combining the magnetic particles and the transposase embedding compound according to a certain proportion to form a complex;
(2) incubating the complex of (1) with a target gene;
(3) separating the complex from the reaction system in (2);
(4) PCR amplifying and purifying the complex in (3) and a primer of a joint sequence with a tag sequence;
wherein the complex comprises a magnetic particle and a transposase-embedded complex; the transposase-embedded complex comprises a transposase, a transposase recognition sequence, a first sequencing linker sequence, and/or a second sequencing linker sequence; the PCR primers include a front primer comprising a first sequencing tag sequence and a back primer comprising a second sequencing tag sequence.
In some embodiments, the method does not include a step of quantifying the target DNA contained in the target DNA sample.
In some embodiments, the magnetic particles are magnetic beads that chelate divalent metal cations; as a preferred embodiment of the present invention, the magnetic particles chelate divalent metal cations by coupling coordinated Nitrilotriacetic Acid (NAT); more preferably, the divalent metal cation is a divalent nickel ion (Ni)2+)。
In some embodiments, the transposase-embedding complex is unpurified prior to contacting with the magnetic particles.
In some embodiments, the transposase is a transposase with a protein purification tag; as a preferred embodiment of the present invention, the protein tag is a polyhistidine tag (His-tag); preferably, the transposase is Tn5 transposase.
In some embodiments, the method further comprises (3) separating the complex from the reaction system of (2) after the incubating; and (4) performing PCR amplification using the complex as a template.
In some embodiments, the PCR uses a pre-primer comprising a first sequencing tag sequence and a post-primer comprising a second sequencing tag sequence
In some embodiments, the transposase-embedding complex is formed by transposase and magnetic particles in a 60U:0.5 mg-2100U: 0.5mg in combination; as a preferred embodiment of the invention, the ratio is 750U:0.5 mg.
In some embodiments, the magnetic particles are incubated with the target DNA sample at low imidazole concentration with shaking at room temperature; as a preferred embodiment of the present invention, the low imidazole concentration is from 15Mm to 50 Mm; preferably 15 Mm.
In some embodiments, the complex and the target DNA sample incubation conditions are oscillation speed of 700-; preferably 1100 rpm; shaking for 20-40 min; preferably 30 min.
The target DNA used in the present invention may be a plasmid, a genomic DNA, an amplified DNA, or the like; the source of the genomic DNA sample may be a cell, a tissue, a trace amount of DNA sample, or the like.
As a preferred embodiment of the invention, the linker sequence and PCR primers are selected from the sequencing linker sequence of Illumina Nextera sequencing protocol.
In a preferred embodiment of the present invention, the tag sequence is a fixed 6-12 base sequence; in a preferred embodiment of the present invention, the tag sequence is an 8-base fixed sequence.
As a preferred embodiment of the present invention, the transposase recognition sequence is a 19bp chimeric end transposon end recognized by transposase Tn 5.
The method can be used for sample processing of a new generation high-throughput Illumina sequencing platform. Among them, the new generation high throughput Illumina sequencing platform includes but is not limited to Miseq, Hiseq, Nextseq sequencing platforms.
As a preferred embodiment of the present invention, a first linker sequence anneals to a transposase recognition sequence complementary sequence to form a first linker, and a second linker sequence anneals to a transposase recognition sequence complementary sequence having a base sequence represented by transposase recognition sequence-inverted (ME-R, i.e., transposase recognition sequence complementary sequence) to form a second linker; the first adaptor sequence has a base sequence shown by Adapter-A; the second linker sequence has a base sequence represented by Adapter-B.
Wherein ME-R is 5 '-phos-CTGTCTCTTATACACATCT-3' (SEQ ID NO: 1); wherein phos is a 5' phosphorylation modification marker.
Wherein Adapter-A is
5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’(SEQ ID NO:2)；
Wherein the transposase recognition sequence is underlined.
Wherein Adapter-B is
5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' (SEQ ID NO: 3); wherein the transposase recognition sequence is underlined.
