CN114686564B - DNA sample preparation method suitable for micro sample - Google Patents
DNA sample preparation method suitable for micro sample Download PDFInfo
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- CN114686564B CN114686564B CN202011642164.6A CN202011642164A CN114686564B CN 114686564 B CN114686564 B CN 114686564B CN 202011642164 A CN202011642164 A CN 202011642164A CN 114686564 B CN114686564 B CN 114686564B
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- PSBOOKLOXQFNPZ-UHFFFAOYSA-M lithium;2-hydroxybenzoate Chemical compound [Li+].OC1=CC=CC=C1C([O-])=O PSBOOKLOXQFNPZ-UHFFFAOYSA-M 0.000 claims abstract description 24
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- 238000012163 sequencing technique Methods 0.000 abstract description 36
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- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 21
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- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
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Abstract
The invention belongs to the technical field of biology, and discloses a DNA sample preparation method suitable for a trace sample, which comprises the following steps: 1) Treating the trace sample with liquid nitrogen; 2) Heating and incubating the sample treated by the liquid nitrogen; 3) The incubated sample is treated with lithium salicylate or purified with magnetic beads to obtain DNA sample of the trace sample. The process of the invention requires only as little as 1mg or 10 4 The order of magnitude of each cell can extract enough DNA for sequencing and banking, and the DNA impurity extracted by the method is less, and the input amount of the sequencing and banking DNA is as low as 2ng.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly provides a DNA sample preparation method suitable for a trace sample, in particular to a trace plant or algae sample.
Background
Trace plants and algae are challenging to obtain enough DNA samples for whole genome sequencing due to the small sample size. Existing plant whole genome library sequencing generally requires that whole genome DNA be extracted from plant tissues by a CTAB (CTAB) method and then a sequencing library be established by breaking and amplifying the DNA fragments by a whole gene shotgun method. The CTAB method generally requires at least 100mg of plant tissue samples for extraction of whole genomic DNA, thus requiring long growth of trace plants or algae to obtain sufficient samples. The whole genome DNA is extracted by using a CTAB method, and the plant sample is subjected to liquid nitrogen grinding and phenol chloroform extraction, so that the extraction process is complex, the operation is complex and the time consumption is long. Moreover, organic solvents such as phenol chloroform used in the CTAB method may cause environmental pollution, and may even harm the health of laboratory personnel, including carcinogenic risks. In addition, the DNA extracted by the CTAB method often contains more impurities such as protein residues, polysaccharide pollution, phenolic substance pollution (such as brown DNA) and the like, and the residues have great influence on the subsequent downstream experiments such as PCR detection, and the input amount of DNA for sequencing and library building after the DNA extraction by the CTAB method is at least in μg level.
Thus, there is a need in the art for DNA preparation methods suitable for microscale plant or algae samples.
Disclosure of Invention
At least to solve the problem that the CTAB method commonly used in sequencing of plant whole genome pool is not suitable for sequencing of micro samples, the invention provides a novel DNA preparation method suitable for micro samples, and the method is particularly suitable for sequencing of micro plant or algae whole genome.
Accordingly, the present invention provides a method of preparing a DNA sample suitable for use in a microsample, the method comprising:
1) Treating the trace sample with liquid nitrogen;
2) Heating and incubating the sample treated by the liquid nitrogen;
3) The incubated sample is treated with lithium salicylate or purified with magnetic beads to obtain DNA sample of the micro sample.
In one embodiment, in 3), the concentration of lithium salicylate in the reaction system is 1-4mM.
In one embodiment, the time of lithium salicylate treatment is from 5 to 15 minutes.
In one embodiment, in 1), the microsample is a microscale plant sample or an algae sample.
In one embodiment, the trace plant sample is treated with a surfactant prior to treatment with liquid nitrogen; preferably, the surfactant is SDS or Triton X-100.
In one embodiment, in 1), the trace plant sample is broken plant tissue.
In one embodiment, in 1), the amount of the microsample is less than 100mg or 10 5 A cell; preferably, the amount of the micro sample is less than 10mg or 2×10 4 A cell; more preferably 1-5mg or (0.8-1).5)×10 4 Individual cells.
