CN114846152B - Cell label microbead containing frameshift base, and preparation method and application thereof - Google Patents
Cell label microbead containing frameshift base, and preparation method and application thereof Download PDFInfo
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Abstract
A cell label microbead containing frameshift base, its preparing process and application are disclosed. The cell label microbead comprises a microbead body and a nucleic acid sequence structure marked on the body and provided with a cell label sequence, wherein different cell label microbeads are inserted with frame shift base sequences with different lengths at the 5' end of the cell label sequence, so that the base distribution balance of a single cell sequencing library during sequencing can be realized, thereby improving the sequencing quality and reducing the sequencing cost.
Description
Technical Field
The invention relates to the technical field of high-flux single-cell libraries, in particular to a cell tag microbead containing a frameshift base, a preparation method and application thereof.
Background
It is well known that a complex living organism is composed of many cells having specific properties, and the kinds and amounts of nucleic acids and proteins expressed by each cell are different in a specific state, so that detection of nucleic acid and protein indicators at a single cell level is of great importance for biomedical research. Single cell sequencing mainly comprises single cell genome sequencing, single cell RNA sequencing, single cell epigenomic sequencing and spatial single cell sequencing from the detection index; from the detection flux, it is largely classified into low-flux single cell sequencing (1-500 cells were detected at a time) and high-flux single cell sequencing (1000-10000 cells were detected at a time).
The high-throughput single-cell RNA detection mainly comprises a water-in-oil-based droplet separation technology and a magnetic bead labeling implementation mode based on a micro-pore plate, such as 10X Genomics, drop-Seq platform, inDrop platform, BD Rhapsody, seqwell, microwell-Seq and other technologies. The basic principle is that single cells are spatially close to microbeads with cell label labels based on a water-in-oil or micropore array and other separation technologies, mRNA released after the cells are cracked is specifically labeled, and finally cDNA (complementary deoxyribonucleic acid) labels with different cell sources are labeled with different cell labels and used for later mixed library construction and sequencing analysis.
The current microbeads with cell tag labels contain at least a cell tag base sequence and an RNA action base sequence, which are identical in cell tag base sequence on the same microbead and have a variety of cell tag base sequences on different microbeads, which generally require between half a million to several tens of millions. The methods for making such diverse cell label-labeled microbeads are currently divided into two types, one type is to directly synthesize bases onto microbeads to form oligonucleotide chains, for example, chemgenes CPG microbeads used in DropSeq and SeqWell, and the technical scheme is limited by base synthesis matrix materials, and has narrow applicability and high price; another class is nucleic acid-labeled microbeads, to which oligonucleotide chains are labeled onto microbeads by a segmented labeling method using an amplification method or ligation method, such as applied in 10X Genomics, BD Rhapsody, and microwell-seq.
The nucleic acid modified microbeads used by BD Rhapsody or Microwell at present consist of a universal primer sequence, a three-segment Cell tag (Cell battery), a molecular tag and an oligo dT, wherein the three-segment Cell tag contains a linker with a fixed base sequence. The fixed base sequence linker has the same base at the same position on different microbeads, which results in a serious degradation of sequencing quality at the linker position when sequencing the constructed transcriptome library. The main reason for this degradation of sequencing quality is the high throughput sequencer principle: in high throughput sequencing, because the sequence is read based on fluorescent signals or electrical signals, it is necessary to model the equalization of four different base signals prior to reading the sequence, and a common statistical assumption is that the number of all four bases is substantially equivalent, and at least the numbers of G and C bases and A and T bases are respectively equal. While the modeling process typically occurs during the first few cycles of sequence reads, a sequencer such as Illumina typically selects the first 25 sequencing cycles for base modeling. When there is only one fixed base at the same position in the library, this can lead to a serious base imbalance, leading to erroneous construction of the statistical model and errors in subsequent base recognition.
In order to solve the problem of reduced sequencing quality value, it is common practice to add 10% -30% of balance library during sequencing or sequence together with other base balance libraries, which has the disadvantage of greatly increasing the sequencing cost or reducing the experimental flexibility.
Targeting RNA libraries are also often constructed at present when single cell nucleic acid information is analyzed using high throughput sequencing, e.g., using BD Rhapsody TM Immune Response Targeted Panel or BD Rhapsody TM T-Cell Targeted Panel constructs mRNA libraries of genes associated with immune responses or T cells. The targeted RNA library construction procedure involves the amplification of the cDNA Sequence that has been labeled by Cell Barcode using gene specific primers and universal primers (typically Sequence primers) located at the Cell Barcode end, and finally adding the complete linker Sequence carrying the sample index through amplification.
The disadvantages of the above technique are: targeting RNA libraries constructed by the prior art, such as Immune Response Targeted Panel and 10 x Genomics VDJ libraries of BD Rhapsody, can suffer from base imbalance not only at the cell barcode of Read 1 but also at the gene specific primer sequence of Read 2 when sequenced at high throughput, mainly because the sequences of primers designed for the same gene are fixed at the same sequencing position. There are two existing solutions, one is to add a certain proportion of base balance library to the sequencing library to reduce the influence of the base imbalance targeting library, which will raise the sequencing cost; the other is to adjust the position of the primer on the gene to meet the relative base balance at different sequencing positions as much as possible when designing the primers of different target genes, but this can increase the difficulty of designing the multiple target primers.
At present, another scheme can realize the base balance of a high-throughput single-cell RNA sequencing library, as described in CN111051523A, four batches of magnetic beads are respectively manufactured by designing connecting sequences with 4 different bases, and finally, the 4 batches of magnetic beads are mixed, so that the purpose of relatively balancing the bases at the same sequencing position is achieved. The method has complex operation flow and is not suitable for manufacturing microbeads with more sections of cell labels.
Therefore, a more efficient sequencing method is needed to avoid base imbalance and thus improve sequencing quality.
Disclosure of Invention
It is an object of the present invention to provide a sequencing technique that improves sequencing quality.
It is another object of the present invention to provide a cell-tagged microbead containing a frameshift base, which allows for the balanced base distribution of single-cell sequencing libraries during sequencing by adding a frameshift base to the cell-tagged microbead.
Another object of the present invention is to provide a method for producing the cell-labeled microbead.
It is a further object of the present invention to provide the use of the cell-tagged microbeads described above.
To achieve the above object, in one aspect, the present invention provides a composition of cell tag microbeads, the composition comprising:
First cell tag microbeads, each first cell tag microbead comprising a microbead body and a first nucleic acid sequence structure with a first cell tag sequence, wherein a first frame shift base sequence is inserted into the 5' end of the first cell tag sequence in the first nucleic acid sequence structure, and the first nucleic acid sequence structure is marked on the body; and
a second cell tag microbead, each second cell tag microbead comprising a microbead body and a second nucleic acid sequence structure with a second cell tag sequence marked on the body, wherein a second frame shift base sequence is inserted into the 5' end of the second cell tag sequence in the second nucleic acid sequence structure; the first frameshift base sequence is different in length from the second frameshift base sequence.
According to some embodiments of the invention, the composition further comprises:
a third cell tag microbead, each third cell tag microbead comprising a microbead body and a third nucleic acid sequence structure with a third cell tag sequence marked on the body, wherein a third frameshift base sequence is inserted into the 5' end of the third cell tag sequence in the third nucleic acid sequence structure; and/or
Fourth cell tag microbeads, each of which comprises a microbead body and a fourth nucleic acid sequence structure with a fourth cell tag sequence, wherein the fourth nucleic acid sequence structure is marked on the body, and a fourth frame shift base sequence is inserted into the 5' end of the fourth cell tag sequence; the lengths of the third frame shift base sequence and the fourth frame shift base sequence are different from those of the first frame shift base sequence and the second frame shift base sequence.
According to some embodiments of the invention, each frameshift base sequence is independently selected from a random non-repeating permutation and combination of any one or more of the A, T, C, G four bases; preferably, the last base of each frameshift base sequence in the 5 'to 3' direction is identical.
According to some embodiments of the invention, each cell-tagged bead may be labeled with 10 6 -10 9 Of the same structure as the stripsNucleic acid sequence structure. The specific data can also be adjusted according to the actual situation.
According to some embodiments of the invention, each nucleic acid sequence structure is independently in the 5 'to 3' direction: universal primer sequences, frameshift bases, cell tags, molecular tags, and RNA reaction sequences; the 5' end of the nucleic acid sequence structure is marked on the body of the microbead.
According to some embodiments of the invention, the cell tag is used to label different cells, the cell tag sequence on each cell tag bead being different. Preferably, each cell tag sequence independently comprises a plurality of cell sub-tags (i.e., a multi-segment cell tag) and a linker sequence linked between the plurality of cell sub-tags.
According to some embodiments of the invention, each of the linker sequences may independently be 2-19bp in length. More specifically, each linking sequence may be independently selected from one or two or more of the following sequences: ACTGGTGA, GGTAGTGACA, a non-repetitive 4-base cyclic repeat sequence. Wherein the non-repeating 4 base cyclic repeat sequence comprises: a cyclic repeat sequence of GCAT, a cyclic repeat sequence of CGAT, a cyclic repeat sequence of GCTA, a cyclic repeat sequence of CGTA, a cyclic repeat sequence of GACT, a cyclic repeat sequence of GTCA; wherein the cycle repeat portion (without the non-repeat 4 base portion) is 2-15bp in length. Preferably, the non-repeating 4 base cyclic repeat sequence comprises: GCATGCAT, GCATGC, GACTGA, ACTGAC, CTGACT and TGACTG.
According to some specific embodiments of the invention, the universal primer sequence is a synthetic DNA sequence which can be complementary to one end of any DNA strand template strand and has a length ranging from about 18 bp to 33bp and is used as a PCR amplification primer; preferably, the universal primer sequence is a binding region of a sequencer compatible primer; further preferably, the primers include, but are not limited to, sequencing primers of a sequencing platform such as illumina, MGIseq, life Ion, pacbio or Nanopore, e.g., illumina Sequencing Reads1 primer sequence ACACTCTTTCCCTACACGACGCTCTTCCGATCT.
