阅读说明：本技术 高通量单细胞多组学 (High-throughput unicellular multiple groups ) 是由 R·范 B·杜拉 于 2018-02-13 设计创作，主要内容包括：在一些实施方式中,本文提供了用于高通量单细胞多组学(例如,基因组学、表观基因组学、蛋白质组学和/或表型谱)分析的装置、系统和方法。(In some embodiments, there is provided herein the devices, systems, and methods for high-throughput unicellular multiple groups (for example, genomics, epigenomics, proteomics and/or phenotypic spectrum) analysis.)

1. a kind of multiple groups multiplex machine, comprising:

Substrate, including arranging the X row intersected with Y to form X*Y patch, wherein each of X*Y patch includes to be fixed to the base Piece and include more T-sequences unique nucleic acid bar code,

Wherein each row includes the different subsets of the bar coded nucleic acid chains of first group of nucleic acid chains, and respectively column include second group of nucleic acid The different subsets of the bar coded nucleic acid chains of chain, and

Wherein first group of the nucleic acid chains form unique nucleic acid bar code in conjunction with second group of the nucleic acid chains.

2. multiple groups multiplex machine according to claim 1, which is characterized in that X is at least 10.

3. multiple groups multiplex machine according to claim 1, which is characterized in that X is at least 20, at least 50, at least 100, at least 1000, at least 10000 or at least 20000.

4. multiple groups multiplex machine according to claim 1, which is characterized in that X is 10 to 20000.

5. multiple groups multiplex machine described in any one of -4 according to claim 1, which is characterized in that Y is at least 10.

6. multiple groups multiplex machine described in any one of -4 according to claim 1, which is characterized in that Y is at least 20, extremely Few 50, at least 100, at least 1000, at least 10000 or at least 20000.

7. multiple groups multiplex machine described in any one of -4 according to claim 1, which is characterized in that Y be 10 to 20000。

8. multiple groups multiplex machine described in any one of -7 according to claim 1 further includes the Z intersected with X row_n+1Column Form X*Z patch, wherein each of X*Z patch includes the molecular binding partner for being fixed to substrate, and wherein, n is Zero or bigger.

9. multiple groups multiplex machine according to claim 8, which is characterized in that n is at least 1, and Z_n+1In column It each include different molecular binding partners.

10. multiple groups multiplex machine according to claim 9, which is characterized in that n is at least 2, and Z_n+1In column Each of include different molecular binding partners.

11. the multiple groups multiplex machine according to any one of claim 8-10, which is characterized in that the molecule knot Closing gametophyte is antibody.

12. multiple groups multiplex machine described in any one of -11 according to claim 1 further includes being coupled to the substrate Microwell array, wherein each micropore includes one of unique molecular barcode of the substrate.

13. the multiple groups multiplex machine according to any one of claim 8-11 further includes being connected to the substrate Microwell array, wherein each micropore includes in one of unique molecular barcode and the molecular binding partner of the substrate At least one.

14. multiple groups multiplex machine described in 3 or 14 according to claim 1, which is characterized in that the microwell array includes At least 20 micropores.

15. multiple groups multiplex machine described in 3 or 14 according to claim 1, which is characterized in that the microwell array includes At least 50, at least 100, at least 1000 or at least 10000 micropores.

16. multiple groups multiplex machine described in any one of -15 according to claim 1, which is characterized in that described first group The nucleic acid chains of nucleic acid chains include, optionally in 5' to the direction 3': promoter sequence, sequence measuring joints sequence, the first bar code sequence With the first anchor sequence.

17. multiple groups multiplex machine described in any one of -16 according to claim 1, which is characterized in that described second group The nucleic acid chains of nucleic acid chains include, optionally in 5' to the direction 3': more T-sequences, unique molecular marker accord with sequence, the second bar code sequence Column and the second anchor sequence, wherein the second anchor sequence is complementary with the first anchor sequence.

18. multiple groups multiplex machine described in any one of -17 according to claim 1, which is characterized in that unique core Sour bar code includes, optionally in 5' to the direction 3': promoter sequence, sequence measuring joints sequence, the first bar code sequence, Article 2 Shape code sequence, optionally, unique molecular marker symbol and more T-sequences.

19. multiple groups multiplex machine described in any one of -18 according to claim 1, which is characterized in that the substrate packet Include glass, silicon or silica.

20. multiple groups multiplex machine described in any one of -19 according to claim 1, which is characterized in that the substrate applies It is covered with poly-L-Lysine.

21. multiple groups multiplex machine described in any one of -20 according to claim 1, which is characterized in that each column and/or Capable width is 50-200 microns.

22. multiple groups multiplex machine according to claim 21, which is characterized in that the width of each columns and/or rows is 100 microns.

23. multiple groups multiplex machine described in any one of -22 according to claim 1, which is characterized in that the face of each patch Product is 400-40,000 μm²。

24. multiple groups multiplex machine according to claim 23, which is characterized in that the area of each patch is 10,000 μm²。

25. multiple groups multiplex machine described in any one of -24 according to claim 1, which is characterized in that in uniline and/ Or single-row interior patch is spaced each other 20-200 microns.

26. multiple groups multiplex machine according to claim 25, which is characterized in that in uniline is interior and/or single-row Patch is spaced each other 100 microns.

27. multiple groups multiplex machine described in any one of -26 according to claim 1, which is characterized in that between adjacent rows And/or the patch between adjacent column is spaced each other 20-200 microns.

28. multiple groups multiplex machine according to claim 27, which is characterized in that between adjacent rows and/or adjacent Patch between column is spaced each other 100 microns.

29. a kind of multiple groups multiplex machine, comprising:

Microwell array, it includes at least 20 micropores, wherein each micropore of the array includes point specific to single micropore Sub-barcode, and wherein each molecular barcode includes the nucleic acid bar code that (a) includes more T-sequences, and (b) at least one anti- Body.

30. multiple groups multiplex machine according to claim 29, it includes at least two kinds of, at least three kinds of or at least four kinds of Different antibody.

31. the multiple groups multiplex machine according to claim 29 or 30, which is characterized in that the microwell array includes At least 50, at least 100, at least 1000 or at least 10000 micropores.

32. the multiple groups multiplex machine according to any one of claim 29-31, which is characterized in that described unique Nucleic acid bar code includes, optionally in 5' to the direction 3': promoter sequence, sequence measuring joints sequence, the first bar code sequence, second Bar code sequence, optionally, unique molecular marker symbol and more T-sequences.

