CN109680062A - A method of detection minimal residual disease MRD - Google Patents

A method of detection minimal residual disease MRD Download PDF

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CN109680062A
CN109680062A CN201811549203.0A CN201811549203A CN109680062A CN 109680062 A CN109680062 A CN 109680062A CN 201811549203 A CN201811549203 A CN 201811549203A CN 109680062 A CN109680062 A CN 109680062A
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artificial sequence
dna
sequence
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primer
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CN109680062B (en
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孙涛
余莹莹
张天骄
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Hangzhou Aimuanthene Biotechnology Co Ltd
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Abstract

It is hematologic cancers such as T/B system leukaemia the present invention provides a kind of leaching based on high-flux sequence research and development, lymthoma, the detection method of myeloma minimal residual disease, this method passes through addition internal reference and House Keeping gene, using with UMB (Unique Molecular Barcode, unimolecule label) multiple PCR primer group carry out IGH (VDJ) respectively to sample, IGH (DJ), IGK, IGL, TCR β, TCR γ, TCR δ, BCL1/IGH, the library construction of BCL2/IGH, resulting DNA library is sequenced by Illumina HiSeq platform, and pass through bioinformatic analysis high-flux sequence result.This method can find the cancer cell of new mutation while detecting cancer cell, and apply high throughput sequencing technologies combination bioinformatic analysis, and the detection sensitivity of MRD can at least detect a cancer cell in 1,000,000 cells, i.e., 10‑6(0.0001%).This method not only can reach minimal residual disease testing goal with quantitative analysis cancer cell number, and can correct the sequence read mistake generated in series jump caused by the base mispairing of PCR amplification generation and library sequencing procedure.

Description

A method of detection minimal residual disease MRD
Technical field
The invention belongs to field of biotechnology, and in particular to a method of detection minimal residual disease MRD.
Background technique
Leaching is that hematologic cancers mainly include T/B lymphocytic leukemia, lymthoma and Huppert's disease, and small residual Disease (Minimal Residual Disease, MRD) is stayed to refer to that leukaemia/lymthoma/patients with malignant myeloma is alleviated through inductive treatment Afterwards or after bone-marrow transplantation, the state of a small amount of cancer cell is still remained in vivo, may finally cause palindromia.Leukaemia can be divided into Lymphocytic leukemia, myelocytic leukemia, cell mixing leukaemia are a kind of candidate stem cell malignant clone diseases.Gram Mechanism are a large amount of in marrow and other hematopoietic tissues because proliferation out of control, dysdifferentiation, apoptosis are obstructed etc. by grand property leukaemia cell Proliferation accumulation, and other non-hematopoietic tissues and organ are infiltrated, while inhibiting normal hematopoiesis function.Lymthoma is initiated by lymph and makes The malignant tumour of blood system can be divided into B cell, T cell, NK cell lymphoma according to different lymphocytes origin.Multiple bone Myeloma are a kind of malignant tumours of Clonal thick liquid cell abnormality proliferation, and tumour cell is starched originating from the thick liquid cell in marrow Cell is cell of the bone-marrow-derived lymphocyte development to final function phases.
With continuously improving for the technologies such as chemotherapy, selectively targeted treatment, hematopoietic stem cell transplantation (HSCT) treatment, blood The therapeutic effect of cancer is even up to hematology complete incidence graph (hematologic complete remission, HCR), but It obtains HCR and is not sufficient to guarantee patient's Long-term disease-free survival, therefore recurrence is still the difficult point for perplexing cancer and curing, and studies carefully its original Because mainly related with hematologic cancer cell minimal residual disease (MRD).
Recent study shows that hematologic cancers recurrence is closely related with MRD, and MRD raising can indicate hematologic cancers in advance Recurrence comprehensively.Therefore, hematologic cancers patient is carried out using high sensitivity, high specificity and reliable and stable experimental method regular MRD detection to assessment morbid state, judges that curative effect, prediction recurrence, guiding treatment have important clinical meaning.Hematologic cancers Therapeutic scheme and novel effective drug cooperation, patient disease alleviation doubles, MRD monitoring time point and detection spirit Sensitivity is particularly important for recurrence prediction.
A large amount of V (variable region) on B/T cytogene seat, D (variable region), J (bonding pad) genetic fragment B/T cell by Diversity recombination can be generated in the forming process of body.The recombination of this V-D-J gene imparts each B/T cell oneself uniqueness Cell receptor (BCR/TCR) so that the sequence of each BCR/TCR can effectively become B/T cell clone Specific biological marker.Therefore BCR/TCR gene order is sequenced, can be very good to position each B/T cell, wherein B/T cell including canceration, and MRD detection sensitivity is at least up to 10-6(0.0001%).
Since the maximum feature of BCR/TCR gene is the random recombination of V, D, J genetic fragment, according to various known V, D, J The multi-primers of the conservative region design of gene expand different receptor chains, then add sequencing label.But in cancer patient, cancer is thin The gene mutation of born of the same parents be it is very common, if the BCR/TCR of cancer cell produces mutation, the primer of known array just has can Gene after capable of can not identifying mutation is easy for causing false negative for testing result.
In the prior art, document " Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombination in suspect lymphoproliferations:Report of the BIOMED-2Concerted Action BMH4-CT98-3936 " in devise 3 VH-JH, 2 DH-JH, 2 IGK, an IGL, 3 TCRB, 2 TCRG, 1 TCRD, 3 BCL1-IGH, a BCL2-IGH have 18 multi-PRC reactions altogether, relate to 107 different primers, This set primer is finally named as BIOMED2.But primer does not cover all V, J genetic fragments, is easy to produce multiplex PCR and draws Object Preference is not directly suitable for high-flux sequence, and can not carry out PCR and sequencing amplification mispairing reparation.Document 《Deep-sequencing approach for minimal residual disease detection in acute Lymphoblastic leukemia " in using internal reference establish the computation model of cancer cell.
The method of detection minimal residual disease (MRD) includes that multi-parameter flow cytomery (FC), polymerase chain are anti-at present Answer (PCR), fusion detection real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), allele oligonucleotide hybridization method (AS0-PCR) and (IG/TR NGS) etc. is sequenced in immune group high-throughput two generations.The most commonly used is the streams based on molecular immunology Formula cell instrument art (Flow Cytometry), but its sensitivity is only up to 10-4The order of magnitude (0.01%), and this method ties MRD The accuracy of fruit judgement depends greatly on the experiment condition in each laboratory and the personal experience of operator, standardizes Degree is low, and some researches show that the influences in chemotherapy process due to chemotherapeutics to become the immunophenotype of cancer cell Change, i.e. " immunological drift " phenomenon, the reliability and accuracy of MRD testing result can be influenced.The method of polymerase chain reaction (PCR) Without standard measure, the case where can only see the gene rearrangement of IG/TCR.Fusion real-time fluorescence quantitative polymerase chain reaction (RT- QPCR) detection method can be only applied to that the source of leukaemia cell cannot be prompted there are the acute leukemia of fusion exception. The application of allele oligonucleotide hybridization method (AS0-PCR) is relatively prevalent in Europe, and this method is needed according to each trouble Person customizes a set of primer, is easy to appear non-specific amplification, and experiment condition and operating technology are more demanding, time-consuming consumption manpower, and And cancer cell nucleic acid mutates and will result in the false negative of MRD detection.
Summary of the invention
The technical problem to be solved by the present invention is to the conservative region design for known BCR/TCR V, J genetic fragment is special Property primer amplification there is the region CDR3 of great diversity, the cancer cell of new mutation is found while can detecte cancer cell.It answers With high throughput sequencing technologies combination bioinformatic analysis, the detection sensitivity of MRD at least detects one in 1,000,000 cells A cancer cell, i.e., 10-6(0.0001%).
