CN108300805A - A method of confirming life entity - Google Patents
A method of confirming life entity Download PDFInfo
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- CN108300805A CN108300805A CN201810034010.5A CN201810034010A CN108300805A CN 108300805 A CN108300805 A CN 108300805A CN 201810034010 A CN201810034010 A CN 201810034010A CN 108300805 A CN108300805 A CN 108300805A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The present invention provides a kind of method confirming life entity and the applications in terms of medicine and scientific research, confirm that life entity, this method save the time, improve efficiency, exclude the prior art by signal to waveform again to generated algorithmic error in A, T, C, G sequence transfer process specifically by the nucleic acid determination signal for comparing life entity.
Description
Technical field
The present invention relates to nucleic acid sequencing fields, and in particular to it is a kind of confirm life entity method and in medicine and scientific research side
The application in face.
Background technology
DNA sequence dna is the blueprint of life, contains the hereditary information of entire organism.Rapidly and accurately understand these heredity
Information i.e. sequencing technologies, progress and bioenvironmental development to life science have great importance.Sequencing technologies pass through
Go through first generation sequencing technologies dideoxy chain termination and chemical degradation method to shotgun sequencing again to being sequenced when synthesizing
Second generation PCR sequencing PCR and Performances of Novel Nano-Porous metre hole PCR sequencing PCR.
Performances of Novel Nano-Porous metre hole PCR sequencing PCR is to use electrophoretic techniques, drives single DNA or RNA molecule one by one by receiving by electrophoresis
Metre hole realizes sequencing.The accuracy of nano-pore technology is high, can be carried out detecting and detecting holding without carrying out amplification and label
Continuous property is good, this low cost and to be rapidly performed by DNA sequencing.Nano-pore technology constantly development and it is complete
It is kind, to as third generation sequencing technologies attack.
Patent CN104694649A discloses the nano-pore sequencing method of the low punching rate of a kind of nucleic acid molecules and its special
Nano-pore device, which has effectively slowed down nucleic acid molecules in nano-pore under the premise of not influencing measuring signal signal-to-noise ratio
In movement velocity, to improve temporal resolution, method simple, easily operated.
Patent CN106596645A discloses a kind of grapheme nano-pore DNA sequencer of single molecule manipulation, including nanometer
Steerable system, nano-pore sequencing system, data acquisition and analysis system and central control system, the DNA sequencer survey nano-pore
The basic principle of sequence is organically combined with unimolecule nano-manipulation technology, is had overlength read-write length, can be carried out single-molecule DNA reality
When control, can realize single base identification ultrahigh resolution.
Patent CN106255551A disclose it is a kind of for it is long read, it is unmarked, the sequencing of optical nano long-chain molecule
Method and apparatus is used for using the novelty of simple gluey nano particle, orientation self-assembled nanometer fabrication scheme on nanochannel top
Upper formation nanohole array allows single base to parse to make long-chain molecule be unfolded.
Patent CN102313769A discloses a kind of FET nanopore sensor, using including the field being embedded in nano-pore
Effect transistor sensor detects being worn corresponding at least one of biomolecule and DNA DNA in sensor
The drain current variation for crossing nano-pore, improves the sensitivity of nano-pore sequencing.
Nano-pore sequencing is mainly to pass through the rate of nano-pore, expansion DNA long-chains, the single base of parsing from control at this stage
Etc. improvement, and processing for follow-up signal and comparing is not paid attention to.Sequencing technologies genomes all at present
Analysis is to convert after A, T, C, G the obtained electric wave signal of sequencing to carry out sequence alignment again, determines organism.However repeatedly
Conversion process in can fault in enlargement so that comparison result accuracy is inadequate.Therefore, the present invention provides a kind of confirmations of life entity
Method, by electric signal waveform that nucleic acid is obtained by nano-pore directly with the electric signal waveform library of the sequence built in advance into
Row compares, and the electric signal waveform of sequence has completely included the base arrangement information of surveyed nucleic acid sequence, therefore can be by sequence
Electric signal waveform is applied to other experiments instead of base sequence.
Invention content
A kind of method confirmed the present invention provides life entity represents nucleic acid sequence and known core using nucleic acid determination signal
Searching analysis is compared in acidity test signal, eliminates by signal to waveform again to produced in A, T, C, G sequence transfer process
Algorithmic error, the effectively save time, improve efficiency and accuracy；The present invention also provides a kind of structures of nucleic acid determination signal library
Construction method.