According to the inventionIn some embodiments, the RNA reaction sequence is selected from oligo dT, random base sequence (dN) x And one or more than two of template conversion sequences; the molecular tag is a random nucleic acid sequence with the length of 8-12bp or a random degenerate base sequence NNNNNNNNNNN with the length of at least partially being a random nucleic acid sequence, preferably 8 bp; it is further preferred that the single cell tag microbeads have different molecular tag sequences thereon for labeling molecules derived from all mRNA in the same cell.
In another aspect, the present invention also provides a method for preparing a cell tag microbead containing a frameshift base sequence, which comprises: providing an oligonucleotide chain with a cell tag sequence, wherein the oligonucleotide chain contains at least one section of cell tag and a frame shift base sequence is contained at the 5' end of the cell tag sequence; and marking the 5' end of the oligonucleotide chain with the cell label sequence on the body of the microbead to prepare the cell label microbead containing the frame shift base sequence.
According to a specific embodiment of the present invention, the preparation method of the cell tag microbead of the present invention specifically comprises:
synthesizing an oligonucleotide chain comprising the following structure in 5 'to 3' order: a universal primer sequence, a frameshift base, a cell sub-tag and all or part of a connecting sequence, and then modifying the 5' end of the oligonucleotide chain to the microbead; preferably, the 5' end of the universal primer sequence has a modification group selected from one or more of a hydroxyl group, an aldehyde group, an epoxy group, an amino group, a carboxyl group or an activated form thereof, a phosphate group, an alkynyl group, an azide, a sulfhydryl group, an alkene group, a biotin group or an avidin group;
optionally, further subsequent cellular sub-tags, molecular tags and RNA reaction sequences are sequentially attached in 5' to 3' order at the 5' end of the oligonucleotide strand by chemical bonds, enzymatic reactions or DNA polymerase. Wherein the enzymatic reaction includes, but is not limited to, a click chemistry based ligation reaction.
On the other hand, the invention also provides the application of the composition of the cell label microbeads or the preparation method in constructing a high-throughput single cell library.
In another aspect, the invention also provides a method of constructing a high throughput single cell sequencing library, comprising:
mRNA released after single cell lysis is marked by the composition of the cell label microbeads, which is used for constructing a high-throughput single cell sequencing library.
According to some embodiments of the invention, the high throughput single cell library comprises a base balanced 3' single cell RNA library and/or a base balanced targeted RNA amplification library.
According to some embodiments of the invention, a method of constructing a base-balanced 3' single cell RNA library comprises: (1) Capturing mRNA released after single cell lysis in a BD Rhapsody microporous chip of a single cell analysis platform by using the cell tag microbead composition with a frameshift base, and reversely transcribing the mRNA into cDNA; (2) Performing two-strand synthesis by using a random primer 5' containing a sequencing primer; (3) construction of a 3' single cell RNA library by cDNA amplification.
According to some embodiments of the invention, the construction of the base-balanced targeted RNA amplification library comprises:
capturing mRNA released after single cell lysis in a microporous chip of a single cell analysis platform by using the composition of the cell label microbead with the frameshift base, and reversely transcribing the mRNA into cDNA to obtain cDNA magnetic beads;
the cDNA magnetic beads were amplified using the BD Rhapsody technique using targeting primers to construct a targeted RNA amplification library.
According to a specific embodiment of the present invention, in the process of constructing the above-mentioned base-balanced targeted RNA amplification library, the sequence of the targeting primer of the BD rhepsody technology includes a gene-specific primer sequence and a universal primer sequence located at the Cell barcode end, and a frame shift base is provided between the universal primer sequence and the gene-specific primer sequence. Specifically, the manner of setting the frame shift bases between the universal primer sequence and the gene-specific primer sequence refers to the design of the frame shift bases on the cell-tag microbeads of the present invention, and the specific sequence or length thereof may be the same as or different from the frame shift bases on the cell-tag microbeads containing the frame shift base sequences. Thereby constructing a base-balanced RNA amplification library. The amplified library can have more base diversity at the same sequencing position to improve base balance.
The invention has the beneficial effects that:
according to the invention, by adding the frame shift base on the cell label microbeads and the specific positions of the gene specific primers, the base distribution balance of the single-cell sequencing library during sequencing is realized, so that the sequencing quality is improved and the sequencing cost is reduced.
Drawings
FIG. 1 is a block diagram of a cell tag microbead having a frameshift base according to the present invention.
FIG. 2A is sequencing data without frameshift bases according to the invention.
FIG. 2B is a schematic representation of the effect of the base balance of frame shift base pair sequencing data of the present invention.
FIG. 3 is a schematic diagram showing a scheme for preparing a magnetic cell-labeled bead containing a frameshift base according to the present invention.
FIG. 4A is a schematic diagram of specific gene primers used in constructing a base balanced targeted single cell amplification library of the present invention.
FIG. 4B is sequencing data of a targeted single cell RNA amplification library without frameshift bases according to the present invention.
FIG. 4C is a schematic representation of the effect of frame shift base pair targeting on the base balance of a single cell RNA amplification library of the present invention.
FIG. 5A shows the base distribution 50bp prior to Read1 sequencing data for a single cell transcriptome library constructed using BD Rhapsody original magnetic beads without frameshift bases.
FIG. 5B shows the base distribution of the first 50bp of Read1 sequencing data of a single cell transcriptome library constructed using cell-labeled magnetic beads containing frameshift bases according to the present invention.
FIG. 6 is an electrophoretogram of a completed base-balanced targeted single cell amplification library constructed in accordance with the present invention, showing a major library peak between 600-700 bp.
FIG. 7 shows the first 50bp base distribution of Read1 sequencing data for a single cell transcriptome library constructed using the cell-labeled magnetic beads containing another set of frameshift bases of the present invention.
FIG. 8 shows the first 50bp base distribution of Read1 sequencing data for a single cell transcriptome library constructed using the cell-labeled magnetic beads containing another set of frameshift bases of the present invention.
Detailed Description
The objects, technical solutions and advantageous effects of the present invention will be described in further detail below with reference to specific embodiments and accompanying drawings. It will be apparent that the embodiments described are merely examples
Some, but not all embodiments of the invention. It should be understood that these embodiments are not intended to limit the scope of the present invention, and any modifications or equivalent substitutions made within the spirit and principles of the present invention, all without departing from the spirit and scope of the present invention, and modifications thereof should be covered by the scope of the present invention. Experimental methods for which specific conditions are not noted in the examples are conventional methods and conventional conditions well known in the art or according to the conditions suggested by the manufacturer.
Example 1
The embodiment provides a cell label microbead with a frame shift base and a preparation method thereof, and the cell label microbead comprises the following specific steps:
based on the synthesis of the cell tag bead structure of fig. 1, a theoretical manufacturing method of the cell tag bead is provided, specifically as shown in fig. 3: the microbeads are modified by a first nucleic acid sequence, a second nucleic acid sequence and a third nucleic acid sequence in sequence, wherein the modification can be based on chemical bond connection or can be completed by enzymatic reaction, including but not limited to click chemistry-based connection reaction, template-based DNA polymerase reaction and DNA connection reaction, and the second nucleic acid sequence and the third nucleic acid sequence in FIG. 3 are formed by DNA polymerase amplification; wherein the first nucleic acid sequence structure comprises, in 5' to 3' order, a universal primer sequence, a frameshift base, a cell tag 1 and all or part of a linker sequence 1, wherein the 5' end of the first nucleic acid sequence has a modification which may be hydroxyl, aldehyde, epoxy, amino, carboxyl and activated forms thereof, phosphate, alkynyl, azide, sulfhydryl, alkene, biotin, avidin, etc.; the second nucleic acid sequence structure comprises a part or all of the connecting sequence 1, the cell tag 2 and the connecting sequence 2 in the sequence from 5 'to 3'; the third nucleic acid sequence structure comprises all or part of the connecting sequence 2, the cell tag 3, the molecular tag and the RNA reaction sequence from 5 'to 3'. Wherein cell tag 1, cell tag 2, cell tag 3 are collectively referred to as cell sub-tags.
The method for manufacturing the cell label microbeads in the actual operation process comprises the following steps:
the following method can be used to produce magnetic beads with a cell tag of 884736 varieties, and the magnetic beads can be classified into 4 kinds according to the difference of frame shift bases (A, CA, TCA, GTCA).
1.1 synthesis of single-stranded nucleic acids of the following sequences, respectively: wherein "nnnnnnnnnn" is an 8bp cell tag, and "n ' n ' n ' n ' n '" is a reverse complement of "nnnnnnnn"; according to different cell labels, 24 kinds of CB1-1, 24 kinds of CB1-2, 24 kinds of CB1-3, 24 kinds of CB1-4, 96 kinds of CB2 'and 96 kinds of CB3' are synthesized respectively.
| SEQ ID No. | Name of the name | Sequence(s) |
| 1 | CB1-1 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTAnnnnnnnnACTGGCCTGCGA |
| 2 | CB1-2 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTCAnnnnnnnnACTGGCCTGCGA |
| 3 | CB1-3 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTTCAnnnnnnnnACTGGCCTGCGA |
| 4 | CB1-4 | ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTCAnnnnnnnnACTGGCCTGCGA |
| 5 | CB2’ | GTCACCGCTACCn’n’n’n’n’n’n’n’TCGCAGGCCAGT |
| 6 | CB3’ | AAAAAAAAAAAAAAAAAANNNNNNNNn’n’n’n’n’n’n’n’TGTCACCGCTACC |
1.2 96 kinds of 300pmol of amino-modified CB1 single-stranded nucleic acids (24 kinds of CB1-1: SEQ ID No.1, 24 kinds of CB1-2: SEQ ID No.2, 24 kinds of CB1-3: SEQ ID No.3 and 24 kinds of CB1-4: SEQ ID No. 4) were mixed with 6 ten thousand pieces of 30 μm carboxyl magnetic beads at a EDC concentration of 0.25M for 3 hours at room temperature, respectively, and washed twice to obtain 96 kinds of nucleic acid-labeled magnetic beads (equivalent to the aforementioned cell-labeled microbeads).
