CN112538657A - Cerebrospinal fluid gene sequencing library building and detecting method and application thereof - Google Patents
Cerebrospinal fluid gene sequencing library building and detecting method and application thereof Download PDFInfo
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Abstract
A cerebrospinal fluid gene sequencing library construction method, a detection method and application thereof are disclosed, wherein the library construction method specifically comprises the steps of extracting cfDNA from a sample to be detected of cerebrospinal fluid and detecting the concentration of fragments of the sample by adopting Labchip GX Touch, so that the PCR cycle number of a constructed library is determined to meet the requirement of amplification; and then carrying out enrichment amplification by adopting the concentration of the PCR amplification primer with the concentration of at least 20 mu mol/L so as to improve the library construction success rate.
Description
Technical Field
The invention relates to the field of gene detection, in particular to cerebrospinal fluid gene sequencing library building, a detection method and application thereof.
Background
Cerebrospinal fluid is mainly distributed in the ventricles of the brain, the subarachnoid space and the spine, flows in a certain direction among the three, and is finally absorbed into blood by arachnoid granules. The tight junction between epithelial cells between the blood-cerebrospinal fluid and the glial limiting membrane formed by the astrocytes constitute a filtering barrier between cerebrospinal fluid and blood, called the blood-cerebrospinal fluid barrier. Most of the cns tumors directly contact the cerebrospinal fluid, releasing ctDNA into the cerebrospinal fluid, while due to the filtering effect of the blood-cerebrospinal fluid barrier ctDNA cannot enter the blood, plasma cfDNA may appear false negative. Therefore, for central nervous system diseases, the detection of ctDNA (circulating tumor DNA)) in the cerebrospinal fluid can reflect the central nervous system diseases more truly than the detection of plasma ctDNA.
In the detection of free cfDNA in liquid biopsies, there is a need to increase the starting DNA amount and the amount of sequencing data due to the low proportion of valid data. For cerebrospinal fluid with a rare sample amount, the amount of nucleic acid extracted from separated supernatant is mostly lower than the detection lower limit, so that normal library building cannot be performed, and the ex-library concentration of the sample after library building is low, so that low-frequency detection cannot be guaranteed.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide a cerebrospinal fluid gene sequencing and library building method which is short in library building time and low in cost and can be applied to liquid biopsy. The second technical problem to be solved by the present invention is to provide a method for sequencing and detecting cerebrospinal fluid genes by using the DNA sequencing library prepared by the library construction method, which can be applied to liquid biopsy of trace samples to be detected. The invention also provides a cerebrospinal fluid gene sequencing detection kit, which comprises a DNA sequencing library prepared by the library construction method. The fourth technical problem to be solved by the invention is to provide the application of the construction method or the kit in cerebrospinal fluid genome liquid biopsy.
According to the first aspect, the method for sequencing and constructing the cerebrospinal fluid gene comprises the steps of extracting cfDNA from a sample to be detected of the cerebrospinal fluid and performing quality control on the sample by adopting Labchip GX Touch, so that the PCR cycle number for constructing the library is determined to meet the requirement of amplification. And then carrying out enrichment amplification by adopting the concentration of the PCR amplification primer with the concentration of at least 20 mu mol/L so as to improve the library construction success rate.
Specifically, the cerebrospinal fluid gene sequencing and banking method comprises the following steps of:
A. extraction and quality control: centrifuging a sample to be detected of cerebrospinal fluid at least twice, extracting cfDNA of the sample to be detected from a supernatant, and detecting the concentration of fragments of the cfDNA by adopting Labchip GX Touch;
B. constructing a library: and performing end-segment repair and A addition, joint connection and purification on the cfDNA, performing PCR amplification on a purified product, determining the PCR amplification cycle number according to the detection result of the fragment concentration of the cfDNA, and purifying the PCR amplification product to obtain the gene sequencing library of the cerebrospinal fluid cfDNA.