The first aspect of the present invention is related to a kind of method confirming life entity, and the nucleic acid determination by comparing life entity is believed
Number confirm life entity.
Preferably, the life entity is selected from ancient bacterium domain, bacterium domain, eucaryote domain, virus and viroid.
Preferably, the nucleic acid determination signal of the life entity is electric signal waveform, it is further preferred that the electrical signal wave
Shape is formed by nucleic acid sequence by nanopore sensor.
In the specific embodiment of the present invention, the method for the confirmation life entity, including measure life to be measured
The electric signal waveform of life entity to be measured is carried out waveform with existing electric signal waveform and compared by the electric signal waveform of body.
Existing electric signal waveform of the present invention is preferably the existing electric signal waveform in electric signal waveform library, described
Electric signal waveform library in include at least one electric signal waveform from known life entity.The known life entity is selected from
Ancient bacterium domain, bacterium domain, eucaryote domain, virus and viroid.Those skilled in the art are it is understood that existing electricity of the present invention
Signal waveform can be obtained from the electric signal waveform of known life entity by measuring, and can also be obtained by simulating.This
The electric signal waveform of the invention existing electric signal waveform or known life entity be whole genome sequence electric signal waveform and/
Or the electric signal waveform and/or RNA sequence of partial genome sequence.
Life entity to be measured of the present invention is selected from ancient bacterium domain, bacterium domain, eucaryote domain, virus and viroid, described
The method of the electric signal waveform of life entity to be measured is measured as, by nanopore sensor, described waits for by the nucleic acid of life entity to be measured
The electric signal waveform for surveying life entity is the electric signal waveform of whole genome sequence and/or the electric signal waveform of partial genome sequence
And/or the electric signal waveform of RNA sequence.
Waveform comparison of the present invention can be by the entire electric signal waveform of life entity to be measured and existing electrical signal wave
Shape compares, and can also compare the part electric signal waveform of life entity to be measured with existing electric signal waveform.
The nucleic acid determination signal of the present invention for comparing life entity is preferably the nucleic acid determination signal for comparing life entity
Similarity, it is furthermore preferred that the nucleic acid determination signal of the present invention for comparing life entity includes by the electric signal waveform base of sequence
In the contraction and stretch processing of time scale, Euclidean distance is calculated, to determine the similarity between sequence electric signal waveform.
In the specific implementation mode of the present invention, the similarity comparison method of the nucleic acid determination signal of the life entity selects
From Edit distance, ANN, LSH, Dynamic time warp or Smith Waterman；Further preferably Dynamic
time warp。
The method of the electric signal waveform of the present invention for measuring life entity to be measured is preferably by the nucleic acid of life entity to be measured
Electric signal waveform is obtained by nanopore sensor.
The second aspect of the present invention is related to a kind of method of the nucleic acid determination electric signal waveform of life entity to be measured, including such as
Lower step：
1) from life entity to be measured, extraction, purification of nucleic acid；
2) it is the nucleic acid Ghana metre hole library connector obtained in step 1), obtains the nucleic acid with connector；
3) nucleic acid with connector obtained in step 2) is mixed with containing nano-pore sequencing required buffer liquid, is mixed
Close liquid；
4) detection chip containing nano-pore, access sequencing circuit are added in the mixed liquor obtained in step 3) so that
Life entity nucleic acid to be measured passes through nano-pore；
5) nano-pore current signal is read, the electric signal waveform of life entity sequence to be measured is obtained.
Preferably, extraction, purification of nucleic acid pass through enzyme dissolving, the side of surfactant and/or mechanical force in the step 1)
Formula smudge cells, the then extraction purification in such a way that affinity column precipitating or organic solvent are precipitated；It is furthermore preferred that passing through existing skill
The extracts kit of DNA or RNA directly extracts in art.Bacterium, plant, the animal difficulty in clasmatosis are not quite similar.
Preferably, the method in the step 2) being nucleic acid Ghana metre hole library connector is that end is repaired-linked enzyme process, turns
Seat enzyme process, universal primer sequence PCR amplification method or RNA are by reverse transcription at RNA-DNA complexs and adjunction head.
Preferably, buffer solution described in the step 3) is electrolyte solution.
Preferably, nano-pore described in the step 4) be micrococcus pyogenes alpha-hemolysin nano-pore, MspA nanometers
Hole, Csgg nano-pores, phi29 nano-pores or solid nano hole；Further preferably MspA nano-pores and Csgg nano-pores.
Preferably, detection chip described in the step 4) is biological nano-pore chip, solid nano hole chip, tunnel electricity
Flow detecting system, field effect transistor guard system or microfluidic system；Further preferably biological nano hole chip.