1.3 uniformly mixing the 96 nucleic acid-labeled magnetic beads obtained in 1.2, and uniformly dividing into 96-well plates.
1.4 96 kinds of CB2' (SEQ ID No. 5) were added as templates to 96 well plates containing magnetic beads, respectively, in the following proportions.
| Reagent(s) | 50 mu L system |
| 2 x high fidelity hot start polymerase premix | 25μL |
| CB2' nucleic acids | 3μL |
| Enzyme-free water | Supplement to 50. Mu.L |
1.5 amplification was carried out using the following conditions.
1.6 after the reaction, 96 magnetic beads are uniformly mixed, and after the complementary strand is removed by high-temperature treatment at 95 ℃, the mixture is uniformly distributed into a 96-well plate again.
1.7 96 kinds of CB3' (SEQ ID No. 6) were added as templates to 96 well plates containing magnetic beads in the following proportions, respectively.
| Reagent(s) | 50 mu L system |
| 2 x high fidelity hot start polymerase premix | 25μL |
| CB3' nucleic acid | 3μL |
| Enzyme-free water | Supplement to 50. Mu.L |
1.8 amplification was carried out using the following conditions.
1.9, after the reaction is completed, 96 kinds of magnetic beads are uniformly mixed, and the mixture is treated at a high temperature of 95 ℃ and placed at a temperature of 4 ℃ for standby. The structure of the cell tag microbead with frameshift base is shown in detail in FIG. 1.
EXAMPLE 2 construction of base-equilibrated 3' single cell RNA library
The embodiment provides a construction scheme of a base-balanced 3' single-cell RNA library, which comprises the following specific steps:
a construction scheme of a base-balanced 3' single-cell RNA library was constructed using the magnetic beads containing frameshift bases prepared in example 1 and the BD Rhapsody single-cell library construction kit.
2.1 peripheral blood was drawn and fresh PBMC cells were obtained and resuspended in PBS.
2.2 microwell chip instructions provided in the BD Rhapsody single cell library construction kit were processed chip and 1 ten thousand incubated PBMC cells were added.
2.3 50 ten thousand of the beads prepared in example 1 were added to the microplate, and the excess beads were washed off after the magnetic attraction into the wells.
2.4 adding the self-contained lysate into the kit, magnetically sucking out the magnetic beads after 2min, and cleaning.
2.5 reverse transcription was performed as described in the BD Rhapsody single cell library construction kit and the unused primers on the beads were excised with ExoI.
2.6 random primer two-strand extension: the following hybridization reaction system was prepared and the magnetic beads obtained in 2.5 were suspended.
| Kit component | For 1library(μL) |
| WTA extension buffer (Cat. No. 91-1114) | 20 |
| Nuclease-free water (Cat.No. 650000076) | 134 |
| WTA extension primer | 20 |
| Sum up | 174 |
2.7 hybridization was performed under the following temperature conditions.
2.8 the following extension reagents were added.
| Kit component | 1 library (mu L) |
| 10mM dNTP(Cat.No.650000077) | 8 |
| Magnetic bead RT/PCR enhancer (Cat. No. 91-1082) | 12 |
| WTA extension (Cat. No. 91-1117) | 6 |
| Sum up | 26 |
2.9 extension according to BD kit specified conditions (following conditions).
2.10 washing the magnetic beads after extension, configuring the following reaction system and suspending the magnetic beads.
| Kit component | 1 library (mu L) |
| PCR reaction mixture (Cat. No. 91-1118) | 60 |
| Universal oligonucleotides (Cat.No. 650000074) | 10 |
| WTA amplification primers (Cat. No. 91-1116) | 10 |
| Enzyme-free water | 40 |
2.11 PCR was performed according to the following reaction conditions, followed by 0.9Xpurification using SPRI magnetic beads, 30. Mu.L elution.
2.12 the following Index PCR mixtures were configured.
| PCR reaction mixture (Cat. No. 91-1118) | 25 |
| Library upstream primer (Cat. No. 91-1085) | 5 |
| Library downstream primer Cat. Nos.650000080 | 5 |
| Nuclease-free water | 5 |
| Eluent in 3.2.11 | 10 |
| Sum up | 50 |
2.13 after amplification using SPRI magnetic beads 0.5/0.15Xsorting, 30. Mu.L sterile water was used to obtain a 3' single cell RNA library.
2.14illumina NovaSeq 6000 sequencing, comparing the obtained data with the data obtained from BD Rhody original magnetic beads, analyzing the GC content distribution of the cell tag and the connection region in Read1, as shown in FIG. 5A and FIG. 5B, wherein the base is obviously unbalanced at the 10-21 and 31-43 sequencing positions in FIG. 5A, the base is not obviously unbalanced at the 1-50 sequencing positions in FIG. 5B, and the four base distribution is relatively uniform. It can thus be concluded that: sequencing libraries containing frameshift bases showed significantly less variation in base content than the library obtained with BD Rhapsody.
Further, by comparing with sequencing data without frame shift base, the base can be well balanced when the frame shift base exists, and specific comparison results are shown in FIG. 2A and FIG. 2B, wherein bases at the sequencing positions 10-21 and 31-43 in FIG. 2A are respectively a connecting sequence 1 and a connecting sequence 2, and the bases at each sequencing position are extremely unbalanced (the base diversity is 1); as shown in FIG. 2B, if 1-4 barcoding bases are added before different cell tags 1, the base diversity of the same sequencing positions 10-21 and 31-43 is greatly increased to 2-4 bases.
In the present invention, a scheme of a frame shift base ATC, GATC, CGATC, TCGATC is also provided, and the structure of a cell tag microbead having this frame shift base is shown in FIG. 1 (microbead structure is the same as in example 1 except for the frame shift base sequence), and according to the construction scheme of a base balanced 3' single cell RNA library of this example 2, illuminea NovaSeq 6000 sequencing is performed, and the obtained data shows no obvious imbalance of bases at 1-50 sequencing positions (FIG. 7).
In the present invention, a scheme of a frame shift base T, AT, GAT, CGAT is also provided, and the structure of a cell tag microbead having this frame shift base is shown in FIG. 1 (microbead structure is the same as in example 1 except for the frame shift base sequence), and according to the construction scheme of a base balanced 3' single cell RNA library of this example 2, illuminea NovaSeq 6000 sequencing is performed, and the obtained data shows no obvious imbalance of bases at 1-50 sequencing positions (FIG. 8).
EXAMPLE 3 construction of base-equilibrated targeted RNA amplification library
The embodiment provides a base balance targeted RNA amplification library construction scheme, which comprises the following specific steps:
a base-balanced 3' single-cell RNA library construction protocol was constructed using the cDNA magnetic beads obtained in step 2.5 of example 2 with a BD Rhapsody Immune Response Targeted Panel kit.
3.1 Synthesis of primers SEQ ID No.7-SEQ ID No.496 in the nucleic acid sequences listed below, mixing the concentrations of SEQ ID No.7-SEQ ID No.104 etc. to a primer mix PCR1 primer plate at a concentration of 50. Mu.M, and mixing SEQ ID No.105-SEQ ID No.496 to a primer mix PCR2 primer plate at a concentration of 50. Mu.M.
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3.2 the PCR1 reaction mixture was prepared and the cDNA beads obtained in step 2.5 were suspended according to the following system.
| Composition of the composition | 1 library (mu L) |
| PCR reaction mixture (Cat. No. 91-1083) | 100.0 |
| Universal oligonucleotide 00074) | 20.0 |
| Microbead RT/PCR enhancer (Cat. No. 91-1082) | 12.0 |
| PCR1 primer plate | 40.0 |
| BD TM Abseq primer (Cat. No. 91-1086) | 12.0 |
| Nuclease-free water (Cat.No. 650000076) | Up to 16.0 |
| Sum up | 200.0 |
3.3 200. Mu.L of the reaction system was equally divided into 4 parts and amplified using the following procedure.
3.4 after amplification was completed, 0.7Xpurification using SPRI magnetic beads, 30. Mu.l elution.
3.5 the PCR2 system was configured according to the following table.
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3.6 after mixing and centrifugation, amplification was performed using the following procedure.
3.7 after amplification was completed, 1 Xpurification was performed using SPRI magnetic beads, and 30. Mu.l elution was performed.
3.8 Index PCR system configuration.
| Composition of the composition | 1 library (mu L) |
| PCR reaction mixture (Cat. No. 91-1083) | 25.0 |
| Library upstream primer (Cat. No. 91-1085) | 2.0 |
| Library downstream primer | 2.0 |
| Nuclease-free water (Cat.No. 650000076) | 18.0 |
| PCR2 purification of the product | 3.0 |
| Sum up | 50.0 |
3.9 after mixing and centrifugation, amplification was performed using the following procedure.
3.10 purification using SPRI magnetic beads 0.7Xafter amplification was completed, library quality control after 30. Mu.l elution, library results are shown in FIG. 6 with library peaks between 600-700 bp. In the construction process of the amplified library, frame shift bases with different lengths are added between a 5 'sequencing primer and a 3' gene specific sequence aiming at the same gene fragment primer (figure 4A), and finally, the RNA amplified library with balanced bases is constructed, and the amplified library can have more base diversity at the same sequencing position so as to improve the base balance capability, as shown in figures 4B and 4C.
