CN106148482B - A kind of sequencing approach suitable for small-sized sequenator - Google Patents
A kind of sequencing approach suitable for small-sized sequenator Download PDFInfo
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- CN106148482B CN106148482B CN201510130536.XA CN201510130536A CN106148482B CN 106148482 B CN106148482 B CN 106148482B CN 201510130536 A CN201510130536 A CN 201510130536A CN 106148482 B CN106148482 B CN 106148482B
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Abstract
This application discloses a kind of sequencing approaches suitable for small-sized sequenator, including sample is sequenced using microarray sequence testing chip, at least two libraries are used in loading, at least two library includes sequencing library and occupy-place library, occupy-place library is the library that can be connect with sequence testing chip, does not have the region hybridized with sequencing primer；Sequencing library can be connect with sequence testing chip, and connect sample to be tested sequence, have the region hybridized with sequencing primer.The sequencing approach of the application, the spacing of sequencing library is changed under the premise of ensureing sequencing stability and uniformity using the mixed solution that uniformly mixed occupy-place library and sequencing library form when loading in library, to adapt to different optical systems, particularly suitable for small-sized sequenator.
Description
Technical field
This application involves gene sequencing fields, more particularly to a kind of sequencing approach suitable for small-sized sequenator.
Background technique
Illumina company uses glass flow chamber chip, forms tool biology point at regular intervals by surface treatment
Son, biomolecule connect DNA sequence dna to be measured, carry out amplification and form DNA cluster, these clusters have a fixed spacing so as to optical system
It can recognize.In patent CN101415839A, they utilize the solid support comprising " functionalized " inert substrate or matrix
Object, support use polyacrylamide hydrogel, and functionalization is realized by the layer or coating of application intermediate materials, intermediate wood
Comprising the reactive group of covalent bond can be formed with biomolecule in material, to realize the connection with DNA sequence dna to be measured.Patent
In CN102597256A, template terminal fragment having the same is reacted using bridge amplification, formed cluster or clone by multiple
" the cluster array " that the polynucleotide chain of identical fixation and the complementary polynucleotide chain of multiple identical fixations are formed.It is clustering
In the preparation of array, usually used template concentrations are higher, and the cluster density generated on cluster array will be higher.If cluster
Density is too high, it may be difficult to individually analyze each cluster.It can be titrated to determine optimal Template concentration, on array
Reach best cluster density, wherein each cluster can be individually analyzed.Therefore, Illumina company is with this control template
The spacing of the method control dot matrix of concentration.Complete Genomics company uses micro- battle array in its patent CN101910399A
The mode for arranging (Patterned Array), controls the spacing of dot matrix, and cooperate electron multiplication CCD (i.e. EMCCD), high numerical aperture
The optical system of diameter accurately obtains the signal of each point.
With the development of biotechnology, the demand to nucleic acid sequencing increasingly increases, and the small-sized sequenator of small throughput is also more next
More it is taken seriously.In the R&D process of the small-sized sequenator based on glass flow chamber chip, the spacing for controlling dot matrix is one big
Difficult point.Although the mode of Illumina company dilution template concentrations can effectively control lattice distance, for small-sized survey
For sequence instrument, its stability and uniformity will affect, both ends will appear non-uniform problem to runner end to end.Complete
Genomics company is handled the signal of microarray using the optical system of electron multiplication CCD and high-NA, although
Lattice distance can effectively be controlled, and obtain the accurate signal of each point, still, minimize sequenator itself with it is at low cost,
Cheap, easy to operate famous, electron multiplication CCD and high-NA optical system are not suitable for minimizing sequenator.
Therefore, it is badly in need of a kind of method of effectively control lattice distance to meet the growing demand to small-sized sequenator.
Summary of the invention
The purpose of the application is to provide a kind of suitable for small-sized sequenator, can effectively control the sequencing side of lattice distance
Method.
To achieve the goals above, the application uses following technical scheme:
This application discloses a kind of sequencing approaches suitable for small-sized sequenator, including are treated using microarray sequence testing chip
Sample product are sequenced, and at least two libraries are used in loading, which includes sequencing library and occupy-place library,
Occupy-place library is the ineffective law, rule, etc. library for being only capable of connecting with sequence testing chip, is not connected to sample to be tested sequence, is not also had miscellaneous with sequencing primer
The region of friendship；Sequencing library can be connect with sequence testing chip, and connect sample to be tested sequence, have the area hybridized with sequencing primer
Domain.
