CN110438572A - The banking process of unicellular sequencing - Google Patents

The banking process of unicellular sequencing Download PDF

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CN110438572A
CN110438572A CN201910868822.4A CN201910868822A CN110438572A CN 110438572 A CN110438572 A CN 110438572A CN 201910868822 A CN201910868822 A CN 201910868822A CN 110438572 A CN110438572 A CN 110438572A
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round
barcode
dna
artificial sequence
primer
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孔培涛
李奇
陈亚庆
杨圳
蔡建飞
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SHANGHAI GENMINIX INFORMATICS CO Ltd
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    • C12Q1/6869—Methods for sequencing
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    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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Abstract

The invention discloses a kind of banking process of unicellular sequencing comprising the fixed cell of (a) PARA FORMALDEHYDE PRILLS(91,95) carries out intracellular reversion record mark first round bar code (barcode) using the first reverse transcription primer；(b) using connection reaction, the second wheel primer and the wheel of third round primer mark second and third round barcode are used；(c) it is cracked to form sublibrary, after template switch, PCR is utilized to be enriched with template；(d) fourth round barcode is marked using index PCR.Banking process of the invention, as experimental material, is not limited solely to fresh sample, allows the complexity of sample source, can more effectively utilize sample using fixed cell or nucleus.

Description

The banking process of unicellular sequencing
Technical field
The present invention relates to field of biotechnology, and in particular to unicellular sequencing field is particularly related to a kind of based on cell The unicellular method for building library is carried out after fixation.
Background technique
In past 10 years, as (next generation sequencing, NGS) technology and third is sequenced in the second generation The rapid development of generation sequencing (third generation sequencing, TGS) technology, causes the huge of life science It changes.Previous research needs to obtain enough nucleic acid from a large amount of cells and is sequenced, therefore sequencing result often indicates Be cell colony characterization, and the exclusive cell characteristics of individual cells are often ignored.In order to solve above-mentioned confinement problems, Unicellular sequencing technologies come into being.
Unicellular sequencing achieves achievement abundant in fields such as tumour, Developmental Biology, Neuscience.And it is slender The research of born of the same parents originally more can rapidly expand scientific payoffs, but unicellular sequencing technologies are there is also many problems, than if you need to Using fresh cell, sample utilisation is not high, expensive equipment and related reagent etc., this research to unicellular sequencing technologies Bring many inconvenience with popularization, and also have for the extensive development of unicellular life science it is many unfavorable.Therefore optimize The new unicellular sequencing technologies of exploitation, just seem very urgent.
Summary of the invention
Technical purpose of the invention is to provide a kind of banking process of unicellular sequencing, it can overcome existing unicellular Disadvantage present in sequencing technologies, e.g., be limited to fresh cell, need the pertinent instruments, the cell capture rate that customize low with And sample treatment is complicated for operation, which can simplify sample treatment, improve cell utilization rate, and complicated sample is allowed Source, and finally make unicellular transcript profile sequencing popular.
The present invention provides a kind of banking process of unicellular sequencing comprising following steps:
(a) the fixed cell of PARA FORMALDEHYDE PRILLS(91,95) carries out the intracellular reversion record mark first round (Round using the first reverse transcription primer 1) bar code (barcode)；
(b) using connection reaction, (Round 2), third round are taken turns using the second wheel primer and third round primer mark second (Round 3)barcode；
(c) lytic cell generates sublibrary, after template switch, PCR is utilized to be enriched with template；
(d) fourth round barcode is marked using index PCR.
In a specific embodiment, the cell in step (a) can be multiple cells, include described in multiple cells The cell of different types of cell or different tissue sources.
The present invention gives each cell to tag at random using more wheel combination barcode, when the number of combinations of label is far longer than When cell number, each cell can be preferably distinguished.
In a specific embodiment, in step (a), first reverse transcription primer successively draws including 5 ' by 5 ' to 3 ' Object synthesizes phosphorylation label, for the fixed structure of the linker sequence with the second wheel connection, for distinguishing the work of cell origin Poly (A) tail for the varistructure of barcode sequence, and for mRNA is specifically bound to guarantee reverse transcription The fixed structure of specificity.About each part mentioned above structure particular sequence and length range, those skilled in the art can basis Actual needs is designed.For example, the fixed structure for the linker sequence with the second wheel connection can be AGGCCAGAGCATTCG, the varistructure as barcode sequence for distinguishing cell origin can be AACGTGAT, be used for Being specifically bound with poly (A) tail of mRNA can be with the fixed structure for guaranteeing the specificity of reverse transcription TTTTTTTTTTTTTTTVN。
In a specific embodiment, in step (b), the second wheel primer successively includes that 5 ' primers close by 5 ' to 3 ' It is marked at phosphorylation, the fixed structure of the linker sequence for being connected with third round, for distinguishing the conduct of cell origin The varistructure of barcode sequence, and the fixed structure of the linker sequence for being connected with the first round.Wherein, described The varistructure as barcode sequence in two wheel primers respectively correspond tos the conduct in first reverse transcription primer The varistructure of barcode sequence, for example, the two is identical.About each part mentioned above structure particular sequence and length range, ability Field technique personnel can be designed according to actual needs.For example, the fixed knot of the linker sequence for being connected with third round Structure can be CATCGGCGTACGACT, and the varistructure as barcode sequence for distinguishing cell origin can be AACGTGAT, and the fixed structure of the linker sequence for connecting with the first round can be ATCCACGTGCTTGAG.
In a specific embodiment, in step (b), the third round primer successively includes that 5 ' primers close by 5 ' to 3 ' It is marked at phosphorylation, plus the fixed structure of fourth round while for index PCR reaction, for marking intracellular molecules Fixed structure, for marking the varistructure as barcode sequence of cell, and the linker for connecting with the second wheel The fixed structure of sequence.Wherein, the varistructure as barcode sequence in the third round primer respectively correspond tos described Barcode sequence is used as in the varistructure and first reverse transcription primer as barcode sequence in second wheel primer The varistructure of column, for example, three is identical.About each part mentioned above structure particular sequence and length range, those skilled in the art Member can be designed according to actual needs.For example, fixed structure while for index PCR reaction plus fourth round can It can be the UMI sequence that length is 10bp for marking the fixed structure of intracellular molecules for CAGACGTGTGCTCTTCCGATCT (NNNNNNNNNN), for mark cell the varistructure as barcode sequence can for AACGTGAT, and for and the The fixed structure of the linker sequence of two wheel connections can be GTGGCCGATGTTTCG.
In a specific embodiment, in step (d), a P5 and P7 (P7-1~P7-8) group become pair of primers, It is adapted to the sequencing of Illumina microarray dataset, the library built is sequenced.Wherein, P5 and P7 is Illumina microarray dataset Use conventional tags primer.For example, P5 sequence are as follows: AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGC AGCGTCAGATGTGTATAAGAGACAG；P7-1 sequence is CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAG TTCAGACGTGTGCTCTTCCGATCT。
Beneficial effect
Compared with unicellular sequencing technologies compare, ICB-scSeq (Intelligent of the invention Combinatorial Barcoding-single cell Sequencing) unicellular sequencing technologies, using fixed cell Or nucleus is not limited solely to fresh sample as experimental material, allows complexity (including the cell category of sample source Complexity and tissue-derived complexity), can more efficiently utilize sample.ICB-scSeq of the invention is unicellular Sequencing technologies will realize the breakthrough of unicellular sequencing technologies, which is not necessarily to expensive customization microfluid and micropore cell Screening installation can carry out transcript profile sequencing to individual cells with cheap cost, be expected to really realize that unicellular transcript profile is surveyed Sequence it is popular, be conducive to the extensive development of unicellular life science.
