CN111394799A - Method for constructing meningitis pathogen metagenome second-generation sequencing library and kit thereof - Google Patents
Method for constructing meningitis pathogen metagenome second-generation sequencing library and kit thereof Download PDFInfo
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Abstract
The invention discloses a method for constructing a meningitis pathogen metagenome second-generation sequencing library and a kit thereof. The invention firstly provides a method for constructing a meningitis pathogen metagenome second-generation sequencing library, which comprises the steps of performing end repair, phosphorylation and A addition, joint connection and purification, PCR enrichment and purification on fragmented meningitis pathogen DNA through one-step reaction, and obtaining the meningitis pathogen metagenome second-generation sequencing library. The method and the kit thereof can realize the completion of end repair, phosphorylation and addition of A in one step, the library building steps are few, the time is short, the cost is low, the problem that the library building is difficult to succeed due to extremely low nucleic acid amount of a cerebrospinal fluid sample is solved by adding exogenous genome DNA, the joint connection and the library amplification obtain a second-generation sequencing library of a macro genome with extremely high specificity, and the effective library proportion is high; therefore, the method or the kit has wide application prospect in the construction of a meningitis pathogen metagenome next-generation sequencing library.
Description
Technical Field
The invention belongs to the technical field of sequencing library construction. More particularly, relates to a method for constructing a meningitis pathogen metagenome second-generation sequencing library and a kit thereof.
Background
With the development of sequencing technology, the second generation sequencing has become the mainstream of sequencing technology with the advantages of high accuracy, high flux and low price. In the aspects of personalized medicine, genetic diseases, clinical diagnosis and the like, the second-generation sequencing has become a common technical means; for example, tumor whole exome sequencing, whole exome genetic testing. Metagenomic sequencing (metagenomic sequencing) is high-throughput sequencing of the genome of a microbial community in an environmental sample, and mainly researches the microbial population structure, gene function activity, the mutual cooperation relationship among microorganisms and the relationship between the microorganisms and the environment. In recent years, metagenome sequencing has become a hotspot, and few effective library-building kits for metagenome second-generation sequencing exist in the market.
The existing method for constructing the next generation sequencing library of the metagenome mainly comprises the following five steps: (1) fragmenting and purifying DNA; (2) carrying out end repairing and purification on the fragmentation product; (3) adding A to the end repairing product and purifying; (4) performing joint connection on the product A, and purifying; (5) and enriching the joint connection product. In the existing five steps for constructing a second-generation sequencing library of the metagenome, four steps of purification are needed, a product after fragmentation, a terminal repair product, an A adding product and a joint connection product all need to be purified, the time consumption is long in multiple times of magnetic bead purification, the steps are complex, the terminal repair step and the A adding step cannot be integrated, and the requirements on the initial quantity and the quality of a DNA sample are high. As can be seen, the existing method for constructing the metagenome next-generation sequencing library not only has high operation cost and long time consumption, but also inevitably increases the loss of the DNA sample through multi-step purification, and the library construction can not be successful for the DNA sample with lower initial quantity and poorer quality.
Therefore, it is of great significance to develop a simplified construction method of metagenomic next-generation sequencing library, which is especially suitable for DNA samples with low initial amount and poor quality (the nucleic acid amount of cerebrospinal fluid samples is extremely low).
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of the existing construction method of the metagenome second-generation sequencing library and provide a construction method of the meningitis pathogen metagenome second-generation sequencing library and a kit thereof.
The invention aims to provide a method for constructing a meningitis pathogen metagenome second-generation sequencing library.
The invention also aims to provide a kit for constructing a meningitis pathogen metagenome next-generation sequencing library.
The invention further aims to provide application of the method or the kit in construction of a meningitis pathogen metagenome next-generation sequencing library.
The above purpose of the invention is realized by the following technical scheme:
the invention firstly provides a method for constructing a meningitis pathogen metagenome second-generation sequencing library, which comprises the steps of performing end repair, phosphorylation and A addition, joint connection and purification, PCR enrichment and purification on fragmented meningitis pathogen DNA through one-step reaction, and obtaining the meningitis pathogen metagenome second-generation sequencing library.