In a preferred embodiment of the present invention, the PCR forward Primer has a base sequence represented by Primer-F, and the PCR reverse Primer has a base sequence represented by Primer-R.
Wherein Primer-F is
5 '-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCA GCGTC-3' (SEQ ID NO: 4); wherein NNNNNNNN is a first tag sequence, and each N can be selected from any one of A, T, C and G.
Wherein Primer-R is
5 '-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCG G-3' (SEQ ID NO: 5); wherein NNNNNNNN is a second tag sequence, and each N can be selected from any one of A, T, C and G.
In the present invention, the terms "first" and "second" are used only for distinguishing different objects, and are understood to have technical meanings or sequentially defined meanings.
Advantageous effects
The sequencing library construction method based on immobilized transposase interruption is based on the combination of magnetic beads and protein, and optimizes the existing sequencing library construction method based on transposase interruption, so that the size of the finally obtained library fragment and the quality of the library are basically not influenced by the initial amount of target DNA, and the problems of library quality homogenization and library size homogenization of large-scale NGS library construction are effectively solved. The conventional DNA library homogenization needs a quantitative-calculation-absorption process, the time consumption is long when the operation is carried out on a large-scale sample, and the cost is high. In general, the immobilized transposase-interrupted NGS library homogenization method provided by the invention solves the problems of short plates such as high cost, long time consumption, complex operation and the like of sample homogenization in large-scale construction of the NGS library, and has unique design and simple and convenient operation.
Drawings
FIG. 1 is a scheme of conventional transposase-based DNA library construction.
FIG. 2 is a process of transposase-based pooling of a normalized DNA library of the present invention.
FIG. 3 is a graph showing the comparison of the sizes of the DNA library fragments obtained by adjusting the ratio of the magnetic beads to the transposase-embedded complex in the sample of the present invention, and performing library construction using the magnetic bead complex, according to the change in the ratio.
FIG. 4 is a graph comparing the sizes of DNA library fragments obtained by using RCA samples of the present invention with different initial amounts, and performing the method of the present invention and a conventional transposase disruption-based library construction method simultaneously.
FIG. 5 is a comparison of DNA library quality obtained by simultaneous performance of the method of the present invention and a conventional transposase disruption-based library construction method using different starting amounts for plasmid samples of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings.
As shown in fig. 1, the conventional DNA library construction process based on transposase includes DNA template quantification, transposase-embedded linker, transposase-embedded complex and a certain amount of DNA template transposition reaction, PCR enrichment, magnetic bead purification, library quantification, and the like.
As shown in FIG. 2, the transposase-embedded complex formed by embedding the sequencing adaptor and the transposase in the present invention is formed by the poly-histidine tag (His-tag) of the transposase and Ni on the surface of the magnetic beads2+Combining, namely controlling the size of a DNA fragment formed after target DNA breaking by a transposase embedding compound by adjusting the proportion of the input amount of the transposase embedding compound and the target DNA; meanwhile, because the quantity of the transposase embedding compound attached to the magnetic beads is fixed, the quantity of DNA corresponding to the quantity of the transposase embedding compound in a fixed quantity can be obtained by grabbing the magnetic beads out of the solution. Due to the two points, DNA libraries with similar fragment size range and same quality can be finally obtained.
Exemplary method of the invention
1. Preparing a joint:
(1) the following linker sequences were synthesized:
ME-R：5’-phos-CTGTCTCTTATACACATCT-3’
Adapter-A：5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3’
Adapter-B：5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3’
(2) dissolving ME-R, Adapter-A, Adapter-B to 100. mu.M with nuclease-free water;
(3) the corresponding first and second linker sequences are mixed according to the following system:
(4) the above mixture was placed on a PCR instrument and the following procedure was run:
| temperature (. degree.C.) | Time (min) |
| 75 | 15 |
| 60 | 10 |
| 50 | 10 |
| 40 | 10 |
| 25 | 30 |
| 4 | ∞ |
(5) After the procedure was completed, equal volumes of Adapter 1 and Adapter 2 were mixed into the annealing joint mixture and diluted to a concentration of 10. mu.M for each annealing joint.