Specific embodiment
Hereinafter, by specific embodiment, the present invention is described in detail, so that those skilled in the art is more preferable Ground understands the present invention, however, the range that these specific embodiments are not intended to restrict the invention, but, the scope of the present invention It should include on the basis of the present invention, those skilled in the art can understand that or being easy the following specific embodiments made Various equivalent or variations.Technical term used herein has normally understood meaning in this field.
Embodiment
It should be noted that library construction hereinafter is carried out based on the cell after being fixed with formaldehyde.
It is as shown in Table 1 below using reagent, instrument and material in the present invention.
Table 1
The present invention illustrates unicellular sequencing library construction method of the invention by following steps.
(I) intracellular reversion record mark first round barcode (SEQ ID NO:1-96) is utilized
1. solution is formulated as follows, and the solution is assigned in 48 holes:
The solution composition is as follows:
5 × RT buffer --- --- --- --- --- --- --- --- --- --- --- --- -- 4 μ L
Water without RNase --- --- --- --- --- --- --- --- --- --- --- --- 2.625 μ L
RNase inhibitor --- --- --- --- --- --- --- --- --- --- --- --- -- 0.125 μ L
SuperaseIn RNase inhibitor --- --- --- --- --- --- --- --- --- 0.25 μ L
10mM dNTPs---------------------------------------1μL
25μM poly(dT)15Barcoded RT primer (SEQ ID NO:1-96) --- -2 μ L
Maxima H Minus reverse transcriptase --- --- --- --- --- --- --- --- --- 2 μ L
Total volume --- --- --- --- --- --- --- --- --- --- --- --- --- --- -12 μ L
2. by mouse embryonic fibroblasts (1X10 of the 10 μ L formaldehyde after fixed6/ mL) it is resuspended into the solution of 384 μ L, Then it is dispersed in 48 hole, every 8 μ L of hole.
3. carrying out RT-PCR:
50 DEG C, 10 minutes
3 circulation: 8 DEG C 12 seconds, 15 DEG C 45 seconds, 20 DEG C 45 seconds, 30 DEG C 30 seconds, 42 DEG C 2 minutes, 50 DEG C 3 minutes,
50 DEG C, 5 minutes.
4. RT-PCR product is recycled to same 15mL Fu Erken pipe (falcon tube).
5. 9.6 μ L 10%Triton X-100 are added, at 500g, 4 DEG C of centrifugation 3min abandon supernatant.
6. cell is resuspended with 1 × NEB of 2mL buffer 3.1 containing 20 μ L Enzymatics RNase inhibitor.
With a first reverse transcription primer sequence in Round 1:
For "/5Phos/AGGCCAGAGCATTCGAACGTGATTTTTTTTTTTTTTTTVN ", illustrate anti-in Round 1 The construction of transcription primers:
/ 5Phos/: fixed structure, primer synthesize phosphorylation label
AGGCCAGAGCATTCG: fixed structure, linker sequence with Round2 for connecting
AACGTGAT: varistructure, 1 sequence of barcode, length 8bp, for distinguishing cell origin
TTTTTTTTTTTTTTTVN: fixed structure is specifically bound for poly (A) tail with mRNA, is guaranteed anti- The specificity of transcription.
(II) prepare oligonucleotides connection
The connection for carrying out second (Round 2) and third round (Round 3) barcode is reacted, and each round is with different 96 Orifice plate.Ligation rounds has a general connection chain and the second chain part complementary.
1. the second 96 orifice plates of wheel:
Second wheel connection chain (SEQ ID NO:289) --- --- --- --- --- --- --- -11 μM of --- ---
Second wheel Barcodes (SEQ ID NO:97-192) --- --- --- --- --- --- 12 μM of --- --
2. 96 orifice plate of third round:
Third round connection chain (SEQ ID NO:290) --- --- --- --- --- --- --- -13 μM of --- ---
Third round Barcodes (SEQ ID NO:193-288) --- --- --- --- --- -14 μM of --- ---
Connection chain is connected with Round 2 with the primer of 3Barcodes, annealing.95 DEG C, 2min, cool to 20 DEG C, per second- 0.1℃。
Second wheel dropping chain rupture R2 (SEQ ID NO:291) --- --- --- --- --- --- --- 26.4 μM of --- --
Third round blocks chain R3 (SEQ ID NO:292) --- --- --- --- --- --- --- 30.8 μM of --- --
Second wheel dropping chain rupture blocks chain 150mM EDTA molten with 2.5 × T4 DNA ligase buffer (NEB) third round Liquid (terminates connection enzymatic activity)
Block chain at 37 DEG C, under conditions of 50rpm and cell is incubated with 30min.
(III) intracellular the second wheel of connection and third round barcode
1. it is as follows to prepare ligation mix:
Water without RNase --- --- --- --- --- --- --- --- --- 1,287.5 μ L
10 × T4 Ligase buffer --- --- --- --- --- --- --- 500 μ L
T4 DNA ligase --- --- --- --- --- --- --- --- --- -100 μ L
RNase inhibitor --- --- --- --- --- --- --- --- --- -- 40 μ L
SuperaseIn RNase inhibitor --- --- --- --- --- --- 12.5 μ L
5%Triton-X100--------------------------100 μ L
Total volume --- --- --- --- --- --- --- --- --- --- --- -2040 μ L
2. the cell in 2.04mL ligation mix and (I) above in 1 × NEB of 2mL buffer 3.1 is mixed into The connection cell mixing of 4.04mL.
3. 40 μ L connection cell mixings are added separately in 96 orifice plate of the second wheel barcoding (in each hole with the volley of rifle fire Include 10 μ L DNA barcodes).
4. 37 DEG C, 50rpm is incubated for 30min hybridization connection.
5. 10 μ L the second wheel dropping chain ruptures are separately added into 96 orifice plates.
6. 37 DEG C, 50rpm is incubated for 30min, extra connector is blocked to combine.
7. cell in 96 holes is merged, 40 μm of filter filterings, the merging cell after collection is added 100 μ L T4 DNA and connects Connect enzyme.
8. 50 μ L merging cell is separately added into 96 orifice plate of third round barcoding (comprising 10 μ L DNA in each hole Barcodes the) (total volume in each hole to 60 μ L).
9. 37 DEG C, 30min being incubated under 50rpm and carries out hybridization connection.
10. 10 μ L third rounds blocking chain is separately added into 96 orifice plates.
11. 37 DEG C, 50rpm is incubated for 30min, extra connector is blocked to combine.
12. 96 hole cells are merged, 40 μm of filters are collected by filtration.
With 2 primer First ray of Round: "/5Phos/CATCGGCGTACGACTAACGTGATATCCACGTGCTT For GAG ", illustrate that 2 primer of Round is constructed:
/ 5Phos/: fixed structure, primer synthesize phosphorylation label
CATCGGCGTACGACT: fixed structure, linker sequence, length 15bp are used for and the connection of Round 3
AACGTGAT: varistructure, 2 sequence of barcode, length 8bp, for distinguishing cell origin
ATCCACGTGCTTGAG: fixed structure, linker sequence, length 15bp are used for and the connection of Round 1.
With 3 primer First ray of Round:
"/5Biosg/CAGACGTGTGCTCTTCCGATCTNNNNNNNNNNAACGTGA TGTGGCCGATGTTTCG " is Example illustrates that 3 primer of Round is constructed:
/ 5Biosg/: fixed structure, primer synthesize phosphorylation label
CAGACGTGTGCTCTTCCGATCT: fixed structure, length 22bp, for being added while index PCR reaction Round 4
NNNNNNNNNN: fixed structure, length is the UMI sequence of 10bp, for marking intracellular molecules
AACGTGAT: varistructure, length is the barcode3 sequence of 8bp, for marking cell
GTGGCCGATGTTTCG: fixed structure, linker sequence, length 15bp are used for and the connection of Round 2.