SEQUENCE LISTING
<110> Beijing petiole biotechnology Co., ltd
<120> cell tag microbeads containing frameshift base, and preparation method and application thereof
<130> WPI20CN1270X-CNB
<160> 496
<170> PatentIn version 3.3
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agcaacgtgg agtcagtctg t 21
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<400> 48
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tgctattgac cgatgcttca 20
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<400> 52
gcagccaacc taagcaagat 20
<210> 53
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 53
gaggatgtgt ggcattttca 20
<210> 54
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 54
catccaattc ccaaggacag 20
<210> 55
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 55
tgtgtgaggt gtctggcttc 20
<210> 56
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 56
cccggagaag tatgtgacca 20
<210> 57
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 57
ccccaaccac ttcattcttg 20
<210> 58
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 58
agcaggctgt acacagcaga 20
<210> 59
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 59
aaaatgaaac cctccccaaa 20
<210> 60
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 60
ctgcatcaat gctcaaggaa 20
<210> 61
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 61
aaatcaagct cccaaggtca 20
<210> 62
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 62
atgccccaaa gcgattttt 19
<210> 63
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 63
tccctcattg aaagatgcaa 20
<210> 64
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 64
actgtgccct catccagaac 20
<210> 65
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 65
gacatctttg ctgcctcca 19
<210> 66
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 66
tgcctagagg tgctcattca 20
<210> 67
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 67
cagtgagaat gagggccaag 20
<210> 68
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 68
tcctcacacc acgaatctga 20
<210> 69
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 69
gctcttcctc aaaccacgaa 20
<210> 70
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 70
agagctgtct agcccaggtg 20
<210> 71
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 71
gcctggcacg taatagcttg 20
<210> 72
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 72
agaccgatgc acagtcttcc 20
<210> 73
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 73
ctgctggccc tgtacctg 18
<210> 74
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 74
tgacgtgtgt tgcttttgtg 20
<210> 75
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 75
acatcctacg gtcccaaggt 20
<210> 76
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 76
tcgaataatc cagggaaacc 20
<210> 77
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 77
ccctgcagtc acagctacac 20
<210> 78
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 78
tgcatagttc ccatgttaaa tcc 23
<210> 79
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 79
accctctctc cctccctttc 20
<210> 80
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 80
ccagctcacc tgttctccag 20
<210> 81
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 81
gggggaggga tgtgaagtta 20
<210> 82
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 82
tgcaagccat ttatgggaat 20
<210> 83
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 83
atttccatgg tgctccagtc 20
<210> 84
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 84
taaggcccaa agtgggtaca 20
<210> 85
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 85
cacattcctg tgcattgagg 20
<210> 86
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 86
gggtgttggt gaacttggtt 20
<210> 87
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 87
tgcaaaaccc agaagctaaa a 21
<210> 88
<211> 24
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 88
caacaaagca cagtgttaaa tgaa 24
<210> 89
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 89
ggtaaacacg cctgcaaac 19
<210> 90
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 90
gactgcggat ctctgtgtca 20
<210> 91
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 91
ggctctgcct tgcactattt 20
<210> 92
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 92
ccattccgca gtactccatt 20
<210> 93
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 93
tgcagtgagc tgagatcgag 20
<210> 94
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 94
ttggttgact tcatggatgc 20
<210> 95
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 95
catcatgcag ttcaacaagc 20
<210> 96
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 96
gggtgtgttt ccatgtctca 20
<210> 97
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 97
tcaggctgtt gcatgaagaa 20
<210> 98
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 98
atgcgcagta aaaactcgtg 20
<210> 99
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 99
gcctgtactt tcagctgggt a 21
<210> 100
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 100
ggtgagctct gattgcttca 20
<210> 101
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 101
gaccgggtca gtggtctct 19
<210> 102
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 102
gctttccact cccagctatg 20
<210> 103
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 103
acatcaagct ccattgtttc g 21
<210> 104
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 104
tgatcaagca cctgagaacg 20
<210> 105
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 105
gttccttggc acccgagaat tccaagaaat ggagtctcaa agcttca 47
<210> 106
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 106
gttccttggc acccgagaat tccacagaaa tggagtctca aagcttca 48
<210> 107
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 107
gttccttggc acccgagaat tccatcagaa atggagtctc aaagcttca 49
<210> 108
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 108
gttccttggc acccgagaat tccagtcaga aatggagtct caaagcttca 50
<210> 109
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 109
gttccttggc acccgagaat tccaagagca cctgtgatat gtgcg 45
<210> 110
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 110
gttccttggc acccgagaat tccacagagc acctgtgata tgtgcg 46
<210> 111
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 111
gttccttggc acccgagaat tccatcagag cacctgtgat atgtgcg 47
<210> 112
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 112
gttccttggc acccgagaat tccagtcaga gcacctgtga tatgtgcg 48
<210> 113
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 113
gttccttggc acccgagaat tccaaaggtg gaaaaataga tgccagtc 48
<210> 114
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 114
gttccttggc acccgagaat tccacaaggt ggaaaaatag atgccagtc 49
<210> 115
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 115
gttccttggc acccgagaat tccatcaagg tggaaaaata gatgccagtc 50
<210> 116
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 116
gttccttggc acccgagaat tccagtcaag gtggaaaaat agatgccagt c 51
<210> 117
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 117
gttccttggc acccgagaat tccaaagttt tcaatgatgg gcgag 45
<210> 118
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 118
gttccttggc acccgagaat tccacaagtt ttcaatgatg ggcgag 46
<210> 119
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 119
gttccttggc acccgagaat tccatcaagt tttcaatgat gggcgag 47
<210> 120
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 120
gttccttggc acccgagaat tccagtcaag ttttcaatga tgggcgag 48
<210> 121
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 121
gttccttggc acccgagaat tccaatttgt aactgtgcag ggcag 45
<210> 122
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 122
gttccttggc acccgagaat tccacatttg taactgtgca gggcag 46
<210> 123
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 123
gttccttggc acccgagaat tccatcattt gtaactgtgc agggcag 47
<210> 124
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 124
gttccttggc acccgagaat tccagtcatt tgtaactgtg cagggcag 48
<210> 125
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 125
gttccttggc acccgagaat tccaatgtgc ctcactggac ttgtc 45
<210> 126
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 126
gttccttggc acccgagaat tccacatgtg cctcactgga cttgtc 46
<210> 127
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 127
gttccttggc acccgagaat tccatcatgt gcctcactgg acttgtc 47
<210> 128
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 128
gttccttggc acccgagaat tccagtcatg tgcctcactg gacttgtc 48
<210> 129
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 129
gttccttggc acccgagaat tccaaaacca ggcaacttgg gaact 45
<210> 130
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 130
gttccttggc acccgagaat tccacaaacc aggcaacttg ggaact 46
<210> 131
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 131
gttccttggc acccgagaat tccatcaaac caggcaactt gggaact 47
<210> 132
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 132
gttccttggc acccgagaat tccagtcaaa ccaggcaact tgggaact 48
<210> 133
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 133
gttccttggc acccgagaat tccaaactgg tccttagccc catct 45
<210> 134
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 134
gttccttggc acccgagaat tccacaactg gtccttagcc ccatct 46
<210> 135
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 135
gttccttggc acccgagaat tccatcaact ggtccttagc cccatct 47
<210> 136
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 136
gttccttggc acccgagaat tccagtcaac tggtccttag ccccatct 48
<210> 137
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 137
gttccttggc acccgagaat tccaatcagg aggaaggctt acacc 45
<210> 138
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 138
gttccttggc acccgagaat tccacatcag gaggaaggct tacacc 46
<210> 139
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 139
gttccttggc acccgagaat tccatcatca ggaggaaggc ttacacc 47
<210> 140
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 140
gttccttggc acccgagaat tccagtcatc aggaggaagg cttacacc 48
<210> 141
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 141
gttccttggc acccgagaat tccaaactca gctcttggct ccact 45
<210> 142
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 142
gttccttggc acccgagaat tccacaactc agctcttggc tccact 46
<210> 143
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 143
gttccttggc acccgagaat tccatcaact cagctcttgg ctccact 47
<210> 144
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 144
gttccttggc acccgagaat tccagtcaac tcagctcttg gctccact 48
<210> 145
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 145
gttccttggc acccgagaat tccaatcaag tttgggtctg tgctg 45
<210> 146
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 146
gttccttggc acccgagaat tccacatcaa gtttgggtct gtgctg 46
<210> 147
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 147
gttccttggc acccgagaat tccatcatca agtttgggtc tgtgctg 47
<210> 148
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 148
gttccttggc acccgagaat tccagtcatc aagtttgggt ctgtgctg 48
<210> 149
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 149
gttccttggc acccgagaat tccaagcaga caccttggac atcct 45
<210> 150
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 150
gttccttggc acccgagaat tccacagcag acaccttgga catcct 46
<210> 151
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 151
gttccttggc acccgagaat tccatcagca gacaccttgg acatcct 47
<210> 152
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 152
gttccttggc acccgagaat tccagtcagc agacaccttg gacatcct 48
<210> 153
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 153
gttccttggc acccgagaat tccaaataac ctgacactgg acggg 45
<210> 154
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 154
gttccttggc acccgagaat tccacaataa cctgacactg gacggg 46
<210> 155
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 155
gttccttggc acccgagaat tccatcaata acctgacact ggacggg 47
<210> 156
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 156
gttccttggc acccgagaat tccagtcaat aacctgacac tggacggg 48
<210> 157