2. Embedding transposase:
mu.L of transposase (20U/. mu.L) and 10. mu.L of the diluted adaptor mixture (each annealed adaptor concentration is 10. mu.M) were mixed in equal volume, incubated for 60min at 25 ℃ in a PCR instrument, and then cooled to 4 ℃ to form a transposase-embedded complex, which was stored at-20 ℃ for further use.
3. And (3) magnetic bead binding:
(1) taking out HisPur Ni-NTA magnetic bead of Thermo Fishier company from refrigerator, and standing at room temperature for 30 min;
(2) HisPur Ni-NTA magnetic beads were mixed well with shaking, 40. mu.L was taken out and put into a new 1.5mL centrifuge tube, and 160. mu.L of binding buffer (100mM Na) was added thereto3PO4600mM NaCl, 0.05% Tween20, 30mM imidazole, pH 8.0, 25 ℃), shaking and mixing evenly for 10s, and then placing on a magnetic frame;
(3) after the solution is clarified, the supernatant is discarded, 400 mu L of binding buffer solution is added into the solution, the solution is evenly mixed by oscillation for 10s and is placed on a magnetic frame;
(4) after the solution is clarified, the supernatant is discarded, and the following prepared binding components are added into the magnetic beads:
| composition (I) | Volume (μ L) |
| Transposase embedding complex | 50 |
| |
150 |
| |
200 |
| Total of | 400 |
(5) Oscillating and mixing for 10s, placing on a vortex instrument, and fully oscillating and mixing for 30min at 1100 rpm;
(6) after the oscillation is finished, placing the centrifugal tube on a magnetic frame, and after the solution is clarified, discarding the supernatant;
(7) to the beads 400. mu.L of washing buffer (100mM Na) was added3PO4600mM NaCl, 0.05% Tween20, 50mM imidazole, pH 8.0, 25 ℃), shaking and mixing evenly for 10s, placing on a magnetic frame, and discarding the supernatant after the solution is clarified;
(8) repeating the previous step;
(9) mu.L of Tn5 storage buffer was added to the magnetic beads, and the mixture was thoroughly shaken and mixed for 10 seconds to form a magnetic bead complex, which was stored at 4 ℃.
4. Disruption by transposase:
(1) preparing a magnetic bead complex interrupting system according to the following system:
5x TAPS：200mM TAPS-NaOH(pH 8.5，25℃),25mM MgCl2and 50% DMF (dimethylformamide).
(2) Fully and uniformly blowing, and resuspending magnetic beads;
(3) placing the centrifugal tube on a PCR instrument, and setting and operating according to the following procedures:
| temperature of | Time of day | Number of cycles | |
| 55 | 10min | 1 | |
| 4℃ | ∞ | 1 |
5. Magnetic bead cleaning:
(1) after the reaction is finished, instantly separating, and placing the centrifugal tube on a magnetic frame;
(2) after the solution is clarified, discarding the supernatant;
(3) add 100. mu.L of ddH to the beads2Fully and uniformly blowing and stirring, and resuspending magnetic beads;
(4) placing the centrifuge tube on a magnetic frame, and discarding the supernatant after the solution is clarified;
(5) repeating the previous step, discarding the clean supernatant with a small-range gun, and keeping the magnetic beads on the magnetic rack.
6. PCR enrichment:
(1) the following primers were synthesized:
Primer-F：
5’-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC-3’
Primer-R：
5’-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG-3’
(2) Primer-F, Primer-R was dissolved to 2. mu.M with nuclease-free water;
(3) preparing a PCR reaction system according to the following system, fully blowing, beating and uniformly mixing:
note: the 10x P2 buffer, dNTP, and P2 polymerase used in the examples were manufactured by Genscript.