On the one hand, the present invention provides a kind of multiple PCR primer groups for detecting minimal residual disease MRD, which is characterized in that packet Include following primer pair: the primer pair of detection IgH (VDJ), upstream primer sequence is as shown in SEQ ID NO:1-16, downstream primer sequence Column are as shown in SEQ ID NO:24；Detect IgH (DJ) primer pair, upstream primer sequence as shown in SEQ ID NO:17-23, under Primer sequence is swum as shown in SEQ ID NO:24；Detect the primer pair of IgK, upstream primer sequence such as SEQ ID NO:25-34 institute Show, downstream primer sequence is as shown in SEQ ID NO:35-37；The primer pair of IgL is detected, upstream primer sequence such as SEQ IDNO: Shown in 38-54, downstream primer sequence is as shown in SEQ ID NO:55-57；Detect the primer pair of TRB, upstream primer sequence such as SEQ Shown in ID NO:58-88, downstream primer sequence is as shown in SEQ ID NO:89-100；Detect the primer pair of TRG, upstream primer sequence Column are as shown in SEQ ID NO:101-106, and downstream primer sequence is as shown in SEQ ID NO:107-109；Detect the primer of TRD Right, upstream primer sequence is as shown in SEQ ID NO:110-112, and downstream primer sequence is as shown in SEQ ID NO:113-116；Inspection The primer pair of BCL1/IGH is surveyed, upstream primer sequence is as shown in SEQ ID NO:117, downstream primer sequence such as SEQID NO:118 It is shown；The primer pair of BCL2/IGH is detected, upstream primer sequence is as shown in SEQ ID NO:119-244, and downstream primer sequence is such as Shown in SEQ ID NO:118；Wherein N:A or T or C or G；R:A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
On the other hand, the present invention provides the kits comprising the multiple PCR primer.
On the other hand, the present invention provides the method that a kind of couple of T, B-cell receptor carry out high-flux sequence, feature exists In, comprising the following steps:
(1) sample to be tested is taken；
(2) sample to be tested is expanded using the multiple PCR primer group；
(3) by the second wheel PCR, illumina high-flux sequence instrument is added respectively in the both ends of step 2) amplified production Upper machine sequencing primer and label carry out high-flux sequence to amplified production after purification；
(4) bioinformatic analysis carries out quantitative and qualitative analysis to sample to be tested.
Optionally, according to target gene (IGH (VDJ), IGH (DJ), IGK, IGL, TCR β, TCR γ, TCR in step (2) δ, BCL1/IGH, BCL2/IGH) it is added respectively in PCR reaction system known to three corresponding 1%input DNA profiling amounts The internal reference DNA of sequence carries out specific amplification with sample simultaneously.
Optionally, the internal reference DNA sequence dna is shown in SEQ ID NO:128-226.
Optionally, in the step (2), while House Keeping gene (beta-Actin or GAPDH) is expanded, with Sample carries out specific amplification simultaneously, and the primer sequence that amplification House Keeping gene beta-Actin is used is SEQ IDNO:250-251, the primer sequence that amplification House Keeping gene GAPDH is used is SEQ ID NO:252-253.
Optionally, the sequence of sequencing primer described in step (3) and label be SEQ ID NO:227-247 shown in, wherein Upstream primer sequence is shown in SEQ ID NO:227-238, and downstream primer sequence is shown in SEQ ID NO:239-247.
Optionally, bioinformatic analysis described in step (4): by the immune group library fastq text of NGS high-flux sequence Part removes machine sequencing sequence under low quality, and V (D) J gene is determined after comparing with IMGT database, and assembling obtains complete CDR3 sequence, removing further according to clone's frequency may polluted sequence.MRD value takes two kinds of calculations.
One, using internal reference as quantitative criterion.MRD=(Rc/(Rstd/Nstd)/2)/NTOT, wherein RcIt is that sequencing gained cancer is thin The reads number of born of the same parents, RstdIt is the reads number of sequencing gained internalcontrol sequence, NstdIt is the molecular number for adding internal reference, NTOT=internal reference is total Quality/each reference molecules molal weight, NTOT=MTOT/ K, NTOTIt is the quantity for always having core living cells, MTOTIt is always to have core living thin The quality of born of the same parents, that is, input DNA quality, K are each cell DNA total amount 0.0064ng.
Two, using House Keeping gene as quantitative criterion.There are two types of calculation methods: 1) .MRD=NC/NH, wherein NCIt is sequencing gained cancer cell number according to have passed through UMB analysis treated cancer cell number, NHIt is sequencing gained House Keeping Gene data have passed through UMB analysis treated total template cell number.2) .MRD=(Rc/(RH/TH)/2)/NTOT, wherein RcIt is The reads number of sequencing gained cancer cell, RHIt is the reads number of sequencing gained House Keeping gene, THIt is House Keeping The template number of gene magnification, NTOT=internal reference gross mass/each reference molecules molal weight, NTOT=MTOT/ K, NTOTIt is always to have core living The quantity of cell, MTOTIt is the quality i.e. quality of input DNA for always having core living cells, K is each cell DNA total amount 0.0064ng.
On the other hand, the purposes the present invention also provides multiple PCR primer group in preparation detection minimal residual disease MRD kit
The technology of the present invention effect:
1. adjusting suitable primer concentration by over-designed region multi-primers, solving the preference of multiplexed PCR amplification Property.Increase unimolecule label (UMB) of the random sequence as each template in design of primers, each not phase of every primer sequence Together, not only quantitatively each DNA molecular can also can really be carried out with error correction.
2. this method can not only track the sequence as diagnostic flag, while can also detect emerging cancer cell gram Grand sequence, these may imply the progress or recurrence of disease.MRD detection in high sensitivity can the prediction before clinical symptoms occur Recurrence, and therapeutic effect is assessed, doctor can be helped early stage to take therapeutic intervention to patient more in time.
3. this method imitates the pairing of the V and J of complete BCR/TCR, this composition sequence using the internal reference of a set of synthesis It is all highly stable in each detection process, and quantify to detect the whole karyocytes for including in sample by internal reference (input DNA).This method is vital to the accuracy for ensuring clinical MRD result.
4. this method House Keeping gene stable using expression quantity, this gene are non-in each detection process Often stablize, can quantify to detect the template number for participating in reaction in sample after analyzing by UMB and the shape before PCR amplification can be restored State.This method is to correct quantitatively MRD the result is that vital.
5. the accuracy that this method ensures clinical sample MRD report in conjunction with exclusive bioinformatic analysis.Correct reason " denominator " used in MRD testing result is solved for explaining that MRD tumor load is most important.The testing result of MRD is cancer cell Number/all has core viable count, then denominator " all to have core living cells " number just will affect the result of final MRD.For example, Cancer cell is " 137 ", is if total cell detects background only in accordance with 10000 B cell BCR sequencing results, this means that MRD value is 137/10000, this is clinically to recur；If with 1,000,000 total cells (all mononuclearcells) for background Detection, then MRD value is 137/10000000, then clinically thinking not recur.Other MRD testing result uses It is the percentage of B cell or T cell, then there is MRD result inaccuracy for the patient of lymphocyte depletion after treatment.
Bibliography:
1.David Wu et al.High-Throughput Sequencing Detects Minimal Residual Disease in Acute T Lymphoblastic Leukemia.Sci Transl Med.2012May 16；4(134)
2.Carlson CS et al.Using synthetic templates to design an unbiased multiplex PCR assay.Nat Commun.2013；4:2680.