In one embodiment, in 1), the liquid nitrogen treatment time is 2 to 4 minutes.
In one embodiment, in 2), the temperature of the incubation is 50-70 ℃.
In one embodiment, in 2), the incubation time is from 5 to 10 minutes.
In one embodiment, in 3), the magnetic Beads are AmPure XP Beads.
In one embodiment, in 3), the magnetic bead purification steps are as follows: gently stirring and mixing the incubated sample and magnetic beads for several times, incubating at room temperature, adsorbing on a magnetic rack, and discarding supernatant after the liquid is clarified; washing the magnetic beads with 80% ethanol for 1-2 times, adding water for dissolution, standing, and adsorbing the supernatant by a magnetic rack.
The process of the invention requires only as little as 1mg or 10 4 The order of magnitude of individual cells can extract enough DNA for sequencing and banking, and the DNA impurities extracted by the method are less, and the input amount of the sequencing and banking DNA is as low as 2ng, so that the sequencing can be realized. In addition, the method is simple to operate, the whole genome DNA can be obtained only by 1/3 time of the traditional method, and reagents which pollute the environment and risk human health are not involved.
Drawings
The invention is illustrated by the following figures:
FIG. 1 shows the distribution of amplified fragments after disruption with 0.2. Mu.L Tn5 after treatment of Arabidopsis leaves with an area of 0.2 cm. Times.0.2 cm by the miniextraction-free DNA method.
FIG. 2 is a process 10 utilizing trace amounts of DNA free extraction 4 After disruption of the Chlamydomonas reinhardtii cell samples with 0.2 μl Tn5, the fragment distribution was amplified.
FIG. 3 is a treatment 10 using a method of trace amounts of DNA extraction-free and purification (final concentration of 2.5mM lithium salicylate) 4 After disruption of the chlorella cell samples with 0.2. Mu.L Tn5, the distribution of amplified fragments was followed.
FIG. 4 is a treatment 10 using a method of trace amounts of DNA extraction-free and purification (final concentration of 0.5mM lithium salicylate) 4 After disruption of the chlorella cell samples with 0.2. Mu.L Tn5, the distribution of amplified fragments was followed.
FIG. 5 is a treatment 10 using a method of trace amounts of DNA extraction-free and purification (final concentration 5mM lithium salicylate) 4 After disruption of the chlorella cell samples with 0.2. Mu.L Tn5, the distribution of amplified fragments was followed.
Detailed Description
The present invention provides a method for rapidly obtaining high quality DNA fragments of a minute amount of sample, which requires a sample size as low as 1mg or 10mg 4 The order of magnitude of individual cells, the DNA input amount of sequencing and library building can be as low as 2ng; in contrast, the conventional method requires a plant sample or algae sample input of at least 100mg or more than 10 5 On the order of magnitude, DNA samples prepared using less than the above amounts of samples often fail to meet library construction, on-machine sequencing requirements. The method is particularly suitable for trace plants or algae, can sample early in plant development, greatly advances the detection time node of engineering plants, is quick and simple to acquire DNA, greatly reduces labor and time cost, and has important significance for realizing high-throughput whole genome sequencing of plants and algae. In the present invention, the amount of the trace sample is less than 100mg or 10 5 Individual cells, for example 90mg, 80mg, 70mg, 60mg, 50mg, 40mg, 30mg, 20mg, 10mg, 9mg, 8mg, 7mg, 6mg, 5mg, 4mg, 3mg, 2mg, 1mg, 0.9mg, 0.8mg, 0.7mg, 0.6mg, 0.5mg, 0.4mg, 0.3mg, 0.2mg, 0.1mg or 9X 10mg 5 、8×10 5 、7×10 5 、6×10 5 、5×10 5 、4×10 5 、3×10 5 、2×10 5 、1×10 5 、9×10 4 、8×10 4 、7×10 4 、6×10 4 、5×10 4 、4×10 4 、3×10 4 、2×10 4 、1×10 4 、9×10 3 、8×10 3 、7×10 3 、6×10 3 、5×10 3 、4×10 3 、3×10 3 、2×10 3 、1×10 3 A cell; preferably, the amount of the micro sample is less than 10mg or 2×10 4 A cell; more preferably 1-5mg or (0.8-1.5). Times.10 4 Individual cells. In the present invention, the amount of the trace plant sample may be expressed in mg and the amount of the algae sample may be expressed in terms of the number of cells.