(4) Taking the magnetic beads down from the magnetic frame, resuspending the magnetic beads by using the PCR reaction system, and fully and uniformly blowing;
(5) the PCR tube was placed on a PCR instrument, and the following procedures were set up and run:
7. magnetic bead purification
(1) Placing the PCR tube on a magnetic frame, and transferring all supernatants to a new centrifuge tube after the solution is clarified;
(2) adding 30 μ L of purified magnetic beads (Hieff NGS DNA sorting magnetic beads produced by Yeasen), thoroughly stirring, and standing for 5 min;
(3) placing the centrifuge tube on a magnetic frame, and discarding the supernatant after the solution is clarified;
(4) adding 200 mu L of prepared 80% ethanol on the magnetic beads, standing for 30s, and removing the supernatant;
(5) repeating the previous step, and discarding the clean residual supernatant by using a small-range gun;
(6) standing the centrifugal tube at room temperature for 2-4 min, taking the magnetic beads off the magnetic frame after the magnetic beads are slightly dried, and adding 17 mu L ddH into the magnetic beads2O, fully blowing, beating and uniformly mixing;
(7) incubating at room temperature for 5 min;
(8) and (3) placing the centrifuge tube on a magnetic frame, after the solution is clarified, taking 16 mu L of supernatant, and placing the supernatant into a new centrifuge tube, wherein the supernatant is the constructed DNA library.
To further illustrate the method of the present invention, the present invention is further described with reference to the accompanying drawings and examples.
Example 1：
This example compares the sizes of library fragments obtained from the same sample of an interrupted library of magnetic bead complexes formed by binding different amounts of transposase-embedded complexes to magnetic beads.
The magnetic bead complexes used in this example are shown below:
FIG. 3 shows the results of DNA library fragment sizes obtained after the pooling of target DNA by magnetic bead complexes formed by binding different amounts of transposase-embedding complexes to the same amount of magnetic beads.
The results in FIG. 3 show that a larger amount of transposase-embedded complex is input during the binding process with magnetic beads, and a DNA library with smaller fragment size will be formed.
Example 2：
This example uses the products of rolling circle replication (rolling circle amplification technique, RCA) of the target DNA, together with different initial amounts of library construction using the method of the invention and conventional transposase disruption based library construction methods.
The target DNA used in the test was a sample of the well-known plasmid pUC57, which had a full length of 2710bp and a sequence shown in SEQ ID NO: 6.
Test group one and control group the libraries were created using the methods of the invention (as described above in the "exemplary methods of the invention") and the conventional transposase disruption-based library construction method described below, respectively.
Conventional transposase disruption-based library construction methods:
1. disruption by transposase:
(1) the transposase disruption system was formulated as follows:
| composition (I) | Volume (μ L) |
| | x |
| Transposase | |
| 1 | |
| 5x TAPS | 2 |
| ddH2O | 7-x |
| Total of | 10 |
5x TAPS：200mM TAPS-NaOH(pH 8.5，25℃),25mM MgCl2And 50% DMF (dimethylformamide).
(2) Fully beating and uniformly mixing, and centrifuging for a short time;
(3) placing the centrifugal tube on a PCR instrument, and setting and operating according to the following procedures:
| temperature of | Time of day | Number of cycles | |
| 55 | 10min | 1 | |
| 4℃ | ∞ | 1 |
2. PCR enrichment:
(1) the following primers were synthesized:
Primer-F：
5’-AATGATACGGCGACCACCGAGATCTACACNNNNNNNNTCGTCGGCAGCGTC-3’
Primer-R：
5’-CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGG-3’
(2) Primer-F, Primer-R was dissolved to 2. mu.M with nuclease-free water;
(3) preparing a PCR reaction system according to the following system, fully blowing, beating and uniformly mixing:
| composition (I) | Volume (μ L) |
| Breaking of the product | 10 |
| 10x P2 buffer solution | 3 |
| dNTP(25μM) | 0.75 |
| Primer-F(2μM) | 2 |
| Primer-R(2μM) | 2 |
| |
1 |
| ddH2O | 11.25 |
| Total of | 30 |
Note: the 10x P2 buffer, dNTP, and P2 polymerase used in the examples were manufactured by Genscript.