3.David Wu et al.Detection of minimal residual disease in B lymphoblastic leukemia by high-throughput sequencing of IGH.Clin Cancer Res.2014Sep1；20(17):4540-8
4.Michaela Kotrova1·Jan Trka2·Michael Kneba1·Monika Bru¨ggemann.Is Next-Generation Sequencing the way to go for Residual Disease Monitoring in Acute Lymphoblastic Leukemia? Mol Diagn Ther (2017) 21:481-492
5.Malek Fahamet al.Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia.Blood.2012Dec 20；120(26):5173-80
6.Jinghua Wu et al.Minimal Residual Disease Detection and Evolved IGH Clones Analysis in Acute B Lymphoblastic Leukemia Using IGH Deep Sequencing.Front Immunol.2016Oct 4；7:403
7.Sepideh Shahkarami et al.Minimal residual disease(MRD)detection using rearrangement of immunoglobulin/T cell receptor genes in adult patients with acute lymphoblastic leukemia(ALL).Annals of Hematology(2018)97:585–595
8.Martinez-Lopez,Joaquin Lahuerta,Juan J.et al.Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma.Blood.2014.123:3073-3079
9.A M Sherrod,P Hari,C A Mosse,R C Walker&R F Cornell.Minimal residual disease testing after stem cell transplantation for multiple myeloma.Bone Marrow Transplant.2016Jan；51(1):2-12.
10.Avet-Loiseau H.Minimal Residual Disease by Next-Generation Sequencing:Pros and Cons.Am Soc Clin Oncol Educ Book.2016；35:e425-30.
11.BrentWoodet al.Measurable residual disease detection by high- throughput sequencing improves risk stratification for pediatric B- ALL.Blood.2018 131:1350-1359.
12.Smith T,Heger A,Sudbery I.UMI-tools:modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy.Genome Res.2017Mar；27(3):491-499.
Detailed description of the invention
The process of Fig. 1 immune group high-flux sequence detection minimal residual disease method.
The construction method in the library Fig. 2
2100 quality inspection of Fig. 3 is as a result, peak corresponding to 15bp and 1500bp respectively represents low marker and up on abscissa Marker, multiple peaks, that is, library size in this range.
Two kinds of calculation methods of Fig. 4 MRD
Fig. 5 bioinformatics treated result example.
Result example of the Fig. 6 after bioinformatics and visualization processing, wherein each circle represents a clone, often The size of a circle indicates the frequency size of the clone, and frequency is bigger, and circle is bigger.
Specific embodiment
The present invention is further illustrated with embodiment below, but the present invention is not intended to be limited thereto.It is not infused in the following example The experimental method of bright actual conditions, usually according to normal condition, or according to the normal condition proposed by manufacturer.
Embodiment 1: the acquisition of sample genome
1. can be divided into according to pretherapy and post-treatment and sample specificity:
Sample of bone marrow 100uL, 200uL, 300uL, 400uL, 500uL are in EDTA anticoagulant tube before ruling treatment by men；
Rule by men treatment after sample of bone marrow 100uL, 200uL, 300uL, 400uL, 500uL, 600uL, 700uL, 800uL, 900uL, 1mL, 2mL are in EDTA anticoagulant tube；
Human peripheral sample 5mL, 6mL, 7mL, 8mL, 9mL, 10mL is in EDTA anticoagulant tube.
2. having core living cells using separation after red blood cell in erythrocyte cracked liquid lysed sample.
3. extracting genome gDNA after thering is core living cells to carry out cell count gained.
Embodiment 2: multiplexed PCR amplification and library construction
The primer of multipair V genetic fragment and J genetic fragment with UMB is added in multi-PRC reaction system, and adds Internal reference DNA or House the Keeping gene PCR primer of the known array of any three corresponding 1%input DNA profiling amounts Specific amplification is carried out simultaneously with sample.
Primer sets sequence is as shown in table 1
1 multiple PCR primer of table
Primer sequence structure as shown in Fig. 2,
It is SEQ ID NO:249 that wherein the Read1 sequence, which is SEQ ID NO:248, Read2 sequence,.
Wherein the sequence of the internal reference DNA is shown in SEQ ID NO:128-226.
Wherein the PCR primer sequence of the amplification House Keeping gene is shown in SEQ ID NO:250-253.
Multiplex PCR system (PCR1), including 25 μ L, 50 μ L, the following are 25uL systems:
PCR premixed liquid used in multiplex PCR: Multiplex PCR Master Mix (UNG).
PCR reaction caching liquid is mixed to carry out according to following response procedures:
Embodiment 3: high-flux sequence and bioinformatic analysis
The Hiseq system from illumina company is used in method of the invention, Hiseq is a kind of based on unimolecule cluster While synthesis while sequencing technologies, based on proprietary reversible termination chemical principle.The random fragment of DNA is attached to when sequencing Optically transparent glass surface (Flow cell), these DNA fragmentations are after extension and bridge amplification, the shape on Flow cell It is the unimolecule cluster with thousands of parts of same templates at hundreds of millions of clusters (cluster), each cluster.Then using having (Sequencing by is sequenced in synthesis by what invertibity terminated in four kinds of special deoxyribonucleotides of fluorophor Synthesis, SBS) template DNA to be measured is sequenced in technology.
Firstly, the upper machine sequencing primer and mark of illumina high-flux sequence instrument are added at the both ends of PCR1 product respectively Label, while expanding (referring to fig. 2).
The primer that the specified sequencing of Illumina uses and the label applied to data analysis:
Overstriking is label index.
PCR2 reaction buffer is prepared in the following proportions:
PCR2 reaction caching liquid is mixed to carry out according to following response procedures:
The purifying of amplified production: PCR2 product is purified using magnetic bead recovery method
A, 80 μ L AMPure XP Beads of addition enter PCR2 reaction product, mix.
B, at incubation at room temperature 10 minutes.
C, magnetic bead-PCR2 product mixtures test tube is placed after on magnetic frame, waiting all magnetic beads to be adsorbed on magnetic frame, All supernatants are sucked with pipettor, are abandoned.
D, addition 150 μ L, 70% ethyl alcohol hatches 30 seconds on magnetic bead, sucks all supernatants with pipettor, abandons.
E, D step 2 time is repeated.
F, test tube cap is opened, is waited 5 minutes, air-dries to magnetic bead, remains in test tube without any ethyl alcohol.
G, test tube is removed from magnetic frame, and adds 30 μ L and removes nuclease water, blow and beat suspension magnetic bead using pipettor.
H, tube back magnetic frame, after waiting all magnetic beads to be all adsorbed in magnetic frame, supernatant is shifted in new test tube In.
I, the PCR2 product after purifying is just contained in supernatant.
Using Agilent nucleic acid fragment analyzer 2100 to purified product carry out quality inspection, as Fig. 3 be 2100 quality inspections as a result, Peak corresponding to 15bp and 1500bp respectively represents low marker and up marker on abscissa, more in this range A peak, that is, library size (referring to Fig. 3).
It carries out high-flux sequence: resulting DNA library is sequenced by Illumina HiSeq platform, mode is sequenced For PE150, library denaturant concentration is 2nM, and upper machine concentration is 20pM.Bioinformatic analysis: by exempting from for NGS high-flux sequence Epidemic disease group library fastq file, removes machine sequencing sequence under low quality, and V (D) J gene, group are determined after comparing with IMGT database Dress obtains complete CDR3 sequence, and removing further according to clone's frequency may polluted sequence.