33. a kind of method for producing bar coded array, comprising:

(a) by first group of bar coded nucleic acid chains the first solution flow pattern and it is fixed over the substrate surface, to generate that This column that are parallel and being spatially separated, wherein the X patch of each bar coded nucleic acid chains of the column comprising first group, wherein in each column The patch is spatially separated each other, wherein each column include the different subsets of bar coded nucleic acid chains, and wherein, X is greater than 2 Number；

(b) by second group of bar coded nucleic acid chains the second solution flow pattern and it is fixed over the substrate surface, to generate that This row that is parallel and being spatially separated, wherein each row includes the Y patch of second group of bar coded nucleic acid chains, wherein in each row The patch is spatially separated each other, wherein each row includes the different subsets of bar coded nucleic acid chains, wherein row and column is vertical, and And wherein, Y is greater than 2 number；

To generate X*Y patch array, each patch includes the subset of (i) first group of bar coded nucleic acid chains, is bound to (ii) the The subset of two groups of bar coded nucleic acid chains forms unique nucleic acid bar code.

34. according to the method for claim 33, which is characterized in that first group of bar coded nucleic acid chains include, optionally Ground is in 5' to the direction 3': promoter sequence, sequence measuring joints sequence, the first bar code sequence and the first anchor sequence.

35. according to the method for claim 33 or 34, which is characterized in that second group of bar coded nucleic acid chains include, in 5' To the direction 3': more T-sequences, unique molecular marker symbol sequence, the second bar code sequence and the second anchor sequence, wherein described second Anchor sequence is complementary with the first anchor sequence.

36. method according to any of claims 33-35, further include by second group of bar coded nucleic acid chains with First group of bar coded nucleic acid strand simultaneously generates the patch comprising partially double stranded bar coded nucleic acid.

37. according to the method for claim 36, further including that will be overlapped patch array and polymerase and second bar code The primer and dNTP that sequence combines combine, and generate nucleic acid chains, include on 5' to the direction 3': promoter sequence, sequencing connect Header sequence, the first bar code sequence, the first anchor sequence, the second bar code sequence, unique molecular marker symbol sequence and more T-sequences.

38. further including according to the method for claim 37, removing second group of bar shaped from the overlapping patch array Codeization nucleic acid.

39. the method according to any one of claim 33-38, which is characterized in that the surface be glass surface, silicon or Silica.

40. according to the method for claim 39, which is characterized in that the glass surface is coated with poly-L-Lysine.

41. the method according to any one of claim 33-40 further includes by molecular binding partner group third solution stream It is dynamic to pattern and fix over the substrate surface, column that are parallel and being spatially separated are arranged each other and relative to (b) to generate, In respectively column include molecular binding partner Z patch, wherein each column include different molecular binding partners, and wherein, Z is Number greater than 2.

42. the method according to any one of claim 33-41 further includes the table that microwell array is applied to the substrate Face is with generation device, wherein each micropore of the microwell array includes patch, the patch include unique nucleic acid bar code and At least one optional antibody.

43. the method according to any one of claim 33-42, which is characterized in that the width of each columns and/or rows is 50- 200 microns.

44. according to the method for claim 43, which is characterized in that the width of each columns and/or rows is 100 microns.

45. the method according to any one of claim 33-43, which is characterized in that the area of each patch is 400-40000 μm²。

46. according to the method for claim 45, which is characterized in that the area of each patch is 10000 μm²。

47. the method according to any one of claim 33-46, which is characterized in that uniline and/or it is single-row in patch that 20-200 microns of this interval.

48. according to the method for claim 47, which is characterized in that in uniline and/or single-row interior patch is spaced each other 100 Micron.

49. the method according to any one of claim 33-48, which is characterized in that between adjacent rows and/or adjacent column it Between patch be spaced each other 20-200 microns.

50. according to the method for claim 49, which is characterized in that patch between adjacent rows and/or between adjacent column that 100 microns of this interval.

51. the method according to any one of claim 33-50, which is characterized in that pattern core using microfluidic flow First and/or second group of bar coded nucleic acid chains are patterned and are fixed on the surface of the substrate by piece.

52. a kind of multiple groups multiplex machine, comprising:

At least 20 closed micropores, the substrate by being connected to microwell array are formed, and wherein each micropore of described device includes The unique molecular barcode being fixed on substrate, wherein each unique molecular barcode includes (a) first comprising first antibody Patch, wherein first patch and (b) include that the second patch of secondary antibody is adjacent, wherein second patch and (c) the Three patches are adjacent, and it optionally includes the more T-sequences in end, wherein the third that the third patch, which includes unique nucleic acid bar code, Patch includes that the 4th patch of third antibody is adjacent with (d), wherein the 4th patch and (f) include the 5th spot of the 4th antibody Piece is adjacent, wherein the first antibody is identical as the type of the 4th antibody, the secondary antibody and the third antibody Type is identical.

53. multiple groups device according to claim 52, which is characterized in that unique molecular barcode also includes (g) The 6th patch comprising the 5th antibody, and (h) comprising the 7th patch of the 6th antibody, wherein the 5th antibody and the described 6th The type of antibody is identical.

Summary of the invention

In some embodiments, there is provided herein for high-throughput unicellular multiple groups (for example, genomics, apparently Genomics, proteomics and/or phenotypic spectrum) analysis devices, systems, and methods.For example, technology provided herein is available In unicellular using the parallel processing of certainty molecular barcode tens of thousands of in the array that space limits.Using the technology, it is based on The spatial position of cell and corresponding molecular barcode can be by multiple " group is learned " (multiple groups) informational linkages to identical cell (or cell subsets).For example, can be more than 400,000 unicellular with parallel processing in a microfluidic elements.The flux is high In current sequencing and genome-based technologies (for example, 5-10 times high).

Certainty bar code is used to distribute predetermined molecules (for example, nucleic acid and/or protein) bar code sequence for each cell Column, it is associated with predetermined position, so that multiple tests on same cell (or cell subsets) can pass through bar code and position It links together.

The technology can obtain entire information bank (including low cell number/low quality sample) in the cell of biosystem, So as to obtain the multi-mode molecular regulation layer for constituting organismal complexity like never before, and it is this to can be used for disclosing composition The mechanism (for example, how epigenetic change adjusts transcriptional expression and/or protein signal conduction) of complexity.

For example, the devices, systems, and methods of the disclosure are suitable for clinical setting.The technology can with low quality sample (for example, Including low cell number) it is used together, the sequencing cost of each cell is reduced, and improve and distinguish rare cell subset and detect rare The resolution ratio of pathogenic cell (such as pathogenic cells).