Preferably, the method comprises the steps of:
s1, carrying out one-step reaction on the fragmented meningitis pathogen DNA, a reaction solution A and an enzyme mixed solution B;
s2, performing joint connection reaction on the product obtained in the step S1, the reaction liquid C, the reaction liquid D and the enzyme E, and purifying;
and S3, performing library amplification on the product obtained in the step S2, the reaction liquid F and the reaction liquid G, and purifying to obtain the meningitis pathogen metagenome second-generation sequencing library.
Preference is given toIn step S1, the reaction solution A includes 0.2-0.3M Tris-HCl, 0.02-0.08M MgCl20.02-0.07M DTT (dithiothreitol), 1-1.5 mM ATP (adenine ribonucleotide), 2.2-2.7 mM dATP (adenine deoxyribonucleotide), 2.2-2.7 mM dGTP (guanine deoxyribonucleotide triphosphate), 2.2-2.7 mM dCTP (cytosine deoxyribonucleotide triphosphate), 2.2-2.7 mM dTTP (thymine deoxynucleotide triphosphate) and 0.1-0.15 g/M L PEG4000 (polyethylene glycol-4000).
More preferably, the reaction solution A in step S1 includes 0.25M Tris-HCl, 0.05M MgCl2、0.05M DTT、1.25mM ATP、2.5mM dATP、2.5mM dGTP、2.5mM dCTP、2.5mM dTTP、0.125g/mL PEG4000。
Preferably, the enzyme mixture B in step S1 is a mixture of Taq DNA polymerase, T4 DNA polymerase and T4 nucleotide kinase.
Preferably, the reaction procedure of the one-step reaction in step S1 is: reacting at 5-15 ℃ for 5-15 min, at 30-50 ℃ for 15-25 min, and at 60-70 ℃ for 15-30 min.
More preferably, the reaction procedure of the one-step reaction in step S1 is: the reaction is carried out for 5min at 12 ℃, 15min at 37 ℃ and 15min at 70 ℃.
Preferably, the reaction solution C in the step S2 includes 0.2-0.3M Tris-HCl, 0.02-0.08M MgCl20.02-0.07M DTT, 13.5-14 mM ATP, 2.2-2.7 mM dATP, 2.2-2.7 mM dGTP, 2.2-2.7 mM dCTP and 2.2-2.7 mM dTTP.
More preferably, the reaction solution C in step S2 includes 0.25M Tris-HCl, 0.05M MgCl20.05M DTT, 13.75mM ATP, 2.5mM dATP, 2.5mM dGTP, 2.5mM dCTP and 2.5mM dTTP.
Preferably, the reaction solution D in the step S2 includes a linker A of 6.5 to 8.5 μ M and a linker B of 6.5 to 8.5 μ M.
More preferably, the reaction solution D in step S2 includes 7.5. mu.M linker A and 7.5. mu.M linker B.
Preferably, the enzyme E in step S2 is DNA ligase.
Preferably, the reaction procedure of the linker ligation reaction in step S2 is: reacting at 15-25 ℃ for 15-20 min, and reacting at 65-75 ℃ for 5-10 min.
More preferably, the reaction procedure of the linker ligation reaction in step S2 is: the reaction is carried out for 15min at 25 ℃ and for 5min at 70 ℃.
Preferably, the Reaction solution F in step S3 includes 75-85% 5 × Q5 Reaction buffer Pack, 0.7-0.9 mM dATP, 00.7-0.9 mM dGTP, 0.7-0.9 mM dCTP, 0.7-0.9 mM dTTP and 3-5% polymerase.
More preferably, the Reaction solution F in step S3 includes 80% 5 × Q5 Reaction buffer Pack, 0.8mM dATP, 0.8mM dGTP, 0.8mM dCTP, 0.8mM dTTP and 4% polymerase.
Preferably, the reaction solution G in the step S3 includes 8-12 μ M of the primer A and 8-12 μ M of the primer B.
More preferably, the reaction solution G in step S3 includes 10. mu.M of the primer A and 10. mu.M of the primer B.
Preferably, the procedure for library amplification in step S3 is: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and 10-15 cycles; complete extension at 72 ℃ for 5 min.