It should be noted that the application's it is critical that hybridized by occupy-place library with sequence testing chip to close sequencing core
The moiety site of on piece, to achieve the purpose that control sequencing library lattice distance.Since occupy-place library is to the dilute of sequencing library
It releases, the dot matrix that sequencing library hybridizes on sequence testing chip is necessarily diluted, and expands the spacing of sequencing dot matrix, it will be understood that specific
Occupy-place library and sequencing library can use the conventional use of library in laboratory, be not specifically limited herein.In addition, occupy-place
The amount ratio of library and sequencing library can also be adjusted according to specific required lattice distance；Generally speaking, the point needed
Battle array spacing is bigger, then the dosage in occupy-place library is also necessarily bigger, is not specifically limited herein.In addition, accounting for according to different requirements,
Position library can be a kind of or multiclass library；Sequencing library is also possible to a kind of or multiclass library, is not specifically limited herein.
Preferably, occupy-place library is single connector DNB, and sequencing library is twin adapter DNB, in loading using single connector DNB
Loading is carried out with the mixed solution of twin adapter DNB, single connector DNB and twin adapter DNB can be connect with sequence testing chip；Single connector
DNB is that the library containing single linker DNA ring is formed through rolling circle amplification, and the library containing single linker DNA ring is not connected to sample to be tested sequence,
Single linker DNA ring does not have the region hybridized with sequencing primer；Twin adapter DNB is that the library of the DNA circle containing twin adapter is expanded through rolling ring
Increase, sample to be tested sequence is connected in the library of the DNA circle containing twin adapter, twin adapter DNA circle has to be hybridized with sequencing primer
Region.
It should be noted that during Loading DNB, chip be it is inclined, DNB solution is upper from chip runner
End enters, and lower end outflow, DNB is much excessive during this, therefore, for purposes of this application, dilute using Illumina company
The mode effect for releasing template concentrations is little, for small-sized sequenator, there is also uneven, the shadow of runner ending
The problems such as ringing sequencing stability.Therefore, single connector DNB is mixed in the sequencing library in twin adapter DNB of the application creativeness；It should
Single connector DNB can equally be connect with sequence testing chip, still, but be free of sequencing primer hybridising region, that is to say, that single connector
DNB actually only plays the role of occupy-place；It had both ensured on each point there is DNB in this way, and had reached flux peak, and widened reality
The spacing of the DNB of sequencing, to adapt to different optical systems.The library DNB is the research direction of current sequencing mode Chinese library, this
Application creativeness uses list connector DNB as occupy-place library, and twin adapter DNB is relatively more first at present to meet as sequencing library
Into sequencing demands.
It is appreciated that the dosage of list connector DNB and twin adapter DNB directly affects the spacing of sequencing DNB；And it is sequenced DNB's
The optical system that best spacing depends on specifically using can make to be sequenced if optical system sensitivity and resolution ratio are higher
The spacing of DNB is smaller, and the dosage of twin adapter DNB is then big in the mixed solution of loading, and the dosage of anti-regular twin adapter DNB is small
A bit；Therefore, the dosage of single connector DNB and twin adapter DNB does not do specific limit depending on the optical system actually used herein
It is fixed.
It should also be noted that, DNB refers to DNA nanosphere, i.e. DNAnamoball in the application, it is the segment by being cyclized,
I.e. single linker DNA ring and twin adapter DNA circle, carry out rolling circle amplification and form；The design and DNA circle of DNA circle expand to form DNB's
Process refers to the library construction of full-length genome company (Complete Genomics calls CG company in the following text) third generation sequencing, herein
It is not tired to state.
Preferably, twin adapter DNA circle be once expanded by single linker DNA ring and added a different connector and
At.