In one embodiment of the invention, detection chip described in the step 4) and the sequencing circuit are a set of company
System, the system being used in conjunction can be an entirety, or discrete individual.
The third aspect of the present invention is related to a kind of construction method of nucleic acid determination signal library, especially a kind of nucleic acid determination
The construction method in electric signal waveform library collects electric signal, obtains sequence by known nucleic acid sequence by nanopore sensor
Electric signal waveform.
Preferably, the known nucleic acid sequence is selected from the whole genome sequence and/or portion gene group of known life entity
The electric signal waveform of sequence and/or RNA sequence.
Preferably, the known nucleic acid sequence inquires to obtain by NCBI, and is expanded by chemical synthesis, link and PCR
Increase, or passes through the nucleic acid of life entity known to extraction.
In one particular embodiment of the present invention, all life entity nucleic acid sequences that NCBI is inquired are passed through into nanometer
Hole sensor stores respective electric signal waveform respectively.
Preferably, the electric signal waveform of the acquisition is pre-processed, it is described pretreatment include low-pass filter filter,
Median filter filtering, down-sampling, leveling and/or normalization.
The fourth aspect of the present invention, is related to a kind of construction method in the electric signal waveform library of sequence, including by nucleic acid sequence
By nanopore sensor, obtains electric signal waveform and pass through computer simulation journey using the electric signal waveform height parameter measured
Sequence, simulation obtain a plurality of data waveform, the electric signal waveform library as sequence.
The fifth aspect of the present invention is related to the nucleic acid determination signal of life entity in terms of medicine and scientific research about sequence alignment
With the application in search, it is preferred that the nucleic acid determination signal of the life entity is the nucleic acid determination electric signal waveform of life entity.
The sixth aspect of the present invention, the waveform for being related to sequence are compared in Causal Agent Identification, nucleic acid mutation detection and distribution type
Application in matching.
The method that life entity of the present invention confirms represents core using nucleic acid by the electric signal waveform that nano-pore obtains
Acid sequence, the electric signal waveform sequence and structure in advance of the life entity that life entity nucleic acid to be measured is obtained by nanopore sensor
The electric signal waveform library of good sequence carries out directly comparing searching analysis, eliminates by waveform to A, T, C, G sequence transfer process
In generated algorithmic error；The sequence electric signal waveform that nanopore sensor obtains simultaneously has completely included surveyed nucleic acid sequence
Base arrangement information, therefore directly on electric signal waveform complete data analysis can the effectively save time, improve efficiency with
Accuracy.
Life entity of the present invention is selected from ancient bacterium domain, bacterium domain, eucaryote domain, virus and viroid；It is wherein described
Ancient bacterium domain include Halophiles, acidophil, Thermophilic Bacteria, the bacterium domain includes actinomyces, Chlamydia, mycoplasma, rickettsia
Family name's body, the eucaryote domain include animal, plant, fungi, protist, Eukaryotic Algae, and the virus includes sub-thread
DNA virus, distrand DNA virus, RNA retroviruses, double-stranded rna virus, single-stranded, single-stranded RNA virus, 20S RNA and
Protein virus, the viroid include pospiviroid, hop stunt viroid category, Coconut cadang class
The latent floral leaf viroid category of Tobamovirus, apscaviroid, coleus blumei viroid 1 category, avsunviroid viroid category, peach.Into one
Step is preferred, and the Halophiles includes that Halobacterium, Haloarcula, the richly endowed Pseudomonas of thermophilic salt, Halococcus, thermophilic salt are thermophilic
Alkali bacillus category, thermophilic salt basophilic Coccus, the acidophil includes lactic acid bacteria, and the Thermophilic Bacteria includes facultative thermophile, obligate thermophilic
Hot bacterium, Thermophilic Bacterium, the actinomyces include streptomyces, Nocardia, actinomyces, micromonospora, pink mold cyst
Pseudomonas, actinoplanes, the Chlamydia include chlamydia psittaci, chlamydia trachomatis and chlamydia pneumoniae, and the branch is former
Body includes mycoplasma pneumoniae, mycoplasma hominis, mycoplasma genitalium, and the rickettsia includes Rickettsia prowazekii, not
Family name's rickettsia, Li Kecishi rickettsias, rickettsia akamushi, the animal include fish, reptiles, bird
Class, the plant include seed plant, bryophyte, pteridophyte and quasi- pteridophyte, and the protist includes algae, original
Raw biological species, primary mushroom, the Eukaryotic Algae include Chlorophyta, charophyta, Euglenophyta, Bacillariophyta, Chrysophyta, Pyrrhophyta,
Cryptophyta, Xanthophyta, Phaeophyta, Rhodophyta.