The concentration of extracted cerebrospinal fluid is always far below the lower limit of detection by using the quantit 3.0, and accurate quantification cannot be carried out. Therefore, the method adopts Labchip GX Touch (2 mu L of sample is diluted to 20 mu L) for fragment quality control, and detects the mass concentration of the 80-400bp fragment (fragment interval where cfDNA is located). The quality control result is normal when the mass concentration is more than 0.1 ng/mu L and the total amount of cfDNA is more than 5ng, and the sample to be detected can be used for subsequent detection.
In one embodiment, the quality control of the fragments refers to the detection of the mass concentration of cfDNA with the fragments of 80-400 bp.
Determining the cycle number of PCR amplification according to the detection result of the concentration of the cfDNA fragment, specifically comprising: setting the mass concentration of the cfDNA fragment to be less than 0.2 ng/muL as a risk library, and setting the PCR cycle number to be 12 cycles; the mass concentration of the cfDNA fragment is more than or equal to 0.2 ng/. mu.L, the library is normally constructed, and the PCR cycle number is 8 cycles.
The number of PCR cycles is too small, so that the number of target DNA in a sample is lower than the detection requirement, and the target DNA cannot be accurately detected; too many PCR cycles reduce amplification fidelity and produce large fragment contamination. The number of the existing PCR cycles is generally judged according to the concentration of the Qubit, however, for a cerebrospinal fluid sample to be detected with a low initial sample amount, the quantification using the Qubit 3.0 is always far lower than the lower detection limit, and accurate quantification cannot be carried out. According to the characteristics of low content and difficult detection of the cerebrospinal fluid sample to be detected, the PCR cycle number rule for cerebrospinal fluid library construction is newly formulated, and the cfDNA in all the cerebrospinal fluid sample to be detected can meet the minimum hybridization input amount (500ng) in the library construction link at present.
This application carries out data statistics analysis through the sample accumulation that awaits measuring, cfDNA quality control concentration, PCR cycle number, library concentration in the sample that awaits measuring cerebrospinal fluid, to categorizing that the PCR cycle number under the different storehouse inception of establishing of sample that awaits measuring cerebrospinal fluid goes on, the fragment of formulating complete cfDNA builds storehouse PCR cycle number rule: setting the mass concentration of the cfDNA fragment to be less than 0.2 ng/muL as a risk library, and setting the PCR cycle number to be 12 cycles; the mass concentration of the cfDNA fragment is more than or equal to 0.2 ng/. mu.L, the library is normally constructed, and the PCR cycle number is 8 cycles.
In one embodiment, the concentration of the PCR amplification primer is preferably 20-40 μmol/L.
The concentration of the PCR amplification primer in library construction has an important influence on the concentration of the library construction, the concentration of the primer in the conventional plasma cfDNA library construction is 10 mu mol/L, and if the cfDNA in cerebrospinal fluid is also 10 mu mol/L, the library construction success rate is low (because the sample is less and the cfDNA content is low), so that the clinical requirement cannot be met. To ensure that the ex-warehouse concentration of cfDNA is within a certain range, the number of PCR cycles must be reduced when the concentration of the PCR amplification primers is increased, but this will result in the failure of ex-warehouse of a low initial amount of samples and reduced sample compatibility.
In the application, the sample to be detected of the cerebrospinal fluid is a trace sample (the initial amount is low), the primer concentration is increased to be more than 20 mu mol/L during library establishment, and the sample library establishment power can be improved while the concentration of the PCR amplification primer is increased by determining that the PCR cycle number rule is 8 cycles (the mass concentration of cfDNA is more than or equal to 0.2 ng/mu L) or 12 cycles (the mass concentration of cfDNA is less than 0.2 ng/mu L).