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 157
gttccttggc acccgagaat tccaagtcag ttccaatgag gtggg 45
<210> 158
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 158
gttccttggc acccgagaat tccacagtca gttccaatga ggtggg 46
<210> 159
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 159
gttccttggc acccgagaat tccatcagtc agttccaatg aggtggg 47
<210> 160
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 160
gttccttggc acccgagaat tccagtcagt cagttccaat gaggtggg 48
<210> 161
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 161
gttccttggc acccgagaat tccaaccaat catgaggaag atgca 45
<210> 162
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 162
gttccttggc acccgagaat tccacaccaa tcatgaggaa gatgca 46
<210> 163
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 163
gttccttggc acccgagaat tccatcacca atcatgagga agatgca 47
<210> 164
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 164
gttccttggc acccgagaat tccagtcacc aatcatgagg aagatgca 48
<210> 165
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 165
gttccttggc acccgagaat tccaattgct ccttttgcta tgcct 45
<210> 166
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 166
gttccttggc acccgagaat tccacattgc tccttttgct atgcct 46
<210> 167
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 167
gttccttggc acccgagaat tccatcattg ctccttttgc tatgcct 47
<210> 168
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 168
gttccttggc acccgagaat tccagtcatt gctccttttg ctatgcct 48
<210> 169
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 169
gttccttggc acccgagaat tccaaattca gggccagctt cataa 45
<210> 170
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 170
gttccttggc acccgagaat tccacaattc agggccagct tcataa 46
<210> 171
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 171
gttccttggc acccgagaat tccatcaatt cagggccagc ttcataa 47
<210> 172
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 172
gttccttggc acccgagaat tccagtcaat tcagggccag cttcataa 48
<210> 173
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 173
gttccttggc acccgagaat tccaagattc ctctccaaac ccatg 45
<210> 174
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 174
gttccttggc acccgagaat tccacagatt cctctccaaa cccatg 46
<210> 175
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 175
gttccttggc acccgagaat tccatcagat tcctctccaa acccatg 47
<210> 176
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 176
gttccttggc acccgagaat tccagtcaga ttcctctcca aacccatg 48
<210> 177
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 177
gttccttggc acccgagaat tccaagagtc cctcagtgga gcaag 45
<210> 178
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 178
gttccttggc acccgagaat tccacagagt ccctcagtgg agcaag 46
<210> 179
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 179
gttccttggc acccgagaat tccatcagag tccctcagtg gagcaag 47
<210> 180
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 180
gttccttggc acccgagaat tccagtcaga gtccctcagt ggagcaag 48
<210> 181
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 181
gttccttggc acccgagaat tccaattttg ttcgcatggt cacac 45
<210> 182
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 182
gttccttggc acccgagaat tccacatttt gttcgcatgg tcacac 46
<210> 183
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 183
gttccttggc acccgagaat tccatcattt tgttcgcatg gtcacac 47
<210> 184
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 184
gttccttggc acccgagaat tccagtcatt ttgttcgcat ggtcacac 48
<210> 185
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 185
gttccttggc acccgagaat tccaactcat ctgtgcactc tcccc 45
<210> 186
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 186
gttccttggc acccgagaat tccacactca tctgtgcact ctcccc 46
<210> 187
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 187
gttccttggc acccgagaat tccatcactc atctgtgcac tctcccc 47
<210> 188
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 188
gttccttggc acccgagaat tccagtcact catctgtgca ctctcccc 48
<210> 189
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 189
gttccttggc acccgagaat tccaaccatc caagggagag tgaga 45
<210> 190
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 190
gttccttggc acccgagaat tccacaccat ccaagggaga gtgaga 46
<210> 191
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 191
gttccttggc acccgagaat tccatcacca tccaagggag agtgaga 47
<210> 192
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 192
gttccttggc acccgagaat tccagtcacc atccaaggga gagtgaga 48
<210> 193
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 193
gttccttggc acccgagaat tccaactgtt tgcccagtgt ttgtg 45
<210> 194
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 194
gttccttggc acccgagaat tccacactgt ttgcccagtg tttgtg 46
<210> 195
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 195
gttccttggc acccgagaat tccatcactg tttgcccagt gtttgtg 47
<210> 196
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 196
gttccttggc acccgagaat tccagtcact gtttgcccag tgtttgtg 48
<210> 197
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 197
gttccttggc acccgagaat tccaatggtg cagagctgaa gatttt 46
<210> 198
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 198
gttccttggc acccgagaat tccacatggt gcagagctga agatttt 47
<210> 199
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 199
gttccttggc acccgagaat tccatcatgg tgcagagctg aagatttt 48
<210> 200
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 200
gttccttggc acccgagaat tccagtcatg gtgcagagct gaagatttt 49
<210> 201
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 201
gttccttggc acccgagaat tccaaggagt cttctgcttt gctgg 45
<210> 202
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 202
gttccttggc acccgagaat tccacaggag tcttctgctt tgctgg 46
<210> 203
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 203
gttccttggc acccgagaat tccatcagga gtcttctgct ttgctgg 47
<210> 204
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 204
gttccttggc acccgagaat tccagtcagg agtcttctgc tttgctgg 48
<210> 205
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 205
gttccttggc acccgagaat tccaagcatg taagttgtcc cccat 45
<210> 206
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 206
gttccttggc acccgagaat tccacagcat gtaagttgtc ccccat 46
<210> 207
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 207
gttccttggc acccgagaat tccatcagca tgtaagttgt cccccat 47
<210> 208
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 208
gttccttggc acccgagaat tccagtcagc atgtaagttg tcccccat 48
<210> 209
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 209
gttccttggc acccgagaat tccaatcctc cagaccattc aggac 45
<210> 210
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 210
gttccttggc acccgagaat tccacatcct ccagaccatt caggac 46
<210> 211
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 211
gttccttggc acccgagaat tccatcatcc tccagaccat tcaggac 47
<210> 212
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 212
gttccttggc acccgagaat tccagtcatc ctccagacca ttcaggac 48
<210> 213
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 213
gttccttggc acccgagaat tccaattttg tagccaacat tcattcaa 48
<210> 214
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 214
gttccttggc acccgagaat tccacatttt gtagccaaca ttcattcaa 49
<210> 215
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 215
gttccttggc acccgagaat tccatcattt tgtagccaac attcattcaa 50
<210> 216
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 216
gttccttggc acccgagaat tccagtcatt ttgtagccaa cattcattca a 51
<210> 217
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 217
gttccttggc acccgagaat tccaattttc tatggagcct tccga 45
<210> 218
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 218
gttccttggc acccgagaat tccacatttt ctatggagcc ttccga 46
<210> 219
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 219
gttccttggc acccgagaat tccatcattt tctatggagc cttccga 47
<210> 220
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 220
gttccttggc acccgagaat tccagtcatt ttctatggag ccttccga 48
<210> 221
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 221
gttccttggc acccgagaat tccaattagt ttgggggttt tgctg 45
<210> 222
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 222
gttccttggc acccgagaat tccacattag tttgggggtt ttgctg 46
<210> 223
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 223
gttccttggc acccgagaat tccatcatta gtttgggggt tttgctg 47
<210> 224
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 224
gttccttggc acccgagaat tccagtcatt agtttggggg ttttgctg 48
<210> 225
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 225
gttccttggc acccgagaat tccaatgtgc aatatgtgat gtggc 45
<210> 226
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 226
gttccttggc acccgagaat tccacatgtg caatatgtga tgtggc 46
<210> 227
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 227
gttccttggc acccgagaat tccatcatgt gcaatatgtg atgtggc 47
<210> 228
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 228
gttccttggc acccgagaat tccagtcatg tgcaatatgt gatgtggc 48
<210> 229
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 229
gttccttggc acccgagaat tccaaatggt acaagcaatg cctgc 45
<210> 230
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 230
gttccttggc acccgagaat tccacaatgg tacaagcaat gcctgc 46
<210> 231
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 231
gttccttggc acccgagaat tccatcaatg gtacaagcaa tgcctgc 47
<210> 232
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 232
gttccttggc acccgagaat tccagtcaat ggtacaagca atgcctgc 48
<210> 233
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 233
gttccttggc acccgagaat tccaatgctt aacccatgga tcctg 45
<210> 234
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 234
gttccttggc acccgagaat tccacatgct taacccatgg atcctg 46
<210> 235
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 235
gttccttggc acccgagaat tccatcatgc ttaacccatg gatcctg 47
<210> 236
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 236
gttccttggc acccgagaat tccagtcatg cttaacccat ggatcctg 48
<210> 237
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 237
gttccttggc acccgagaat tccaatgctc ttgtcatacc ccca 44
<210> 238
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 238
gttccttggc acccgagaat tccacatgct cttgtcatac cccca 45
<210> 239
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 239
gttccttggc acccgagaat tccatcatgc tcttgtcata ccccca 46
<210> 240
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 240
gttccttggc acccgagaat tccagtcatg ctcttgtcat accccca 47
<210> 241
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 241
gttccttggc acccgagaat tccaacattc tggcactcag gtgaa 45
<210> 242
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 242
gttccttggc acccgagaat tccacacatt ctggcactca ggtgaa 46
<210> 243
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 243
gttccttggc acccgagaat tccatcacat tctggcactc aggtgaa 47
<210> 244
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 244
gttccttggc acccgagaat tccagtcaca ttctggcact caggtgaa 48
<210> 245
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 245
gttccttggc acccgagaat tccaatgtca gtggaagaag cagatg 46
<210> 246
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 246
gttccttggc acccgagaat tccacatgtc agtggaagaa gcagatg 47
<210> 247
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 247
gttccttggc acccgagaat tccatcatgt cagtggaaga agcagatg 48
<210> 248
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 248
gttccttggc acccgagaat tccagtcatg tcagtggaag aagcagatg 49
<210> 249
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 249
gttccttggc acccgagaat tccaaccaca aatagtgctc gctttc 46
<210> 250
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 250
gttccttggc acccgagaat tccacaccac aaatagtgct cgctttc 47
<210> 251
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 251
gttccttggc acccgagaat tccatcacca caaatagtgc tcgctttc 48
<210> 252
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 252