In the present invention, the trace sample comprises an algae sample or a plant sample, which is treated with a surfactant, which may be SDS or Triton X-100, prior to treatment with liquid nitrogen. In general, the plant sample needs to be broken up, for example by grinding, before being treated with the surfactant. The trace plant sample can enable the prepared DNA sample to contain more DNA after being treated by the surfactant. In the case of algae samples, the step of surfactant treatment is not required. The inventors found that, although a surfactant such as SDS has a function of releasing DNA, the surfactant such as SDS significantly affects an enzyme reaction in the subsequent sequencing library construction, so that the reaction efficiency is lowered, and therefore, it is preferable that a sample treated with the surfactant needs to be subjected to magnetic bead purification to remove the surfactant, reducing the influence of the surfactant residue on the subsequent enzyme reaction.
According to the preparation method of the DNA sample suitable for the micro sample, disclosed by the invention, the lithium salicylate can be added after liquid nitrogen is frozen and thawed, and the supernatant is taken, so that the DNA sample suitable for the preparation of a subsequent sequencing library is obtained. The inventors have found that lithium salicylate can be used to treat any sample containing DNA, such as plant samples, algae samples, since lithium salicylate can disrupt DNA-protein interactions in cells, thereby releasing DNA from histones, and that lithium salicylate has no significant effect on subsequent sequencing library construction, samples treated with lithium salicylate do not require further bead purification to obtain samples containing higher DNA. Preferably, the concentration of lithium salicylate in the reaction system is 1-4mM, such as 1.5mM, 2mM, 2.5mM or 3mM. It is also preferred that the time to rest after the addition of lithium salicylate is 5-15 minutes, for example 8 minutes, 10 minutes or 12 minutes. Also preferably, the stationary sample is centrifuged to take a supernatant.
Example 1
1. DNA sample preparation
a) Sample preparation of trace plant DNA extraction-free (magnetic bead method)
About 0.2 cm. Times.0.2 cm area of cotyledons or leaves (about 1 mg) were placed in 20. Mu.L of 1% SDS solution, the tissue was mashed with a stirring bar (10 s), placed in liquid nitrogen for 3min, and then rapidly placed in a 60℃water bath for 7min. Subsequently, the supernatant was transferred to a new centrifuge tube by centrifugation at 12000rpm for 3 min. The supernatant was fixed to 50. Mu.L with TE, 1.5× (75. Mu.L) of AmPure XP Beads were added to purify the diluted product, and the Beads were equilibrated at room temperature for 30min. Mixing supernatant fluid and magnetic beads in the centrifuge tube for 10 times, incubating for 10min at room temperature, then placing the mixture on a magnetic rack for adsorption for 5min, and after the liquid is clarified, absorbing supernatant fluid. 200 μL of 80% ethanol was added without disturbing the beads, and after standing for 30s, the supernatant was aspirated and the wash was repeated. The centrifuge tube was removed from the magnetic rack and dried at room temperature for 3-5min, and 21. Mu.L of ddH was added 2 O, blowing and mixing uniformly, and standing for 5min at room temperature; and (3) adsorbing for 2min by a magnetic rack, sucking the supernatant into a new centrifuge tube, and taking 1 mu L to detect the DNA concentration, wherein the solution is the trace plant DNA preparation solution.