(IV) cracking and sublibrary generate.
1. 70 μ L 10%Triton-X100 are added in the merging cell in (III), at 1000g, 4 DEG C of centrifugations 5min。
2. carefully absorbing supernatant, about 30 μ L liquid are stayed to avoid precipitating is drawn onto.
3. cell is resuspended with 4mL cleaning buffer solution.
Cleaning buffer solution: (4mL of 1 × PBS, 40 μ L of 10%Triton X-100 and 10 μ L of SUPERase In RNase inhibitor)
4. at 1000g, 4 DEG C of centrifugation 5min (spin down).
5. absorbing supernatant, cell is resuspended with 50 μ L PBS-RI.
6. a pair cell counts, divide exactly respectively cell to sublibrary in 1.7ml centrifuge tube.
System is complemented into 50 μ L 7. 1 × PBS is added.
8. cell cracking: (50 2 × lysis buffers of μ L and 10 μ L Proteinase Ks are added)
2 × lysis buffer (20mM Tris (pH 8.0), 400mM NaCl, 100mM EDTA
(pH 8.0) and 4.4%SDS) --- --- --- --- --- --- --- --- --- --- --- --- --- -50 μ L
Proteinase K Solution (20mg/mL) --- --- --- --- --- --- --- --- --- --- --- --- --- -10 μ L
9. under 200rpm, 55 DEG C of incubation 2h reverse formaldehyde crosslinking.
10. in -80 DEG C of preservation lysates.
(V) cDNA is purified
1. each library prepares 40 μ L Dynabeads MyOne Streptavidin C1 magnetic beads.
2. cleaning magnetic bead three times with 800 μ 1 × B&W of L buffers (containing 0.05%Tween-20).
3. being resuspended with 100 μ 2 × B&W of L buffers (containing 2 μ L of SUPERase In Rnase inhibitor).
4. 5 μ L, 100 μM of PMSF are added in each sublibrary (lysate after defrosting).
5. incubation at room temperature 10 minutes, protease inhibition K activity.
6. each library is added 100 μ L and magnetic bead is resuspended.
7. on constant temperature blending instrument, room temperature mixes 60min.(being stirred on micro-pipe foam inserted sheet)
8. removing, cleaned once with 800 μ 1 × B&W of L buffers (containing 0.05%Tween-20).
9. room temperature mixes 5 minutes.
10. being cleaned once with 800 μ L 10mM Tris (containing 0.1%Tween-20) again.
11. room temperature mixes 5 minutes.
(VI) template switch
The streptavidin magnesphere that constraint obtained in (V) has cDNA molecule is resuspended with following solution system (Streptavidin beads)
1. system is as follows:
5 × Maxima RT buffer --- --- --- --- --- --- --- --- --- --- -- 44 μ L
20%Ficoll PM-400 solution --- --- --- --- --- --- --- --- --- -- 44 μ L
10mM dNTPs----------------------------------------22μL
RNase inhibitor --- --- --- --- --- --- --- --- --- --- --- --- --- 5.5 μ L
Maxima H Minus reverse transcriptase --- --- --- --- --- --- --- --- --- -11 μ L
100 μM of template switch primer (SEQ ID NO:293 (AAGCAGTGGTATCAACGCAGAGTGAATrGrG+ G))-------------------------------------------------------5.5μL
Total volume --- --- --- --- --- --- --- --- --- --- --- --- --- --- -- 132 μ L
Note: template switch primer contains in prime end there are two ribonucleic acid guanine, the nucleic acid bird being then locked out Purine.
2. being incubated at room temperature 30min.
3.42 DEG C of mild shake are incubated for 90min.
(VII) template is enriched with using PCR:
1. template 10mM Tris and the 0.1%Tween-20 solution and water that obtain in (VI) are respectively washed once.
2. magnetic bead is resuspended, agents useful for same system is as follows:
2×Kapa HiFi HotStart Master Mix--------------------110μL
10μM stocks of primers PCR primer F(SEQ ID NO:294)--8.8μL
10μM stocks of primers PCR primer R(SEQ ID NO:295)--8.8μL
Water --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -- 92.4 μ L
Total volume --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -220 μ L
3.PCR program is as follows:
95 DEG C of 3min,
5 circulation: 98 DEG C 20 seconds, 65 DEG C 45 seconds, 72 DEG C 3 minutes,
4. removing immunomagnetic beads from PCR solution, 1 × EvaGreen dye liquor is added and carries out qPCR.
5.qPCR response procedures:
95 DEG C 3 minutes,
Circulation: 98 DEG C of (once qPCR signal starts to stablize, then stopping reacting) 20 seconds, 65 DEG C 20 seconds, 72 DEG C 3 minutes,
72 DEG C 5 minutes.
6. after reaching plateau, stopping reaction.
(VIII) labeling
1. by the 0.8 × SPRI magnetic beads for purifying of the PCR product in (VII), Qubit is quantitative.
2. cDNA is diluted to 600pg/5 μ L.
3. 10 μ L TD buffers are added, 5 μ L expand subtab enzyme (Nextera XT DNA Library Preparation Kit), piping and druming mixes.
It is mixed 4. being blown and beaten with rifle, 55 DEG C of incubation 5min.
5. 5 μ L neutralization buffers are added, piping and druming is mixed, and is incubated at room temperature 5min.
6.Index amplification system is as follows:
Nextera PCR mix----------------------15μL
P5(10μM)(SEQ ID NO:296)--------------1μL
P7(10μM)(SEQ ID NO:297-304)----------1μL
Water --- --- --- --- --- --- --- --- --- --- --- -- 8 μ L
Total volume --- --- --- --- --- --- --- --- --- --- -25 μ L
System is merged, 50 μ L of final volume.
7.PCR response procedures:
95 DEG C 30 seconds,
12 circulation: 95 DEG C 10 seconds, 55 DEG C 30 seconds, 72 DEG C 30 seconds,
72 DEG C 5 minutes.
8. 40 μ L PCR product 0.7 × SPRI magnetic beads for purifying are removed, to generate the compatible sequencing library of Illumina.
In Round 4, a P5 and P7 (P7-1~P7-8) group become pair of primers, are adapted to Illumina microarray dataset Sequencing.
With P7-1 " CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCT TCCGA For TCT ", illustrate P7 structure.
CAAGCAGAAGACGGCATACGAGAT: fixed structure
GATCTG: varistructure, reverse complementary sequence index
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT: fixed structure
P5 sequence AATGATACGGCGACCACCGAGATCTACACTAGATCGCTCGTCGGCAGCGTCAGATG TGTATA AGAGACAG is fixed structure.
(IX) library built is sequenced.
Above oligonucleotides used, Barcodes sequence are see sequence table.