MRD value takes following two calculation: one, using internal reference as quantitative criterion.MRD=(Rc/(Rstd/Nstd)/ 2)/NTOT, wherein RcIt is the reads number of sequencing gained cancer cell, RstdIt is the reads number of sequencing gained internalcontrol sequence, NstdIt is Add the molecular number of internal reference, NTOT=internal reference gross mass/each reference molecules molal weight, NTOT=MTOT/ K, NTOTIt is always to have core The quantity of living cells, MTOTIt is the quality i.e. quality of input DNA for always having core living cells, K is each cell DNA total amount 0.0064ng.Two, using House Keeping gene as quantitative criterion, there are two types of calculation methods: 1) .MRD=NC/NH, wherein NCIt is sequencing gained cancer cell number according to have passed through UMB analysis treated cancer cell number, NHIt is sequencing gained HouseKeeping base Total template cell number after have passed through at UMB analysis because of data.2) .MRD=(Rc/(RH/TH)/2)/NTOT, wherein RcIt is sequencing The reads number of gained cancer cell, RHIt is the reads number of sequencing gained House Keeping gene, THIt is House Keeping base The template number of gene-amplification, NTOT=internal reference gross mass/each reference molecules molal weight, NTOT=MTOT/ K, NTOTIt is always to have core living The quantity of cell, MTOTIt is the quality i.e. quality of input DNA for always having core living cells, K is each cell DNA total amount 0.0064ng (referring to fig. 4).Acute lymphatic leukaemia high-flux sequence detects MRD, if MRD > 10-4It (0.01%) is then the positive, If MRD < 10-4It (0.01%) is then feminine gender.Huppert's disease high-flux sequence detects MRD, if MRD > 10-5(0.001%) then For the positive, if MRD < 10-5It (0.001%) is then feminine gender.The Hiseq system from illumina company is used in method of the invention System.
Bioinformatic analysis is carried out to lower machine data.Fig. 5 is that treated as a result, result includes for bioinformatics clone count、clone fraction、V segment、D segment、J segment、CDR3clone sequence、 The information such as CDR3AA sequence.Fig. 6 is after bioinformatics and visualization processing as a result, wherein each circle represents One clone, the size of each circle indicate the frequency size of the clone, and frequency is bigger, and circle is bigger.
It should be understood that those skilled in the art can make the present invention various after having read above content of the invention Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
SEQUENCE LISTING
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<120>a kind of method for detecting minimal residual disease MRD
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<213>artificial sequence (artificial sequence)
<400> 3
tcgtgggctc ggagatgtgt ataagagaca gagcagcctg agatctgagg ac 52
<210> 4
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 4
tcgtgggctc ggagatgtgt ataagagaca gacatggacc ctgtggacac a 51
<210> 5
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 5
tcgtgggctc ggagatgtgt ataagagaca gctgtatctg caaatgaaca g 51
<210> 6
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 6
tcgtgggctc ggagatgtgt ataagagaca ggcagattca ccatctccag a 51
<210> 7
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 7
tcgtgggctc ggagatgtgt ataagagaca gagcctgaaa accgaggaca c 51
<210> 8
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 8
tcgtgggctc ggagatgtgt ataagagaca gggccgattc accatctcca ga 52
<210> 9
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 9
tcgtgggctc ggagatgtgt ataagagaca gatcgcctat ctgcaaatga ac 52
<210> 10
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 10
tcgtgggctc ggagatgtgt ataagagaca gagcaaattc accatctcca a 51
<210> 11
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 11
tcgtgggctc ggagatgtgt ataagagaca gagagacaat tccaggaact 50
<210> 12
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 12
tcgtgggctc ggagatgtgt ataagagaca gtgtatctgc aaacgaata 49
<210> 13
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 13
tcgtgggctc ggagatgtgt ataagagaca gctgaagctg agctctgtga c 51
<210> 14
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 14
tcgtgggctc ggagatgtgt ataagagaca gtacctgcag tggagcagcc t 51
<210> 15
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 15
tcgtgggctc ggagatgtgt ataagagaca gtgaactctg tgactcccga g 51
<210> 16
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 16
tcgtgggctc ggagatgtgt ataagagaca gagcctaaag gctgaggac 49
<210> 17
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 17
tcgtgggctc ggagatgtgt ataagagaca ggcggaatgt gtgcaggc 48
<210> 18
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 18
tcgtgggctc ggagatgtgt ataagagaca gactgggctc agagtcctct 50
<210> 19
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 19
tcgtgggctc ggagatgtgt ataagagaca gtggccctgg gaatataa 48
<210> 20
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 20
tcgtgggctc ggagatgtgt ataagagaca gatccccagg acgcagc 47
<210> 21
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 21
tcgtgggctc ggagatgtgt ataagagaca gagggggaca ctgtgcatgt 50
<210> 22
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 22
tcgtgggctc ggagatgtgt ataagagaca gtgaccccag caagggaa 48
<210> 23
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 23
tcgtgggctc ggagatgtgt ataagagaca gacaggcccc ctaccagc 48
<210> 24
<211> 58
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (47)..(47)
<223> r is a, or g
<220>
<221> misc_feature
<222> (53)..(53)
<223> r is a, or g
<400> 24
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnctgargag acrgtgac 58
<210> 25
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 25
tcgtgggctc ggagatgtgt ataagagaca gttcactctc acaatcagca gc 52
<210> 26
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 26
tcgtgggctc ggagatgtgt ataagagaca gtactttcac catcagcagc 50
<210> 27
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 27
tcgtgggctc ggagatgtgt ataagagaca gggatctggg acggatt 47
<210> 28
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 28
tcgtgggctc ggagatgtgt ataagagaca ggatttcaca ctgaaaatca gc 52
<210> 29
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 29
tcgtgggctc ggagatgtgt ataagagaca gactgaaaat cagcagagtg ga 52
<210> 30
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 30
tcgtgggctc ggagatgtgt ataagagaca gtggaggctg aggatgttgg 50
<210> 31
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 31
tcgtgggctc ggagatgtgt ataagagaca gttcactctc accatcagca g 51
<210> 32
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 32
tcgtgggctc ggagatgtgt ataagagaca gtctgggaca gatttcac 48
<210> 33
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 33
tcgtgggctc ggagatgtgt ataagagaca gtaataacat agaatctgag 50
<210> 34
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 34
tcgtgggctc ggagatgtgt ataagagaca gacaattaat cctgtggaag ct 52
<210> 35
<211> 58
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (46)..(46)
<223> w is a, or t
<220>
<221> misc_feature
<222> (52)..(52)
<223> y is c, or t
<400> 35
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nngatwtcca cyttggtc 58
<210> 36
<211> 58
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (51)..(51)
<223> s is c, or g
<400> 36
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nngatctcca scttggtc 58
<210> 37
<211> 58
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 37
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnaatctcca gtcgtgtc 58
<210> 38
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 38
tcgtgggctc ggagatgtgt ataagagaca ggaggatgag gctgattatt ac 52
<210> 39
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 39
tcgtgggctc ggagatgtgt ataagagaca gatcactggc ctctggcctg a 51
<210> 40
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 40
tcgtgggctc ggagatgtgt ataagagaca gatcaccgga ctccagact 49
<210> 41
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 41
tcgtgggctc ggagatgtgt ataagagaca gaccaattct ctggttccaa g 51
<210> 42
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 42
tcgtgggctc ggagatgtgt ataagagaca gctccaggct gaggacgag 49
<210> 43
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 43
tcgtgggctc ggagatgtgt ataagagaca gatccctgag cgattctctg g 51
<210> 44
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 44
tcgtgggctc ggagatgtgt ataagagaca gggatgaggc tgactatta 49
<210> 45
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 45
tcgtgggctc ggagatgtgt ataagagaca ggaggatgag gctgattatt ac 52
<210> 46
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 46
tcgtgggctc ggagatgtgt ataagagaca gttctctggc tccagctca 49
<210> 47
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 47
tcgtgggctc ggagatgtgt ataagagaca gatcagcagg gtcctgac 48
<210> 48
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 48
tcgtgggctc ggagatgtgt ataagagaca gggatgaggc tgactatta 49
<210> 49
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (37)..(37)
<223> y is c, or t
<220>
<221> misc_feature
<222> (42)..(42)
<223> r is a, or g
<400> 49
tcgtgggctc ggagatgtgt ataagagaca gtccagyctg argatgaggc t 51
<210> 50
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 50
tcgtgggctc ggagatgtgt ataagagaca gatctctgga ctgaagactg a 51
<210> 51
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 51
tcgtgggctc ggagatgtgt ataagagaca gggcaaagct gccctgac 48
<210> 52
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 52
tcgtgggctc ggagatgtgt ataagagaca gatgatgaat ctgattatta 50
<210> 53
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 53