Preferably, the purification in step S2 and step S3 are both magnetic bead purification.
The invention also provides a kit for constructing the meningitis pathogen metagenome second-generation sequencing library, which comprises a reaction liquid A, an enzyme mixed liquid B, a reaction liquid C, a reaction liquid D, an enzyme E, a reaction liquid F and a reaction liquid G.
Preferably, the use method of the kit is a construction method of the meningitis pathogen metagenome second-generation sequencing library.
In addition, the application of the method or the kit in the construction of a meningitis pathogen metagenome next-generation sequencing library also belongs to the protection scope of the invention.
The invention has the following beneficial effects:
(1) the method and the reagent for constructing the meningitis pathogen metagenome next-generation sequencing library can realize one-step completion of end repair, phosphorylation and A addition, and purification is not needed before joint connection of a product after fragmentation, a product after end repair, phosphorylation and A addition product, so that the library construction steps are few, the time is short, and the cost is low.
(2) Exogenous genome DNA is added in the construction process of the metagenome next-generation sequencing library creatively, the library construction success rate of the cerebrospinal fluid sample is obviously increased, and the problem that the library construction is difficult to succeed due to extremely low nucleic acid quantity of the cerebrospinal fluid sample is solved; the second-generation sequencing library of the macro genome with extremely high specificity is obtained through adaptor connection and library amplification, the effective library proportion is high, the requirement of sequencing on a computer is met, and the detection and analysis results of samples are not influenced; therefore, the method or the kit disclosed by the invention can be suitable for low-initial-quantity (0-50 ng) and low-quality or degraded DNA samples, and has a wide application prospect in construction of a meningitis pathogen metagenome next-generation sequencing library.
Drawings
FIG. 1 is an electrophoretogram of 6 cerebrospinal fluid samples after pooling amplification.
FIG. 2 is a 2100 map of a successful pooling cerebrospinal fluid sample; wherein (A) is a 2100 map of a cerebrospinal fluid sample of group B-1 without the addition of an exogenous nucleic acid; (B) FIGS. (C), (D) are 2100 maps of cerebrospinal fluid samples of the exogenous nucleic acid-added groups (A-2, B-2, C-2), respectively.
FIG. 3 is a 2100 map of a meningitis pathogen metagenomic second-generation sequencing library constructed with the kit of the present invention.
FIG. 4 is a 2100 map of a meningitis pathogen metagenomic second-generation sequencing library constructed with a kit.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 construction method of meningitis pathogen metagenome second-generation sequencing library
A method for constructing a meningitis pathogen metagenome second-generation sequencing library comprises the following steps:
s1.DNA fragmentation and one-step reaction
(1) Instrument preparation and setup
Preparing an instrument: turning on a power switch of a Bior μ ptor (TM) UCD-200 breaking instrument, taking broken ice to a position half of a water tank of the breaking instrument, and standing for 15min to freeze the bottom of the water tank; ultrapure water was added to the water tank (not exceeding the scale lines).
The instrument is set up: ultrasonic frequency is ultrasonic 30s, pause is 30s, ultrasonic power is selected: h: high, the interruption can be started when a layer of floating ice floats on the surface of the water tank.
(2) Nucleic acid sampling and fragmentation
Nucleic acid was quantified (total amount of nucleic acid was 40u L) and sampled as shown in Table 1. the samples were taken at 4min for physical disruption (8 disruptions), and taken out every 1min for 1 (two disruptions), centrifuged instantaneously, and ice was added once (water level could not exceed the scale line, and a suitable amount of water could be removed before ice addition) to obtain fragmented DNA.
Note that: when the water tank is interrupted, floating ice is guaranteed, otherwise, the instrument is easy to damage; after the interruption, one-step reaction (end repair, phosphorylation, and addition of A) is required immediately, the power supply of the interruption instrument is turned off, the water in the water tank is poured out, the water is wiped dry by a paper towel, and the sound insulation box is closed.