SEQUENCE LISTING
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<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 22
<400> 22
aggccagagc attcgacgta tcattttttt tttttttt 38
<210> 23
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 23
<400> 23
aggccagagc attcgactat gcattttttt tttttttt 38
<210> 24
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 24
<400> 24
aggccagagc attcgagagt caattttttt tttttttt 38
<210> 25
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 25
<400> 25
aggccagagc attcgagatc gcattttttt tttttttt 38
<210> 26
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 26
<400> 26
aggccagagc attcgagcag gaattttttt tttttttt 38
<210> 27
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 27
<400> 27
aggccagagc attcgagtca ctattttttt tttttttt 38
<210> 28
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 28
<400> 28
aggccagagc attcgatcct gtattttttt tttttttt 38
<210> 29
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 29
<400> 29
aggccagagc attcgattga ggattttttt tttttttt 38
<210> 30
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 30
<400> 30
aggccagagc attcgcaacc acattttttt tttttttt 38
<210> 31
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 31
<400> 31
aggccagagc attcggacta gtattttttt tttttttt 38
<210> 32
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 32
<400> 32
aggccagagc attcgcaatg gaattttttt tttttttt 38
<210> 33
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 33
<400> 33
aggccagagc attcgcactt cgattttttt tttttttt 38
<210> 34
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 34
<400> 34
aggccagagc attcgcagcg ttattttttt tttttttt 38
<210> 35
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 35
<400> 35
aggccagagc attcgcatac caattttttt tttttttt 38
<210> 36
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 36
<400> 36
aggccagagc attcgccagt tcattttttt tttttttt 38
<210> 37
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 37
<400> 37
aggccagagc attcgccgaa gtattttttt tttttttt 38
<210> 38
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 38
<400> 38
aggccagagc attcgccgtg agattttttt tttttttt 38
<210> 39
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 39
<400> 39
aggccagagc attcgcctcc tgattttttt tttttttt 38
<210> 40
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 40
<400> 40
aggccagagc attcgcgaac ttattttttt tttttttt 38
<210> 41
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 41
<400> 41
aggccagagc attcgcgact ggattttttt tttttttt 38
<210> 42
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 42
<400> 42
aggccagagc attcgcgcat acattttttt tttttttt 38
<210> 43
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 43
<400> 43
aggccagagc attcgctcaa tgattttttt tttttttt 38
<210> 44
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 44
<400> 44
aggccagagc attcgctgag ccattttttt tttttttt 38
<210> 45
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 45
<400> 45
aggccagagc attcgctggc atattttttt tttttttt 38
<210> 46
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 46
<400> 46
aggccagagc attcggaatc tgattttttt tttttttt 38
<210> 47
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 47
<400> 47
aggccagagc attcgcaaga ctattttttt tttttttt 38
<210> 48
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 48
<400> 48
aggccagagc attcggagct gaattttttt tttttttt 38
<210> 49
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 49
<400> 49
aggccagagc attcggatag acattttttt tttttttt 38
<210> 50
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 50
<400> 50
aggccagagc attcggccac atattttttt tttttttt 38
<210> 51
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 51
<400> 51
aggccagagc attcggcgag taattttttt tttttttt 38
<210> 52
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 52
<400> 52
aggccagagc attcggctaa cgattttttt tttttttt 38
<210> 53
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 53
<400> 53
aggccagagc attcggctcg gtattttttt tttttttt 38
<210> 54
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 54
<400> 54
aggccagagc attcgggaga acattttttt tttttttt 38
<210> 55
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 55
<400> 55
aggccagagc attcgggtgc gaattttttt tttttttt 38
<210> 56
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 56
<400> 56
aggccagagc attcggtacg caattttttt tttttttt 38
<210> 57
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 57
<400> 57
aggccagagc attcggtcgt agattttttt tttttttt 38
<210> 58
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 58
<400> 58
aggccagagc attcggtctg tcattttttt tttttttt 38
<210> 59
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 59
<400> 59
aggccagagc attcggtgtt ctattttttt tttttttt 38
<210> 60
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 60
<400> 60
aggccagagc attcgtagga tgattttttt tttttttt 38
<210> 61
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 61
<400> 61
aggccagagc attcgtatca gcattttttt tttttttt 38
<210> 62
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 62
<400> 62
aggccagagc attcgtccgt ctattttttt tttttttt 38
<210> 63
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 63
<400> 63
aggccagagc attcgtcttc acattttttt tttttttt 38
<210> 64
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 64
<400> 64
aggccagagc attcgtgaag agattttttt tttttttt 38
<210> 65
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 65
<400> 65
aggccagagc attcgtggaa caattttttt tttttttt 38
<210> 66
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 66
<400> 66
aggccagagc attcgtggct tcattttttt tttttttt 38
<210> 67
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 67
<400> 67
aggccagagc attcgtggtg gtattttttt tttttttt 38
<210> 68
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 68
<400> 68
aggccagagc attcgttcac gcattttttt tttttttt 38
<210> 69
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 69
<400> 69
aggccagagc attcgaactc accttttttt tttttttt 38
<210> 70
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 70
<400> 70
aggccagagc attcgaagag atcttttttt tttttttt 38
<210> 71
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 71
<400> 71
aggccagagc attcgaagga cacttttttt tttttttt 38
<210> 72
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 72
<400> 72
aggccagagc attcgaatcc gtcttttttt tttttttt 38
<210> 73
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 73
<400> 73
aggccagagc attcgaatgt tgcttttttt tttttttt 38
<210> 74
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 74
<400> 74
aggccagagc attcgacacg accttttttt tttttttt 38
<210> 75
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 75
<400> 75
aggccagagc attcgacaga ttcttttttt tttttttt 38
<210> 76
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 76
<400> 76
aggccagagc attcgagatg tacttttttt tttttttt 38
<210> 77
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 77
<400> 77
aggccagagc attcgagcac ctcttttttt tttttttt 38
<210> 78
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 78
<400> 78
aggccagagc attcgagcca tgcttttttt tttttttt 38
<210> 79
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 79
<400> 79
aggccagagc attcgaggct aacttttttt tttttttt 38
<210> 80
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 80
<400> 80
aggccagagc attcgatagc gacttttttt tttttttt 38
<210> 81
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 81
<400> 81
aggccagagc attcgatcat tccttttttt tttttttt 38
<210> 82
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 82
<400> 82
aggccagagc attcgattgg ctcttttttt tttttttt 38
<210> 83
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 83
<400> 83
aggccagagc attcgcaagg agcttttttt tttttttt 38
<210> 84
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 84
<400> 84
aggccagagc attcgcacct tacttttttt tttttttt 38
<210> 85
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 85
<400> 85
aggccagagc attcgccatc ctcttttttt tttttttt 38
<210> 86
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 86
<400> 86
aggccagagc attcgccgac aacttttttt tttttttt 38
<210> 87
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 87
<400> 87
aggccagagc attcgcctaa tccttttttt tttttttt 38
<210> 88
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 88
<400> 88
aggccagagc attcgcctct atcttttttt tttttttt 38
<210> 89
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 89
<400> 89
aggccagagc attcgcgaca cacttttttt tttttttt 38
<210> 90
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 90
<400> 90
aggccagagc attcgcggat tgcttttttt tttttttt 38
<210> 91
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 91
<400> 91
aggccagagc attcgctaag gtcttttttt tttttttt 38
<210> 92
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 92
<400> 92
aggccagagc attcggaaca ggcttttttt tttttttt 38
<210> 93
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 93
<400> 93
aggccagagc attcggacag tgcttttttt tttttttt 38
<210> 94
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 94
<400> 94
aggccagagc attcggagtt agcttttttt tttttttt 38
<210> 95
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 95
<400> 95