gttccttggc acccgagaat tccagtcacc acaaatagtg ctcgctttc 49
<210> 253
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 253
gttccttggc acccgagaat tccaagctct gaggactgca ccatt 45
<210> 254
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 254
gttccttggc acccgagaat tccacagctc tgaggactgc accatt 46
<210> 255
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 255
gttccttggc acccgagaat tccatcagct ctgaggactg caccatt 47
<210> 256
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 256
gttccttggc acccgagaat tccagtcagc tctgaggact gcaccatt 48
<210> 257
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 257
gttccttggc acccgagaat tccaaacgag gaagccctaa gacgt 45
<210> 258
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 258
gttccttggc acccgagaat tccacaacga ggaagcccta agacgt 46
<210> 259
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 259
gttccttggc acccgagaat tccatcaacg aggaagccct aagacgt 47
<210> 260
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 260
gttccttggc acccgagaat tccagtcaac gaggaagccc taagacgt 48
<210> 261
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 261
gttccttggc acccgagaat tccaactcac cttctcttgc cttgg 45
<210> 262
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 262
gttccttggc acccgagaat tccacactca ccttctcttg ccttgg 46
<210> 263
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 263
gttccttggc acccgagaat tccatcactc accttctctt gccttgg 47
<210> 264
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 264
gttccttggc acccgagaat tccagtcact caccttctct tgccttgg 48
<210> 265
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 265
gttccttggc acccgagaat tccaacatcc ccatgatatt tggga 45
<210> 266
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 266
gttccttggc acccgagaat tccacacatc cccatgatat ttggga 46
<210> 267
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 267
gttccttggc acccgagaat tccatcacat ccccatgata tttggga 47
<210> 268
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 268
gttccttggc acccgagaat tccagtcaca tccccatgat atttggga 48
<210> 269
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 269
gttccttggc acccgagaat tccaactgtg agcatggtgc ttcat 45
<210> 270
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 270
gttccttggc acccgagaat tccacactgt gagcatggtg cttcat 46
<210> 271
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 271
gttccttggc acccgagaat tccatcactg tgagcatggt gcttcat 47
<210> 272
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 272
gttccttggc acccgagaat tccagtcact gtgagcatgg tgcttcat 48
<210> 273
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 273
gttccttggc acccgagaat tccaagcaac gggctacagc tttat 45
<210> 274
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 274
gttccttggc acccgagaat tccacagcaa cgggctacag ctttat 46
<210> 275
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 275
gttccttggc acccgagaat tccatcagca acgggctaca gctttat 47
<210> 276
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 276
gttccttggc acccgagaat tccagtcagc aacgggctac agctttat 48
<210> 277
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 277
gttccttggc acccgagaat tccaacagca agagcacaag aggaa 45
<210> 278
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 278
gttccttggc acccgagaat tccacacagc aagagcacaa gaggaa 46
<210> 279
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 279
gttccttggc acccgagaat tccatcacag caagagcaca agaggaa 47
<210> 280
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 280
gttccttggc acccgagaat tccagtcaca gcaagagcac aagaggaa 48
<210> 281
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 281
gttccttggc acccgagaat tccaaggaca acctgactgg ctttg 45
<210> 282
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 282
gttccttggc acccgagaat tccacaggac aacctgactg gctttg 46
<210> 283
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 283
gttccttggc acccgagaat tccatcagga caacctgact ggctttg 47
<210> 284
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 284
gttccttggc acccgagaat tccagtcagg acaacctgac tggctttg 48
<210> 285
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 285
gttccttggc acccgagaat tccaaatcca gttactgccg gtttg 45
<210> 286
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 286
gttccttggc acccgagaat tccacaatcc agttactgcc ggtttg 46
<210> 287
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 287
gttccttggc acccgagaat tccatcaatc cagttactgc cggtttg 47
<210> 288
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 288
gttccttggc acccgagaat tccagtcaat ccagttactg ccggtttg 48
<210> 289
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 289
gttccttggc acccgagaat tccaaggttc ctaggtgagc aggtg 45
<210> 290
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 290
gttccttggc acccgagaat tccacaggtt cctaggtgag caggtg 46
<210> 291
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 291
gttccttggc acccgagaat tccatcaggt tcctaggtga gcaggtg 47
<210> 292
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 292
gttccttggc acccgagaat tccagtcagg ttcctaggtg agcaggtg 48
<210> 293
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 293
gttccttggc acccgagaat tccaaggtga aggtgccgag ctata 45
<210> 294
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 294
gttccttggc acccgagaat tccacaggtg aaggtgccga gctata 46
<210> 295
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 295
gttccttggc acccgagaat tccatcaggt gaaggtgccg agctata 47
<210> 296
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 296
gttccttggc acccgagaat tccagtcagg tgaaggtgcc gagctata 48
<210> 297
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 297
gttccttggc acccgagaat tccaaaggag caccacgttc tgg 43
<210> 298
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 298
gttccttggc acccgagaat tccacaagga gcaccacgtt ctgg 44
<210> 299
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 299
gttccttggc acccgagaat tccatcaagg agcaccacgt tctgg 45
<210> 300
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 300
gttccttggc acccgagaat tccagtcaag gagcaccacg ttctgg 46
<210> 301
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 301
gttccttggc acccgagaat tccaagtact tcgcccacag catc 44
<210> 302
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 302
gttccttggc acccgagaat tccacagtac ttcgcccaca gcatc 45
<210> 303
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 303
gttccttggc acccgagaat tccatcagta cttcgcccac agcatc 46
<210> 304
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 304
gttccttggc acccgagaat tccagtcagt acttcgccca cagcatc 47
<210> 305
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 305
gttccttggc acccgagaat tccaattcaa ttcctctggg aatgtt 46
<210> 306
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 306
gttccttggc acccgagaat tccacattca attcctctgg gaatgtt 47
<210> 307
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 307
gttccttggc acccgagaat tccatcattc aattcctctg ggaatgtt 48
<210> 308
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 308
gttccttggc acccgagaat tccagtcatt caattcctct gggaatgtt 49
<210> 309
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 309
gttccttggc acccgagaat tccaagacac taggcacatt ggctg 45
<210> 310
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 310
gttccttggc acccgagaat tccacagaca ctaggcacat tggctg 46
<210> 311
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 311
gttccttggc acccgagaat tccatcagac actaggcaca ttggctg 47
<210> 312
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 312
gttccttggc acccgagaat tccagtcaga cactaggcac attggctg 48
<210> 313
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 313
gttccttggc acccgagaat tccaatcctt tggagattaa ggccc 45
<210> 314
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 314
gttccttggc acccgagaat tccacatcct ttggagatta aggccc 46
<210> 315
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 315
gttccttggc acccgagaat tccatcatcc tttggagatt aaggccc 47
<210> 316
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 316
gttccttggc acccgagaat tccagtcatc ctttggagat taaggccc 48
<210> 317
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 317
gttccttggc acccgagaat tccaaccaag gctgctctgt ttctt 45
<210> 318
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 318
gttccttggc acccgagaat tccacaccaa ggctgctctg tttctt 46
<210> 319
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 319
gttccttggc acccgagaat tccatcacca aggctgctct gtttctt 47
<210> 320
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 320
gttccttggc acccgagaat tccagtcacc aaggctgctc tgtttctt 48
<210> 321
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 321
gttccttggc acccgagaat tccaatgtga atgacttggt ccctg 45
<210> 322
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 322
gttccttggc acccgagaat tccacatgtg aatgacttgg tccctg 46
<210> 323
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 323
gttccttggc acccgagaat tccatcatgt gaatgacttg gtccctg 47
<210> 324
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 324
gttccttggc acccgagaat tccagtcatg tgaatgactt ggtccctg 48
<210> 325
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 325
gttccttggc acccgagaat tccaacaaag gaaaccaatg ccact 45
<210> 326
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 326
gttccttggc acccgagaat tccacacaaa ggaaaccaat gccact 46
<210> 327
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 327
gttccttggc acccgagaat tccatcacaa aggaaaccaa tgccact 47
<210> 328
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 328
gttccttggc acccgagaat tccagtcaca aaggaaacca atgccact 48
<210> 329
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 329
gttccttggc acccgagaat tccaatagaa tcattaggcc aggcg 45
<210> 330
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 330
gttccttggc acccgagaat tccacataga atcattaggc caggcg 46
<210> 331
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 331
gttccttggc acccgagaat tccatcatag aatcattagg ccaggcg 47
<210> 332
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 332
gttccttggc acccgagaat tccagtcata gaatcattag gccaggcg 48
<210> 333
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 333
gttccttggc acccgagaat tccaaaacga cgccaaggtg atact 45
<210> 334
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 334
gttccttggc acccgagaat tccacaaacg acgccaaggt gatact 46
<210> 335
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 335
gttccttggc acccgagaat tccatcaaac gacgccaagg tgatact 47
<210> 336
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 336
gttccttggc acccgagaat tccagtcaaa cgacgccaag gtgatact 48
<210> 337
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 337
gttccttggc acccgagaat tccaaatgag aaggacactc gctgc 45
<210> 338
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 338
gttccttggc acccgagaat tccacaatga gaaggacact cgctgc 46
<210> 339
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 339
gttccttggc acccgagaat tccatcaatg agaaggacac tcgctgc 47
<210> 340
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 340
gttccttggc acccgagaat tccagtcaat gagaaggaca ctcgctgc 48
<210> 341
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 341
gttccttggc acccgagaat tccaagttga tgctggaggc agaat 45
<210> 342
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 342
gttccttggc acccgagaat tccacagttg atgctggagg cagaat 46
<210> 343
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 343
gttccttggc acccgagaat tccatcagtt gatgctggag gcagaat 47
<210> 344
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 344
gttccttggc acccgagaat tccagtcagt tgatgctgga ggcagaat 48
<210> 345
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 345
gttccttggc acccgagaat tccaagaatg agggccaaga aagag 45
<210> 346
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 346
gttccttggc acccgagaat tccacagaat gagggccaag aaagag 46
<210> 347
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 347
gttccttggc acccgagaat tccatcagaa tgagggccaa gaaagag 47
<210> 348