Therefore, some aspects of the disclosure provide multiple groups multiplex machine comprising substrate, the substrate include The X intersected with Y row is arranged to form X*Y patch, and wherein each of X*Y patch includes and is fixed on substrate and includes more T (polyT) unique nucleic acid bar code of sequence, wherein difference of each bar coded nucleic acid chains of the column comprising first group of nucleic acid chains Collection, and the different subsets of bar coded nucleic acid chains of each row comprising second group of nucleic acid chains, and wherein first group of nucleic acid chains To form unique nucleic acid bar code in conjunction with second group of nucleic acid chains.See, e.g., Figure 15 D.

In some embodiments, X is at least 10.Therefore, in some embodiments, device includes at least 10 column.In In some embodiments, X is at least 20, at least 50, at least 100, at least 1000, at least 10000 or at least 20000.Some In embodiment, X is 10 to 20000.

In some embodiments, Y is at least 10.Therefore, in some embodiments, device includes at least 10 rows.In In some embodiments, Y is at least 20, at least 50, at least 100, at least 1000, at least 10000 or at least 20000.Some In embodiment, Y is 10 to 20000.

In some embodiments, device also includes the Z intersected with Y row_n+1Column form Y*Z patch, wherein in Y*Z patch Each include molecular binding partner (for example, antibody) fixed to substrate, and wherein n is 0 or bigger (for example, n is 1,2,3,4 or 5).In some embodiments, n is at least 1, and Z_n+1It each include different molecular binding partners in column (for example, different antibody, such as antibody A, antibody B etc.).In some embodiments, n is at least 2, and Z_n+1It is each in column Include different molecular binding partners (for example, different antibody, such as antibody A, antibody B, antibody C etc.).In some implementations In mode, molecular binding partner is antibody.Term " antibody " includes complete antibody and antibody fragment (such as scFv and/or Fab Segment).

In some embodiments, device also includes the microwell array that couples with substrate (for example, making each micropore and base Piece forms sealing), wherein each micropore includes one of unique molecular barcode of substrate.See, e.g., Figure 15 D.In some realities It applies in mode, device also includes the microwell array coupled with substrate, wherein each micropore includes unique molecular barcode of substrate One of and at least one molecular binding partner.Therefore, each micropore can be encoded by molecular label comprising nucleic acid and antibody Unique combinations.

It should be appreciated that term " unique " is component about single device, and mean specific components (or the group of device The subset of part) " only one ".Therefore, the patch comprising unique nucleic acid bar code (or unique sub-set of nucleic acid bar code) is dress The unique patch set comprising specific unique nucleic acid bar code (or unique sub-set of nucleic acid bar code), so that based on only One nucleic acid bar code (or unique sub-set of nucleic acid bar code) can identify patch (and any micropore relevant to patch and micropore Interior any cell).

In some embodiments, microwell array (and therefore, device) includes at least 20 micropores.For example, microwell array It may include at least 50, at least 100, at least 1000 or at least 10000 micropores.In some embodiments, microwell array include 10, 20、40、50、60、70、80、90、100、200、300、400、500、600、700、800、900、1000、2000、3000、4000、 5000,6000,7000,8000,9000,10000,20000,30000 or 40000 micropore.

In some embodiments, the nucleic acid chains of first group of nucleic acid chains include, optionally in 5' to the direction 3': promoter sequence (for example, T7 promoter sequence), sequence measuring joints sequence, the first bar code sequence are arranged (for example, being unique for first group of nucleic acid chains And/or be unique to the nucleic acid chains subset in first group) and the first anchor sequence.In some embodiments, second group of nucleic acid chains Nucleic acid chains include, optionally in 5' to the direction 3': more T-sequences, unique molecular marker accord with sequence, the second bar code sequence (example Such as, it is unique for second group of nucleic acid chains and/or is unique to the nucleic acid chains subset in second group) and the second anchor sequence, wherein Second anchor sequence is complementary with the first anchor sequence.In some embodiments, unique nucleic acid bar code includes, optionally in 5' to 3' Direction: promoter sequence, sequence measuring joints sequence, the first bar code sequence, the second bar code sequence, optional unique molecular marker Symbol and more T-sequences.

In some embodiments, substrate includes glass, silicon or silica.In some embodiments, substrate is coated with Poly-L-Lysine.

In some embodiments, the width of each columns and/or rows is 50-200 microns.In some embodiments, each column And/or the width of row is 100 microns.

In some embodiments, each patch have 400-40,000 μm²Area.In some embodiments, often A patch has 10,000 μm²Area.

In some embodiments, in uniline and/or it is single-row in patch be spaced each other 20-200 microns.In some implementations In mode, in uniline and/or single-row interior patch is spaced each other 100 microns.

In some embodiments, the patch between adjacent rows and/or between adjacent column is spaced each other 20-200 microns.In In some embodiments, the patch between adjacent rows and/or between adjacent column is spaced each other 100 microns.

The some aspects of the disclosure provide multiple groups multiplex machine, and it includes microwell array, microwell array includes At least 20 micropores, wherein each micropore of array includes the molecular barcode specific to single micropore, and wherein each point Sub-barcode includes (a) nucleic acid bar code, and it includes more T-sequences, and (b) at least antibody.In some embodiments, device Include at least two kinds of, at least three kinds of or at least four kinds of different antibody.In some embodiments, micropore (and therefore, device) includes At least 50, at least 100, at least 1000 or at least 10000 micropores.In some embodiments, unique nucleic acid bar code includes and appoints Selection of land is in 5' to the direction 3': promoter sequence, sequence measuring joints sequence, the first bar code sequence, the second bar code sequence, optional Unique molecular marker symbol and more T-sequences.

Other aspects of the disclosure provide a kind of method for producing bar coded array, include (a) first group of bar shaped The first solution of codeization nucleic acid chains flow pattern (flow patterning) is simultaneously fixed on the surface of substrate, to generate that This column that are parallel and being spatially separated, wherein the X patch of each bar coded nucleic acid chains of the column comprising first group, wherein in each column Patch is spatially separated each other, wherein each column include the different subsets of bar coded nucleic acid chains, and wherein X is greater than 2 number； (b) by second group of bar coded nucleic acid chains the second solution flow pattern and be fixed to substrate surface on, thus generate each other Row that is parallel and being spatially separated, wherein each row includes the Y patch of second group of bar coded nucleic acid chains, wherein the spot in each row Piece is spatially separated each other, wherein each row includes the different subsets of bar coded nucleic acid chains, and wherein Y is greater than 2 number, from And X*Y patch array is generated, each patch includes the subset of the bar coded nucleic acid chains of first group of (i), is bound to second group of (ii) Bar coded nucleic acid chains subset to forming unique nucleic acid bar code.