Because the cerebrospinal fluid sample to be tested generally contains nucleated cells, in order to avoid gDNA pollution during extraction, at least two times of centrifugation and refreezing or extraction are required immediately after the sample is obtained. The purpose of centrifugation is to separate nucleated cells, and thus conventional centrifugation procedures can be applied to the present application. In one embodiment of the present application, the sample to be tested is preferably centrifuged twice, and the centrifugal force during the first centrifugation is 1500-1700 rcf for 5-30 min; the centrifugal force during the second centrifugation is 15000-17000 rcf, and the time is 5-30 min. In a more preferred embodiment, the centrifugal force at the first centrifugation is 1608rcf for 10 min; the centrifugal force during the second centrifugation was 16000rcf for 10 min. After centrifugation, the supernatant was extracted and further processed. And if the sample is frozen and thawed after twice centrifugation, flocculent substances appear, and normal extraction can be carried out without re-centrifugation.
In one embodiment, a free DNA extraction reagent is used for extracting a sample to be detected, wherein the free DNA extraction reagent comprises proteinase K, a sodium dodecyl sulfate solution, ethanol and a magnetic bead mother solution containing magnetic beads; wherein the adding ratio of the sample to be detected to the magnetic bead mother solution is 1mL to 15 mu L-1 mL to 20 mu L. Namely, the dosage of the magnetic beads in each 1mL of sample to be detected is 15-20 mu L, and the concentration of the mother solution of the magnetic beads is 35-45 mg/mL. The total amount of cfDNA extracted can be effectively increased in the range of the addition ratio under the condition of avoiding incomplete reaction or excessive magnetic beads.
cfDNA extraction was performed using the Thermo MagMAX Cell-Free DNA Isolation Kit magnetic bead method, the magnetic beads suitable for use in the present invention, also known as magnetic beads, can be magnetic microspheres commonly used in the art. Preferably, the magnetic ball used in the present invention is a superparamagnetic nano magnetic ball prepared by using a nanotechnology. The magnetic bead can be specifically identified and efficiently combined with nucleic acid molecules on a micro interface, and DNA and RNA can be separated from samples such as blood, animal tissues, food, pathogenic microorganisms and the like under the action of Chaotropic salts (guanidine hydrochloride, guanidine isothiocyanate and the like) and an external magnetic field.
In one embodiment, due to the current limitation of the highest input amount for hybridization, in order to ensure the fragment abundance during hybridization, the input amount of the DNA sequencing library volume is ensured to be more than 50%, and single hybridization is generally selected. Hybridization can also be carried out if the input is such that > 50% of the volume of the DNA sequencing library is satisfied and the total amount of multiple libraries hybridized together is less than the highest input for hybridization.
Subsequent off-machine depth data shows that the dup average sequencing depth of the cerebrospinal fluid samples with the same proportion and multiple impurities is obviously improved, and the omission of low-frequency mutation is avoided.
In some embodiments, the PCR amplification primers are selected from any one of Illumina sequencing platform primers, huada gene MGI sequencing platform primers, and gieni plus sequencing platform primers.
In some embodiments, after library amplification, the constructed library can be applied to a sequencing platform of Illumina, DNBSEQ-T7 of Shenzhen Huada Gene technology, Inc., Gene + Seq 2000, Gene + Seq200, and other high throughput sequencing platforms of Guijing Gen plus technology, Inc. The sequencing platform to which the library of the present invention is applicable is not limited, and the above sequencing platforms are merely exemplary.
According to a second aspect, the present application provides a cerebrospinal fluid gene sequencing detection method, which uses a DNA sequencing library prepared by the library construction method of the first aspect to perform high-throughput sequencing detection on cfDNA in cerebrospinal fluid.
According to a third aspect, the present application provides a cerebrospinal fluid gene sequencing detection kit, which comprises a DNA sequencing library prepared by the library construction method of the first aspect.
According to a fourth aspect, the present application provides a use of the method of the first aspect or the kit of the third aspect for constructing a cerebrospinal fluid genome second-generation sequencing library.