tcgtgggctc ggagatgtgt ataagagaca gaccatcaag aacatccagg 50
<210> 54
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 54
tcgtgggctc ggagatgtgt ataagagaca gctccagcct gaggacgagg 50
<210> 55
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (51)..(51)
<223> s is c, or g
<400> 55
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnaggacggt saccttggt 59
<210> 56
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (56)..(56)
<223> b is t, c, or g
<400> 56
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnaggacggt cagctbggt 59
<210> 57
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 57
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnaaaatgat cagctgggt 59
<210> 58
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 58
tcgtgggctc ggagatgtgt ataagagaca gagatccggt ccacaaagct gga 53
<210> 59
<211> 55
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 59
tcgtgggctc ggagatgtgt ataagagaca gatcaattcc ctggagcttg gtgac 55
<210> 60
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 60
tcgtgggctc ggagatgtgt ataagagaca gagtcgcttc tcacctgaat gc 52
<210> 61
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 61
tcgtgggctc ggagatgtgt ataagagaca ggctctgaga tgaatgtgag 50
<210> 62
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 62
tcgtgggctc ggagatgtgt ataagagaca gtctgagctg aatgtgaacg cct 53
<210> 63
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 63
tcgtgggctc ggagatgtgt ataagagaca gggctacaat gtctccaga 49
<210> 64
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 64
tcgtgggctc ggagatgtgt ataagagaca ggctgctccc tcccagacat ct 52
<210> 65
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (38)..(38)
<223> r is a, or g
<400> 65
tcgtgggctc ggagatgtgt ataagagaca gggatccrtc tccactctga 50
<210> 66
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 66
tcgtgggctc ggagatgtgt ataagagaca gcggttctct gcagagagg 49
<210> 67
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 67
tcgtgggctc ggagatgtgt ataagagaca ggtctctact ctgaagatc 49
<210> 68
<211> 55
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 68
tcgtgggctc ggagatgtgt ataagagaca ggacttgcac tctgaactaa acctg 55
<210> 69
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 69
tcgtgggctc ggagatgtgt ataagagaca ggctcccaga catctgtgta 50
<210> 70
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 70
tcgtgggctc ggagatgtgt ataagagaca gctcactctg gagtctgctg cct 53
<210> 71
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 71
tcgtgggctc ggagatgtgt ataagagaca gagtagactc cactctcaag at 52
<210> 72
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 72
tcgtgggctc ggagatgtgt ataagagaca gcagggactc agctgtgta 49
<210> 73
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 73
tcgtgggctc ggagatgtgt ataagagaca gactatcatt ctgaactgaa ca 52
<210> 74
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 74
tcgtgggctc ggagatgtgt ataagagaca gaactggagg attctggagt tta 53
<210> 75
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 75
tcgtgggctc ggagatgtgt ataagagaca ggacatccgc tcaccaggcc tg 52
<210> 76
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 76
tcgtgggctc ggagatgtgt ataagagaca ggagatccag gctacgaagc ttga 54
<210> 77
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 77
tcgtgggctc ggagatgtgt ataagagaca gaacggaacg tcttccacgc tg 52
<210> 78
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 78
tcgtgggctc ggagatgtgt ataagagaca gagcatcctg aggatccagc ag 52
<210> 79
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 79
tcgtgggctc ggagatgtgt ataagagaca gctcactgtg acatcggccc aa 52
<210> 80
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 80
tcgtgggctc ggagatgtgt ataagagaca gtctgacagt gaccagtgcc cat 53
<210> 81
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 81
tcgtgggctc ggagatgtgt ataagagaca gtgtaccttg gagatccagt cca 53
<210> 82
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 82
tcgtgggctc ggagatgtgt ataagagaca gtgcagcctg gcaatcctgt 50
<210> 83
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 83
tcgtgggctc ggagatgtgt ataagagaca ggatggatac agtgtctctc ga 52
<210> 84
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (41)..(41)
<223> r is a, or g
<220>
<221> misc_feature
<222> (49)..(49)
<223> y is c, or t
<400> 84
tcgtgggctc ggagatgtgt ataagagaca gctgaccctg ragtctgcya g 51
<210> 85
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 85
tcgtgggctc ggagatgtgt ataagagaca ggaagggtac aaagtctctc ga 52
<210> 86
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 86
tcgtgggctc ggagatgtgt ataagagaca gctgattctg gagtccgcca gca 53
<210> 87
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 87
tcgtgggctc ggagatgtgt ataagagaca gtcaactctg actgtgagca aca 53
<210> 88
<211> 50
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 88
tcgtgggctc ggagatgtgt ataagagaca gcaggaccgg cagttcatcc 50
<210> 89
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 89
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacaactg tgagtctgg 59
<210> 90
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 90
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacaacgg ttaacctgg 59
<210> 91
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 91
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacaacag tgagccaac 59
<210> 92
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 92
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacaagac agagagctg 59
<210> 93
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (30)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (35)..(38)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<400> 93
tcggcagcgt cagatgtgta taagagacan nnntnnnntn ntacaggatg gagagtcga 59
<210> 94
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (30)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (35)..(38)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<400> 94
tcggcagcgt cagatgtgta taagagacan nnntnnnntn ntacacagtg agcctggtc 59
<210> 95
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 95
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacacggt gagccgtgt 59
<210> 96
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (48)..(48)
<223> y is c, or t
<400> 96
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacagyac ggtcagcct 59
<210> 97
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 97
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacactgt cagccgggt 59
<210> 98
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 98
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacactga gagccgggt 59
<210> 99
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 99
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntacaccag gagccgcgt 59
<210> 100
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (30)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (35)..(38)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<400> 100
tcggcagcgt cagatgtgta taagagacan nnntnnnntn ntacaccgtg agcctggtg 59
<210> 101
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 101
tcgtgggctc ggagatgtgt ataagagaca gactccagga aagtatgaca ct 52
<210> 102
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 102
tcgtgggctc ggagatgtgt ataagagaca gaaatctaat tgaaaatga 49
<210> 103
<211> 48
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 103
tcgtgggctc ggagatgtgt ataagagaca gttggaatca ggaatcag 48
<210> 104
<211> 49
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 104
tcgtgggctc ggagatgtgt ataagagaca ggaaacgtct acatccact 49
<210> 105
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 105
tcgtgggctc ggagatgtgt ataagagaca gacttcaatc cttaccatca agt 53
<210> 106
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 106
tcgtgggctc ggagatgtgt ataagagaca gttcttagag aaagaagatg ag 52
<210> 107
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 107
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntgttgttc cactgccaa 59
<210> 108
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 108
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntgttccgg gaccaaata 59
<210> 109
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 109
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntgttacta tgagcctag 59
<210> 110
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 110
tcgtgggctc ggagatgtgt ataagagaca gaatccgtcg ccttaaccat ttc 53
<210> 111
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 111
tcgtgggctc ggagatgtgt ataagagaca ggatattgca aagaacctgg ct 52
<210> 112
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 112
tcgtgggctc ggagatgtgt ataagagaca gatctctcca gtaaggactg aa 52
<210> 113
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 113
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntccacagt cacacgggt 59
<210> 114
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 114
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntccacgaa gagtttgat 59
<210> 115
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 115
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntccacgaa gagtttgat 59
<210> 116
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 116
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nntccagata ggttccttt 59
<210> 117
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 117
tctcgtgggc tcggagatgt gtataagaga cagataaagg cgaggagcat a 51
<210> 118
<211> 61
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (30)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (35)..(38)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<400> 118
tcggcagcgt cagatgtgta taagagacan nnntnnnntn nttacctgag gagacggtga 60
c 61