In some embodiments, first group of bar coded nucleic acid chains include, optionally the direction from 5' to 3': promoter Sequence, sequence measuring joints sequence, the first bar code sequence and the first anchor sequence.In some embodiments, second group of bar code Changing nucleic acid chains in 5' to the direction 3' includes: more T-sequences, unique molecular marker symbol sequence, the second bar code sequence and the second anchor sequence Column, wherein the second anchor sequence is complementary with the first anchor sequence.

In some embodiments, method further includes by second group of bar coded nucleic acid chains and first group of bar coded nucleic acid Chain hybridizes and generates the patch comprising partially double stranded bar coded nucleic acid.

In some embodiments, method further includes that will be overlapped patch array and polymerase and the second bar code sequence knot The primer and dNTP of conjunction combine, and generate nucleic acid chains, include on 5' to the direction 3': promoter sequence, sequence measuring joints sequence, First bar code sequence, the first anchor sequence, the second bar code sequence, unique molecular marker symbol sequence and more T-sequences.

In some embodiments, method further includes removing second group of bar coded nucleic acid from overlapping patch array.

In some embodiments, method further includes by molecular binding partner group third solution flow pattern and fixation Over the substrate surface, column that are parallel and being spatially separated are arranged each other and relative to (b) to generate, wherein each column include molecule The Z patch of binding partners, wherein each column include different molecular binding partners, and wherein, Z is greater than 2 number.

In some embodiments, this method further include couple microwell array to substrate surface with generation device, wherein Each micropore of microwell array includes patch, and patch includes unique nucleic acid bar code and an optional at least antibody.

In some embodiments, chip is patterned by first and/or second group of bar coded core using microfluidic flow Sour chain and/or molecular binding partner (such as antibody) are patterned and are fixed over the substrate surface.

Multiple groups multiplex machine is also provided herein, it includes at least 20 (for example, at least 50, at least 100 It is a, at least 1000, at least 10000 or at least 20000) closed micropore, micropore is by being coupled to the substrate of microwell array It is formed, wherein each micropore of device includes the unique molecular barcode being fixed on substrate, wherein each unique molecule bar shaped First patch of the code comprising (a) comprising first antibody, wherein the first patch includes that the second patch of secondary antibody is adjacent with (b), Wherein the second patch is adjacent with (c) third patch, and it optionally includes the more T sequences in end that third patch, which includes unique nucleic acid bar code, Column, wherein third patch includes that the 4th patch of third antibody is adjacent with (d), wherein the 4th patch and (f) include the 4th antibody The 5th patch it is adjacent, wherein first antibody is identical as the type of the 4th antibody, and secondary antibody is identical as the type of third antibody. In some embodiments, it includes that the 6th patch of the 5th antibody and (h) resist comprising the 6th that unique molecular barcode, which also includes (g), 7th patch of body, wherein the 5th antibody is identical as the type of the 6th antibody.

In some embodiments, the micropore of device includes individual cells or single subgroup (for example, 2 or 3) cell.Carefully Born of the same parents can obtain from biological sample, such as blood, urine or saliva sample.Other biological sample is included herein.

In some embodiments, using single device as provided herein test (for example, for specific nucleic acid and/or resisting The presence of body) 100 to 1000 (for example, 100,200,300,400,500,600,700,800,900 or 1000) cells.

Brief Description Of Drawings

Fig. 1: the extensive different molecular bar code patch (each chip 40,000) for the capture of unicellular transcript profile Design and manufacture.Upper figure depicts exemplary flow patterning processes.Bottom panel show the flowings that microchannel guides on a left side Patterned viewgraph of cross-section.Bottom-right graph is shown in conjunction with bar code A, bar code B and other sequences (for example, promoter, sequencing Connector, unique molecular marker symbol (UMI) and more T-sequences) compound molecule bar code example.

Fig. 2: the microfluidic flow designed for generating molecular barcode patch patterns chip.The example includes having (bar coded patch is 100 × 100 to 42 × 42 arrays (flux~300-600 is unicellular) in 100 microns of flow pattern channels μm²).Corresponding micropore is spaced each other 100 μm, allows and does not share identical bar code there are two hole.

Fig. 3: bar code immobilized reactant.It the use of the first microfluidic flow patterning chip will include T7 promoter, sequencing The oligonucleotide sequence of connector and row bar code (bar code A) is patterned and is fixed on the glass surface.Then, by column bar code Sequence (bar code B) hybridization is simultaneously extended through primer extension reaction.The second shorter chain is removed with sodium hydroxide quenching reaction, And molecular barcode terminal is completed with poly- (dT) sequence.Bar code sequence is a priori known, can be realized certainty bar code.

Fig. 4: pass through the flow pattern for the molecular barcode that the hybridization probe of fluorescent marker confirms.Row bar code (bar shaped Code A, upper left) fixation.The patterning of column bar code and the hybridization probe for extending the fluorescent marker by being directed to poly- (dT) sequence are (right On) confirmation, show bar coded patch.Use the specific item for being directed to row bar code 20 (bottom right) and column bar code 19 (bottom right) The hybridization probe of the fluorescent marker of shape code sequence carries out quality inspection to different bar code sequences.Only specific marker carries the 19th column With the oligonucleotide sequence of the 20th row bar code sequence.

Fig. 5: the microwell array device for unicellular capture.In an example, drilling is (left on the surface of the substrate Figure).In another example, through-hole microwell array is created on thin substrate (middle graph and right figure).Substrate can be glass, Polymer or plastics.

Fig. 6: it is used for the misalignment plan of high-density bar code array and individual cells interaction (interfacing) Slightly.Micropore random loading cell, so that the hole of~50-70% is occupied by individual cells.Then micropore is sealed with bar code array And it is misaligned.After mechanical grip is with stabilized seal, fluorescence and light field imaging are acquired to determine hole and bar coded patch position It sets.Patch is patterned, so that a patch at most can only include at most 4 holes.This misalignment method generation~10- 30% unicellular corresponding determining bar coded patch.

Fig. 7: the certainty Pointing strategy for high-density bar code array and individual cells to interact.Micropore carries out Through-hole manufacture, then using the alignment stage alignment of customization and in conjunction with high-density bar code array, so that each bar code Change patch and corresponds to single hole.Once random loading cell seals micropore with the second slide glass.Mechanical grip with stabilized seal it Afterwards, light field imaging is carried out to determine hole occupancy and match bar code with cell.It is expected that yield is bar coded patch sum ~50-70%.