The cerebrospinal fluid gene sequencing, library building and detection method has the following advantages and beneficial effects:
1. the kit is suitable for detecting free DNA (cfDNA) in plasma, and is particularly suitable for detecting fragments of the cfDNA in cerebrospinal fluid with low initial amount of a sample (the sample lower than a detection lower line can also be subjected to risk detection after extraction);
2. centrifuging the collected cerebrospinal fluid sample (containing nucleated cells) for more than two times, so that gDNA pollution during extraction can be avoided;
3. performing fragment quality control by adopting Labchip GX Touch, and determining PCR cycle number according to the mass concentration of cfDNA in a sample to be detected;
4. by the library construction method, the quality control qualification rate of the library can be improved by 14%, the proportion of the DNA detection library (the concentration of the library constructed output is more than 40 ng/mu L) is improved by 29%, and more samples can flow into a hybridization link; meanwhile, the reservoir building power (mass concentration is more than 0.1 ng/mu L) is more than 98%, the dup removing depth (more than or equal to 500 x) is 100%, and the clinical detection requirement (the qualification rate is more than 95%) is met.
Drawings
FIG. 1 is a comparison graph of the mass concentration of DNA at 87.5. mu.L and 75. mu.L for the mother solution of magnetic beads based on Labchip detection in the first example;
FIG. 2 is a comparison graph of the PCR amplification primer concentrations of 20. mu. mol/L and 10. mu. mol/L for the library quantification in example two;
FIG. 3 is a comparison graph of the average depth of dup removal with multiple hybrid (5 hybrid) and single hybrid for DNA sequencing library in the third example.
Detailed Description
Chemical specific examples: the invention will be further illustrated by means of specific embodiments in conjunction with the accompanying drawings. It should be understood that the examples are illustrative only and are not to be construed as limiting the scope of the invention. The experimental procedures in the following examples are carried out in the usual manner unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. Any methods and materials similar or equivalent to those described below can be used in the present invention.
Example 1:
the embodiment provides a method for extracting cfDNA from cerebrospinal fluid supernatant, which comprises the following specific steps:
extraction was performed using Thermo MagMAX Cell-Free DNA Isolation Kit (Free DNA extraction reagents including proteinase K, sodium dodecyl sulfate solution, ethanol, and mother solution of magnetic beads containing magnetic beads, hereinafter described using magnetic beads instead of mother solution of magnetic beads for convenience).
29 cerebrospinal fluid test samples were collected and centrifuged twice immediately thereafter. First centrifugation: centrifuging at room temperature for 10min at a centrifugal force of 1608rcf, transferring the supernatant to a new centrifuge tube, and centrifuging for the second time; and (3) second centrifugation: the centrifugal force is 16000rcf, the time is 10min, the centrifugation is carried out at normal temperature, and the supernatant is transferred to a new centrifugal tube. Each sample to be tested is divided into two parts to be tested in a 15mL/50mL centrifuge tube respectively, and the serial numbers are 1-29 and 30-58. After the split, the initial amount of extraction was 5mL, and the sample with insufficient volume was extracted after adding PBS (1X, pH 7.4) to make up the volume to 5 mL.
To the sample to be tested, 75. mu.L of proteinase K was added, and after vortexing for 30 seconds, 250. mu.L of SDS (SDS means a 20% sodium dodecyl sulfate solution) was added. After all additions, vortex for 30s (note: SDS inactivates proteinase K, so addition of proteinase K simultaneously with 20% SDS is to be avoided).
And (3) putting the centrifuge tube into a 60 ℃ water bath pot for incubation for 20min, and quickly turning up and down and uniformly mixing every 5min (note: the speed of turning over and uniformly mixing is high, so that the temperature reduction of a cracking system is avoided).
Preparing 80% ethanol (at least 2mL for each sample to be tested); taking out the centrifuge tube from the water bath, quickly putting the centrifuge tube into ice, and incubating for 5min to cool the sample to be measured; adding 6.25mL of cfDNA Binding Solution into a centrifuge tube, and adding 1-29 samples to be detected
MyOneTMSILANE, adding sample to be tested 30-58
MyOneTMSILANE, vortex for 30s, place on the reverse mixer, and reverse at room temperature for 10 min.