Nucleic acid of the present invention includes DNA and RNA.
Nucleic acid determination signal of the present invention refers to the base signal obtained when nucleic acid passes through nanopore sensor, institute
The base signal stated can be the base signal of composition DNA or RNA, or the base of the composition DNA or RNA by modification
Signal.
Description of the drawings
Hereinafter, carry out the embodiment that the present invention will be described in detail in conjunction with attached drawing, wherein：
Fig. 1：Two electric signal waveforms alignment thereof of similarity degree in time by way of distortion；
Fig. 2：Electric signal waveform Data Serialization；
Fig. 3：The matching result of the electric signal waveform of sequence and the electric signal waveform library of comparing；
Fig. 4：The handling result of measured data, wherein the first behavior patch-clamp measured data waveform, the second behavior is through low pass
As a result, third behavior result after median filter is handled after filter filtering；
Fig. 5：The one section of waveform waveform to be matched intercepted in Wave data；
Fig. 6：It is filtered flow chart, wherein data processing is carried out by MATLAB, according to actual waveform data sampling rate
Fs generates low-pass filter and median filter.
Specific implementation mode
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation describes, it is clear that and described embodiment is only the section Example of the present invention, rather than all.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
1 analog waveform of embodiment simultaneously compares
1, principle (Dynamic Time Warp) is compared
DTW can be by nonlinear distortion, by stretching and shrinking, and delay countershaft finds the best alignment of two waveforms
Pattern, this distortion can be used for finding the similarity of the matching area and two time waveforms of two time waveforms.
The best mode for weighing two waveform similarity degrees is to calculate Euclidean distance, waveform n one of by calculating
The distance to n point of another waveform is put to carry out numerical value measurement, while DTW also solves two time serieses in time span
Upper inconsistent problem, two waveforms can be flexible there are global and local in time span or be stretched, and traditional letter is overcome
Number defect of the matching algorithm on time series mismatch problem, Fig. 1 are that two waveforms are similar in time by way of distortion
The alignment thereof of degree.
Dynamic time warping DTW is with Time alignment function W (n) the description test templates for meeting certain condition and to refer to mould
The corresponding time relationship of plate, warping function when solving two template matches corresponding to cumulative distance minimum.
Assuming that there are two time series Q and C, length is n and m respectively：(during actual speech matching is used, a sequence is
Reference template, a sequence are test template, and the value that each of sequence is put is the characteristic value of each frame in voice sequence.Such as
Voice sequence Q shares n frames, and the characteristic value (number or a vector) of the i-th frame is qi.
Q=q1, q2 ..., qi ..., qn；
C=c1, c2 ..., cj ..., cm；
If n=m, the distance of two sequences is directly calculated.But if n carries out linear scale alignment not equal to m.Short
Sequences are amplified to the length as long sequence and compare again, or the long linear length shortened to as short sequence
Compare again.
In order to be aligned the two sequences, need the matrix grid for constructing a n x m, matrix element (i, j) indicate qi and
The distance d (qi, cj) of two points of cj (get over by the similarity between each point and each point of C of namely sequence Q, distance
Small then similarity is higher.Here first no matter sequence), generally use Euclidean distance, d (qi, cj)=(qi-cj) 2 (it also will be understood that
For the distortion factor).Each matrix element (i, j) indicates the alignment of point qi and cj.DP algorithm can be attributed to searching one and pass through
The path of several lattice points in this grid, path by lattice point be the alignment that two sequences are calculated point.
This paths is defined as the regular paths warping path, is indicated with W, k-th of element definition of W is wk
=(i, j) k, define the mapping of sequence Q and C.
W=w1,w2,...,wk,...wKMax (m, n)≤K ＜ m+n-1
First, path needs to meet following constraint：
1) boundary condition：w1=(1,1) and wK=(m, n).The pronunciation speed of any type voice is likely to change, but
It is the precedence of its each section to change, therefore selected path must be terminated in the upper right corner from the lower left corner.
2) continuity：If wk-1=(a ', b '), then for next point w in pathk=(a, b) needs to meet (a-
a’)<=1 and (b-b ')<=1.It namely can not possibly go to match across some point, point alignment that can only be adjacent with oneself.In this way
It can ensure that each coordinate in Q and C occurs in W.