It should be noted that the rolling circle amplification of DNA circle is actually the unlimited repetition to DNA circle, the twin adapter of the application
DNA circle is preferably once expanded by single linker DNA ring and adds a different connector and formed, that is twin adapter DNA
Ring is about twice of size of single linker DNA ring；Therefore, the DNB of identical number of rings, the twin adapter of twin adapter DNA circle amplification are expanded
DNB is bigger than single connector DNB that single linker DNA circle amplification obtains, and different sizes is to single connector DNB and twin adapter DNB mixed
The distribution closed in solution has influence, and then influences its absorption, and twin adapter DNA circle is designed as single linker DNA ring by the application
About twice is big, can achieve the purpose that the spacing for widening sequencing DNB and the stability for ensureing small-sized sequenator.
It is appreciated that as long as meeting twin adapter DNB for sequencing DNB, single connector DNB is that occupy-place DNB can reach the application
Basic demand, twin adapter DNA circle is once to be expanded by single linker DNA ring and added a different connector to form, only
It is that a kind of of the application more preferably selects, in the case where not considering cost and meeting compatibility, directly can also in addition designs
One twin adapter DNA circle unrelated with single linker DNA ring.
Preferably, the volume ratio of list connector DNB and twin adapter DNB is 1:0.25-4 in mixed solution.
It is appreciated that since the dosage of single connector DNB and twin adapter DNB directly affects the spacing of sequencing DNB, and be sequenced
The spacing of DNB depends on used optical system again；Therefore, the application passes through to more conventional use of optical system at present
It is studied, proposes that the volume ratio of list connector DNB and twin adapter DNB is mixed solution prepared by 1:0.25-4, can satisfy
General sequencing demands, specific ratio can be excellent in the proportional region that the application limits according to the optical system specifically used
Change.
Due to using the technology described above, the beneficial effects of the present application are as follows:
The sequencing approach of the application, in library using the mixed solution being made of occupy-place library and sequencing library when loading,
Under the premise of ensureing sequencing stability and uniformity, the spacing of sequencing library is changed, effectively to adapt to different optics
System, particularly suitable for small-sized sequenator.
In the preferred embodiment of the application, especially carried out using single connector DNB of the twin adapter DNB of sequencing and occupy-place
Sample effectively changes the spacing of sequencing DNB.
Detailed description of the invention
Fig. 1 is after the twin adapter DNB in the embodiment of the present application using 100% carries out Loading sequencing, using fluorescence microscopy
The image of sem observation；
Fig. 2 is to carry out Loading sequencing using list connector DNB and twin adapter DNB volume ratio 4:1 in the embodiment of the present application
Afterwards, using the image of fluorescence microscope；
Fig. 3 is to be carried out in another test of the embodiment of the present application using list connector DNB and twin adapter DNB volume ratio 4:1
After Loading sequencing, using the image of fluorescence microscope；
Fig. 4 is to be carried out in another test of the embodiment of the present application using list connector DNB and twin adapter DNB volume ratio 4:1
After Loading sequencing, using the image of fluorescence microscope；
Fig. 5 is to be carried out in another test of the embodiment of the present application using list connector DNB and twin adapter DNB volume ratio 6:1
After Loading sequencing, using the image of fluorescence microscope；
Fig. 6 is to be carried out in another test of the embodiment of the present application using list connector DNB and twin adapter DNB volume ratio 9:1
After Loading sequencing, using the image of fluorescence microscope；
Fig. 7 is the point that the luminous point area detected in the embodiment of the present application accounts for total area ratio Yu twin adapter DNB concentration
Figure；
Fig. 8 is the line that the luminous point area detected in the embodiment of the present application accounts for total area ratio Yu twin adapter DNB concentration
Sexual intercourse figure.
Specific embodiment
The sequencing approach of the application is to be widened particular for small-sized sequenator in DNB loading by diluting template concentrations
DNB spacing ineffective is sequenced and proposes, the key of the application is have as occupy-place DNB by addition list connector DNB
Effect has widened the spacing of sequencing DNB, and has also ensured that each point has DNB, makes sequencing that can reach flux peak, to adapt to
Different optical systems.