Placing a 15mL/50mL centrifuge tube on a magnetic frame, standing for 15min, and adsorbing magnetic beads until the liquid is clear and transparent; absorbing and removing the supernatant, adding 1mL of DNA Wash Solution, oscillating for 30s, carrying out instantaneous separation, and transferring all liquid into a corresponding 1.5mL centrifuge tube; after the liquid in the 1.5mL centrifuge tube is clarified, taking the supernatant and adding the supernatant into the 15mL/50mL centrifuge tube, re-suspending the residual magnetic beads and transferring all the liquid into the 1.5mL centrifuge tube; after the liquid in the centrifugal tube of 1.5mL is clarified, discarding the supernatant, adding 1mL of DNA Wash Solution, carrying out vortex oscillation for 30s, carrying out instantaneous separation, placing on a magnetic frame of 1.5mL for standing for 1min until the Solution is clarified, and then sucking and discarding the supernatant; adding 1mL of freshly prepared 80% ethanol, performing vortex oscillation for 30s, performing instantaneous separation, standing on a 1.5mL magnetic frame for 1min, clarifying the solution, and then absorbing and removing a supernatant; then 1mL of freshly prepared 80% ethanol was added, vortexed and shaken for 30s, instantaneously separated, and the solution was left on a 1.5mL magnetic stand for 1min to clarify, and then the supernatant was discarded. Keeping a 1.5mL centrifuge tube on a magnetic frame, and drying the magnetic beads at room temperature (the magnetic beads are only matte surfaces, so that excessive drying is avoided); taking down a 1.5mL centrifuge tube from the magnetic frame, adding 60 μ L of Solution, vortexing and oscillating for 5min, and resuspending the magnetic beads; the 1.5mL centrifuge tube was placed on a 1.5mL magnetic rack immediately after centrifugation for 2min until the liquid cleared and the supernatant transferred to the corresponding 1.5mL cfDNA storage tube.
Diluting 2 mu L of sample to be detected to 20 mu L, detecting Labchip GX Touch for fragment quality control, and detecting the mass concentration of the 80-400bp fragment. And the rest samples to be tested are used for building a library or are stored at the temperature of between 25 ℃ below zero and 15 ℃ below zero.
The results of the Labchip assay are shown in FIG. 1 below. As can be seen from FIG. 1, the total amount of the samples with 87.5. mu.L of magnetic beads (i.e., the ratio of the sample to be detected to the magnetic beads is 1mL: 17.5. mu.L) and 75. mu.L of magnetic beads (i.e., the ratio of the sample to be detected to the magnetic beads is 1mL: 15. mu.L) was increased by 19%. In the present application, the amount of the magnetic beads is preferably 15 μ L to 20 μ L per 1mL of the sample to be tested.
Example 2:
and selecting a plurality of samples to be detected for library building. Use of
UltraTMII, carrying out end repair, A adding and joint connection on the DNA Library Prep Kit, wherein the Library construction method is divided into two methods, one PCR amplification primer concentration is 10P, and the other PCR amplification primer concentration is 20P. P is expressed in units of. mu. mol/L. The method comprises the following specific steps:
end repair plus a: the following single sample reagent reaction system (i.e., single hybrid reaction system) was configured: mu.L of NEBNext Ultra II End Prep Reaction Buffer (Reaction conditions: 20 ℃ for 30min, 65 ℃ for 30 min).
Connecting a joint: the linker was added singly (in order to avoid self-ligation of the linker, the linker could not be added to mix in the reaction, but the linker was added to the sample alone), the linker solution was a solution of the linker mother liquor diluted to a concentration of 15. mu. mol/L with TE buffer, the volume of the linker solution added in this step was 2. mu.L, and the linker sequence and the preparation method of the linker mother liquor were as described in example 2 of the Chinese patent application No. 202011061421.7. The following single sample reagent reaction system was configured: 30 μ L NEBNext Ultra II Ligation Master Mix, 1 μ L NEBNext Ligation Enhancer. Reaction conditions are as follows: incubate at 20 ℃ for 15 h.