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 348
gttccttggc acccgagaat tccagtcaga atgagggcca agaaagag 48
<210> 349
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 349
gttccttggc acccgagaat tccaacactc ctttgcttag cccac 45
<210> 350
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 350
gttccttggc acccgagaat tccacacact cctttgctta gcccac 46
<210> 351
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 351
gttccttggc acccgagaat tccatcacac tcctttgctt agcccac 47
<210> 352
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 352
gttccttggc acccgagaat tccagtcaca ctcctttgct tagcccac 48
<210> 353
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 353
gttccttggc acccgagaat tccaacacac tcctttgctt agccc 45
<210> 354
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 354
gttccttggc acccgagaat tccacacaca ctcctttgct tagccc 46
<210> 355
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 355
gttccttggc acccgagaat tccatcacac actcctttgc ttagccc 47
<210> 356
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 356
gttccttggc acccgagaat tccagtcaca cactcctttg cttagccc 48
<210> 357
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 357
gttccttggc acccgagaat tccaatggtg tcctttctct gctcc 45
<210> 358
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 358
gttccttggc acccgagaat tccacatggt gtcctttctc tgctcc 46
<210> 359
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 359
gttccttggc acccgagaat tccatcatgg tgtcctttct ctgctcc 47
<210> 360
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 360
gttccttggc acccgagaat tccagtcatg gtgtcctttc tctgctcc 48
<210> 361
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 361
gttccttggc acccgagaat tccaaaggaa agaaatgccc ttggt 45
<210> 362
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 362
gttccttggc acccgagaat tccacaagga aagaaatgcc cttggt 46
<210> 363
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 363
gttccttggc acccgagaat tccatcaagg aaagaaatgc ccttggt 47
<210> 364
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 364
gttccttggc acccgagaat tccagtcaag gaaagaaatg cccttggt 48
<210> 365
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 365
gttccttggc acccgagaat tccaacttca cgtctggcct cagtc 45
<210> 366
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 366
gttccttggc acccgagaat tccacacttc acgtctggcc tcagtc 46
<210> 367
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 367
gttccttggc acccgagaat tccatcactt cacgtctggc ctcagtc 47
<210> 368
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 368
gttccttggc acccgagaat tccagtcact tcacgtctgg cctcagtc 48
<210> 369
<211> 43
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 369
gttccttggc acccgagaat tccaactcca ccctggccaa gat 43
<210> 370
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 370
gttccttggc acccgagaat tccacactcc accctggcca agat 44
<210> 371
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 371
gttccttggc acccgagaat tccatcactc caccctggcc aagat 45
<210> 372
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 372
gttccttggc acccgagaat tccagtcact ccaccctggc caagat 46
<210> 373
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 373
gttccttggc acccgagaat tccaaacttg ggagaaaaca ggggt 45
<210> 374
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 374
gttccttggc acccgagaat tccacaactt gggagaaaac aggggt 46
<210> 375
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 375
gttccttggc acccgagaat tccatcaact tgggagaaaa caggggt 47
<210> 376
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 376
gttccttggc acccgagaat tccagtcaac ttgggagaaa acaggggt 48
<210> 377
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 377
gttccttggc acccgagaat tccaagcaga agtgcaggca ccta 44
<210> 378
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 378
gttccttggc acccgagaat tccacagcag aagtgcaggc accta 45
<210> 379
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 379
gttccttggc acccgagaat tccatcagca gaagtgcagg caccta 46
<210> 380
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 380
gttccttggc acccgagaat tccagtcagc agaagtgcag gcaccta 47
<210> 381
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 381
gttccttggc acccgagaat tccaaaccaa agcatcacgt tgaca 45
<210> 382
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 382
gttccttggc acccgagaat tccacaacca aagcatcacg ttgaca 46
<210> 383
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 383
gttccttggc acccgagaat tccatcaacc aaagcatcac gttgaca 47
<210> 384
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 384
gttccttggc acccgagaat tccagtcaac caaagcatca cgttgaca 48
<210> 385
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 385
gttccttggc acccgagaat tccaatcagg gctggtcttt tagga 45
<210> 386
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 386
gttccttggc acccgagaat tccacatcag ggctggtctt ttagga 46
<210> 387
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 387
gttccttggc acccgagaat tccatcatca gggctggtct tttagga 47
<210> 388
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 388
gttccttggc acccgagaat tccagtcatc agggctggtc ttttagga 48
<210> 389
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 389
gttccttggc acccgagaat tccaatacca ggaatggatg tcgct 45
<210> 390
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 390
gttccttggc acccgagaat tccacatacc aggaatggat gtcgct 46
<210> 391
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 391
gttccttggc acccgagaat tccatcatac caggaatgga tgtcgct 47
<210> 392
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 392
gttccttggc acccgagaat tccagtcata ccaggaatgg atgtcgct 48
<210> 393
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 393
gttccttggc acccgagaat tccaatagtt ggctatgctg gcatg 45
<210> 394
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 394
gttccttggc acccgagaat tccacatagt tggctatgct ggcatg 46
<210> 395
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 395
gttccttggc acccgagaat tccatcatag ttggctatgc tggcatg 47
<210> 396
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 396
gttccttggc acccgagaat tccagtcata gttggctatg ctggcatg 48
<210> 397
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 397
gttccttggc acccgagaat tccaagaatc ccgtacctgc tgct 44
<210> 398
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 398
gttccttggc acccgagaat tccacagaat cccgtacctg ctgct 45
<210> 399
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 399
gttccttggc acccgagaat tccatcagaa tcccgtacct gctgct 46
<210> 400
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 400
gttccttggc acccgagaat tccagtcaga atcccgtacc tgctgct 47
<210> 401
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 401
gttccttggc acccgagaat tccaaatctt gtctgtgatt ccggg 45
<210> 402
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 402
gttccttggc acccgagaat tccacaatct tgtctgtgat tccggg 46
<210> 403
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 403
gttccttggc acccgagaat tccatcaatc ttgtctgtga ttccggg 47
<210> 404
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 404
gttccttggc acccgagaat tccagtcaat cttgtctgtg attccggg 48
<210> 405
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 405
gttccttggc acccgagaat tccaaccttg ggttttcttt tcaattc 47
<210> 406
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 406
gttccttggc acccgagaat tccacacctt gggttttctt ttcaattc 48
<210> 407
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 407
gttccttggc acccgagaat tccatcacct tgggttttct tttcaattc 49
<210> 408
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 408
gttccttggc acccgagaat tccagtcacc ttgggttttc ttttcaattc 50
<210> 409
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 409
gttccttggc acccgagaat tccaaagaga agcagaagtc gctcg 45
<210> 410
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 410
gttccttggc acccgagaat tccacaagag aagcagaagt cgctcg 46
<210> 411
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 411
gttccttggc acccgagaat tccatcaaga gaagcagaag tcgctcg 47
<210> 412
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 412
gttccttggc acccgagaat tccagtcaag agaagcagaa gtcgctcg 48
<210> 413
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 413
gttccttggc acccgagaat tccaatagga agcacgagga aagga 45
<210> 414
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 414
gttccttggc acccgagaat tccacatagg aagcacgagg aaagga 46
<210> 415
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 415
gttccttggc acccgagaat tccatcatag gaagcacgag gaaagga 47
<210> 416
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 416
gttccttggc acccgagaat tccagtcata ggaagcacga ggaaagga 48
<210> 417
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 417
gttccttggc acccgagaat tccaaatact cagttcggaa ggggc 45
<210> 418
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 418
gttccttggc acccgagaat tccacaatac tcagttcgga aggggc 46
<210> 419
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 419
gttccttggc acccgagaat tccatcaata ctcagttcgg aaggggc 47
<210> 420
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 420
gttccttggc acccgagaat tccagtcaat actcagttcg gaaggggc 48
<210> 421
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 421
gttccttggc acccgagaat tccaatttaa tatggatgcc gtggg 45
<210> 422
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 422
gttccttggc acccgagaat tccacattta atatggatgc cgtggg 46
<210> 423
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 423
gttccttggc acccgagaat tccatcattt aatatggatg ccgtggg 47
<210> 424
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 424
gttccttggc acccgagaat tccagtcatt taatatggat gccgtggg 48
<210> 425
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 425
gttccttggc acccgagaat tccaagttct gtgcaaatgg cattc 45
<210> 426
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 426
gttccttggc acccgagaat tccacagttc tgtgcaaatg gcattc 46
<210> 427
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 427
gttccttggc acccgagaat tccatcagtt ctgtgcaaat ggcattc 47
<210> 428
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 428
gttccttggc acccgagaat tccagtcagt tctgtgcaaa tggcattc 48
<210> 429
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 429
gttccttggc acccgagaat tccaatgtgt cagctactgc ggaaa 45
<210> 430
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 430
gttccttggc acccgagaat tccacatgtg tcagctactg cggaaa 46
<210> 431
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 431
gttccttggc acccgagaat tccatcatgt gtcagctact gcggaaa 47
<210> 432
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 432
gttccttggc acccgagaat tccagtcatg tgtcagctac tgcggaaa 48
<210> 433
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 433
gttccttggc acccgagaat tccaacagga ctcagaagcc tctgg 45
<210> 434
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 434
gttccttggc acccgagaat tccacacagg actcagaagc ctctgg 46
<210> 435
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 435
gttccttggc acccgagaat tccatcacag gactcagaag cctctgg 47
<210> 436
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 436
gttccttggc acccgagaat tccagtcaca ggactcagaa gcctctgg 48
<210> 437
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 437
gttccttggc acccgagaat tccaatctgc actattcctt tgccc 45
<210> 438
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 438
gttccttggc acccgagaat tccacatctg cactattcct ttgccc 46
<210> 439
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 439
gttccttggc acccgagaat tccatcatct gcactattcc tttgccc 47
<210> 440
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 440
gttccttggc acccgagaat tccagtcatc tgcactattc ctttgccc 48
<210> 441
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 441
gttccttggc acccgagaat tccaactctt cctcccttcc atgc 44
<210> 442
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 442
gttccttggc acccgagaat tccacactct tcctcccttc catgc 45
<210> 443
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 443
gttccttggc acccgagaat tccatcactc ttcctccctt ccatgc 46