aggccagagc attcggatga atcttttttt tttttttt 38
<210> 96
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 1 Barcode 96
<400> 96
aggccagagc attcggccaa gacttttttt tttttttt 38
<210> 97
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 1
<400> 97
catcggcgta cgactaacgt gatatccacg tgcttgag 38
<210> 98
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 2
<400> 98
catcggcgta cgactaaaca tcgatccacg tgcttgag 38
<210> 99
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 3
<400> 99
catcggcgta cgactatgcc taaatccacg tgcttgag 38
<210> 100
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 4
<400> 100
catcggcgta cgactagtgg tcaatccacg tgcttgag 38
<210> 101
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 5
<400> 101
catcggcgta cgactaccac tgtatccacg tgcttgag 38
<210> 102
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 6
<400> 102
catcggcgta cgactacatt ggcatccacg tgcttgag 38
<210> 103
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 7
<400> 103
catcggcgta cgactcagat ctgatccacg tgcttgag 38
<210> 104
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 8
<400> 104
catcggcgta cgactcatca agtatccacg tgcttgag 38
<210> 105
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 9
<400> 105
catcggcgta cgactcgctg atcatccacg tgcttgag 38
<210> 106
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 10
<400> 106
catcggcgta cgactacaag ctaatccacg tgcttgag 38
<210> 107
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 11
<400> 107
catcggcgta cgactctgta gccatccacg tgcttgag 38
<210> 108
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 12
<400> 108
catcggcgta cgactagtac aagatccacg tgcttgag 38
<210> 109
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 13
<400> 109
catcggcgta cgactaacaa ccaatccacg tgcttgag 38
<210> 110
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 14
<400> 110
catcggcgta cgactaaccg agaatccacg tgcttgag 38
<210> 111
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 15
<400> 111
catcggcgta cgactaacgc ttaatccacg tgcttgag 38
<210> 112
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 16
<400> 112
catcggcgta cgactaagac ggaatccacg tgcttgag 38
<210> 113
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 17
<400> 113
catcggcgta cgactaaggt acaatccacg tgcttgag 38
<210> 114
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 18
<400> 114
catcggcgta cgactacaca gaaatccacg tgcttgag 38
<210> 115
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 19
<400> 115
catcggcgta cgactacagc agaatccacg tgcttgag 38
<210> 116
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 20
<400> 116
catcggcgta cgactacctc caaatccacg tgcttgag 38
<210> 117
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 21
<400> 117
catcggcgta cgactacgct cgaatccacg tgcttgag 38
<210> 118
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 22
<400> 118
catcggcgta cgactacgta tcaatccacg tgcttgag 38
<210> 119
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 23
<400> 119
catcggcgta cgactactat gcaatccacg tgcttgag 38
<210> 120
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 24
<400> 120
catcggcgta cgactagagt caaatccacg tgcttgag 38
<210> 121
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 25
<400> 121
catcggcgta cgactagatc gcaatccacg tgcttgag 38
<210> 122
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 26
<400> 122
catcggcgta cgactagcag gaaatccacg tgcttgag 38
<210> 123
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 27
<400> 123
catcggcgta cgactagtca ctaatccacg tgcttgag 38
<210> 124
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 28
<400> 124
catcggcgta cgactatcct gtaatccacg tgcttgag 38
<210> 125
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 29
<400> 125
catcggcgta cgactattga ggaatccacg tgcttgag 38
<210> 126
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 30
<400> 126
catcggcgta cgactcaacc acaatccacg tgcttgag 38
<210> 127
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 31
<400> 127
catcggcgta cgactgacta gtaatccacg tgcttgag 38
<210> 128
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 32
<400> 128
catcggcgta cgactcaatg gaaatccacg tgcttgag 38
<210> 129
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 33
<400> 129
catcggcgta cgactcactt cgaatccacg tgcttgag 38
<210> 130
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 34
<400> 130
catcggcgta cgactcagcg ttaatccacg tgcttgag 38
<210> 131
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 35
<400> 131
catcggcgta cgactcatac caaatccacg tgcttgag 38
<210> 132
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 36
<400> 132
catcggcgta cgactccagt tcaatccacg tgcttgag 38
<210> 133
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 37
<400> 133
catcggcgta cgactccgaa gtaatccacg tgcttgag 38
<210> 134
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 38
<400> 134
catcggcgta cgactccgtg agaatccacg tgcttgag 38
<210> 135
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 39
<400> 135
catcggcgta cgactcctcc tgaatccacg tgcttgag 38
<210> 136
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 40
<400> 136
catcggcgta cgactcgaac ttaatccacg tgcttgag 38
<210> 137
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 41
<400> 137
catcggcgta cgactcgact ggaatccacg tgcttgag 38
<210> 138
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 42
<400> 138
catcggcgta cgactcgcat acaatccacg tgcttgag 38
<210> 139
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 43
<400> 139
catcggcgta cgactctcaa tgaatccacg tgcttgag 38
<210> 140
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 44
<400> 140
catcggcgta cgactctgag ccaatccacg tgcttgag 38
<210> 141
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 45
<400> 141
catcggcgta cgactctggc ataatccacg tgcttgag 38
<210> 142
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 46
<400> 142
catcggcgta cgactgaatc tgaatccacg tgcttgag 38
<210> 143
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 47
<400> 143
catcggcgta cgactcaaga ctaatccacg tgcttgag 38
<210> 144
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 48
<400> 144
catcggcgta cgactgagct gaaatccacg tgcttgag 38
<210> 145
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 49
<400> 145
catcggcgta cgactgatag acaatccacg tgcttgag 38
<210> 146
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 50
<400> 146
catcggcgta cgactgccac ataatccacg tgcttgag 38
<210> 147
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 51
<400> 147
catcggcgta cgactgcgag taaatccacg tgcttgag 38
<210> 148
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 52
<400> 148
catcggcgta cgactgctaa cgaatccacg tgcttgag 38
<210> 149
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 53
<400> 149
catcggcgta cgactgctcg gtaatccacg tgcttgag 38
<210> 150
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 54
<400> 150
catcggcgta cgactggaga acaatccacg tgcttgag 38
<210> 151
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 55
<400> 151
catcggcgta cgactggtgc gaaatccacg tgcttgag 38
<210> 152
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 56
<400> 152
catcggcgta cgactgtacg caaatccacg tgcttgag 38
<210> 153
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 57
<400> 153
catcggcgta cgactgtcgt agaatccacg tgcttgag 38
<210> 154
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 58
<400> 154
catcggcgta cgactgtctg tcaatccacg tgcttgag 38
<210> 155
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 59
<400> 155
catcggcgta cgactgtgtt ctaatccacg tgcttgag 38
<210> 156
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 60
<400> 156
catcggcgta cgacttagga tgaatccacg tgcttgag 38
<210> 157
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 61
<400> 157
catcggcgta cgacttatca gcaatccacg tgcttgag 38
<210> 158
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 62
<400> 158
catcggcgta cgacttccgt ctaatccacg tgcttgag 38
<210> 159
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 63
<400> 159
catcggcgta cgacttcttc acaatccacg tgcttgag 38
<210> 160
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 64
<400> 160
catcggcgta cgacttgaag agaatccacg tgcttgag 38
<210> 161
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 65
<400> 161
catcggcgta cgacttggaa caaatccacg tgcttgag 38
<210> 162
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 66
<400> 162
catcggcgta cgacttggct tcaatccacg tgcttgag 38
<210> 163
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 67
<400> 163
catcggcgta cgacttggtg gtaatccacg tgcttgag 38
<210> 164
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 68
<400> 164
catcggcgta cgactttcac gcaatccacg tgcttgag 38
<210> 165
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 69
<400> 165
catcggcgta cgactaactc accatccacg tgcttgag 38
<210> 166
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 70
<400> 166
catcggcgta cgactaagag atcatccacg tgcttgag 38
<210> 167
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 71
<400> 167
catcggcgta cgactaagga cacatccacg tgcttgag 38
<210> 168
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 72