It is appreciated that the key of the application is arbitrarily change the spacing of sequencing DNB as needed, surveyed without influencing
The stability and uniformity of sequence, for other sequenators, such as CG sequenator, although its use electron multiplication CCD and
The optical system of high-NA does not need to carry out DNB dilution, still, under special circumstances, between it needs to widen DNB
Away from when, can equally use the present processes.That is, although the sequencing approach of the application is particular for small-sized sequencing
Instrument and propose, but it is not limited in using on small-sized sequenator；The sequencing approach of the application, essence be by using
The mixed solution of single connector DNB composition of the twin adapter DNB and occupy-place of sequencing, widens the spacing of sequencing DNB, any required
The sequenator regulated and controled to the spacing in sequencing site can use the present processes.
In addition, it is necessary to explanation, single connector DNB and twin adapter DNB is respectively adopted as occupy-place library and sequencing library,
Only a kind of preferred implementation of the application, it was confirmed that it is feasible that the occupy-place library of the application and sequencing library, which are used in mixed way,
Scheme, the application are not only limited in using list connector DNB and twin adapter DNB, and other common libraries in laboratory can also be according to
Present invention thinking is respectively as occupy-place library and sequencing library, to realize the purpose for widening sequencing site spacing, thus
Adapt to different optical systems.
The application is described in further detail below by specific embodiments and the drawings.Following embodiment is only to the application
It is further described, should not be construed as the limitation to the application.
Embodiment
One, experimental material
Single i.e. 1adaptor DNB of connector DNB, twin adapter DNB i.e. 2adaptor DNB has been respectively adopted in this example, and surveys
Sequence chip, that is, Patterned array.
Wherein, sequence testing chip is provided by Complete Genomics.
1adaptor DNB is single connector DNB in the library NIFTY, and the library NIFTY is the DNA library of fetus dissociative,
The adaptor of 1adaptor DNB is 141 types, is ordered from idt.
2adaptor DNB is the library of human genome, and 2adapter DNB is that another builds library, 2adaptor DNB
Two adaptor be respectively 141 types and 142 types, wherein the adaptor type of connection Primer is 142 types.It needs to illustrate
It is that the twin adapter DNB in this example can be two different connectors, can also individually uses 142 type joints.
The sequence of 141 types is sequence shown in Seq ID No.1,
Seq ID No.1:
5’-AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAAGCTCGAGCTCGAGCGATCGGGCTTCGACTGGAG
ACTTCAGCCTCCGGTTCGCCAGAATCCTTCTGTTCGAGCTCGAGCTCGCTAGCCCGAAGCTGACCTCTG-3’
The sequence of 142 types is sequence shown in Seq ID No.2,
Seq ID No.2:
5’-ACTGCTGACGTACTGCATGTTTCCCAGCACGAGACGTTCTCGACTCAGCAGATGACGACTGCATG ACACAGTATTTATCGTGCTCTGCAAGAGCTGAGTCGTCT-3’
Wherein, underscore area, i.e. the 65th bit base to the 104th bit base of sequence shown in Seq ID No.2 are and primer
Complementary region；In addition, the 16th bit base to the 25th bit base of sequence shown in Seq ID No.2 is for marking different samples
The combined sequence in source, the 68th bit base to the 77th bit base are the sequence groups that another is used to mark different samples sources
It closes.It should be noted that different samples sources can use different flags sequence.
Primer sequence is designed according to the Primers complementary region in 142 type sequences and its reverse complemental in Primers complementary region
Sequence.
Two, experimental method and result
There are eight chip runners on the sequence testing chip that this example uses, the concentration of single connector DNB and twin adapter DNB are
0.44ng/uL prepares mixed solution in ratio shown in table 1, and mixed solution is loaded in the specified chip runner of table 1, table
Middle lane indicates chip runner.The operation of DNB loading is operated with reference to the DNBLoading of CG company, not tired herein to state.
1 mixed solution of table proportion
| lane1 | 15uL1adaptorDNB |
| lane2 | 12uL1adaptorDNB+3uL2adaptorDNB |
| lane3 | 10uL1adaptorDNB+5uL2adaptorDNB |
| lane4 | 9uL1adaptorDNB+6uL2adaptorDNB |
| lane5 | 7.5uL1adaptorDNB+7.5uL2adaptorDNB |
| lane6 | 5uL1adaptorDNB+10uL2adaptorDNB |
| lane7 | 3uL1adaptorDNB+12uL2adaptorDNB |
| lane8 | 15uL2adaptorDNB |
After completion of the sample, sequencing primer is added into sequence testing chip, hybridizes 30 minutes, sequencing primer is made to be integrated to twin adapter
On DNB, the sequencing approach of Complete Genomics company is then utilized, completes a sequencing circulation, specific sequencing approach ginseng
Examine the operation instruction of CG sequenator.