<210> 444
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 444
gttccttggc acccgagaat tccagtcact cttcctccct tccatgc 47
<210> 445
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 445
gttccttggc acccgagaat tccaaatgga aaacatcctc atggc 45
<210> 446
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 446
gttccttggc acccgagaat tccacaatgg aaaacatcct catggc 46
<210> 447
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 447
gttccttggc acccgagaat tccatcaatg gaaaacatcc tcatggc 47
<210> 448
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 448
gttccttggc acccgagaat tccagtcaat ggaaaacatc ctcatggc 48
<210> 449
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 449
gttccttggc acccgagaat tccaaaagga aaagagcaaa cgtgg 45
<210> 450
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 450
gttccttggc acccgagaat tccacaaagg aaaagagcaa acgtgg 46
<210> 451
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 451
gttccttggc acccgagaat tccatcaaag gaaaagagca aacgtgg 47
<210> 452
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 452
gttccttggc acccgagaat tccagtcaaa ggaaaagagc aaacgtgg 48
<210> 453
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 453
gttccttggc acccgagaat tccaaggaaa cagcacaaat gaacttaa 48
<210> 454
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 454
gttccttggc acccgagaat tccacaggaa acagcacaaa tgaacttaa 49
<210> 455
<211> 50
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 455
gttccttggc acccgagaat tccatcagga aacagcacaa atgaacttaa 50
<210> 456
<211> 51
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 456
gttccttggc acccgagaat tccagtcagg aaacagcaca aatgaactta a 51
<210> 457
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 457
gttccttggc acccgagaat tccaaatgca ctctgtttgc gaaga 45
<210> 458
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 458
gttccttggc acccgagaat tccacaatgc actctgtttg cgaaga 46
<210> 459
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 459
gttccttggc acccgagaat tccatcaatg cactctgttt gcgaaga 47
<210> 460
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 460
gttccttggc acccgagaat tccagtcaat gcactctgtt tgcgaaga 48
<210> 461
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 461
gttccttggc acccgagaat tccaattgaa agtgtgtgtg tccgc 45
<210> 462
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 462
gttccttggc acccgagaat tccacattga aagtgtgtgt gtccgc 46
<210> 463
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 463
gttccttggc acccgagaat tccatcattg aaagtgtgtg tgtccgc 47
<210> 464
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 464
gttccttggc acccgagaat tccagtcatt gaaagtgtgt gtgtccgc 48
<210> 465
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 465
gttccttggc acccgagaat tccaagtatg cccttgctgg accta 45
<210> 466
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 466
gttccttggc acccgagaat tccacagtat gcccttgctg gaccta 46
<210> 467
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 467
gttccttggc acccgagaat tccatcagta tgcccttgct ggaccta 47
<210> 468
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 468
gttccttggc acccgagaat tccagtcagt atgcccttgc tggaccta 48
<210> 469
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 469
gttccttggc acccgagaat tccaatacag ttccacgctg agctg 45
<210> 470
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 470
gttccttggc acccgagaat tccacataca gttccacgct gagctg 46
<210> 471
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 471
gttccttggc acccgagaat tccatcatac agttccacgc tgagctg 47
<210> 472
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 472
gttccttggc acccgagaat tccagtcata cagttccacg ctgagctg 48
<210> 473
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 473
gttccttggc acccgagaat tccaaaaggt gtttgtgcca tttgg 45
<210> 474
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 474
gttccttggc acccgagaat tccacaaagg tgtttgtgcc atttgg 46
<210> 475
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 475
gttccttggc acccgagaat tccatcaaag gtgtttgtgc catttgg 47
<210> 476
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 476
gttccttggc acccgagaat tccagtcaaa ggtgtttgtg ccatttgg 48
<210> 477
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 477
gttccttggc acccgagaat tccaatatca ggaggcaggg atcac 45
<210> 478
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 478
gttccttggc acccgagaat tccacatatc aggaggcagg gatcac 46
<210> 479
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 479
gttccttggc acccgagaat tccatcatat caggaggcag ggatcac 47
<210> 480
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 480
gttccttggc acccgagaat tccagtcata tcaggaggca gggatcac 48
<210> 481
<211> 44
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 481
gttccttggc acccgagaat tccaaggtga tcctgagccc tgac 44
<210> 482
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 482
gttccttggc acccgagaat tccacaggtg atcctgagcc ctgac 45
<210> 483
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 483
gttccttggc acccgagaat tccatcaggt gatcctgagc cctgac 46
<210> 484
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 484
gttccttggc acccgagaat tccagtcagg tgatcctgag ccctgac 47
<210> 485
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 485
gttccttggc acccgagaat tccaatgctt tgagtgctac ggaga 45
<210> 486
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 486
gttccttggc acccgagaat tccacatgct ttgagtgcta cggaga 46
<210> 487
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 487
gttccttggc acccgagaat tccatcatgc tttgagtgct acggaga 47
<210> 488
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 488
gttccttggc acccgagaat tccagtcatg ctttgagtgc tacggaga 48
<210> 489
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 489
gttccttggc acccgagaat tccaatttgc ctgcactctt tgtagg 46
<210> 490
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 490
gttccttggc acccgagaat tccacatttg cctgcactct ttgtagg 47
<210> 491
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 491
gttccttggc acccgagaat tccatcattt gcctgcactc tttgtagg 48
<210> 492
<211> 49
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 492
gttccttggc acccgagaat tccagtcatt tgcctgcact ctttgtagg 49
<210> 493
<211> 45
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 493
gttccttggc acccgagaat tccaatacca gtgcaccatc tgcac 45
<210> 494
<211> 46
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 494
gttccttggc acccgagaat tccacatacc agtgcaccat ctgcac 46
<210> 495
<211> 47
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 495
gttccttggc acccgagaat tccatcatac cagtgcacca tctgcac 47
<210> 496
<211> 48
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<220>
<223> primer
<400> 496
gttccttggc acccgagaat tccagtcata ccagtgcacc atctgcac 48
Claims (5)
1. A method of constructing a high throughput single cell sequencing library comprising a base balanced 3 'single cell RNA library and/or a base balanced targeted RNA amplification library, wherein the construction of the base balanced 3' single cell RNA library comprises:
(1) Capturing mRNA released after single cell lysis in a single cell analysis platform microporous chip by using a composition of cell tag microbeads with frame shift bases and reversely transcribing the mRNA into cDNA;
(2) Performing two-strand synthesis by using a random primer 5' containing a sequencing primer;
(3) Constructing a 3' single-cell RNA library through cDNA amplification;
wherein, the construction process of the base balance targeted RNA amplification library comprises the following steps:
capturing mRNA released after single cell lysis in a single cell analysis platform microporous chip by using a composition of cell label microbeads with frame shift bases, and performing reverse transcription to obtain cDNA (complementary deoxyribonucleic acid) magnetic beads;
Amplifying the cDNA magnetic beads using a targeting primer using BD Rhapsody technology to construct a targeted RNA amplification library; the sequence of the targeted primer of the BD Rhapsody technology comprises a gene specific primer sequence and a universal primer sequence positioned at the cell tag end, and a frame shift base is arranged between the universal primer sequence and the gene specific primer sequence, so that an RNA amplification library with balanced bases is constructed;
wherein the composition of cell tag microbeads comprises:
first cell tag microbeads, each first cell tag microbead comprising a microbead body and a first nucleic acid sequence structure with a first cell tag sequence, wherein a first frame shift base sequence is inserted into the 5' end of the first cell tag sequence in the first nucleic acid sequence structure, and the first nucleic acid sequence structure is marked on the body; a second cell tag microbead, each second cell tag microbead comprising a microbead body and a second nucleic acid sequence structure with a second cell tag sequence marked on the body, wherein a second frame shift base sequence is inserted into the 5' end of the second cell tag sequence in the second nucleic acid sequence structure; the first frameshift base sequence and the second frameshift base sequence are different in length; a third cell tag microbead, each third cell tag microbead comprising a microbead body and a third nucleic acid sequence structure with a third cell tag sequence marked on the body, wherein a third frameshift base sequence is inserted into the 5' end of the third cell tag sequence in the third nucleic acid sequence structure; and fourth cell tag microbeads, each of which comprises a microbead body and a fourth nucleic acid sequence structure with a fourth cell tag sequence, wherein the fourth nucleic acid sequence structure is provided with a fourth frame shift base sequence inserted into the 5' end of the fourth cell tag sequence; and, each frame shift base sequence is independently selected from any one or more than two random non-repeated permutation and combination of A, T, C, G bases, the lengths of the first frame shift base sequence, the second frame shift base sequence, the third frame shift base sequence and the fourth frame shift base sequence are respectively 1-4nt; the last base of each frame shift base sequence from 5 'to 3' is the same;
Wherein each nucleic acid sequence structure independently comprises, in order from 5 'to 3': a universal primer sequence, a frameshift base, a cell tag, a molecular tag and an RNA reaction sequence, wherein the RNA reaction sequence is oligo dT; the 5' end of the nucleic acid sequence structure is marked on the microsphere body; the cell tag sequence on each cell tag microbead is different; each cell tag sequence independently comprises a plurality of cell sub-tags and a linker sequence linked between the plurality of cell sub-tags;
wherein the universal primer sequence is ACACTCTTTCCCTACACGACGCTCTTCCGATCT;
wherein each linking sequence is independently selected from one or more of the following sequences: ACTGGTGA, GGTAGTGACA, GCATGCAT, GCATGC, GACTGA, ACTGAC, CTGACT and TGACTG.
2. The method of claim 1, wherein each cell tag microbead is labeled with 10 6 -10 9 Nucleic acid sequence structure of identical structure.
3. The method of claim 1, wherein the molecular tag is a random nucleic acid sequence of 8-12nt length or is at least partially a random nucleic acid sequence.
4. The method of claim 3, wherein the molecular tag is a random degenerate base sequence of 8 nt.
5. The method of claim 4, wherein the single cell tag microbeads have different molecular tag sequences thereon.
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| PCT/CN2020/142012 WO2022141366A1 (en) | 2020-12-31 | 2020-12-31 | Cell barcode microbead containing frameshift base, preparation method therefor and application thereof |
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