Fig. 8: the deterministic policy of the substitution for high-density bar code array and individual cells to interact.Be not by It on molecular barcode patterning to plate slide glass, but by bar code pattern and generates onto micropore platform, so that different Bar code is directly anchored in each hole.Cell load and measurement are similar with other versions.

Fig. 9: exploitation Cell capture strategy, wherein improving the loading efficiency of micropore using two micropore platforms.First will Cell is loaded on lesser microwell array, wherein due to size exclusion (each pore size~cell dia), nearly all hole All it is loaded with individual cells.Once saturation, lesser micropore is aligned with biggish microwell array, and cell is transferred to biggish Kong Zhong.This makes it possible to efficiently load biggish microwell array, for example, it provides bigger region for mRNA capture.

Figure 10: the example of freeze-thaw method cell cracking in microwell array is used.Top image depicts the visual field of micropore, micro- Hole loads unicellular (cell is invisible in bright field image) of useful green fluorescence dye marker.Two images of bottom are aobvious Cell after having shown Frozen-thawed cycled twice.It is diffused into the whole volume of single hole by fluorescent dye, cell cracking is obvious 's.Sealing keeps the cell lysate in each hole, prevents from leaking.Other cleavage methods, including but not limited to base can be used In detergent, heat painting or spraying lysis buffer.

Figure 11: the quality examination for the library preparation procedure applied in the system of description.~500,000 cells will be come from A large amount of cell lysates be applied to high-density bar code array.As control, the water of no RNA enzyme is applied to high density bar shaped On code array.Library is prepared according to CelSeq2 agreement high-sensitivity biological analyzer.Item as the result is shown from lysate application Prepared by the successful library of shape code array, wherein sample RNA and DNA shows that mean size is the expecting curve of 300bp and 400bp. Control bar code array does not generate detectable RNA or DNA.

Figure 12: the example tested by follow-on test from the multiple groups of identical individual cells.After cell load, by various Microscope imaging technology to cell imaging, by recording surface marker expression, in the form of, motility physical table relevant with other Type.Then with antibody bar code array sealing hole (referring to international publication number WO 2014/ disclosed on 2 27th, 2014 031997, be incorporated herein by reference), to test the secretion of~45 kinds of albumen.It is used in combination next, removing antibody bar code array High-density bar code array for transcription group test replaces,.It then will be from identical individual cells using customization software Data Integration is simultaneously analyzed.

Figure 13: multiple groups of credit analysis are combined by certainty bar code.Embodiment 1: unicellular transcript profile and Microrna are total Analysis.It is left: the schematic diagram of cell cracking on loading cells and device assembling and chip.It is right: using upper and lower part slide glass into The identical single celled Microrna of row and transcript profile test.Microrna and mRNA can link and with identical individual cells phase Associated mechanism is due to certainty molecular barcode, wherein the sequence of each molecular barcode and position in each microchamber (micropore) It is known for setting.

Figure 14 A-14B: multiple groups credit analysis is combined by certainty bar code.Embodiment 2: entire transcript profile and one are detected altogether Intracellular/surface protein (Figure 14 B) that group discharges after the protein (Figure 14 A) or cell cracking that cell is secreted.The former makes It is carried out with above-mentioned molecular barcode method with bottom glass slide glass, contains molecular barcode microplate array.The latter is at top Patterned antibody characteristic array carries out on slide glass.Protein and mRNA can be linked and associated with identical individual cells Mechanism be due to deterministic molecular barcode, wherein the sequence of each molecular barcode and position are known in each microchamber 's.

Figure 15 A-15D depicts the example of the device of the disclosure comprising substrate, it includes the bar coded of the first subset Nucleic acid arranges (Figure 15 A), and the bar coded nucleic acid row (Figure 15 B) of second subset, the other column of the subset of different antibodies are (by " wave Shape " flow pattern is formed) (Figure 15 C) and it is coupled to the micropore (black square) (Figure 15 C) of substrate.

Specific embodiment

Unicellular sequencing, especially for gene expression profile and phenotypic analysis entire transcript profile sequencing, be almost institute There is the advantageous scientific discovery tool in field of biology.However, however it remains several main problems.Firstly, in order to fixed The phenotype and functional heterogeneity of amount analysis complex cell group, it is necessary to while sequencing is unicellular more than 10,000.So far, do not have There is technology that this target may be implemented.Secondly, the technology should apply to low input sample (example in order to use in clinical setting Such as, < 50,000 cell) and the rare cell group that is separated from clinical samples.Third, the field are still unable to test same thin Multiple groups information in born of the same parents, for example, directly (for example, Microrna, apparently by gene expression (transcript profile sequencing) and controlling element Genetic modification) it is associated, this is important the mechanism for understanding cell heterogeneity.The technology of the disclosure solves above three Problem.

Method

There is provided herein the methods for producing bar coded array, include (a) first group of bar coded nucleic acid (such as DNA) chain First solution flow pattern is simultaneously fixed on substrate surface, so that column that are parallel to each other and being spatially separated are generated, wherein respectively X patch of the column comprising first group of bar coded nucleic acid chains, wherein the patch in each column is spatially separated each other, wherein each column packet Different subsets containing bar coded nucleic acid chains, and wherein, X is greater than 2 number；(b) by second group of bar coded nucleic acid (such as DNA) chain the second solution flow pattern is simultaneously fixed on substrate surface, to generate parallel to each other and be spatially separated Row, wherein each row includes the Y patch of second group of bar coded nucleic acid chains, wherein the patch in each row is spatially separated each other, Wherein each row includes the different subsets of bar coded nucleic acid chains, wherein row is vertical relative to column, and wherein, Y is greater than 2 number Word；It is each to be overlapped the subset that patch includes the bar coded nucleic acid chains of first group of (i) to generate X*Y overlapping patch array, and (ii) subset of second group of bar coded nucleic acid chains.

The conventional method of flow pattern is known, and is patterned including such as streamline flow, is fluid Flowing, wherein its speed at any point is constant or changes in regular fashion.

In some embodiments, first group of bar coded nucleic acid chains include, in 5' to the direction 3': promoter sequence, Sequence measuring joints sequence, the first bar code sequence and the first anchor sequence.In some embodiments, first group of bar coded nucleic acid Chain includes, in 3' to the direction 5': promoter sequence, sequence measuring joints sequence (" sequence measuring joints "), the first bar code sequence and first Anchor sequence.