3) monotonicity：If wk-1=(a ', b '), then for next point w in pathk=(a, b) needs satisfaction 0<=
(a-a ') and 0<=(b-b ').Point above this limitation W must be as time dullness carries out.
In conjunction with continuity and monotonicity constraint, just only there are three directions in the path of each lattice point.If path is
Passed through lattice point (i, j), then it is next by lattice point be only possible to be one of following three situation：(i+1, j), (i, j+1)
Or (i+1, j+1).
The path for meeting the above constraints can have index, and then selection makes following regular Least-cost
Path：
Wherein, K indicates the compensation to the regular path of different length.
Here an accumulation distance (cumulative distances) is defined.The two sequences are matched since being put (0,0)
Q and C, every point are arranged, the distance that before all points calculate can all add up.It reaches home after (n, m), this Cumulative Distance
It is exactly above said last total distance, that is, the similarity of sequence Q and C.
Cumulative Distance γ (i, j) can indicate in following way, Cumulative Distance γ (i, j) be current lattice point distance d (i,
J), that is, the Euclidean distance of point qi and cj (similitude) and can reach the point minimum adjacent elements Cumulative Distance it
With：
γ (i, j)=d (qi,cj)+min{γ(i-1,j-1),γ(i-1,j),γ(i,j-1)}
2, experimental method
A plurality of data waveform is simulated by simulation program, forms Data Serialization, the data ratio as waveform to be detected
To library.Since waveform modelling is to be directed to DTW processing, the influence of noise in practice is not accounted for, so not being filtered.
It is used as data waveform to be matched by intercepting one piece of data waveform at random, then one by one to the waveform of Data Serialization
It is compared.
3, experimental result
Fig. 2 serializes for Wave data, and the matching result in wave sequence and comparing library is shown in Fig. 3, each waveform
It will obtain a matching maximum of points, the matching maximum of points of each waveform is compared, find wherein that maximum value to obtain the final product
To the highest target waveform of similarity.
2 measured waveform of embodiment simultaneously compares
1, experimental method
1) it from life entity to be measured, directly extracted by kit, purify DNA；
2) it is DNA Ghana metre hole library connector after purification to repair-link enzyme process by end；
3) nucleic acid with connector is mixed with containing electrolyte solution needed for MspA nano-pore sequencings, obtains mixed liquor；
4) the microfluidic system detection chip containing MspA nano-pores, access sequencing circuit will be added in mixed liquor so that
Life entity nucleic acid to be measured passes through nano-pore；
5) nano-pore current signal is read, life entity patch-clamp measured data waveform to be measured is obtained.
2, experimental result
Patch-clamp measured data waveform is shown in shown in Fig. 4 the first rows, it can be seen that the data-signal shake of patch-clamp actual measurement is very
Greatly, high frequency component noise produces prodigious influence to the signal-to-noise ratio of entire signal bandwidth, and measured data waveform is carried out low pass
Filter (see the second rows of Fig. 4), median filter are handled (see Fig. 4 the third lines).It can be seen that after low-pass filter
The high dither of Wave data has been filtered greatly, and useful signal in entire signal bandwidth is largely improved
The interior signal-to-noise ratio of band, but at this time partially or there are the larger spike of some step evolutions and burr interference signals, then again
Waveform shown in the third line is obtained by medium filtering, it can be seen that by the spike and burr that median filter last transition is larger
Interference signal can filter substantially, and be not destroyed by the original signal of signal after two stage filter, the saltus step at edge also base
This reservation is complete.
As shown in figure 5, intercepting one section of waveform in Wave data as waveform to be matched, then by being introduced in Fig. 6
Method is handled, it can be seen that the signal similitude can be found after being handled by DTW.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail can carry out a variety of simple variants to technical scheme of the present invention within the scope of the technical concept of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (23)
1. a kind of method confirming life entity, which is characterized in that the nucleic acid determination signal by comparing life entity confirms life entity.
2. a kind of method confirming life entity according to claim 1, which is characterized in that the nucleic acid determination of the life entity
Signal is electric signal waveform.
3. a kind of method confirming life entity according to claim 2, which is characterized in that the electric signal waveform is by core
Acid sequence is formed by nanopore sensor.
4. according to the method described in claim 1, it is characterized in that, the nucleic acid determination signal for comparing life entity includes measuring
The electric signal waveform of life entity to be measured is carried out waveform with existing electric signal waveform and compared by the electric signal waveform of life entity to be measured.