TABLE 1 nucleic acid sampling
(3) One-step reaction (end repair, phosphorylation, addition A)
Taking out the reaction liquid A and the enzyme mixed liquid B from a refrigerator at the temperature of-20 ℃, unfreezing the reaction liquid A at room temperature, reversing, uniformly mixing, centrifuging for a short time for later use, unfreezing the enzyme mixed liquid B on ice, reversing, uniformly mixing, centrifuging for a short time, placing on an ice box for later use, and preparing a reaction system for one-step reaction in a PCR tube (table 2); slightly vortexing, mixing and centrifuging for a short time; the PCR tube was then placed in a PCR machine and a one-step reaction was performed according to the reaction procedure shown in Table 3.
Wherein the reaction solution A comprises 0.25M Tris-HCl and 0.05M MgCl2、0.05M DTT、1.25mM ATP、2.5mMdATP、2.5mM dGTP、2.5mM dCTP、2.5mM dTTP、0.125g/mL PEG4000。
The enzyme mixture B is a mixture of Taq DNA polymerase, T4 DNA polymerase and T4 nucleotide kinase.
TABLE 2 reaction System for one-step reaction
| Composition of | Volume of |
| Fragmented DNA obtained in step (2) | 37.5μL |
| Reaction solution A | 10μL |
| Enzyme mixture B | 2.5μL |
| Total | 50μL |
TABLE 3 reaction sequence for one-step reaction
| Step (ii) of | Temperature of | Time of |
|
| 1 | 12 | 5min | |
| 2 | 37℃ | 15min | |
| 3 | 70℃ | 15min | |
| 4 | 4℃ | Hold |
S2, connecting and purifying by joints
(1) Joint connection
Taking out the reaction solution C and the reaction solution D from a refrigerator at the temperature of-20 ℃, thawing at room temperature, mixing uniformly, and centrifuging for a short time for later use; taking out the enzyme E from a refrigerator at the temperature of-20 ℃, centrifuging for a short time, and putting the enzyme E on an ice box for later use.
Adding a reaction solution C, a reaction solution D and an enzyme E to the reaction product of the one-step reaction in step S1 (the system of linker ligation reaction is shown in Table 4); slightly vortexing, mixing and centrifuging for a short time; the linker ligation reaction was carried out according to the reaction procedure shown in Table 5.
After the termination of the linker ligation reaction, the resulting linker-ligated product may be stored for 24 hours at-20 ℃ or below, if subsequent tests cannot be performed in time.
Wherein the reaction solution C comprises 0.25M Tris-HCl and 0.05M MgCl20.05M DTT, 13.75mM ATP, 2.5mM dATP, 2.5mM dGTP, 2.5mM dCTP and 2.5mM dTTP.
Reaction solution D included 7.5. mu.M linker A and 7.5. mu.M linker B.
Enzyme E is DNA ligase.
TABLE 4 systems for linker ligation reactions
TABLE 5 reaction sequence for linker ligation reaction
| Step (ii) of | Temperature of | Time of |
|
| 1 | 25 | 15min | |
| 2 | 70℃ | 5min | |
| 3 | 4℃ | Hold |
(2) Purification (fragment sorting of adaptor ligation products)
1) Taking out the DNA purification magnetic beads from a refrigerator at 2-8 ℃ 30min in advance, standing, balancing the temperature to room temperature, reversing or carrying out vortex oscillation to fully and uniformly mix the DNA purification magnetic beads, and subpackaging 60 mu L magnetic beads into 1.5m L low-adsorption centrifuge tubes;
2) adding the adaptor connection product obtained in the step (1) into magnetic beads, repeatedly blowing, uniformly mixing (the suction head does not leave the liquid surface during uniform mixing, so as to prevent excessive bubbles from being generated), and incubating at room temperature for 5min to enable DNA to be combined on the magnetic beads;
3) keeping the sample on the magnetic frame all the time, adding 200 mu L of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30s, carefully removing the supernatant, and rinsing for 2 times;
4) taking out a 1.5m L tube from the magnetic frame, placing the tube on the magnetic frame after instantaneous separation, removing residual 80% ethanol, and uncovering the cover at room temperature to dry the magnetic beads for about 2-5 min;
5) and taking the sample out of the magnetic frame, adding 37 mu L ribozyme-removing water, gently sucking and uniformly mixing by using a pipette, standing at room temperature for 2min, then placing on the magnetic frame, and after the solution is clarified (5min), finishing the fragment sorting (purification) of the adaptor ligation product.