After completing a sequencing circulation, is excited under fluorescence microscope with green light, take pictures and obtain sequencing image, as a result show
Show, using the chip runner of mixed solution loading, with the increase of single connector DNB dosage, the density of luminous point is reduced；As it can be seen that
After single connector DNB dilution, the spacing of the twin adapter DNB of sequencing is widened.In table 1, all using twin adapter DNB loading
The luminous dot density of chip runner 8 is maximum, and is all then not detected using the chip runner 1 of single connector DNB loading luminous
Point.Parts of images result is as depicted in figs. 1 and 2, Fig. 1 be table 1 chip runner 8 as a result, i.e. all using twin adapter DNB into
Row loading as a result, Fig. 2 be table 1 chip runner 2 as a result, i.e. list connector DNB and twin adapter DNB according to 4:1 ratio mix
Close the result of the mixed solution loading of preparation；Obviously, phosphor dot ratio Fig. 2 in Fig. 1 is more dense, and the spacing quilt of DNB is sequenced in Fig. 2
It widens.
After completion of the sample, the primer only hybridized with occupy-place DNB, hybridization 30 is added into sequence testing chip using same method
Minute, it is integrated to primer on single connector DNB, then utilizes the sequencing approach of Complete Genomics company, complete one
Sequencing circulation, specific sequencing approach refer to the operation instruction of CG sequenator.At this point, the twin adapter DNB of script then plays occupy-place work
With.It after completing a sequencing circulation, is excited under fluorescence microscope with green light, takes pictures and obtain sequencing image, the results show that shining
The density of point is directly proportional to single connector DNB dosage, and result is opposite with the image result that sequencing primer pair twin adapter DNB is expanded.
Statistical analysis is shown, is equally the proportion and specified runner loading according to table 1 for sequence testing chip identical for two, point
Not Cai Yong sequencing primer pair twin adapter DNB carry out sequencing circulation, using single connector DNB primer pair, its one sequencing of progress is followed
Therefore ring can be recognized in the summation that the sum of two all luminous points of image of the same runner are approximately equal to sequence testing chip microarray
For each point has DNB on sequence testing chip microarray, has reached the flux peak of sequence testing chip.
According to the above test result, it is molten according to the ratio preparation mixing of 4:1 that this example selectes list connector DNB and twin adapter DNB
Liquid, with measuring stability.The concentration of single connector DNB and twin adapter DNB is 0.44ng/uL, prepares and mixes in ratio shown in table 2
Solution, and mixed solution is loaded in the specified chip runner of table 2, lane indicates chip runner in table.DNB loading operation ginseng
The DNB Loading operation of CG company is examined, it is not tired herein to state.Wherein, chip runner 3 and 4, in DNB loading, runner 4
5uL correlation assisted solution is added in the normal process provided using Comlpete Genomics company, and it is related that 10uL is added in runner 3
Assisted solution.
2 mixed solution of table proportion
| lane1 | 15uL2adaptorDNB |
| lane2 | 12uL1adaptorDNB+3uL2adaptorDNB |
| lane3 | 12uL1adaptorDNB+3uL2adaptorDNB |
| lane4 | 12uL1adaptorDNB+3uL2adaptorDNB |
| lane5 | 12.5uL1adaptorDNB+2.5uL2adaptorDNB |
| lane6 | 12.9uL1adaptorDNB+2.1uL2adaptorDNB |
| lane7 | 13.5uL1adaptorDNB+1.5uL2adaptorDNB |
| lane8 | 15uL2adaptorDNB |
After completion of the sample, sequencing primer is added into sequence testing chip, hybridizes 30 minutes, sequencing primer is made to be integrated to twin adapter
On DNB, the sequencing approach of Complete Genomics company is then utilized, completes a sequencing circulation, specific sequencing approach ginseng
Examine the operation instruction of CG sequenator.