Promoter sequence is DNA sequence dna, limits polymerase (such as RNA polymerase) and starts open gene or other downstreams The position of nucleotide sequence.The example of promoter sequence includes but is not limited to that T7 promoter sequence, T3 promoter sequence and SP6 are opened Promoter sequences.

Sequence acceptor is added to short (known) nucleotide (such as DNA) sequence of target nucleic acid end.Complementation sequencing is drawn Object is in conjunction with sequence acceptor.The length of sequence acceptor can change.For example, sequence acceptor can have the length of 10 to 50 nucleotide Degree.In some embodiments, sequence acceptor has the length of 10,20,30,40 or 50 nucleotide.

Anchor sequence makes bar coded nucleic acid be bonded to each other.As shown in Fig. 3 (step 2), bar code A has anchor sequence, item Shape code B has the anchor sequence complementary with the anchor sequence of bar code A, and bar code A and B are bonded to each other.

Bar code sequence is nucleotide sequence (for example, deoxyribonucleotide), and group or subset to nucleic acid chains are special Anisotropic.For example, as shown in Figure 1, the nucleic acid chains of subset A1 (bar code A) are encoded with specific bar code sequence, and subset A2, The bar code sequence coding that A3, A4 etc. be each to use by oneself different, each bar code sequence is specific to subset.Similarly, subset B1 (bar shaped Code B) nucleic acid chains particular bar sequential coding, and each different bar code sequences coding of using by oneself such as subset B2, B3, B4, often A bar code is specific to subset.Therefore, each overlapping patch comprising unique group of bar code A subset and bar code B subset It closes, includes unique composite bar code (bar code A+ bar code B).For example, the overlapping patch comprising Al+B1 bar code is relative to it For adjacent overlapping patch by unique encodings, adjacent overlapping patch includes A2+B1 bar code, A1+B2 bar code, A2+B2 bar shaped Code etc., as shown in Figure 1.

In some embodiments, second group of bar coded nucleic acid chains include, in 5' to the direction 3': more T-sequence (examples Such as, T19V), unique molecular marker symbol (UMI) sequence, the second bar code sequence and the second anchor sequence, wherein the second anchor sequence with First anchor sequence is complementary.In some embodiments, second group of bar coded nucleic acid chains include, in 3' to the direction 5': more T sequences Column, unique molecular marker symbol sequence, the second bar code sequence and the second anchor sequence, wherein the second anchor sequence and first anchor sequence It is complementary.Kivioja T et al., natural method Nature Methods 9,72-74 (2012) describes the example of UMI, as ginseng It examines and is incorporated herein.

Method, which may further include, is causing second group of bar coded nucleic acid chains and first group of bar coded nucleic acid chains miscellaneous (incubation) is maintained to be overlapped patch array under conditions of friendship, to generate the patch for including partially double stranded bar coded nucleic acid.Nucleic acid is miscellaneous Friendship condition is known.

Method can also include polymerase, the primer in conjunction with the second bar code sequence and dNTP (for example, dATP, DTTP, dCTP and dGTP) in the presence of, overlapping patch battle array is maintained under conditions of causing DNA to polymerize (generation/synthesis of DNA chain) Column, to generate nucleic acid chains, it includes (for example, in 5' to the direction 3'): promoter sequence, sequence measuring joints sequence, the first bar code Sequence, the first anchor sequence, the second bar code sequence, unique molecular marker symbol sequence and more T-sequences.Nucleic acid synthesizing conditions are known 's.

In some embodiments, method includes that (for example, washing) second group of bar code is removed from overlapping patch array Change nucleic acid.In some embodiments, with sodium hydroxide quenching polymerization/synthetic reaction to remove the bar coded core of shorter second Sour chain.

Surface can be glass surface, silicon face or silica surface.The disclosure covers other surfaces.In some implementations In mode, glass surface is coated with poly-L-Lysine.

In some embodiments, substrate is microwell array or is coupled to microwell array, and wherein each overlapping patch It occupies the single micropore of microwell array or is aligned with the single micropore of microwell array.

In some embodiments, X is equal to 20-20000.For example, X can be equal to 20-50,20-100,20-500,20- 1000、20-5000、20-10000、50-100、50、500、50-1000、50-5000、50-10000、50-20000、100- 500,100-1000,100-5000,100-10000 or 100-20000.In some embodiments, X is equal to 100-20000.

In some embodiments, Y is equal to 20-20000.For example, Y can be equal to 20-50,20-100,20-500,20- 1000、20-5000、20-10000、50-100、50、500、50-1000、50-5000、50-10000、50-20000、100- 500,100-1000,100-5000,100-10000 or 100-20000.In some embodiments, Y is equal to 100-20000.

At least one (for example, at least 2,3,4,5,10,20) column or each (all) rows can have 10-500 microns or 50- 200 microns of width.For example, the width of column can be 10,20,30,40,50,60,70,80,90,100,110,120,130, 140,150,160,170,180,190 or 200 microns.In some embodiments, the width of column is 100 microns.

At least one (for example, at least 2,3,4,5,10,20) column or each (all) rows can have 10-500 microns or 50- 200 microns of width.For example, row can have 10,20,30,40,50,60,70,80,90,100,110,120,130,140, 150,160,170,180,190 or 200 microns of width.In some embodiments, capable width is 100 microns.

In general, at least one or each (all) overlapping patches have 100-40000 μm²Area.For example, overlapping patch Can have 100,200,300,400,500,600,700,800,900,1000,2000,3000,4000,5000,6000, 7000,8000,9000,10000,20000,30000 or 40000 μm²Area.In some embodiments, overlapping patch tool There are 10000 μm²Area.Therefore, in some embodiments, the size of patch can be 10 × 10 μm to 200 × 200 μm. The disclosure covers greater or lesser overlapping patch.

In some embodiments, the overlapping patch in uniline is spaced each other 10-500 microns or 20-200 microns.For example, It is micro- that overlapping patch in uniline can be spaced each other 10,20,30,40,50,60,70,80,90,100,125,150,175 or 200 Rice.In some embodiments, the overlapping patch in uniline is spaced each other 100 microns.

In some embodiments, the overlapping patch in single row is spaced each other 10-500 microns or 20-200 microns.Example Such as, the overlapping patch in single row can be spaced each other 10,20,30,40,50,60,70,80,90,100,125,150,175 or 200 microns.In some embodiments, the overlapping patch in single row is spaced each other 100 microns.