5. according to the method described in claim 4, it is characterized in that, the life entity to be measured is selected from ancient bacterium domain, bacterium domain, eukaryon
The method of biocycle, virus and viroid, the electric signal waveform for measuring life entity to be measured is by the nucleic acid of life entity to be measured
Pass through nanopore sensor.
6. according to the method described in claim 4, it is characterized in that, the electric signal waveform of the life entity to be measured is full-length genome
The electric signal waveform of the electric signal waveform of sequence and/or the electric signal waveform of partial genome sequence and/or RNA sequence.
7. according to the method described in claim 4, it is characterized in that, the existing electric signal waveform is in electric signal waveform library
Existing electric signal waveform, include at least one electrical signal wave from known life entity in the electric signal waveform library
Shape.
8. according to the method described in claim 4, it is characterized in that, the existing electric signal waveform is to measure to obtain or simulate
It obtains, the existing electric signal waveform is the electric signal waveform of whole genome sequence and/or the telecommunications of partial genome sequence
The electric signal waveform of number waveform and/or RNA sequence.
9. according to the method described in claim 4, it is characterized in that, it is by the entire electricity of life entity to be measured that the waveform, which compares,
Signal waveform and existing electric signal waveform compare or by the part electric signal waveforms of life entity to be measured and existing electrical signal wave
Shape compares.
10. according to the method described in claim 4, it is characterized in that, the waveform comparison method be Edit distance,
ANN, LSH, Dynamic time warp or Smith Waterman.
11. a kind of method obtaining life entity electric signal waveform, which is characterized in that include the following steps：
1) from life entity to be measured, extraction, purification of nucleic acid；
2) it is the nucleic acid Ghana metre hole library connector obtained in step 1), obtains the nucleic acid with connector；
3) nucleic acid with connector obtained in step 2) is mixed with containing nano-pore sequencing required buffer liquid, is mixed
Liquid；
4) detection chip containing nano-pore, access sequencing circuit so as to be measured are added in the mixed liquor obtained in step 3)
Life entity nucleic acid passes through nano-pore；
5) nano-pore current signal is read, the electric signal waveform of life entity nucleic acid sequence to be measured is obtained.
12. according to the method for claim 11, which is characterized in that be nucleic acid Ghana metre hole library connector in the step 2)
Method is that enzyme process, swivel base enzyme process, universal primer sequence PCR amplification method or RNA are repaired-linked in end by reverse transcription into RNA-
DNA complex and adjunction head.
13. according to the method for claim 11, which is characterized in that buffer solution described in the step 3) is that electrolyte is molten
Liquid.
14. according to the method for claim 11, which is characterized in that nano-pore described in the step 4) is golden yellow grape
Coccus alpha hemolysin nano-pore, MspA nano-pores, Csgg nano-pores, phi29 nano-pores or solid nano hole.
15. according to the method for claim 11, which is characterized in that detection chip described in the step 4) is biological nano
Hole chip, solid nano hole chip, tunnel current detecting system, field effect transistor guard system or microfluidic system.
16. a kind of construction method of nucleic acid determination signal library, which is characterized in that sense known nucleic acid sequence by nano-pore
Device collects electric signal, obtains electric signal waveform.
17. according to the method for claim 16, which is characterized in that the known nucleic acid sequence is selected from known life entity
The electric signal waveform of whole genome sequence and/or partial genome sequence and/or RNA sequence.
18. according to the method for claim 16, which is characterized in that the known nucleic acid sequence is inquired by NCBI
It arrives, and by link, PCR amplification or chemical synthesis, or passes through the nucleic acid of life entity known to extraction.
19. according to the method for claim 16, which is characterized in that all life entity nucleic acid sequences for inquiring NCBI
By nanopore sensor, respective electric signal waveform is stored respectively.
20. according to the method for claim 16, which is characterized in that the electric signal waveform of the acquisition is pre-processed,
The pretreatment includes low-pass filter filtering, median filter filtering, down-sampling, leveling and/or normalization.
21. a kind of construction method in the electric signal waveform library of sequence, which is characterized in that including passing nucleic acid sequence by nano-pore
Sensor obtains electric signal waveform, and using the electric signal waveform height parameter measured, by computer simulator, simulation obtains
A plurality of data waveform, the electric signal waveform library as sequence.
22. the nucleic acid determination signal of life entity is in terms of medicine and scientific research about the application in sequence alignment and search.
23. the waveform of sequence compares the application in microorganism classification, Causal Agent Identification, nucleic acid mutation detection and distribution type matching.
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