b) Sample preparation of trace algae DNA extraction-free (magnetic bead method)
Taking the concentration of the logarithmic phase at 10 7 1. Mu.L of Chlamydomonas reinhardtii algae solution on the order of cells/mL was placed in a centrifuge tube (less than 10. Mu.L of ddH 2 O complement). Sealing, placing in liquid nitrogen for 3min, and rapidly placing in a water bath kettle at 60 ℃ for 7min. Subsequently, the supernatant was transferred to a new centrifuge tube by centrifugation at 12000rpm for 3 min. The supernatant was fixed to a volume of 50. Mu.L with TE, and the diluted product was purified by adding 1.5× (75. Mu.L) of AmPure XP Beads, which were equilibrated for 30min at room temperature. Mixing supernatant fluid and magnetic beads in the centrifuge tube for 10 times, incubating for 10min at room temperature, then placing the mixture on a magnetic rack for adsorption for 5min, and after the liquid is clarified, absorbing supernatant fluid. 200 μL of 80% ethanol was added without disturbing the beads, and after standing for 30s, the supernatant was aspirated and the wash was repeated. The centrifuge tube was removed from the magnetic rack and dried at room temperature for 3-5min, and 21. Mu.L of ddH was added 2 O, blowing and mixing uniformly, and standing for 5min at room temperature; and (3) adsorbing for 2min by a magnetic rack, sucking the supernatant into a new centrifuge tube, and taking 1 mu L to detect the DNA concentration, wherein the solution is the trace algae DNA preparation solution.
c) Sample preparation of trace algae DNA extraction and purification-free (lithium salicylate method)
Taking the concentration of the logarithmic phase at 10 7 Chlorella algae liquid 1. Mu.L of cell/mL order was placed in a centrifuge tube (less than 10. Mu.L of ddH 2 O complement). Sealing the centrifuge tube, placing the centrifuge tube in liquid nitrogen for 3min, and then rapidly placing the centrifuge tube in a water bath kettle at 60 ℃ for 7min. The centrifuge tube was removed and left at room temperature, and an equal volume of 5mM C was added 7 H 5 LiO 3 (lithium salicylate, lithium o-hydroxybenzoate) solution, and standing for 10min. Subsequently, the supernatant was transferred to a new centrifuge tube by centrifugation at 12000rpm for 3min, and 1. Mu.L of the supernatant was used for DNA concentration detection, and the solution was a microalgae DNA preparation solution.
d) The control conventional DNA sample preparation used a CTAB-based method, which was as follows:
(1) Preheating 2% CTAB extraction buffer in a water bath at 65 ℃;
(2) A small amount of the experimental material (about 300 mg) was placed in a mortar and ground to powder with liquid nitrogen;
(3) 700. Mu.L of 2% CTAB extraction buffer was added and gently stirred;
(4) Pouring the ground liquid into a 1.5mL sterilizing centrifuge tube, wherein the height of the ground liquid is about two thirds of that of the tube;
(5) Placing the mixture in a water bath or a constant temperature box at 65 ℃ and slightly shaking the mixture every 10min, and taking out the mixture after 30 to 60 min;
(6) Cooling for 2min, adding chloroform-isoamyl alcohol (24:1) to the full tube, and shaking vigorously for 2-3 min (if the total genome is extracted, the shaking cannot be violent), so that the chloroform-isoamyl alcohol and the full tube are mixed uniformly;
(7) Centrifuging at 10000rpm in a centrifuge for 10min, and simultaneously adding 600 μl isopropanol into another new sterilizing centrifuge tube;
(8) After centrifugation at 10000rpm for 1min, the supernatant was gently pipetted by a pipette and transferred into a centrifuge tube containing isopropyl alcohol, which was slowly shaken up and down for 30sec to thoroughly mix isopropyl alcohol with the water layer until DNA floc was visible;
(9) Immediately pouring out the liquid after centrifugation at 10000rpm for 1min, taking care not to pour out the white DNA precipitate, and inverting the centrifuge tube on the spread paper towel;
(10) After 60sec, the tube was stood upright, 720. Mu.L of 75% ethanol and 80. Mu.L of 5M sodium acetate were added, and the tube was gently turned and the tip was flicked with a finger to float the pellet and DNA cake at the bottom of the tube in the liquid;
(11) Standing for 30min to dissolve impurities in the DNA block;
(12) After centrifugation at 10000rpm for 1min, the liquid was decanted, 800. Mu.L of 75% ethanol was added, and the DNA was rinsed for another 30min;
(13) Immediately pouring out the liquid after centrifugation at 10000rpm for 30sec, inverting the centrifuge tube on the spread paper towel; after a few minutes, the tube is upright and the DNA is dried (naturally air-dried or blow-dried by a dryer);
(14) mu.L of 0.5 XTE (RNase-containing) buffer was added to dissolve the DNA, and the mixture was placed in a 37℃incubator for about 15 hours to digest the RNA.