Purifying after joint connection: a shallow well plate was taken and 87. mu.L of Axygen beads were added to each well, leaving at least one column empty between each column of beads for gun walking. Adding the adaptor connection reaction product into a corresponding reaction hole, blowing, uniformly mixing, incubating at room temperature for 10min to ensure that the magnetic beads are fully combined with the DNA fragments, and preparing 80% ethanol during the incubation period. The shallow hole plate was placed on a plate magnetic stand and allowed to stand for 10 min. After the magnetic beads are fully adsorbed, the supernatant is discarded. Add 200. mu.L of 80% ethanol to each well, blow gently 3 times, and pipette out 300. mu.L of eight-well pipette the ethanol from the bottom of the plate. Then, 200. mu.L of 80% ethanol was added to each well and the wells were rinsed again, and the ethanol on the bottom of the plate was removed by pipetting with 300. mu.L of eight-well pipette and 10. mu.L of eight-well pipette in this order. Placing the shallow-hole plate on a 38 ℃ drier, heating and drying until the surfaces of the magnetic beads are not reflected, taking down the shallow-hole plate from the drier, placing the shallow-hole plate on a plate type magnetic frame, adding 22 mu L of TE into the shallow-hole plate, blowing and uniformly mixing the magnetic beads by using a pipettor, and incubating for 5min at room temperature; after incubation, the plate was placed on a magnetic rack until clear. The supernatant was transferred to 20. mu.L to eight rows.
Amplification: the amplification reaction for each sample was as follows: 20 μ L of sample, 5 μ L of primer, 25 μ L of 2 XKAPA HiFi HotStart ReadyMix. Here, 5. mu.L of the primer means that the total amount of the primer added is 5. mu.L. The concentration of the PCR amplification primer of the sample is 10P or 20P. The primer sequence is the sequence in the specification table 1 in the Chinese patent with the application number of 202011061421.7.
Rule for PCR cycle number: establishing a library for the risk that the mass concentration of the cfDNA of the cerebrospinal fluid supernatant is less than 0.2 ng/mu L, and the PCR cycle number is 12 cycles; and (4) normally constructing a library when the mass concentration of the cfDNA of the cerebrospinal fluid supernatant is more than or equal to 0.2 ng/mu L, and performing PCR cycle number of 8 cycles. The PCR reaction conditions are shown in Table 1 below.
TABLE 1
Library purification: a shallow well plate was taken and 45. mu.L of Axygen beads were added to each reaction well, leaving at least one column empty between each column of beads for gun walking. Adding the adaptor connection reaction product into a corresponding reaction hole, blowing, uniformly mixing, incubating at room temperature for 10min to ensure that the magnetic beads are fully combined with the DNA fragments, and preparing 80% ethanol during the incubation period. The shallow hole plate was placed on a plate magnetic stand and allowed to stand for 10 min. After the magnetic beads are fully adsorbed, the supernatant is discarded. Add 200. mu.L of 80% ethanol to each well, blow gently 3 times, and pipette out 300. mu.L of eight-well pipette the ethanol from the bottom of the plate. Then, 200. mu.L of 80% ethanol was added to each well and the wells were rinsed again, and the ethanol on the bottom of the plate was removed by pipetting with 300. mu.L of eight-well pipette and 10. mu.L of eight-well pipette in this order. Placing the shallow-hole plate on a 38 ℃ drier, heating and drying until the surfaces of the magnetic beads are not reflected, taking down the shallow-hole plate from the drier, placing the shallow-hole plate on a plate type magnetic frame, adding 31 mu L of TE into the shallow-hole plate, blowing and uniformly mixing the magnetic beads by using a pipettor, and incubating for 5min at room temperature; after incubation, the plate was placed on a magnetic rack until clear. Supernatants were transferred 30 μ L to eight rows.