(4) The PCR tube was placed on a PCR instrument, and the following procedures were set up and run:
3. magnetic bead purification
(1) Adding 30 μ L of purified magnetic beads (Hieff NGS DNA sorting magnetic beads produced by Yeasen), thoroughly stirring, and standing for 5 min;
(3) placing the centrifuge tube on a magnetic frame, and discarding the supernatant after the solution is clarified;
(4) adding 200 mu L of prepared 80% ethanol on the magnetic beads, standing for 30s, and removing the supernatant;
(5) repeating the previous step, and discarding the clean residual supernatant by using a small-range gun;
(6) standing the centrifugal tube at room temperature for 2-4 min, taking the magnetic beads off the magnetic frame after the magnetic beads are slightly dried, and adding 17 mu L ddH into the magnetic beads2O, fully blowing, beating and uniformly mixing;
(7) incubating at room temperature for 5 min;
(8) and (3) placing the centrifuge tube on a magnetic frame, after the solution is clarified, taking 16 mu L of supernatant, and placing the supernatant into a new centrifuge tube, wherein the supernatant is the constructed DNA library.
FIG. 4 shows the results of the final resulting DNA library fragment sizes for different starting amounts of target DNA by the method of the present invention and by the conventional transposase disruption-based library construction method.
The results in FIG. 4 show that the method of the present invention can be used to efficiently input target DNA of different initial amounts, and the resulting library fragments are of similar size.
Example 3：
In this example, the same plasmid sample was used for three times of library construction of target DNA with different initial amounts by the method of the present invention, and in contrast, the library construction was performed by the conventional library construction method based on transposase disruption. The plasmid samples and the library construction method used were the same as in example 2.
FIG. 5 shows the results of the quality of the resulting DNA library for different initial amounts of target DNA using the method of the present invention and a conventional transposase disruption-based library construction method.
The results in FIG. 5 show that, with the method of the present invention, DNA libraries of the same quality can still be obtained with different initial amounts of target DNA.
Example 4：
This example compares the total time required to library the same batch of 96 plasmids using the method of the invention and using a conventional transposase disruption based library construction method. It can be seen that the process of the invention is significantly less time consuming.
The foregoing is a more detailed description of the present invention that is presented in conjunction with specific embodiments, and the practice of the invention is not to be considered limited to those descriptions. It will be apparent to those skilled in the art that a number of simple derivations or substitutions can be made without departing from the inventive concept.
SEQUENCE LISTING
<110> Jiangsu Kinry Biotechnology Ltd
<120> construction method of gene sequencing library
<130>C18P0098
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accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
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tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggcg 480
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 540
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 600
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 660
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 720
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 780
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 840
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 900
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 960
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 1020
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 1080
tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1140
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1200
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1260
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1320
tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 1380
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1440
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1500
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1560
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1620
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1680
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1740
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 1800
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1860
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1920
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1980
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 2040
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 2100
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 2160
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 2220
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2280
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 2340
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 2400
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 2460
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 2520
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2580
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2640
gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 2700
ccctttcgtc 2710
Claims (12)
1. A method of constructing a gene sequencing library, the method comprising:
(1) contacting the magnetic particles with the transposase-embedded complex such that the magnetic particles and the transposase-embedded complex form a complex;
wherein each transposase-embedding complex comprises (a) a transposase and (b) a first linker sequence and/or a second linker sequence; the first linker sequence comprises a first sequencing linker sequence and a transposase recognition sequence, and the second linker sequence comprises a second sequencing linker sequence and a transposase recognition sequence;
wherein nickel ions (Ni) are passed between the magnetic particles and the transposase in the complex2+) -histidine interaction binding;
(2) incubating the complex obtained in (1) with a target DNA sample to generate a DNA library having linkers at both ends.
2. The method according to claim 1, wherein the method does not comprise a step of quantifying the target DNA contained in the target DNA sample.