<400> 168
catcggcgta cgactaatcc gtcatccacg tgcttgag 38
<210> 169
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 73
<400> 169
catcggcgta cgactaatgt tgcatccacg tgcttgag 38
<210> 170
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 74
<400> 170
catcggcgta cgactacacg accatccacg tgcttgag 38
<210> 171
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 75
<400> 171
catcggcgta cgactacaga ttcatccacg tgcttgag 38
<210> 172
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 76
<400> 172
catcggcgta cgactagatg tacatccacg tgcttgag 38
<210> 173
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 77
<400> 173
catcggcgta cgactagcac ctcatccacg tgcttgag 38
<210> 174
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 78
<400> 174
catcggcgta cgactagcca tgcatccacg tgcttgag 38
<210> 175
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 79
<400> 175
catcggcgta cgactaggct aacatccacg tgcttgag 38
<210> 176
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 80
<400> 176
catcggcgta cgactatagc gacatccacg tgcttgag 38
<210> 177
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 81
<400> 177
catcggcgta cgactatcat tccatccacg tgcttgag 38
<210> 178
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 82
<400> 178
catcggcgta cgactattgg ctcatccacg tgcttgag 38
<210> 179
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 83
<400> 179
catcggcgta cgactcaagg agcatccacg tgcttgag 38
<210> 180
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 84
<400> 180
catcggcgta cgactcacct tacatccacg tgcttgag 38
<210> 181
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 85
<400> 181
catcggcgta cgactccatc ctcatccacg tgcttgag 38
<210> 182
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 86
<400> 182
catcggcgta cgactccgac aacatccacg tgcttgag 38
<210> 183
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 87
<400> 183
catcggcgta cgactcctaa tccatccacg tgcttgag 38
<210> 184
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 88
<400> 184
catcggcgta cgactcctct atcatccacg tgcttgag 38
<210> 185
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 89
<400> 185
catcggcgta cgactcgaca cacatccacg tgcttgag 38
<210> 186
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 90
<400> 186
catcggcgta cgactcggat tgcatccacg tgcttgag 38
<210> 187
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 91
<400> 187
catcggcgta cgactctaag gtcatccacg tgcttgag 38
<210> 188
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 92
<400> 188
catcggcgta cgactgaaca ggcatccacg tgcttgag 38
<210> 189
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 93
<400> 189
catcggcgta cgactgacag tgcatccacg tgcttgag 38
<210> 190
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 94
<400> 190
catcggcgta cgactgagtt agcatccacg tgcttgag 38
<210> 191
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 95
<400> 191
catcggcgta cgactgatga atcatccacg tgcttgag 38
<210> 192
<211> 38
<212> DNA
<213>artificial sequence
<220>
<223> Round 2 Barcode 96
<400> 192
catcggcgta cgactgccaa gacatccacg tgcttgag 38
<210> 193
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 1
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 193
cagacgtgtg ctcttccgat ctnnnnnnnn nnaacgtgat gtggccgatg tttcg 55
<210> 194
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 2
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 194
cagacgtgtg ctcttccgat ctnnnnnnnn nnaaacatcg gtggccgatg tttcg 55
<210> 195
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 3
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 195
cagacgtgtg ctcttccgat ctnnnnnnnn nnatgcctaa gtggccgatg tttcg 55
<210> 196
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 4
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 196
cagacgtgtg ctcttccgat ctnnnnnnnn nnagtggtca gtggccgatg tttcg 55
<210> 197
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 5
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 197
cagacgtgtg ctcttccgat ctnnnnnnnn nnaccactgt gtggccgatg tttcg 55
<210> 198
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 6
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 198
cagacgtgtg ctcttccgat ctnnnnnnnn nnacattggc gtggccgatg tttcg 55
<210> 199
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 7
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 199
cagacgtgtg ctcttccgat ctnnnnnnnn nncagatctg gtggccgatg tttcg 55
<210> 200
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 8
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 200
cagacgtgtg ctcttccgat ctnnnnnnnn nncatcaagt gtggccgatg tttcg 55
<210> 201
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 9
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 201
cagacgtgtg ctcttccgat ctnnnnnnnn nncgctgatc gtggccgatg tttcg 55
<210> 202
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 10
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 202
cagacgtgtg ctcttccgat ctnnnnnnnn nnacaagcta gtggccgatg tttcg 55
<210> 203
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 11
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 203
cagacgtgtg ctcttccgat ctnnnnnnnn nnctgtagcc gtggccgatg tttcg 55
<210> 204
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 12
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 204
cagacgtgtg ctcttccgat ctnnnnnnnn nnagtacaag gtggccgatg tttcg 55
<210> 205
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 13
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 205
cagacgtgtg ctcttccgat ctnnnnnnnn nnaacaacca gtggccgatg tttcg 55
<210> 206
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 14
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 206
cagacgtgtg ctcttccgat ctnnnnnnnn nnaaccgaga gtggccgatg tttcg 55
<210> 207
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 15
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 207
cagacgtgtg ctcttccgat ctnnnnnnnn nnaacgctta gtggccgatg tttcg 55
<210> 208
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 16
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 208
cagacgtgtg ctcttccgat ctnnnnnnnn nnaagacgga gtggccgatg tttcg 55
<210> 209
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 17
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 209
cagacgtgtg ctcttccgat ctnnnnnnnn nnaaggtaca gtggccgatg tttcg 55
<210> 210
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 18
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 210
cagacgtgtg ctcttccgat ctnnnnnnnn nnacacagaa gtggccgatg tttcg 55
<210> 211
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 19
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 211
cagacgtgtg ctcttccgat ctnnnnnnnn nnacagcaga gtggccgatg tttcg 55
<210> 212
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 20
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 212
cagacgtgtg ctcttccgat ctnnnnnnnn nnacctccaa gtggccgatg tttcg 55
<210> 213
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 21
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 213
cagacgtgtg ctcttccgat ctnnnnnnnn nnacgctcga gtggccgatg tttcg 55
<210> 214
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 22
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 214
cagacgtgtg ctcttccgat ctnnnnnnnn nnacgtatca gtggccgatg tttcg 55
<210> 215
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 23
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 215
cagacgtgtg ctcttccgat ctnnnnnnnn nnactatgca gtggccgatg tttcg 55
<210> 216
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 24
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 216
cagacgtgtg ctcttccgat ctnnnnnnnn nnagagtcaa gtggccgatg tttcg 55
<210> 217
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 25
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 217
cagacgtgtg ctcttccgat ctnnnnnnnn nnagatcgca gtggccgatg tttcg 55
<210> 218
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 26
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 218
cagacgtgtg ctcttccgat ctnnnnnnnn nnagcaggaa gtggccgatg tttcg 55
<210> 219
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 27
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 219
cagacgtgtg ctcttccgat ctnnnnnnnn nnagtcacta gtggccgatg tttcg 55
<210> 220
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 28
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 220
cagacgtgtg ctcttccgat ctnnnnnnnn nnatcctgta gtggccgatg tttcg 55
<210> 221
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 29
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 221
cagacgtgtg ctcttccgat ctnnnnnnnn nnattgagga gtggccgatg tttcg 55
<210> 222
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 30
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 222
cagacgtgtg ctcttccgat ctnnnnnnnn nncaaccaca gtggccgatg tttcg 55
<210> 223
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 31
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 223
cagacgtgtg ctcttccgat ctnnnnnnnn nngactagta gtggccgatg tttcg 55
<210> 224
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 32