In some embodiments, the overlapping patch between adjacent rows is spaced each other 20-200 microns.For example, adjacent rows it Between overlapping patch can be spaced each other 20,50,75,100,125,150,175 or 200 microns.In some embodiments, phase Overlapping patch between adjacent rows is spaced each other 20-30,20-50,20-100,50-100 or 50-200 microns.In some embodiment party In formula, the overlapping patch between adjacent rows is spaced each other (about) 100 microns.

In some embodiments, the overlapping patch between adjacent column is spaced each other 20-200 microns.For example, adjacent column it Between overlapping patch can be spaced each other 20,50,75,100,125,150,175 or 200 microns.In some embodiments, phase Overlapping patch between adjacent column is spaced each other 20-30,20-50,20-100,50-100 or 50-200 microns.In some embodiment party In formula, the overlapping patch between adjacent column is spaced each other (about) 100 microns.

Such as microfluidic flow patterning chip can be used first group of bar coded nucleic acid chains is patterned and be fixed to (see, for example, Fig. 2) on the surface of substrate.Likewise it is possible to pattern chip for second group of bar shaped using such as microfluidic flow Codeization nucleic acid chains are patterned and are fixed on the surface of substrate (see, for example, Fig. 2).

Bar coded array and multiplex machine

Bar coded array is also provided herein, for example, generating by any method described herein.For example, bar coded Array can be generated by a method, comprising: (a) is by first group of bar coded nucleic acid chains the first solution flow pattern and fixation Onto the surface of substrate, to generate parallel to each other and column that are being spatially separated, wherein each column are bar coded comprising first group The X patch of nucleic acid chains, wherein the patch in each column is spatially separated each other, wherein each column include the difference of bar coded nucleic acid chains Subset, and wherein X is greater than 2 number；(b) by second group of bar coded nucleic acid chains the second solution flow pattern and solid Determine onto substrate surface, to generate row that is parallel to each other and being spatially separated, wherein each row includes second group of bar coded core The Y patch of sour chain, wherein the patch in each row is spatially separated each other, wherein each row includes difference of bar coded nucleic acid chains Collection, wherein row is vertical relative to column, and wherein Y is greater than 2 number, to generate X*Y overlapping patch array, each overlapping Patch includes the subset of the subset of the bar coded nucleic acid chains of first group of (i) and the bar coded nucleic acid chains of second group of (ii).

Multiplex machine is also provided herein comprising be coupled to the bar coded array of microwell array, wherein each Overlapping patch is aligned with the single micropore of array, so that each overlapping patch corresponds to single micropore.

The multiplex machine comprising bar coded array further provided herein, wherein substrate is microwell array, and And wherein the patch of each overlapping occupies the single micropore of microwell array.

In some embodiments, each micropore of microwell array includes to be no more than 5 cells.For example, microwell array Each micropore may include no more than 4, no more than 3 or be no more than 2 cells.In some embodiments, microwell array Each micropore include be no more than 2 cells.In some embodiments, each micropore of micropore contains individual cells.

Microwell array can be located at, for example, between bar coded array and another substrate, so that the micropore of microarray is close It seals (for example, fluid cannot leave or enter micropore).

In some embodiments, another substrate is coated with dry (such as freeze-drying) lysis buffer.

In some embodiments, another substrate includes trapping nucleic acids array, such as microRNA capture array.

In some embodiments, another substrate include antibody capture array (see, e.g., U.S. Patent number 9,188, 586, be incorporated herein by reference).

The disclosure further includes embodiment described in following number paragraph:

1, a kind of method for producing bar coded array, comprising:

(a) by first group of bar coded nucleic acid chains the first solution flow pattern and it is fixed over the substrate surface, to produce Column that are raw parallel to each other and being spatially separated, wherein the X patch of each bar coded nucleic acid chains of the column comprising first group, wherein each column In patch be spatially separated each other, wherein each column include the different subsets of bar coded nucleic acid chains, and wherein, X is greater than 2 Number；

(b) by second group of bar coded nucleic acid chains the second solution flow pattern and be fixed to substrate surface on, to produce Row that is raw parallel to each other and being spatially separated, wherein each row includes the Y patch of second group of bar coded nucleic acid chains, wherein each row In patch be spatially separated each other, wherein each row include bar coded nucleic acid chains different subsets, wherein row and column is vertical, and And wherein, Y is greater than 2 number；

It is each to be overlapped the son that patch includes the bar coded nucleic acid chains of first group of (i) to generate X*Y overlapping patch array Collection, and second group of (ii) bar coded nucleic acid chains subset.

2, the method according to paragraph 1, wherein first group of bar coded nucleic acid chains include, optionally in 5' to 3' Direction: promoter sequence, sequence measuring joints sequence, the first bar code sequence and the first anchor sequence.

3, the method according to paragraph 1 or 2, wherein second group of bar coded nucleic acid chains include, in 5' to the side 3' To: more T-sequences, unique molecular marker symbol sequence, the second bar code sequence and the second anchor sequence, wherein the second anchor sequence and the One anchor sequence is complementary.

It 4, further include leading to second group of bar coded nucleic acid chains and first group according to any method of paragraph 1-3 Overlapping patch array is maintained under conditions of bar coded nucleic acid strand, to generate the spot for including partially double stranded bar coded nucleic acid Piece.

5, the method according to paragraph 4 further includes in polymerase, primer and dNTP in conjunction with the second bar code sequence In the presence of, overlapping patch array is maintained under conditions of causing DNA to polymerize, to generate nucleic acid chains, it includes in 5' to the side 3' To: promoter sequence, sequence measuring joints sequence, the first bar code sequence, the first anchor sequence, the second bar code sequence, unique molecule Identifier nucleotide sequence and more T-sequences.

6, the method according to paragraph 5 further includes removing second group of bar coded nucleic acid from overlapping patch array.

7, according to the method any in paragraph 1-6, wherein surface is glass surface, silicon or silica.

8, the method according to paragraph 7, wherein glass surface is coated with poly-L-Lysine.

9, according to the method any in paragraph 1-6, wherein substrate is microwell array, and each patch that is overlapped occupies micropore battle array The single micropore of column.

10, according to the method any in paragraph 1-9, wherein X is equal to 20-20000 and/or Y and is equal to 20-20000.

11, the method according to paragraph 10, wherein X is equal to 100-20000 and/or Y and is equal to 100-20000.

12, the method according to paragraph 11, wherein X is equal to 1000-20000 and/or Y and is equal to 1000-20000.