S3.PCR enrichment and purification
(1) PCR enrichment (library amplification)
Taking out reaction solution F and reaction solution G (S1-S40) from a refrigerator at-20 ℃, unfreezing the reaction solution F on ice for 20min, unfreezing the reaction solution G for 20min at room temperature, uniformly mixing, and centrifuging for a short time for later use;
adding reaction solution F and reaction solution G into the product obtained after the linker connection and purification in step S2 (the system of PCR enrichment reaction is shown in Table 6); slightly vortexing, mixing and centrifuging for a short time; PCR enrichment (library amplification) was performed in a thermal cycler following the reaction procedure shown in table 7.
Reaction F contained 80% 5 × Q5 Reaction buffer Pack, 0.8mM dATP, 0.8mM dGTP, 0.8mM dCTP, 0.8mM dTTP and 4% polymerase.
Reaction solution G: includes 10. mu.M of primer A and 10. mu.M of primer B.
TABLE 6 PCR enrichment reaction System
| Composition of | Volume of |
| Product of linker ligation and purification in step S2 | 35μL |
| Reaction solution F | 12.5 mu L (Single adding) |
| Reaction solution G | 2.5 μ L (Single adding) |
| Total | 50μL |
TABLE 7 procedure for PCR enrichment reaction
Note: when the sampling quantity of nucleic acid for establishing the library is less than 20ng, X is 14; when the sampling amount of the nucleic acid for library construction is 20-30 ng, X is 12.
(2) Purification (PCR fragment purification from PCR enrichment)
1) Taking out the DNA purification magnetic beads from a refrigerator at 2-8 ℃ 30min in advance, standing, balancing the temperature to room temperature, reversing or carrying out vortex oscillation to fully and uniformly mix the DNA purification magnetic beads, and subpackaging 62 mu L magnetic beads into 1.5m L low-adsorption centrifuge tubes;
2) supplementing ultrapure water to the PCR fragments obtained by PCR enrichment in the step (1) to 100 mu L, adding the PCR fragments into magnetic beads, repeatedly blowing, uniformly mixing (the suction head does not leave the liquid surface during uniform mixing, so as to prevent excessive bubbles from being generated), and incubating at room temperature for 5min to enable DNA to be combined on the magnetic beads;
3) placing the sample on a magnetic frame, after the solution is clarified (5min), taking the supernatant into a low-adsorption centrifuge tube containing 20 mu L DNA purification magnetic beads, and incubating at room temperature for 5min to enable the DNA to be combined on the magnetic beads;
4) placing the sample on a magnetic frame, carefully removing the supernatant after the solution is clarified (5min), keeping the sample on the magnetic frame all the time, adding 200 mu L of freshly prepared 80% ethanol to rinse the magnetic beads, incubating at room temperature for 30s, carefully removing the supernatant, and rinsing for 2 times;
5) keeping the sample on a magnetic frame all the time, and uncovering the cover at room temperature to dry the magnetic beads for about 2-5 min;
6) and taking the sample out of the magnetic frame, adding 27 mu L ribozyme-removing water, gently sucking and uniformly mixing the sample by using a pipette, standing the mixture at room temperature for 2min, placing the mixture on the magnetic frame, carefully sucking 25 mu L supernatant into a new 1.5m L centrifugal tube after the solution is clarified (5min), and thus completing the purification of the PCR fragment obtained by PCR enrichment.
Example 2 application of method for constructing meningitis pathogen metagenome next-generation sequencing library in cerebrospinal fluid sample library construction
1. Effect of exogenous nucleic acid addition on reservoir building Power
(1) Experimental methods
6 cerebrospinal fluid samples are subjected to library construction according to the construction method of the meningitis pathogen metagenome secondary sequencing library in the embodiment 1, no exogenous nucleic acid group (A-1, B-1 and C-1) and exogenous nucleic acid group (A-2, B-2 and C-2) are arranged, and the influence of the addition of exogenous nucleic acid on the library construction success rate of the cerebrospinal fluid samples is researched; then, a 2100 atlas of the cerebrospinal fluid sample which is successfully built is determined, and off-line data of the cerebrospinal fluid sample is obtained.