2. Sequencing library preparation
The operation flow adopts a commercial kit (MGIEasy general DNA library preparation kit) which is limited in Shenzhen Huazhi Zhi manufacturing technology and technology to prepare the DNA sample.
a) According to the concentration of the DNA preparation prepared in 1, 2ng of DNA was added to the octamer tube by adding X. Mu.L of the DNA preparation, followed by sequentially adding the respective reaction components (Table 1),
TABLE 1 reaction Components
b) Incubation at 55deg.C for 7min, adding 5 μL 0.1% SDS at room temperature for 5min, terminating the reaction, and placing on ice;
c) Eight connecting tubes were placed in an ice bath, each reaction component was added in sequence (table 2),
TABLE 2 reaction Components
d) Lightly blowing for 10 times by using a pipette, and fully and uniformly mixing;
e) Eight-tube was placed in a PCR apparatus, and the following reaction procedure (Table 3) was set,
TABLE 3 reaction conditions
3. Library purification and fragment selection
Each sample was selectively purified using 0.8×+0.2X Agencourt AMPure XP beads.
a) Vortex shaking to uniform AMPure XP beads, sucking 40 mu L volume to 50 mu L PCR product, lightly blowing 10 times by using a pipettor, fully mixing, and incubating for 10min at room temperature;
b) Centrifuging the reaction tube for a short time, placing the reaction tube in a magnetic rack to separate magnetic beads and liquid, carefully transferring supernatant to a clean EP tube after the solution is clarified (about 5 min), discarding the magnetic balls, and removing large fragment nucleic acid adsorbed on the magnetic beads;
c) Vortex shaking to uniform AMPure XP beads, sucking 10 mu L volume into supernatant, gently blowing 10 times by using a pipettor, fully mixing, and incubating for 5min at room temperature;
d) The EP tube was briefly centrifuged and placed in a magnetic rack to separate the beads and liquid, and the supernatant was carefully removed until the solution was clear (about 5 min);
e) The EP tube was kept always in the magnetic rack, 200. Mu.L of freshly prepared 80% ethanol was added to rinse the beads, and after 30s incubation at room temperature the supernatant was carefully removed;
f) Repeating the above steps, and rinsing twice in total;
g) The EP pipe is kept in the magnetic rack all the time, and the cover is opened for air drying for 10min;
h) The EP tube was removed from the magnetic rack, eluted with 25. Mu.L of sterilized ultrapure water, vortexed or gently beaten with a pipette to mix thoroughly, the EP tube was briefly centrifuged and placed in the magnetic rack to separate the beads from the liquid, and the supernatant was carefully aspirated into the clean EP tube until the solution was clear (about 5 min) and stored at-20 ℃.
Fragment quality Standard evaluation (Table 4)
Table 4 quality criteria evaluation
4. Library mixing
a) Mixing according to 390ng of total DNA mix, and determining the mixing volume of each sample according to the mixing base of the samples;
b) At present, the mixing base number can only be a multiple of 8, and the mixing base number is 8*N when 1 flow channel is measured by 8*N samples;
c) The conversion method comprises the following steps: for example, 16 samples were mixed into 1 library, with a total of 390/16=24.375 ng for each sample, and sample 1 at a concentration of 5ng/μl, then sample 1 had a corresponding volume of 24.375/5=4.875 μl; sample 2 was calculated according to this method;
d) The mixed DNA mixture is used for DNA cyclization, and can be frozen to-20 ℃ for standby.
5. Cyclization of DNA
a) Reagents were prepared according to the following formulation (table 5),
TABLE 5 reagent Components
b) The parameters were set according to the following procedure (table 6),
TABLE 6 reaction conditions
c) ssDNA ligation, placing the PCR tube in an ice bath, adding each reaction component in sequence (Table 7),
TABLE 7 reaction Components
d) After the reaction liquid is prepared, vortexing, gently throwing for 5s, incubating for 1h at 37 ℃, and maintaining at 4 ℃; after ligation, 1. Mu.L of the product was used to measure dsDNA concentration.