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 224
cagacgtgtg ctcttccgat ctnnnnnnnn nncaatggaa gtggccgatg tttcg 55
<210> 225
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 33
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 225
cagacgtgtg ctcttccgat ctnnnnnnnn nncacttcga gtggccgatg tttcg 55
<210> 226
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 34
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 226
cagacgtgtg ctcttccgat ctnnnnnnnn nncagcgtta gtggccgatg tttcg 55
<210> 227
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 35
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 227
cagacgtgtg ctcttccgat ctnnnnnnnn nncataccaa gtggccgatg tttcg 55
<210> 228
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 36
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 228
cagacgtgtg ctcttccgat ctnnnnnnnn nnccagttca gtggccgatg tttcg 55
<210> 229
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 37
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 229
cagacgtgtg ctcttccgat ctnnnnnnnn nnccgaagta gtggccgatg tttcg 55
<210> 230
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 38
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 230
cagacgtgtg ctcttccgat ctnnnnnnnn nnccgtgaga gtggccgatg tttcg 55
<210> 231
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 39
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 231
cagacgtgtg ctcttccgat ctnnnnnnnn nncctcctga gtggccgatg tttcg 55
<210> 232
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 40
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 232
cagacgtgtg ctcttccgat ctnnnnnnnn nncgaactta gtggccgatg tttcg 55
<210> 233
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 41
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 233
cagacgtgtg ctcttccgat ctnnnnnnnn nncgactgga gtggccgatg tttcg 55
<210> 234
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 42
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 234
cagacgtgtg ctcttccgat ctnnnnnnnn nncgcataca gtggccgatg tttcg 55
<210> 235
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 43
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 235
cagacgtgtg ctcttccgat ctnnnnnnnn nnctcaatga gtggccgatg tttcg 55
<210> 236
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 44
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 236
cagacgtgtg ctcttccgat ctnnnnnnnn nnctgagcca gtggccgatg tttcg 55
<210> 237
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 45
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 237
cagacgtgtg ctcttccgat ctnnnnnnnn nnctggcata gtggccgatg tttcg 55
<210> 238
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 46
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 238
cagacgtgtg ctcttccgat ctnnnnnnnn nngaatctga gtggccgatg tttcg 55
<210> 239
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 47
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 239
cagacgtgtg ctcttccgat ctnnnnnnnn nncaagacta gtggccgatg tttcg 55
<210> 240
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 48
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 240
cagacgtgtg ctcttccgat ctnnnnnnnn nngagctgaa gtggccgatg tttcg 55
<210> 241
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 49
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 241
cagacgtgtg ctcttccgat ctnnnnnnnn nngatagaca gtggccgatg tttcg 55
<210> 242
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 50
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 242
cagacgtgtg ctcttccgat ctnnnnnnnn nngccacata gtggccgatg tttcg 55
<210> 243
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 51
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 243
cagacgtgtg ctcttccgat ctnnnnnnnn nngcgagtaa gtggccgatg tttcg 55
<210> 244
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 52
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 244
cagacgtgtg ctcttccgat ctnnnnnnnn nngctaacga gtggccgatg tttcg 55
<210> 245
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 53
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 245
cagacgtgtg ctcttccgat ctnnnnnnnn nngctcggta gtggccgatg tttcg 55
<210> 246
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 54
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 246
cagacgtgtg ctcttccgat ctnnnnnnnn nnggagaaca gtggccgatg tttcg 55
<210> 247
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 55
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 247
cagacgtgtg ctcttccgat ctnnnnnnnn nnggtgcgaa gtggccgatg tttcg 55
<210> 248
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 56
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 248
cagacgtgtg ctcttccgat ctnnnnnnnn nngtacgcaa gtggccgatg tttcg 55
<210> 249
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 57
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 249
cagacgtgtg ctcttccgat ctnnnnnnnn nngtcgtaga gtggccgatg tttcg 55
<210> 250
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 58
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 250
cagacgtgtg ctcttccgat ctnnnnnnnn nngtctgtca gtggccgatg tttcg 55
<210> 251
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 59
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 251
cagacgtgtg ctcttccgat ctnnnnnnnn nngtgttcta gtggccgatg tttcg 55
<210> 252
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 60
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 252
cagacgtgtg ctcttccgat ctnnnnnnnn nntaggatga gtggccgatg tttcg 55
<210> 253
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 61
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 253
cagacgtgtg ctcttccgat ctnnnnnnnn nntatcagca gtggccgatg tttcg 55
<210> 254
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 62
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 254
cagacgtgtg ctcttccgat ctnnnnnnnn nntccgtcta gtggccgatg tttcg 55
<210> 255
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 63
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 255
cagacgtgtg ctcttccgat ctnnnnnnnn nntcttcaca gtggccgatg tttcg 55
<210> 256
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 64
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 256
cagacgtgtg ctcttccgat ctnnnnnnnn nntgaagaga gtggccgatg tttcg 55
<210> 257
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 65
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 257
cagacgtgtg ctcttccgat ctnnnnnnnn nntggaacaa gtggccgatg tttcg 55
<210> 258
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 66
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 258
cagacgtgtg ctcttccgat ctnnnnnnnn nntggcttca gtggccgatg tttcg 55
<210> 259
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 67
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 259
cagacgtgtg ctcttccgat ctnnnnnnnn nntggtggta gtggccgatg tttcg 55
<210> 260
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 68
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 260
cagacgtgtg ctcttccgat ctnnnnnnnn nnttcacgca gtggccgatg tttcg 55
<210> 261
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 69
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 261
cagacgtgtg ctcttccgat ctnnnnnnnn nnaactcacc gtggccgatg tttcg 55
<210> 262
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 70
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 262
cagacgtgtg ctcttccgat ctnnnnnnnn nnaagagatc gtggccgatg tttcg 55
<210> 263
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 71
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 263
cagacgtgtg ctcttccgat ctnnnnnnnn nnaaggacac gtggccgatg tttcg 55
<210> 264
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 72
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 264
cagacgtgtg ctcttccgat ctnnnnnnnn nnaatccgtc gtggccgatg tttcg 55
<210> 265
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 73
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 265
cagacgtgtg ctcttccgat ctnnnnnnnn nnaatgttgc gtggccgatg tttcg 55
<210> 266
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 74
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 266
cagacgtgtg ctcttccgat ctnnnnnnnn nnacacgacc gtggccgatg tttcg 55
<210> 267
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 75
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 267
cagacgtgtg ctcttccgat ctnnnnnnnn nnacagattc gtggccgatg tttcg 55
<210> 268
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 76
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 268
cagacgtgtg ctcttccgat ctnnnnnnnn nnagatgtac gtggccgatg tttcg 55
<210> 269
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 77
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 269
cagacgtgtg ctcttccgat ctnnnnnnnn nnagcacctc gtggccgatg tttcg 55
<210> 270
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 78
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 270
cagacgtgtg ctcttccgat ctnnnnnnnn nnagccatgc gtggccgatg tttcg 55
<210> 271
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 79