<210> 119
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 119
tctcgtgggc tcggagatgt gtataagaga cagaccagca gattcaaatc tatg 54
<210> 120
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 120
tctcgtgggc tcggagatgt gtataagaga cagactctgt ggcattattg catt 54
<210> 121
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 121
tctcgtgggc tcggagatgt gtataagaga cagcacctgc tggatacaac actg 54
<210> 122
<211> 53
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 122
tctcgtgggc tcggagatgt gtataagaga cagtgacaga gcaaaacatg aac 53
<210> 123
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 123
tctcgtgggc tcggagatgt gtataagaga cagtaatgac tggggagcaa atct 54
<210> 124
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 124
tctcgtgggc tcggagatgt gtataagaga cagactggtt ggcgtggttt agag 54
<210> 125
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 125
tctcgtgggc tcggagatgt gtataagaga cagttctgaa agaaacgaaa gc 52
<210> 126
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 126
tctcgtgggc tcggagatgt gtataagaga caggagcaag cgcccaataa at 52
<210> 127
<211> 52
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 127
tctcgtgggc tcggagatgt gtataagaga cagaatgcca tctcaaatcc aa 52
<210> 128
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 128
acagcctaca tggagctgag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctcc tcag 134
<210> 129
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 129
acagacctga gcagcctgac aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
tcactgtctc ctcag 135
<210> 130
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 130
agcagcctga gatctgagga caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
tcactgtctc ctcag 135
<210> 131
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 131
acatggaccc tgtggacaca aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctct tcag 134
<210> 132
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 132
ctgtatctgc aaatgaacag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctcc tcag 134
<210> 133
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 133
gcagattcac catctccaga aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctct tcag 134
<210> 134
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 134
agcctgaaaa ccgaggacac aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
cactgtctcc tcag 134
<210> 135
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 135
ggccgattca ccatctccag aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
tcaccgtctc ttcag 135
<210> 136
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 136
atcgcctatc tgcaaatgaa caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
tcaccgtctc ctcag 135
<210> 137
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 137
agcaaattca ccatctccaa aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
cactgtctcc tcag 134
<210> 138
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 138
agagacaatt ccaggaacta agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgtc 120
accgtctctt cag 133
<210> 139
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 139
tgtatctgca aacgaataaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtgtca 120
ccgtctcctc ag 132
<210> 140
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 140
ctgaagctga gctctgtgac aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
cactgtctcc tcag 134
<210> 141
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 141
tacctgcagt ggagcagcct aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctcc tcag 134
<210> 142
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 142
tgaactctgt gactcccgag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtgt 120
caccgtctct tcag 134
<210> 143
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 143
agcctaaagg ctgaggacaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtgtca 120
ctgtctcctc ag 132
<210> 144
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 144
gcggaatgtg tgcaggcaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtgtcac 120
cgtctcctca g 131
<210> 145
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 145
actgggctca gagtcctcta agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgtc 120
actgtctcct cag 133
<210> 146
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 146
tggccctggg aatataaaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtgtcac 120
tgtctcctca g 131
<210> 147
<211> 130
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 147
atccccagga cgcagcaagc gtctgagtac atcagtgccg ttgctaagta actactttgg 60
cgcgacagtt atctacgtag gtcaggactg caatagtgaa tgtcgacgtt gcgtgtcacc 120
gtctcttcag 130
<210> 148
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 148
agggggacac tgtgcatgta agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgtc 120
accgtctcct cag 133
<210> 149
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 149
tgaccccagc aagggaaaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtgtcac 120
cgtctcttca g 131
<210> 150
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 150
acaggccccc taccagcaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtgtcac 120
tgtctcctca g 131
<210> 151
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 151
ttcactctca caatcagcag caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
accaaggtgg aaatc 135
<210> 152
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 152
tactttcacc atcagcagca agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgac 120
caagctggag atc 133
<210> 153
<211> 130
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 153
ggatctggga cggattaagc gtctgagtac atcagtgccg ttgctaagta actactttgg 60
cgcgacagtt atctacgtag gtcaggactg caatagtgaa tgtcgacgtt gcgtgaccaa 120
ggtggaaatc 130
<210> 154
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 154
gatttcacac tgaaaatcag caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
accaaagtgg atatc 135
<210> 155
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 155
actgaaaatc agcagagtgg aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
accaagctgg agatc 135
<210> 156
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 156
tggaggctga ggatgttgga agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgac 120
caaagtggat atc 133
<210> 157
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 157
ttcactctca ccatcagcag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtga 120
ccaaggtgga gatc 134
<210> 158
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 158
tctgggacag atttcacaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtgacac 120
gactggagat t 131
<210> 159
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 159
taataacata gaatctgaga agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtgac 120
caaggtggag atc 133
<210> 160
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 160
acaattaatc ctgtggaagc taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
acacgactgg agatt 135
<210> 161
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 161
gaggatgagg ctgattatta caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ccaaggtcac cgtcct 136
<210> 162
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 162
atcactggcc tctggcctga aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
caagctgacc gtcct 135
<210> 163
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 163
atcaccggac tccagactaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtaccc 120
agctgatcat ttt 133
<210> 164
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 164
accaattctc tggttccaag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
caaggtcacc gtcct 135
<210> 165
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 165
ctccaggctg aggacgagaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtaccg 120
agctgaccgt cct 133
<210> 166
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 166
atccctgagc gattctctgg aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
caagctgacc gtcct 135
<210> 167
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 167
ggatgaggct gactattaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtaccc 120
agctgatcat ttt 133
<210> 168
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 168
gaggatgagg ctgattatta caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ccaaggtcac cgtcct 136
<210> 169
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 169
ttctctggct ccagctcaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtacca 120
agctgaccgt cct 133
<210> 170
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 170
atcagcaggg tcctgacaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtaccga 120
gctgaccgtc ct 132
<210> 171
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 171
ggatgaggct gactattaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtacca 120
aggtcaccgt cct 133
<210> 172
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (6)..(6)
<223> y is c, or t
<220>
<221> misc_feature
<222> (11)..(11)
<223> r is a, or g
<400> 172
tccagyctga rgatgaggct aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
caaggtgacc gtcct 135
<210> 173
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 173
atctctggac tgaagactga aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
ccagctgacc gtcct 135
<210> 174
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 174
ggcaaagctg ccctgacaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtaccaa 120
ggtgaccgtc ct 132
<210> 175
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 175
atgatgaatc tgattattaa agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtacc 120
cagctgaccg tcct 134