6. Exonuclease digestion
a) The reaction solution after the connection was placed in an ice bath, and each reaction component was added in sequence (table 8),
TABLE 8 reaction Components
b) After the reaction liquid is prepared, vortexing, gently throwing for 5s, incubating at 37 ℃ for 30min, and maintaining at 4 ℃; after cleavage, 1. Mu.L of the product was used to measure dsDNA concentration.
7. Magnetic bead purification
a) Adding 170 mu L of PEG32 beads into the reaction liquid, uniformly shaking by vortex, and incubating for 10 minutes at room temperature;
b) The EP tube was briefly centrifuged and placed in a magnetic rack to separate the beads and liquid, and the supernatant was carefully removed until the solution was clear (about 5 min);
c) The EP tube was kept always in the magnetic rack, 200. Mu.L of freshly prepared 80% ethanol was added to rinse the beads, and after 30s incubation at room temperature the supernatant was carefully removed;
d) Repeating the above steps, and rinsing twice in total;
e) The EP pipe is kept in the magnetic rack all the time, and the cover is opened for air drying for 10min;
f) Taking the EP tube out of the magnetic frame, adding 25 mu L of sterilized ultrapure water for eluting, vortex shaking or lightly blowing by using a pipettor, fully and uniformly mixing, briefly centrifuging the EP tube, separating magnetic beads and liquid in the magnetic frame, carefully sucking the supernatant into the clean EP tube after the solution is clarified (about 5 min), and carrying out on-machine sequencing, wherein the EP tube can be stored at the temperature of minus 20 ℃;
g) After purification, 1. Mu.L of the product was taken to measure the ssDNA concentration.
ssDNA quality criteria evaluation (table 9),
TABLE 9 evaluation of ssDNA quality criteria
8. Sequencing
a) Extraction-free DNA whole genome sequencing of trace Arabidopsis leaves
Arabidopsis plants grown on MS medium for 7 days were cut with scissors and 0.2 cm. Times.0.2 cm area of leaves were taken. The leaves were treated in liquid nitrogen by a method of micro extraction-free DNA, centrifuged to remove the precipitate, the supernatant was purified with XP beads, 2ng of DNA was broken with 0.3. Mu.L Tn5 for 5min, and amplified using the adaptor primer with barcode and the amplified fragments recovered (specific procedure was as described previously). Fragments were purified by XP magnetic beads and then detected for fragment size using Agilent High Sensitivity DNA kit (FIG. 1). The experimental result shows that the effective fragments exist in the interval of 180-500bp, and the effective fragments in the interval of 300-500bp are reserved after further screening by a sheet for cyclization and sequencing on the machine.
The data of the arabidopsis leaves after treatment by using CTAB and a trace extraction-free DNA method are shown in Table 10. The values of the machine-off data Q20 and Q30 after the arabidopsis leaves are processed by CTAB and trace extraction-free DNA methods for library construction and sequencing are more than 90, which indicates that the quality control of the machine-off data is excellent. Further comparison with the Arabidopsis reference genome showed that the genome Coverage (Coverage) of the latter two was 92.05% and 90.88%, respectively, indicating extremely high Coverage of the sequencing data with the reference genome. The results show that high-quality data can be obtained by processing Arabidopsis leaves through two methods and performing library construction sequencing. However, compared with the method of micro extraction-free DNA, the CTAB method is used for treating Arabidopsis, which requires growing Arabidopsis leaves after one month, and sampling at least 100mg is used for carrying out a more complex DNA extraction process. The sampling time for carrying out library construction sequencing on Arabidopsis leaves by using the method of trace extraction-free DNA is shorter, the sample input amount is less, the labor and economic cost is lower, and the data quality is also excellent.
Table 10 results of sequencing by different methods on microscale Arabidopsis leaves in pool
| Sample preparation method | Sample size (mg) | DNA input (ng) | Clean read length Q20 | Clean read length Q30 | Coverage rate |
| CTAB | 200 | 50 | 98.97 | 96.76 | 92.05％ |
| Trace extraction-free DNA | 1 | 2 | 99.05 | 94.25 | 90.88％ |
b) Extraction-free DNA whole genome sequencing of trace chlamydomonas reinhardtii
Method for extracting DNA by using trace amount 4 Cells of Chlamydomonas reinhardtii CC400 were treated with liquid nitrogen and centrifuged to remove pellet, the supernatant was purified with XP beads, 2ng of DNA was taken, and the supernatant was broken with 0.3. Mu.LTn 5 for 5min, amplified using the adaptor primer with barcode and the amplified fragment recovered (specific procedure as described above). Fragments pass through XP magnetic beadsFragment size was measured after purification using Agilent High Sensitivity DNAkit (FIG. 2). The experimental result shows that the effective fragments exist in the interval of 180-250bp, and the effective fragments in the interval of 180-250bp are reserved for cyclization and sequencing after further screening.