After completing a sequencing circulation, is excited under fluorescence microscope with green light, take pictures and obtain sequencing image, as a result show
Show, single connector DNB and twin adapter DNB can be stablized according to the ratio mixing of 4:1, uniform image, can satisfy sequencing
Demand；And the luminous point of chip runner 5,6,7 than sparse, i.e. twin adapter DNB spacing is too big, although also can accurately obtain
The fluorescence signal of each point, but do not reach the maximum service efficiency of sequencing.Parts of images result as shown in figures 3 to 6, Fig. 3
For table 2 chip runner 3 as a result, the i.e. mixed solution that is mixed with according to the ratio of 4:1 of list connector DNB and twin adapter DNB
The result of loading；Fig. 4 is the chip runner 4 of table 2 as a result, being equally the ratio of single connector DNB and twin adapter DNB according to 4:1
The mixed solution loading being mixed with as a result, the assisted solution amount of chip runner 3 and 4 is respectively 10uL and 5uL, it is seen that loading
Concentration do not influence density content relationship in a certain range；Fig. 5 is the chip runner 6 of table 2 as a result, Fig. 6 is the chip of table 2
The result of runner 7；As it can be seen that the luminous point of Fig. 6 obviously dilutes very much, Fig. 5 is also sparse compared with Fig. 3 and Fig. 4, indicates the spacing of sequencing DNB
It is very big.
It should be noted that Fig. 3-Fig. 6 is comparable to an amplified image of microexamination grid of Fig. 2, Fig. 2 and
Fig. 3 is the sequencing image used after identical twin adapter DNB concentration loading, from the point of view of the result of analysis software statistics, the two
It is much the same.
With image-adjustment-Threshold order of Image J software, the range of luminance values of selection is adjusted, fluorescence is chosen
Point.After selection phosphor dot, with analysis-analysis particle function, it is 4 to just infinite, selection Summarize, click OK that Size, which is arranged,
The quantity, luminous point area summation, luminous point area summation for obtaining all points within the scope of this Size account for the ratio of the gross area
Example, average area.Wherein, the quantity of point indicates that the sequencing DNB detected points, luminous point area summation indicate that 2ad DNB exists
Area shared by the same region, luminous point area summation account for the ratio of the gross area to characterize 2ad DNB in whole region
DNB proportion, average area indicate 2ad DNB in the average value of different zones proportion.The luminous point area that will be obtained
The ratio that summation accounts for the gross area is mapped in Excel as Y-axis using percentage of the twin adapter DNB in mixed solution as X-axis.
From data are extracted in the image of Fig. 3, Fig. 4, Fig. 5, Fig. 6 and 2 chip runner 8 of table, map in Excel, as a result such as Fig. 7 institute
Show；As it can be seen that the concentration of twin adapter DNB is lower, the area of luminous point is smaller；In conjunction with analysis before, if loading mixed solution
In there is no a twin adapter DNB, the area of only single connector DNB then luminous point is zero.
Partial enlargement is carried out to Fig. 7, is 0 as starting point using light-emitting area, the area summation for choosing Fig. 3, Fig. 5 and Fig. 6 accounts for
The ratio of the gross area is fitted to obtain the linear relationship chart of several points, as shown in figure 8, X-axis is similarly twin adapter DNB in Fig. 8
Percentage in mixed solution, Y-axis are the ratio that luminous point area summation accounts for the gross area, the linear relationship of XY are as follows: y=
46.777x-2.1226.With reference to the linear relationship formula, ratio value in the solution corresponding to the area of needs can be obtained.