3. The method according to claim 1 or 2, the magnetic particles being chelated divalent nickel ions (Ni)2+) Preferably, the magnetic particles chelate divalent nickel ions by coupling of the coordinating Nitrilotriacetic Acid (NAT).
4. The method of any one of claims 1-3, wherein the transposase-embedded complex is unpurified prior to contacting with magnetic particles.
5. The method of any one of claims 1-4, wherein the transposase bears a polyhistidine tag; preferably, the transposase is Tn5 transposase.
6. The method of claim 1, further comprising
(3) Separating the complex from the reaction system of (2) after incubation; and
(4) PCR amplification was performed using the complex as a template.
7. The method of claim 6, wherein the PCR uses a pre-primer comprising a first sequencing tag sequence and a post-primer comprising a second sequencing tag sequence.
8. The method of any one of the preceding claims, wherein the transposase and magnetic particles in the transposase-embedded complex are bound in a ratio of 60U:0.5mg to 2100U:0.5 mg; preferably, the ratio is 750U to 0.5 mg.
9. The method according to any one of claims 1 to 8, wherein the incubation of the compomers with the target DNA sample is performed in the presence of 15-50 mM imidazole; preferably 15 mM.
10. The method according to any one of claims 1 to 9, wherein the incubation of the compomer with the target DNA sample is performed at a shaking speed of 700 and 2000rpm for a shaking time of 20 to 40 min; the preferred oscillation speed is 1100 rpm; the preferred shaking time is 30 min.
11. The method of any one of claims 1-10, wherein the target DNA is a plasmid, genomic DNA, or DNA amplification product.
12. The method of any one of claims 1-11, wherein the target DNA is derived from a cell, a tissue, or a trace DNA sample.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811627007.0A CN111378718A (en) | 2018-12-28 | 2018-12-28 | Construction method of gene sequencing library |
| TW108148063A TW202026430A (en) | 2018-12-28 | 2019-12-27 | A method for constructing a gene sequencing library |
| PCT/CN2019/128947 WO2020135650A1 (en) | 2018-12-28 | 2019-12-27 | Method for constructing a gene sequencing library |
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| CN201811627007.0A CN111378718A (en) | 2018-12-28 | 2018-12-28 | Construction method of gene sequencing library |
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| CN (1) | CN111378718A (en) |
| TW (1) | TW202026430A (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113462748A (en) * | 2021-05-11 | 2021-10-01 | 温氏食品集团股份有限公司 | Preparation method and kit of DNA sequencing library |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101644711A (en) * | 2009-09-04 | 2010-02-10 | 中国科学技术大学 | Reproducible molecule layer of biotin combined with protein and preparation method thereof |
| CN105525357A (en) * | 2014-09-30 | 2016-04-27 | 深圳华大基因股份有限公司 | Sequencing library construction method, and kit and application thereof |
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| CN107446916B (en) * | 2017-09-05 | 2020-09-29 | 大连理工大学 | Method for purifying and directionally immobilizing histidine-tagged protein and application |
| CN108004246A (en) * | 2017-12-25 | 2018-05-08 | 中国人民解放军第四军医大学 | The method that liquid phase target SELEX screenings are quickly carried out using the affine method of metal |
-
2018
- 2018-12-28 CN CN201811627007.0A patent/CN111378718A/en active Pending
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101644711A (en) * | 2009-09-04 | 2010-02-10 | 中国科学技术大学 | Reproducible molecule layer of biotin combined with protein and preparation method thereof |
| CN105525357A (en) * | 2014-09-30 | 2016-04-27 | 深圳华大基因股份有限公司 | Sequencing library construction method, and kit and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| STEPHEN BRUINSMA ET AL: "Bead-linked transposomes enable a normalization-free workflow for NGS library preparation", 《BMC GENOMICS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113462748A (en) * | 2021-05-11 | 2021-10-01 | 温氏食品集团股份有限公司 | Preparation method and kit of DNA sequencing library |
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| WO2020135650A1 (en) | 2020-07-02 |
| TW202026430A (en) | 2020-07-16 |
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