13, the method according to paragraph 12, wherein X is equal to 10000-20000 and/or Y and is equal to 10000-20000.

14, according to the method any in paragraph 1-13, wherein the width of each columns and/or rows is 50-200 microns.

15, the method according to paragraph 14, wherein the width of each columns and/or rows is 100 microns.

16, according to the method any in paragraph 1-15, wherein each overlapping patch has 400-40000 μm²Face Product.

17, the method according to paragraph 16, wherein each overlapping patch has 10000 μm²Area.

18, according to the method any in paragraph 1-17, wherein uniline and/or single-row interior overlapping patch are spaced each other 20-200 microns.

19, the method according to paragraph 18, wherein in uniline and/or it is single-row in overlapping patch to be spaced each other 100 micro- Rice.

20, according to the method any in paragraph 1-19, the wherein overlapping spot between adjacent rows and/or between adjacent column Piece is spaced each other 20-200 microns.

21, the method according to paragraph 20, wherein the overlapping patch between adjacent rows and/or between adjacent column is to each other Every 100 microns.

22, according to the method any in paragraph 1-21, wherein using microfluidic flow patterning chip by first and/ Or second group of bar coded nucleic acid chains is patterned and is fixed on the surface of substrate.

23, the bar coded array generated by method, comprising:

(a) by first group of bar coded nucleic acid chains the first solution flow pattern and it is fixed over the substrate surface, to produce Column that are raw parallel to each other and being spatially separated, wherein the X patch of each bar coded nucleic acid chains of the column comprising first group, wherein each column In patch be spatially separated each other, wherein each column include the different subsets of bar coded nucleic acid chains, and wherein, X is greater than 2 Number；

(b) by second group of bar coded nucleic acid chains the second solution flow pattern and it is fixed over the substrate surface, to produce Row that is raw parallel to each other and being spatially separated, wherein each row includes the Y patch of second group of bar coded nucleic acid chains, wherein each row In patch be spatially separated each other, wherein each row include bar coded nucleic acid chains different subsets, wherein row and column is vertical, and And wherein, Y is greater than 2 number；

It is each to be overlapped the subset that patch includes (i) first group of bar coded nucleic acid chains to generate X*Y overlapping patch array, The subset of (ii) second group of bar coded nucleic acid chains.

24, bar coded array according to claim 23 is appointed wherein first group of bar coded nucleic acid chains include Selection of land is in 5' to the direction 3': promoter sequence, sequence measuring joints sequence, the first bar code sequence and the first anchor sequence.

25, the bar coded array according to claim 23 or 24, wherein second group of bar coded nucleic acid chains packet Contain, in 5' to the direction 3': more T-sequences, unique molecular marker symbol sequence, the second bar code sequence and the second anchor sequence, wherein the Two anchor sequences are complementary with the first anchor sequence.

26, the bar coded array according to any one of claim 23-25 further comprises by second group of item Shape code nucleic acid chains and first group of bar coded nucleic acid strand simultaneously generate the patch comprising partially double stranded bar coded nucleic acid.

27, bar coded array as claimed in claim 26 further includes that will be overlapped patch array and polymerase and second The primer and dNTP that bar code sequence combines combine, and generate nucleic acid chains, and it includes on 5' to the direction 3': promoter sequence, Sequence measuring joints sequence, the first bar code sequence, the first anchor sequence, the second bar code sequence, unique molecular marker symbol sequence and more T Sequence.

28, bar coded array as claimed in claim 27 further includes removing second group of item from overlapping patch array Shape code nucleic acid.

29, the bar coded array according to any one of claim 23-28, wherein surface is glass surface, silicon Or silica.

30, bar coded array as claimed in claim 9, wherein glass surface is coated with poly-1-lysine.

31, the bar coded array according to any one of claim 23-30, further includes being applied to microwell array The surface of substrate is with generation device, wherein each overlapping patch, each row or overlapping patch, or each column overlapping patch occupy it is single micro- The single micropore of hole array.

32, the bar coded array as described in any one of claim 23-31, wherein X is equal to 20-20000 and/or Y It is equal to 100-20000 and/or Y equal to 20-20000, X and is equal to 100-20000, X is equal to 1000-20000 and/or Y and is equal to 1000- 20000 or X is equal to 10000-20000 and/or Y and is equal to 10000-20000.

33, the bar coded array according to any one of claim 23-32, wherein the width of each columns and/or rows Width for 50-200 microns or each columns and/or rows is 100 microns.

34, the bar coded array according to any one of claim 23-33, wherein each overlapping patch has 400-40000μm²Area or each overlapping patch have 10000 μm²Area.

35, the bar coded array according to any one of claim 23-34, it is wherein in uniline and/or single-row interior Overlapping patch be spaced each other in 20-200 microns or uniline and/or be single-row in overlapping patch be spaced each other 100 microns.

36, the bar coded array according to any one of claim 23-35, wherein between adjacent rows and/or phase Overlapping patch between adjacent column be spaced each other the overlapping patch between 20-200 microns or adjacent rows and/or between adjacent column that 100 microns of this interval.

37, the bar coded array according to any one of claim 23-36, wherein using microfluidic flow pattern Change chip first and/or second group of bar coded nucleic acid chains is patterned and be fixed on the surface of substrate.

38, a kind of multiplex machine, including according to the bar coded array any in paragraph 23-37, coupling To microwell array, wherein each overlapping patch is aligned with the single micropore of array, so that each overlapping patch is corresponding to single micro- Hole.

39, a kind of multiplex machine, including according to the bar coded array any in paragraph 23-37, wherein base Piece is microwell array, and wherein each overlapping patch occupies the single micropore of microwell array.

40, the multiplex machine according to paragraph 38 or 39, wherein each micropore of microwell array includes to be no more than 5 A cell.

41, the multiplex machine according to paragraph 40, wherein each micropore of microwell array includes to be no more than 2 carefully Born of the same parents.

42, the multiplex machine according to paragraph 41, wherein each micropore of microwell array includes individual cells.

43, according to the multiplex machine any in paragraph 23-42, wherein microwell array is located at bar coded array Between another substrate, so that the micropore of microarray is sealed.

44, the multiplex machine according to paragraph 43, wherein another substrate is coated with the lysis buffer of freeze-drying.

45, the multiplex machine according to paragraph 43 or 44, wherein another substrate includes trapping nucleic acids array.

46, the multiplex machine according to paragraph 45, wherein trapping nucleic acids array is microRNA capture array.

47, the multiplex machine according to paragraph 45, wherein another substrate includes antibody capture array.