(2) Results of the experiment
The results of the influence of the addition of the exogenous nucleic acid on the reservoir building power of the cerebrospinal fluid samples are shown in table 8, and the electrophoresis chart of the amplified 6 cerebrospinal fluid samples is shown in fig. 1, so that the cerebrospinal fluid samples with the exogenous nucleic acid group are successfully built (the reservoir building power is 100%), the concentration of the library is more than 6.98, the electrophoresis band is clear and bright, and only 1 cerebrospinal fluid sample without the exogenous nucleic acid group is successfully built, and the reservoir building power is 33.3%; indicating that the addition of exogenous nucleic acid can significantly increase the pooling success of cerebrospinal fluid samples (low concentration DNA samples).
TABLE 8 Effect of exogenous nucleic acid addition on cerebrospinal fluid sample pooling success
A2100 map of a cerebrospinal fluid sample with successful library construction is shown in FIG. 2, wherein (A) is a 2100 map of a cerebrospinal fluid sample without adding an exogenous nucleic acid set B-1; (B) FIGS. (C) and (D) are 2100 maps of cerebrospinal fluid samples of the exogenous nucleic acid group (A-2, B-2, C-2), respectively; comparing the graph (A) with the graphs (B), (C) and (D), it can be seen that the addition of exogenous nucleic acid does not affect the 2100 map data; the off-machine data of the cerebrospinal fluid samples successfully constructed in the library are shown in Table 9, and it can be seen that the off-machine data amount of the cerebrospinal fluid samples without the addition of the exogenous nucleic acid group B-1 and the exogenous nucleic acid group has no significant difference in the same data mixture; the exogenous nucleic acid is added, so that the reservoir building success rate of the cerebrospinal fluid sample can be obviously increased, and the information analysis result is not influenced.
TABLE 9 Un-order data for successful cerebrospinal fluid samples for library construction
| Sample name | Off-line data volume/G of equal data mixture |
| B-1 | 0.8268 |
| A-2 | 0.887 |
| B-2 | 0.8937 |
| C-2 | 0.8402 |
2. Cerebrospinal fluid sample bank
(1) Experimental methods
76 cerebrospinal fluid samples were pooled according to the method for constructing the meningitis pathogen metagenomic secondary sequencing library described in example 1.
(2) Results of the experiment
The results of the 76 cerebrospinal fluid sample library construction are shown in table 10, and it can be seen that 76 cerebrospinal fluid samples are successfully constructed by the method for constructing the meningitis pathogen metagenome next-generation sequencing library, and the library construction success rate is 100%; the method of the invention has good application potential.
TABLE 1076 results of pooling of cerebrospinal fluid samples
Example 3 preparation of a kit for constructing a Megeminy second Generation sequencing library of meningitis pathogens
A kit for constructing a meningitis pathogen metagenomic next-generation sequencing library comprising the following components (table 11):
TABLE 11 Components of kit for construction of a meningitis pathogen metagenomic second-generation sequencing library
The components in the table 9 are assembled to obtain the kit for constructing the meningitis pathogen metagenome next-generation sequencing library.
Example 4 comparison of the Performance of the kit of the present invention with other kits
Comparing the performances of the kit for constructing the meningitis pathogen metagenome next-generation sequencing library (the kit of the invention) prepared in the embodiment 3 with the performances of a Novozaki library-establishing kit (a certain kit), specific experimental methods and experimental results are respectively as follows:
1. comparison of 2100 spectra
(1) Experimental methods
The reagent kit and a certain reagent kit are respectively used for constructing a meningitis pathogen metagenome next-generation sequencing library, then 2100 maps are respectively determined, and the determination is repeated for 3 times.
(2) Results of the experiment
The 2100 maps of the meningitis pathogen metagenome second-generation sequencing library constructed by the kit and a certain kit are respectively shown in fig. 3 and fig. 4, and as can be seen by comparing fig. 3 with fig. 4, the library obtained by constructing the kit is a single peak, the fragment is about 400bp, the main band of the library obtained by constructing the kit by a certain kit is about 400bp, but a small peak exists between 700 and 3000bp behind the main peak; the kit of the invention is proved to have better library construction accuracy than a certain kit.