The library quantification results are shown in FIG. 2. As can be seen from FIG. 2, the yield of quality control of the library is improved by 14% when the concentration of the PCR amplification primer is 20P compared with the data with the concentration of 10P, and the ratio of the library is improved by 29% when the concentration of the PCR amplification primer is more than 40 ng/. mu.L, so that more samples can flow into the hybridization process.
Example 3
And selecting a plurality of samples to perform single-hybrid and multi-hybrid data collection so as to determine the hybridization base number of the DNA sequencing library in library construction by adopting single hybrid or multi-hybrid. The method comprises the following specific steps.
Drying Mix by distillation to prepare: and unfreezing the working solution of the double-index joint blocking sequence, the Cot-1 DNA and the library to be hybridized at 4 ℃. After melting, shaking, mixing and centrifuging, adding into a 1.5mL centrifuge tube according to the table 2, shaking, mixing and centrifuging.
TABLE 2
And (3) steaming the Mix tube cover to be punched, putting the Mix tube cover in a vacuum concentrator to be concentrated and steamed at 60 ℃, and sealing the hole on the tube cover by using a sealing film after the Mix tube cover is steamed to be dried. In the process of drying by distillation, the probe to be hybridized is unfrozen at 4 ℃. Placing the xGen 2X Hybridization Buffer and the xGen Hybridization Buffer Enhancer at room temperature for dissolving, and oscillating and centrifuging; preparing denatured Mix according to table 3, shaking, mixing, subpackaging into the mixture, shaking, mixing, centrifuging, and denaturing at 95 deg.C for 10 min.
TABLE 3
2-3 minutes before the sample library denaturation is completed, subpackaging the dissolved probes into 0.2ml PCR tubes, wherein the dosage of each reaction probe is 4 mu L; after the denaturation is finished, centrifuging the sample library for 1min at full speed by using a high-speed centrifuge, then quickly transferring the sample library into a PCR tube, and carrying out oscillation centrifugation; place PCR tube on PCR instrument for hybridization overnight at 65 ℃ (hot lid temperature 75 ℃); elution experiments were performed after overnight incubation. Before the elution experiment, at least 30 minutes ahead of time, the Wash buffers II, III, Stringent Wash Buffer and the Bead Wash Buffer stock solutions are taken out from a refrigerator at the temperature of-20 ℃, unfrozen at room temperature, and elution working solution with the concentration of 1 multiplied by the unit reaction dosage in the table 4 is prepared and is preheated in advance in the corresponding temperature environment. The M-270 magnetic beads and the AMPure XP magnetic beads were equilibrated at room temperature.
TABLE 4
After the M-270 magnetic beads equilibrated to room temperature were sufficiently shaken and mixed, 20. mu.L of the mixture was aspirated into a new 1.5mL centrifuge tube, and the supernatant was discarded by mounting on a magnetic holder. Taking down the magnetic frame, rinsing the magnetic beads for 3 times by using 200 mu L of 1 multiplied by Bead Wash Buffer, adding 100 mu L of 1 multiplied by Bead Wash Buffer to resuspend the magnetic beads after the supernatant is discarded for the last time, and transferring the magnetic beads to a new 0.2mL PCR tube for later use;
the supernatant was removed from the magnetic rack of the PCR tube containing the beads, and the overnight incubated hybridization system was transferred to a bead tube and mixed by shaking and incubated in a 65 ℃ PCR apparatus (hot lid 75 ℃) for 45 minutes. Taking out the reaction tube every 15 minutes during incubation, quickly shaking and uniformly mixing for 1 time; the bead rinses were performed in the order, amounts and times of reagents according to table 5.
TABLE 5
2 XKAPA HiFi HotStart ReadyMix and post-hybridization PCR primers (GF Primer:/5 Phos/TCTCAGTACGTCAGCAGTT; and GRprimer: GGCATGGCGACCTTATCAG;) were thawed at 4 ℃ in advance and centrifuged well with shaking. The post-hybridization PCR Mix was prepared according to Table 6 for use.