The data of the Chlamydomonas reinhardtii CC400 cell samples after treatment and library construction and machine-down by using CTAB and trace extraction-free DNA methods are shown in Table 11. After the Chlamydomonas reinhardtii CC400 cell sample is processed by the CTAB and micro-extraction-free DNA method, the Q20 value is more than 97, the Q30 value is 90.31 and 97.9 respectively, which shows that the quality control of the machine-on data is excellent, and the machine-on data quality of the Chlamydomonas reinhardtii CC400 cell sample is better by the micro-extraction-free DNA method. The genome coverage of the reference genome of Chlamydomonas reinhardtii was 91.85% and 91.82%, respectively, indicating that the sequencing data had very high coverage with the reference genome. The results show that high-quality data can be obtained by processing chlamydomonas reinhardtii through two methods and performing library construction and sequencing. However, compared with the method of trace extraction-free DNA, the cell quantity required by the CTAB method for extracting Chlamydomonas reinhardtii DNA is far more than 10 4 The order of magnitude, the extraction process is also relatively cumbersome. The method for processing chlamydomonas reinhardtii by using trace extraction-free DNA has the advantages of shorter sampling time, smaller sample size, lower labor and economic cost and good data quality for library construction and sequencing.
TABLE 11 library sequencing results after treatment of trace Chlamydomonas reinhardtii cell samples by different methods
c) DNA extraction and purification-free whole genome sequencing of microcystis
Method for extracting DNA by using trace amount 4 Chlorella cells were treated with C after liquid nitrogen treatment 7 H 5 LiO 3 The treatment is carried out for 10min. After centrifugation of the algae solution at 12000rpm for 3min, 2ng DNA of the supernatant was directly broken with 0.3. Mu. LTn5 for 5min, amplified by using the adaptor primer with barcode and amplified fragments were recovered (specific procedure is as described above). Fragments were purified by XP beads and then detected by Agilent High Sensitivity DNAkitSize (fig. 3). The experimental result shows that the effective fragments exist in the interval of 180-250bp, and the effective fragments in the interval of 180-250bp are reserved for cyclization and sequencing after further screening.
The chlorella cell samples were processed by CTAB and trace extraction-free and DNA purification methods and the data were shown in Table 12. After the chlorella cell sample is treated by CTAB and a trace DNA extraction-free and purification method, the Q20 value is more than 97, and the Q30 values are 91.12 and 97.56 respectively, which shows that the quality control of the machine-on data is excellent and the machine-on data quality of the chlorella cell sample treated by the trace DNA extraction-free and purification method is better. The genome coverage rates of the two are 90.27% and 99.17% respectively after further comparison with the chlorella reference genome, which shows that the sequencing data and the reference genome have extremely high coverage, and the coverage of the machine-setting data of the chlorella cell sample processed by the method of trace extraction-free and DNA purification is higher. The results show that high-quality data can be obtained by performing library construction and sequencing on chlorella cell samples processed by two methods, and the quality of the data after chlorella is processed by a method of trace extraction-free and DNA purification is better. Compared with the method of trace extraction-free and DNA purification, the cell quantity required by the extraction of chlorella DNA by using the CTAB method is far more than 10 4 The order of magnitude, the extraction process is also relatively cumbersome. The method for processing chlorella by using the trace extraction-free and DNA purification method has the advantages of shorter sampling time, smaller sample size, lower labor and economic cost and better data quality for library construction sequencing.