<210> 176
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 176
accatcaaga acatccagga agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtacc 120
aaggtcaccg tcct 134
<210> 177
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 177
ctccagcctg aggacgagga agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtacc 120
aagctgaccg tcct 134
<210> 178
<211> 138
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 178
agatccggtc cacaaagctg gaaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
accagactca cagttgta 138
<210> 179
<211> 140
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 179
atcaattccc tggagcttgg tgacaagcgt ctgagtacat cagtgccgtt gctaagtaac 60
tactttggcg cgacagttat ctacgtaggt caggactgca atagtgaatg tcgacgttgc 120
gtaccagact cacagttgta 140
<210> 180
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 180
agtcgcttct cacctgaatg caagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ccagactcac agttgta 137
<210> 181
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 181
gctctgagat gaatgtgaga agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtacc 120
aggttaaccg ttgta 135
<210> 182
<211> 138
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 182
tctgagctga atgtgaacgc ctaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
accagactca cagttgta 138
<210> 183
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 183
ggctacaatg tctccagaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtacca 120
ggttaaccgt tgta 134
<210> 184
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 184
gctgctccct cccagacatc taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ccaggttaac cgttgta 137
<210> 185
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> 1
<222> (7)..(7)
<223> r is a, or g
<400> 185
ggatccrtct ccactctgaa agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtagt 120
tggctcactg ttgta 135
<210> 186
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 186
cggttctctg cagagaggaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgttcga 120
ctctccatcc t 131
<210> 187
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 187
gtctctactc tgaagatcaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtacac 120
ggctcaccgt gta 133
<210> 188
<211> 139
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 188
gacttgcact ctgaactaaa cctaagcgtc tgagtacatc agtgccgttg ctaagtaact 60
actttggcgc gacagttatc tacgtaggtc aggactgcaa tagtgaatgt cgacgttgcg 120
taccagactc acagttgta 139
<210> 189
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 189
gctcccagac atctgtgtaa agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtaca 120
cggctcaccg tgta 134
<210> 190
<211> 138
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 190
ctcactctgg agtctgctgc ctaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
accaggttaa ccgttgta 138
<210> 191
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 191
agtagactcc actctcaaga taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
gttggctcac tgttgta 137
<210> 192
<211> 131
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 192
cagggactca gctgtgtaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgttcga 120
ctctccatcc t 131
<210> 193
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 193
actatcattc tgaactgaac aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ccagactcac agttgta 137
<210> 194
<211> 138
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 194
aactggagga ttctggagtt taaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
agttggctca ctgttgta 138
<210> 195
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (131)..(131)
<223> r is a, or g
<400> 195
gacatccgct caccaggcct gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ggctgaccgt rctg 134
<210> 196
<211> 139
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 196
gagatccagg ctacgaagct tgaaagcgtc tgagtacatc agtgccgttg ctaagtaact 60
actttggcgc gacagttatc tacgtaggtc aggactgcaa tagtgaatgt cgacgttgcg 120
taccaggtta accgttgta 139
<210> 197
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 197
aacggaacgt cttccacgct gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
accaggctca ctgtg 135
<210> 198
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 198
agcatcctga ggatccagca gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
gttggctcac tgttgta 137
<210> 199
<211> 134
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 199
ctcactgtga catcggccca aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtt 120
cgactctcca tcct 134
<210> 200
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 200
tctgacagtg accagtgccc ataagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
caccaggctc acggt 135
<210> 201
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (132)..(132)
<223> r is a, or g
<400> 201
tgtaccttgg agatccagtc caaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
aggctgaccg trctg 135
<210> 202
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 202
tgcagcctgg caatcctgta agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtacg 120
cggctcctgg tg 132
<210> 203
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 203
gatggataca gtgtctctcg aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtg 120
accaggctca ctgtg 135
<210> 204
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (10)..(10)
<223> r is a, or g
<220>
<221> misc_feature
<222> (18)..(18)
<223> y is c, or t
<400> 204
ctgaccctgr agtctgcyag aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgttgcgtac 120
ccggctctca gtg 133
<210> 205
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 205
gaagggtaca aagtctctcg aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
cccagctctc tgtcttg 137
<210> 206
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 206
ctgattctgg agtccgccag caaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
acgcggctcc tggtg 135
<210> 207
<211> 138
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 207
tcaactctga ctgtgagcaa caaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
acccagctct ctgtcttg 138
<210> 208
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 208
caggaccggc agttcatcca agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgcgtcac 120
caggctcacg gt 132
<210> 209
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 209
actccaggaa agtatgacac taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgtt 120
tggcagtgga acaaca 136
<210> 210
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 210
aaatctaatt gaaaatgaaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgttatt 120
tggtcccgga aca 133
<210> 211
<211> 132
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 211
ttggaatcag gaatcagaag cgtctgagta catcagtgcc gttgctaagt aactactttg 60
gcgcgacagt tatctacgta ggtcaggact gcaatagtga atgtcgacgt tgcgtactag 120
gctcatagta ac 132
<210> 212
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 212
gaaacgtcta catccactaa gcgtctgagt acatcagtgc cgttgctaag taactacttt 60
ggcgcgacag ttatctacgt aggtcaggac tgcaatagtg aatgtcgacg ttgcgtttgg 120
cagtggaaca aca 133
<210> 213
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 213
acttcaatcc ttaccatcaa gtaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
tatttggtcc cggaaca 137
<210> 214
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 214
ttcttagaga aagaagatga gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
ctaggctcat agtaac 136
<210> 215
<211> 137
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 215
aatccgtcgc cttaaccatt tcaagcgtct gagtacatca gtgccgttgc taagtaacta 60
ctttggcgcg acagttatct acgtaggtca ggactgcaat agtgaatgtc gacgttgcgt 120
acccgtgtga ctgtgga 137
<210> 216
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 216
gatattgcaa agaacctggc taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
cacaactcat cgtgga 136
<210> 217
<211> 136
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 217
atctctccag taaggactga aaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgcgta 120
tcaaactctt cgtgga 136
<210> 218
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 218
accagcagat tcaaatctat gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acggtcaccg 120
tctcctcagg taa 133
<210> 219
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 219
actctgtggc attattgcat taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgtcac 120
cgtctcctca ggtaa 135
<210> 220
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 220
cacctgctgg atacaacact gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acggtcaccg 120
tctcctcagg taa 133
<210> 221
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 221
tgacagagca aaacatgaac aagcgtctga gtacatcagt gccgttgcta agtaactact 60
ttggcgcgac agttatctac gtaggtcagg actgcaatag tgaatgtcga cgtgtcaccg 120
tctcctcagg taa 133
<210> 222
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 222
taatgactgg ggagcaaatc taagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgtcac 120
cgtctcctca ggtaa 135
<210> 223
<211> 135
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 223
actggttggc gtggtttaga gaagcgtctg agtacatcag tgccgttgct aagtaactac 60
tttggcgcga cagttatcta cgtaggtcag gactgcaata gtgaatgtcg acgttgtcac 120
cgtctcctca ggtaa 135
<210> 224
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 224
ttctgaaaga aacgaaagca agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgtcaccg 120
tctcctcagg taa 133
<210> 225
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 225
gagcaagcgc ccaataaata agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgtcaccg 120
tctcctcagg taa 133
<210> 226
<211> 133