TABLE 6
| Components | Single reaction volume (μ L) |
| 2×KAPA HiFi HotStart ReadyMix | 25 |
| GF Primer(10μM) | 2.5 |
| GR Primer(10μM) | 2.5 |
| Total volume | 30 |
Transferring 20 mu L of rinsed resuspended magnetic beads into post-hybridization PCR Mix, blowing and mixing uniformly by a pipette, placing in a PCR instrument, and performing post-hybridization PCR according to the program in the table 7.
TABLE 7
The PCR product was purified using 60. mu.L of AMPure XP magnetic beads and finally dissolved in 31. mu.L of TE (pH 8.0) (the procedure was the same as for library purification in example 2). The purified library is used for library quality control, sequencing or storing at-20 ℃.
See fig. 3 for subsequent offboard depth data. As can be seen from FIG. 3, the single-impurity cerebrospinal fluid sample has significantly improved dup average speed measurement depth compared with the multi-impurity cerebrospinal fluid sample, and the omission of low-frequency mutation is avoided. Therefore, the hybridization base number of the DNA sequencing library in the library construction of the application adopts single hybridization.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (10)
1. A cerebrospinal fluid gene sequencing and banking method is characterized by comprising the following steps of:
A. extraction and quality control: centrifuging a sample to be detected of cerebrospinal fluid at least twice, extracting cfDNA of the sample to be detected from a supernatant, and detecting the concentration of fragments of the cfDNA by adopting Labchip GX Touch;
B. constructing a library: and performing end-segment repair and A addition, joint connection and purification on the cfDNA, performing PCR amplification on a purified product, determining the PCR amplification cycle number according to the detection result of the fragment concentration of the cfDNA, and purifying the PCR amplification product to obtain the gene sequencing library of the cerebrospinal fluid cfDNA.
2. The cerebrospinal fluid gene sequencing and banking method according to claim 1, wherein determining the number of cycles of PCR amplification based on the detection result of the concentration of the fragment of the cfDNA comprises:
setting the mass concentration of the cfDNA fragment to be less than 0.2 ng/muL as a risk library, and setting the PCR cycle number to be 12 cycles;
the mass concentration of the cfDNA fragment is more than or equal to 0.2 ng/. mu.L, the library is normally constructed, and the PCR cycle number is 8 cycles.
3. The cerebrospinal fluid gene sequencing and banking method according to claim 1, comprising, in step a, two centrifugation steps:
the centrifugal force during the first centrifugation is 1500-1700 rcf, and the time is 5-30 min;
the centrifugal force during the second centrifugation is 15000-17000 rcf, and the time is 5-30 min.
4. The cerebrospinal fluid gene sequencing and banking method according to claim 1, wherein in the step a, a free DNA extraction reagent is used for extracting the sample to be tested, wherein the free DNA extraction reagent comprises proteinase K, a sodium dodecyl sulfate solution, ethanol and a mother solution of magnetic beads containing magnetic beads; wherein the concentration of the mother solution of the magnetic beads is 35-45 mg/mL; the adding ratio of the sample to be detected to the magnetic bead mother solution is 1mL to 15 mu L-1 mL to 20 mu L.
5. The cerebrospinal fluid gene sequencing and banking method according to claim 1, wherein the fragment quality control means detecting the mass concentration of cfDNA with fragments of 80-400 bp.
6. The cerebrospinal fluid gene sequencing and banking method according to claim 1, wherein the concentration of the PCR amplification primer is preferably 20 to 40 μmol/L.
7. The method of claim 1, wherein the number of DNA sequencing library hybrids in the library is single hybrid.
8. A cerebrospinal fluid gene sequencing detection method, characterized in that a DNA sequencing library prepared by the library construction method according to any one of claims 1 to 7 is used for high-throughput sequencing detection of cfDNA in cerebrospinal fluid.
9. A cerebrospinal fluid gene sequencing detection kit, comprising a DNA sequencing library prepared by the library construction method of any one of claims 1 to 7.
10. Use of the method of any one of claims 1 to 7 or the kit of claim 9 for the construction of a cerebrospinal fluid genome next generation sequencing library.
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