TABLE 12 library sequencing results after different methods of processing Chlorella cell samples
Example 2
DNA extraction and purification-free whole genome sequencing of microcystis
To find a more suitable use concentration of lithium salicylate, example 1 used 2.5mM lithium salicylate as the final concentration for DNA extraction and purification of microalgae, while this example used 0.5, 1.5, 3.5 and 5mM lithium salicylate in parallel for treatment of the same microalgae samples. The method comprises the following steps:
will 10 4 After liquid nitrogen treatment of the Chlorella cells, the cells were treated with C at final concentrations of 0.5, 1.5, 3.5 and 5mM, respectively 7 H 5 LiO 3 The treatment is carried out for 10min. After the algae solution is centrifuged at 12000rpm for 3min, 2ng DNA of the supernatant is taken, and is directly broken by 0.3 mu L of Tn5 for 5min, and then amplified by using a joint primer with barcode, and amplified fragments are recovered (specific steps are shown in example 1 for sample preparation of trace algae DNA extraction and purification free by using a lithium salicylate method). Fragments were purified by XP magnetic beads and then detected for fragment size using Agilent High Sensitivity DNA kit. 1.5 and 3.5mM C 7 H 5 LiO 3 With 2.5mM C 7 H 5 LiO 3 Similarly, the minimum requirement of DNA concentration required by the next library establishment can be met>10 ng/ul). Whereas for a final concentration of 0.5mM C 7 H 5 LiO 3 Treated Chlorella sample due to C 7 H 5 LiO 3 Too little amount of DNA released from the histone is insufficient, affecting the efficiency of Tn5 disruption, resulting in a disruption fragment size between 600-3000bp, no effective fragment of 150-250bp was detected (FIG. 4). For a final concentration of 5mM C 7 H 5 LiO 3 The treated chlorella sample, due to excessive usage, may cause degradation of DNA during release, and only DNA fragments of about 98bp in size were detected, which were mainly primer dimer and degraded DNA fragments (FIG. 5). Further Qbit detection is carried out on the chlorella sample treated by C7H5LiO3 with the final concentration of 0.5mM and 5mM, and the DNA concentration is found<1 ng/. Mu.l, and the minimum requirement of the DNA concentration required by the next library establishment is not met>10 ng/ul), so that no further library sequencing can be performed. The results of this example demonstrate that the amount of lithium salicylate used is critical to sample preparation of a trace algal sample.
Claims (11)
1. A method of DNA sample preparation suitable for use in microsamples, the method comprising:
1) Treating a trace sample with liquid nitrogen, wherein the trace sample is a trace plant sample or an algae sample; the amount of the trace sample is 1-100mg or 0.8X10 4 -9×10 5 A cell;
2) Heating and incubating the sample treated by the liquid nitrogen;
3) The incubated samples were treated with lithium salicylate at a concentration of 1.5-3.5mM to obtain DNA samples of the micro samples.
2. The method of claim 1, wherein in 3) the lithium salicylate treatment is for a period of 5 to 15 minutes.
3. The method of claim 1, wherein in 1) the trace plant sample is treated with a surfactant prior to treatment with liquid nitrogen, and further wherein in 3) the purification is performed with magnetic beads.
4. A method according to claim 3, in 1), the trace plant sample is ground upon surfactant treatment.
5. The method according to any one of claims 1 to 4, wherein the amount of the trace sample is 1 to 10mg or (0.8 to 2). Times.10 4 Individual cells.
6. The method according to any one of claims 1-4, wherein in 2) the temperature of the incubation is 50-70 ℃.
7. The method according to any one of claims 1-4, wherein in 2) the incubation time is 5-10 minutes.
8. The method of claim 3, wherein in 3) the magnetic Beads are AmPure XP Beads.
9. A method according to claim 3, wherein in 3), the magnetic bead purification step is as follows: gently stirring and mixing the incubated sample and magnetic beads for several times, incubating at room temperature, adsorbing on a magnetic rack, and discarding supernatant after the liquid is clarified; washing the magnetic beads with 80% ethanol for 1-2 times, adding water for dissolution, standing, and adsorbing the supernatant by a magnetic rack.
10. A method according to claim 3, wherein the surfactant is SDS or Triton X-100.
11. The method according to any one of claims 1 to 4, wherein the amount of the trace sample is 1 to 5mg or (0.8 to 1.5). Times.10 4 Individual cells.
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