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 226
aatgccatct caaatccaaa agcgtctgag tacatcagtg ccgttgctaa gtaactactt 60
tggcgcgaca gttatctacg taggtcagga ctgcaatagt gaatgtcgac gttgtcaccg 120
tctcctcagg taa 133
<210> 227
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 227
caagcagaag acggcatacg agattcgcct tatcgtgggc tcggaga 47
<210> 228
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 228
caagcagaag acggcatacg agatctagta cgtcgtgggc tcggaga 47
<210> 229
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 229
caagcagaag acggcatacg agatttctgc cttcgtgggc tcggaga 47
<210> 230
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 230
caagcagaag acggcatacg agatgctcag gatcgtgggc tcggaga 47
<210> 231
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 231
caagcagaag acggcatacg agataggagt cctcgtgggc tcggaga 47
<210> 232
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 232
caagcagaag acggcatacg agatcatgcc tatcgtgggc tcggaga 47
<210> 233
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 233
caagcagaag acggcatacg agatgtagag agtcgtgggc tcggaga 47
<210> 234
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 234
caagcagaag acggcatacg agatcctctc tgtcgtgggc tcggaga 47
<210> 235
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 235
caagcagaag acggcatacg agatagcgta gctcgtgggc tcggaga 47
<210> 236
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 236
caagcagaag acggcatacg agatcagcct cgtcgtgggc tcggaga 47
<210> 237
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 237
caagcagaag acggcatacg agattgcctc tttcgtgggc tcggaga 47
<210> 238
<211> 47
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 238
caagcagaag acggcatacg agattcctct actcgtgggc tcggaga 47
<210> 239
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 239
aatgatacgg cgaccaccga gatctacact agatcgcgtc ggcagcgtca g 51
<210> 240
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 240
aatgatacgg cgaccaccga gatctacacc tctctatgtc ggcagcgtca g 51
<210> 241
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 241
aatgatacgg cgaccaccga gatctacact atcctctgtc ggcagcgtca g 51
<210> 242
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 242
aatgatacgg cgaccaccga gatctacaca gagtagagtc ggcagcgtca g 51
<210> 243
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 243
aatgatacgg cgaccaccga gatctacacg taaggaggtc ggcagcgtca g 51
<210> 244
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 244
aatgatacgg cgaccaccga gatctacaca ctgcatagtc ggcagcgtca g 51
<210> 245
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 245
aatgatacgg cgaccaccga gatctacaca aggagtagtc ggcagcgtca g 51
<210> 246
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 246
aatgatacgg cgaccaccga gatctacacc taagcctgtc ggcagcgtca g 51
<210> 247
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 247
aatgatacgg cgaccaccga gatctacacg cgtaagagtc ggcagcgtca g 51
<210> 248
<211> 31
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 248
tcgtgggctc ggagatgtgt ataagagaca g 31
<210> 249
<211> 31
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 249
gtcggcagcg tcagatgtgt ataagagaca g 31
<210> 250
<211> 54
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 250
tcgtgggctc ggagatgtgt ataagagaca gagtgtgaca tggtgtatct ctgc 54
<210> 251
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (31)..(34)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (36)..(39)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (41)..(42)
<223> n is a, c, g, or t
<400> 251
gtcggcagcg tcagatgtgt ataagagaca nnnntnnnnt nnctgaggta gtcagtcag 59
<210> 252
<211> 51
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 252
tcgtgggctc ggagatgtgt ataagagaca gctcactcct tttgcagacc a 51
<210> 253
<211> 59
<212> DNA
<213>artificial sequence (artificial sequence)
<220>
<221> misc_feature
<222> (30)..(33)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (35)..(38)
<223> n is a, c, g, or t
<220>
<221> misc_feature
<222> (40)..(41)
<223> n is a, c, g, or t
<400> 253
tcggcagcgt cagatgtgta taagagacan nnntnnnntn nctgaggttt ttctagacg 59

Claims (10)

1. a kind of multiple PCR primer group for detecting minimal residual disease MRD, which is characterized in that including following primer pair:
The primer pair of IgH (VDJ) is detected, upstream primer sequence is as shown in SEQ ID NO:1-16, downstream primer sequence such as SEQ Shown in ID NO:24；
The primer pair of IgH (DJ) is detected, upstream primer sequence is as shown in SEQ ID NO:17-23, downstream primer sequence such as SEQ Shown in ID NO:24；
The primer pair of IgK is detected, upstream primer sequence is as shown in SEQ ID NO:25-34, downstream primer sequence such as SEQ ID Shown in NO:35-37；
The primer pair of IgL is detected, upstream primer sequence is as shown in SEQ ID NO:38-54, downstream primer sequence such as SEQ ID Shown in NO:55-57；
The primer pair of TRB is detected, upstream primer sequence is as shown in SEQ ID NO:58-88, downstream primer sequence such as SEQ ID Shown in NO:89-100；
The primer pair of TRG is detected, upstream primer sequence is as shown in SEQ ID NO:101-106, downstream primer sequence such as SEQ ID Shown in NO:107-109；
The primer pair of TRD is detected, upstream primer sequence is as shown in SEQ ID NO:110-112, downstream primer sequence such as SEQ ID Shown in NO:113-116；
The primer pair of BCL1/IGH is detected, upstream primer sequence is as shown in SEQ ID NO:117, downstream primer sequence such as SEQ ID Shown in NO:118；
The primer pair of BCL2/IGH is detected, upstream primer sequence is as shown in SEQ ID NO:119-127, and downstream primer sequence is such as Shown in SEQ ID NO:118；
Wherein N:A or T or C or G；R:A or G；W:A or T；Y:C or T；S:C or G；B:T or C or G.
2. the kit comprising multiple PCR primer described in claim 1.
3. the method for a kind of couple of T, B-cell receptor progress high-flux sequence, which comprises the following steps:
1) sample to be tested is taken；
2) sample to be tested is expanded using multiple PCR primer group described in claim 1；
3) by the second wheel PCR, the upper machine of illumina high-flux sequence instrument is added respectively in the both ends of step 2) amplified production Sequencing primer and label carry out high-flux sequence to amplified production after purification；
4) bioinformatic analysis carries out quantitative and qualitative analysis to sample to be tested.
4. high-flux sequence method according to claim 3, which is characterized in that according to target gene (IGH in step (2) (VDJ), IGH (DJ), IGK, IGL, TCR β, TCR γ, TCR δ, BCL1/IGH, BCL2/IGH), it is added in PCR reaction system The internal reference DNA of the known array of any three corresponding 1%input DNA profiling amounts, carries out specific amplification with sample simultaneously.
5. high-flux sequence method according to claim 4, which is characterized in that the internal reference DNA sequence dna is SEQ ID Shown in NO:128-226.
6. high-flux sequence method according to claim 3, which is characterized in that in the step (2), expand simultaneously House Keeping gene (beta-Actin or GAPDH) carries out specific amplification with sample simultaneously, and beta-Actin is used Primer sequence be SEQ ID NO:250-251, the primer sequence that uses of amplification GAPDH is SEQ ID NO:252-253.
7. high-flux sequence method according to claim 3, which is characterized in that sequencing primer and mark described in step (3) The sequence of label is shown in SEQ ID NO:227-247, and wherein upstream primer sequence is downstream shown in SEQ ID NO:227-238 Primer sequence is shown in SEQ ID NO:239-247.
8. high-flux sequence method according to claim 3, which is characterized in that used in the bioinformatic analysis Calculation: using internal reference as quantitative criterion；MRD=(Rc/(Rstd/Nstd)/2)/NTOT, wherein RcIt is sequencing gained cancer cell Reads number, RstdIt is the reads number of sequencing gained internalcontrol sequence, NstdIt is the molecular number for adding internal reference, NTOTThe total matter of=internal reference Amount/each reference molecules molal weight, NTOT=MTOT/ K, NTOTIt is the quantity for always having core living cells, MTOTIt is always to have core living cells Quality, that is, input DNA quality, K be each cell DNA total amount 0.0064ng.
9. high-flux sequence method according to claim 3, which is characterized in that used in the bioinformatic analysis Calculation are as follows: using House Keeping gene as quantitative criterion, there are two types of calculation methods；
1) .MRD=NC/NH, wherein NCIt is sequencing gained cancer cell number according to have passed through UMB analysis treated cancer cell number, NHIt is Sequencing gained House Keeping gene data have passed through at UMB analysis after total template cell number；
2) .MRD=(Rc/(RH/TH)/2)/NTOT, wherein RcIt is the reads number of sequencing gained cancer cell, RHIt is sequencing gained The reads number of House Keeping gene, THIt is the template number of House Keeping gene magnification, NTOT=internal reference gross mass/ Each reference molecules molal weight, NTOT=MTOT/ K, NTOTIt is the quantity for always having core living cells, MTOTIt is the matter for always having core living cells Amount is the quality of input DNA, and K is each cell DNA total amount 0.0064ng.
10. purposes of the multiple PCR primer group described in claim 1 in preparation detection minimal residual disease MRD kit.
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