2. Comparison of QPCR and Qubit results
(1) Experimental methods
The QPCR result and the Qubit result obtained by constructing a meningitis pathogen metagenome second-generation sequencing library by using the kit and a certain kit are compared.
(2) Results of the experiment
The comparison table of the QPCR results and the Qubit results is shown in table 12, and it can be seen that although the concentration of the library Qubit obtained by the reagent of a certain kit is slightly higher than that of the library Qubit obtained by the reagent of the present invention, the QPCR concentration thereof is slightly lower than that of the library QPCR obtained by the reagent of the present invention, and the ratio thereof is lower than that of the present invention; the reagent of the kit is proved to have non-specific amplification in the amplification process, and the specificity of the kit is superior to that of the kit.
TABLE 12 comparison of QPCR and Qubit results (ratio QPCR/Qubit concentration)
The above results show that: compared with a certain kit sold in the market, the kit provided by the invention has the advantages of high library building accuracy and strong specificity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A method for constructing a meningitis pathogen metagenome second-generation sequencing library is characterized by comprising the steps of performing end repair, phosphorylation and A addition, joint connection and purification, PCR enrichment and purification on fragmented meningitis pathogen DNA through one-step reaction, and obtaining the meningitis pathogen metagenome second-generation sequencing library.
2. Method according to claim 1, characterized in that it comprises the following steps:
s1, carrying out one-step reaction on the fragmented meningitis pathogen DNA, a reaction solution A and an enzyme mixed solution B;
s2, performing joint connection reaction on the product obtained in the step S1, the reaction liquid C, the reaction liquid D and the enzyme E, and purifying;
and S3, performing library amplification on the product obtained in the step S2, the reaction liquid F and the reaction liquid G, and purifying to obtain the meningitis pathogen metagenome second-generation sequencing library.
3. The method according to claim 2, wherein the reaction solution A in step S1 comprises 0.2-0.3M Tris-HCl, 0.02-0.08M MgCl20.02-0.07M DTT, 1-1.5 mM ATP, 2.2-2.7 mM dATP, 2.2-2.7 mM dGTP, 2.2-2.7 mM dCTP, 2.2-2.7 mM dTTP and 0.1-0.15 g/M L PEG 4000.
4. The method according to claim 2, wherein the enzyme mixture B of step S1 is a mixture of Taq DNA polymerase, T4 DNA polymerase and T4 nucleotide kinase.
5. The method of claim 2, wherein the reaction procedure of the one-step reaction of step S1 is as follows: reacting at 5-15 ℃ for 5-15 min, at 30-50 ℃ for 15-25 min, and at 60-70 ℃ for 15-30 min.
6. The method according to claim 2, wherein the reaction solution C in step S2 comprises 0.2-0.3M Tris-HCl, 0.02-0.08M MgCl20.02 to 0.07M DTT, 13.5 to 14mM ATP, 2.2 to 2.7mM dATP, 2.2 to 2.7mM dGTP, 2.2 to 2.7mM dCTP and 2.2 to 2.7mM dTTP; step S2, the reaction solution D comprises a joint A of 6.5-8.5 mu M and a joint B of 6.5-8.5 mu M; step S2 the enzyme E is DNA ligase.
7. The method of claim 2, wherein the reaction sequence of the linker ligation reaction of step S2 is: reacting at 15-25 ℃ for 15-20 min, and reacting at 65-75 ℃ for 5-10 min.
8. A kit for constructing a meningitis pathogen metagenome next-generation sequencing library is characterized by comprising a reaction liquid A, an enzyme mixed liquid B, a reaction liquid C, a reaction liquid D, an enzyme E, a reaction liquid F and a reaction liquid G.
9. The kit according to claim 8, wherein the method of using the kit is according to any one of claims 1 to 7.
10. Use of the method of any one of claims 1 to 7 or the kit of any one of claims 8 to 9 in the construction of a metagenomic second-generation sequencing library of a meningitis pathogen.
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