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 271
cagacgtgtg ctcttccgat ctnnnnnnnn nnaggctaac gtggccgatg tttcg 55
<210> 272
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 80
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 272
cagacgtgtg ctcttccgat ctnnnnnnnn nnatagcgac gtggccgatg tttcg 55
<210> 273
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 81
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 273
cagacgtgtg ctcttccgat ctnnnnnnnn nnatcattcc gtggccgatg tttcg 55
<210> 274
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 82
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 274
cagacgtgtg ctcttccgat ctnnnnnnnn nnattggctc gtggccgatg tttcg 55
<210> 275
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 83
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 275
cagacgtgtg ctcttccgat ctnnnnnnnn nncaaggagc gtggccgatg tttcg 55
<210> 276
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 84
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 276
cagacgtgtg ctcttccgat ctnnnnnnnn nncaccttac gtggccgatg tttcg 55
<210> 277
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 85
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 277
cagacgtgtg ctcttccgat ctnnnnnnnn nnccatcctc gtggccgatg tttcg 55
<210> 278
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 86
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 278
cagacgtgtg ctcttccgat ctnnnnnnnn nnccgacaac gtggccgatg tttcg 55
<210> 279
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 87
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 279
cagacgtgtg ctcttccgat ctnnnnnnnn nncctaatcc gtggccgatg tttcg 55
<210> 280
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 88
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 280
cagacgtgtg ctcttccgat ctnnnnnnnn nncctctatc gtggccgatg tttcg 55
<210> 281
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 89
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 281
cagacgtgtg ctcttccgat ctnnnnnnnn nncggattgc gtggccgatg tttcg 55
<210> 282
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 90
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 282
cagacgtgtg ctcttccgat ctnnnnnnnn nncggattgc gtggccgatg tttcg 55
<210> 283
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 91
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 283
cagacgtgtg ctcttccgat ctnnnnnnnn nnctaaggtc gtggccgatg tttcg 55
<210> 284
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 92
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 284
cagacgtgtg ctcttccgat ctnnnnnnnn nngaacaggc gtggccgatg tttcg 55
<210> 285
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 93
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 285
cagacgtgtg ctcttccgat ctnnnnnnnn nngacagtgc gtggccgatg tttcg 55
<210> 286
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 94
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 286
cagacgtgtg ctcttccgat ctnnnnnnnn nngagttagc gtggccgatg tttcg 55
<210> 287
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 95
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 287
cagacgtgtg ctcttccgat ctnnnnnnnn nngatgaatc gtggccgatg tttcg 55
<210> 288
<211> 55
<212> DNA
<213>artificial sequence
<220>
<223> Round 3 Barcode 96
<220>
<221> misc_feature
<222> (23)..(32)
<223> n is a, c, g, or t
<400> 288
cagacgtgtg ctcttccgat ctnnnnnnnn nngccaagac gtggccgatg tttcg 55
<210> 289
<211> 30
<212> DNA
<213>artificial sequence
<220>
<223>2 connection chain of Round
<400> 289
cgaatgctct ggcctctcaa gcacgtggat 30
<210> 290
<211> 30
<212> DNA
<213>artificial sequence
<220>
<223>3 connection chain of Round
<400> 290
agtcgtacgc cgatgcgaaa catcggccac 30
<210> 291
<211> 30
<212> DNA
<213>artificial sequence
<220>
<223>Round 2 blocks chain
<400> 291
atccacgtgc ttgagaggcc agagcattcg 30
<210> 292
<211> 30
<212> DNA
<213>artificial sequence
<220>
<223>Round 3 blocks chain
<400> 292
gtggccgatg tttcgcatcg gcgtacgact 30
<210> 293
<211> 32
<212> DNA
<213>artificial sequence
<220>
<223>template switch primer
<400> 293
aagcagtggt atcaacgcag agtgaatrgr gg 32
<210> 294
<211> 22
<212> DNA
<213>artificial sequence
<220>
<223>PCR forward primer
<400> 294
cagacgtgtg ctcttccgat ct 22
<210> 295
<211> 23
<212> DNA
<213>artificial sequence
<220>
<223>PCR reverse primer
<400> 295
aagcagtggt atcaacgcag agt 23
<210> 296
<211> 70
<212> DNA
<213>artificial sequence
<220>
<223>Nextera tagmentation PCR primer N501 (P5)
<400> 296
aatgatacgg cgaccaccga gatctacact agatcgctcg tcggcagcgt cagatgtgta 60
taagagacag 70
<210> 297
<211> 64
<212> DNA
<213>artificial sequence
<220>
<223>Nextera tagmentation PCR primer, subdirectory index #1 (P7)
<400> 297
caagcagaag acggcatacg agatgatctg gtgactggag ttcagacgtg tgctcttccg 60
atct 64
<210> 298
<211> 64
<212> DNA
<213>artificial sequence
<220>
<223>Nextera tagmentation PCR primer, subdirectory index #2 (P7)
<400> 298
caagcagaag acggcatacg agattcaagt gtgactggag ttcagacgtg tgctcttccg 60
atct 64
<210> 299
<211> 64
<212> DNA
<213>artificial sequence
<220>
<223>Nextera tagmentation PCR primer, subdirectory index #3 (P7)
<400> 299
caagcagaag acggcatacg agatctgatc gtgactggag ttcagacgtg tgctcttccg 60
atct 64
<210> 300
<211> 64
<212> DNA
<213>artificial sequence
<220>
<223>Nextera tagmentation PCR primer, subdirectory index #4 (P7)
<400> 300
caagcagaag acggcatacg agataagcta gtgactggag ttcagacgtg tgctcttccg 60
atct 64
<210> 301
<211> 64
<212> DNA
<213>artificial sequence
<220>
<223>Nextera tagmentation PCR primer, subdirectory index #5 (P7)
<400> 301
caagcagaag acggcatacg agatgtagcc gtgactggag ttcagacgtg tgctcttccg 60
atct 64
<210> 302
<211> 64
<212> DNA
<213>artificial sequence
<220>
<223>Nextera tagmentation PCR primer, subdirectory index #6 (P7)
<400> 302
caagcagaag acggcatacg agattacaag gtgactggag ttcagacgtg tgctcttccg 60
atct 64
<210> 303
<211> 64
<212> DNA
<213>artificial sequence
<220>
<223>Nextera tagmentation PCR primer, subdirectory index #7 (P7)
<400> 303
caagcagaag acggcatacg agatttgact gtgactggag ttcagacgtg tgctcttccg 60
atct 64
<210> 304
<211> 64
<212> DNA
<213>artificial sequence
<220>
<223>Nextera tagmentation PCR primer, subdirectory index #8 (P7)
<400> 304
caagcagaag acggcatacg agatggaact gtgactggag ttcagacgtg tgctcttccg 60
atct 64

Claims (9)

1. the banking process of unicellular sequencing comprising following steps:
(a) the fixed cell of PARA FORMALDEHYDE PRILLS(91,95) carries out intracellular reversion record mark first round bar code using the first reverse transcription primer (barcode)；
(b) using connection reaction, the second wheel primer and the wheel of third round primer mark second and third round barcode are used；
(c) it is cracked to form sublibrary, after template switch, PCR is utilized to be enriched with template；
(d) fourth round barcode is marked using index PCR.
2. according to the method described in claim 1, wherein, the cell in step (a) is multiple cells, the multiple cell Cell including different types of cell or different tissue sources.
3. according to the method described in claim 1, wherein, in step (a), first reverse transcription primer is successively wrapped by 5 ' to 3 ' 5 ' primers synthesis phosphorylation label is included, for the fixed structure of the linker sequence with the second wheel connection, for distinguishing cell The varistructure as barcode sequence in source, and specifically bound for poly (A) tail with mRNA to guarantee instead The fixed structure of the specificity of transcription.
4. according to the method described in claim 3, wherein, first reverse transcription primer includes SEQ ID NO:1-96.
5. according to the method described in claim 1, wherein, in step (b), the second wheel primer successively includes 5 ' by 5 ' to 3 ' Primer synthesizes phosphorylation label, the fixed structure of the linker sequence for connecting with third round, for distinguishing cell origin The fixed structure of linker sequence as the varistructure of barcode sequence, and for being connected with the first round.
6. according to the method described in claim 5, wherein, the second wheel primer includes SEQ ID NO:97-192.
7. according to the method described in claim 1, wherein, in step (b), the third round primer successively includes 5 ' by 5 ' to 3 ' Primer synthesizes phosphorylation label, intracellular for marking plus the fixed structure of fourth round while for index PCR reaction The fixed structure of molecule, for marking the varistructure as barcode sequence of cell, and for the second wheel connection The fixed structure of linker sequence.
8. according to the method described in claim 7, wherein, the third round primer includes SEQ ID NO:193-288.
9. according to the method described in claim 1, wherein, in step (d), a P5 becomes a pair with P7-1~P7-8 group respectively Primer, adaptation Illumina microarray dataset sequencing, is sequenced the library built.
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