According to permutation and combination calculation, the value of area y directly affects the probability that two twin adapter DNB are adjoint in sequence testing chip microarray
Z:
Z=y × S × [(Cs1 2+Cs2 2)÷AS 2]
Wherein, Z indicates the adjoint probability of two twin adapter DNB；Y indicates that luminous point area summation accounts for the ratio of the gross area,
It can also be indicated with the ratio of the points Zhan of twin adapter DNB total microarray points；Total point of S expression sequence testing chip microarray
Number, S1 indicate points lateral in microarray, and S2 indicates points longitudinal in microarray, S=S1 × S2；Cs1 2It indicates in transverse direction
Adjacent two o'clock is all the combination of twin adapter DNB, Cs1 2=[S1 × (S1-1)] ÷ 2；Cs2 2It is double for indicating longitudinally adjacent two o'clock all
The combination of connector DNB, Cs2 2=[S2 × (S2-1)] ÷ 2；AS 2Indicate that adjacent two o'clock is all twin adapter DNB in total microarray
Arrangement, AS 2=S × (S-1).The adjoint probability Z of two twin adapter DNB is calculated according to permutation and combination formula, as long as theoretically two
The twin adapter DNB requirement that can satisfy optical system and Basecalling image analysis not adjoint as far as possible；That is,
It is required according to optical system and analysis, as long as probability Z is less than certain value, double accordingly connect can be extrapolated according to the Z value
Head DNB concentration, to obtain that DNB spacing most preferably is sequenced.
There are 600000000 dot matrix on the sequence testing chip one chip runner of this example, it is horizontal and vertical to be respectively
30000 and 20000 points.In the case where having twin adapter DNB on the point of optical system 1/8 according to the present example, two twin adapter DNB
Adjoint probability is Z are as follows:
Z=1/8 × 6 × 108×[(C30000 2+C20000 2)÷A600000000 2]=0.1354.
That is, point not adjoint in this example accounts for the 86.46% of twin adapter DNB sum.
The foregoing is a further detailed description of the present application in conjunction with specific implementation manners, and it cannot be said that this Shen
Specific implementation please is only limited to these instructions.For those of ordinary skill in the art to which this application belongs, it is not taking off
Under the premise of from the application design, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the protection of the application
Range.
Claims (4)
1. a kind of sequencing approach suitable for small-sized sequenator, including measuring samples are surveyed using microarray sequence testing chip
Sequence, it is characterised in that: at least two libraries are used in loading, at least two library includes sequencing library and occupy-place text
Library, the occupy-place library are the library that can be connect with sequence testing chip, do not have the region hybridized with sequencing primer；The sequencing text
Library can be connect with sequence testing chip, and connect sample to be tested sequence, have the region hybridized with sequencing primer；The occupy-place library
For single connector DNB, the sequencing library is sequencing DNB.
2. sequencing approach according to claim 1, it is characterised in that: the occupy-place library is single connector DNB, the sequencing
Library is twin adapter DNB, carries out loading, the list connector using the mixed solution of single connector DNB and twin adapter DNB in loading
DNB and twin adapter DNB can be connect with the sequence testing chip；The list connector DNB is the library containing single linker DNA ring through rolling
Circle amplification forms, and the library containing single linker DNA ring is not connected to sample to be tested sequence, and single linker DNA ring does not have and is sequenced
The region of primer hybridization；The twin adapter DNB is that the library of the DNA circle containing twin adapter is formed through rolling circle amplification, described to contain twin adapter
Sample to be tested sequence is connected in the library of DNA circle, twin adapter DNA circle has the region hybridized with sequencing primer.
3. sequencing approach according to claim 2, it is characterised in that: the twin adapter DNA circle is by single linker DNA
Ring is once expanded and adds a different connector and formed.
4. sequencing approach according to claim 2, it is characterised in that: list connector DNB and twin adapter in the mixed solution
The volume ratio of DNB is 1:0.25-4.
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| CN108070642B (en) * | 2016-11-17 | 2020-10-27 | 深圳华大智造科技股份有限公司 | Method for improving DNB double-end sequencing quality and DNB double-end sequencing method and kit |
| WO2018214036A1 (en) * | 2017-05-23 | 2018-11-29 | 深圳华大基因股份有限公司 | Enrichment method for genomic target region based on rolling circle amplification and application thereof |
| CN115927566B (en) * | 2017-09-20 | 2025-02-18 | 深圳华大智造科技股份有限公司 | A sequencing method, sequencing reagent and application for Small RNA |
| CN111041077A (en) * | 2018-10-15 | 2020-04-21 | 深圳华大生命科学研究院 | Sample loading method, sequencing method and sequencing system for high-throughput sequencing |
| CN119487209A (en) * | 2022-12-30 | 2025-02-18 | 深圳华大生命科学研究院 | Spatiotemporal transcriptome sequencing method, sequencing library construction method and gene sequencing system |
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