CN114242167B - Product and method for absolute quantification of metagenomic blood sample and application of product and method - Google Patents

Abstract

The invention relates to the technical field of metagenome sequencing, and particularly discloses a product, a method and application of absolute quantification of a metagenome blood sample.

Description

Product and method for absolute quantification of metagenomic blood sample and application of product and method

Technical Field

The invention relates to the technical field of gene sequencing, in particular to a product and a method for absolute quantification of metagenomic blood samples and application thereof.

Technical Field

A retrospective study in the J.He.Sonchi.infectious agent showed that 76 cases of hematological diseases, namely 538 cases in 2012-2013, had an infection rate of 14.13%. Because inflammation is not easily limited due to light inflammatory reaction, local infection often spreads to systemic infection, so that severe infection forms such as septicemia or toxic shock in infection are caused, the death rate is high, and the treatment difficulty is high, so that a metagenomic detection technology (mNGS) has been developed. In order to provide reasonable medication guidance for patients more quickly at an earlier stage, the accuracy of the mNGS detection technology is particularly important.

At present, most mNGS detection reports on the market only show the number and relative abundance (belonging to the whole) of detected pathogenic bacteria in blood samples, and the cell number of actual pathogenic bacteria is unknown, so that a clinician has a certain limit on interpretation and analysis of results. The invention has been developed in view of the above in order to better achieve the role of metagenome in diagnosis of the degree of infection in the blood stream of different patients and dynamic monitoring of the course of treatment of the same patient.

Disclosure of Invention

The core technical problem to be solved by the present invention is to find a method that enables absolute quantification of microbial infections in metagenomic samples, in particular blood samples.

In order to solve the technical problems, the invention provides the following technical scheme:

the invention firstly provides a method for absolute quantification of metagene samples, which comprises the following steps:

1) Taking a metagenome sample to be detected, labeling the sequence mixture, and uniformly mixing to obtain a mixed sample;

2) Mixing sample DNA extraction, library establishment and sequencing to obtain machine-on data;

3) Analyzing respective detection proportions of pathogenic bacteria and tag sequences in the off-machine data, wherein the detection proportions are the proportion of the number of pathogenic bacteria or tag detection sequences to the total number of sequences;

4) Establishing a sample fitting linear relation, and fitting a sample linear relation formula by taking the detection proportion of each tag sequence as an abscissa and the known mass of the corresponding tag as an ordinate: y=kx;

5) Quantification of pathogenic bacteria: calculating the mass of the corresponding pathogenic bacteria according to a sample fitting linear relation y=kx and a pathogenic bacteria detection proportion x; defining a quality and copy number conversion formula, and calculating the copy number of pathogenic bacteria;

further, the copy number=6.02×10≡23×10+ (-9) ×mass (ng)/(genome length bp×660 g/mol).

Further, any one or more tags selected from SEQ ID NO.1-10 are included in the tag sequence mixture;

preferably, the number of the tag sequences is 3;

more preferably, the tag sequence is shown in SEQ ID NO.1-3.

Further, the tag concentration ratio of SEQ ID NO.1 to 2:3 in the tag sequence mixture is 50-100:5-10:0.5-1;

preferably, the concentration ratio of the SEQ ID NO.1 to the 2:3 tag is 100:10:1.

Further, the preparation method of the tag sequence mixture comprises the steps of preparing the mixture of tags shown in SEQ ID NO.1, SEQ ID NO. 2 and SEQ ID NO. 3 according to the concentration ratio of 100:10:1, and sub-packaging the mixture into a 30ul system for later use.

Further, the sample is a blood sample.

The invention also provides a tag sequence for absolute quantification of a metagenomic blood sample, the tag sequence comprising any one or more of SEQ ID nos. 1-10;

preferably, the number of the tags is three;

more preferably, the tag is SEQ ID NO.1-3.

Further, the concentration ratio of SEQ ID NO.1 to 2:3 is 50-100:5-10:0.5-1;

preferably, the concentration ratio is 100:10:1.

The invention also provides a product for absolute quantification of a metagenomic blood sample, said product comprising a tag sequence as defined in any one of the above.

The final concentrations of A, B, C (SEQ ID NO.1, 2, 3) in the pudendum, in the products were 0.0011ng/ul, 0.00011ng/ul, 0.000011ng/ul, respectively.

The invention also provides any one of the following applications of the tag sequence or the product described in any one of the above:

1) The application in the detection of blood sample infection;

2) Application in absolute quantification of metagenomic blood samples;

3) The method is applied to monitoring environmental and reagent background bacteria pollution;

4) The application of the method in judging false positive and false negative in auxiliary detection results.

The invention has the beneficial technical effects that:

1) The method can realize absolute quantification of the metagenome blood sample and has the advantages of simplicity, accuracy and the like.

2) The invention designs and screens the label sequence and is obtained by self-grinding and optimizing screening, and experiments prove that the label has good specificity, less background bacteria and high quantitative accuracy, can be prepared in a large amount at one time, has low cost and is convenient for production and use; meanwhile, the optimal quantitative system is obtained through label selection and label input quantity and input proportion optimization.

3) At present, most of blood sample metagenome detection results are presented to clinical sequences and relative abundance indexes which are only detected by pathogenic bacteria, and absolute quantitative indexes of the pathogenic bacteria can be increased by the method, so that the method plays a key role in judging and reading infection conditions of clinical patients.

4) The self-grinding prepared label system can not only realize absolute quantification of pathogenic bacteria and human sources in blood samples, but also monitor the pollution condition of background bacteria such as environment, reagents and the like and assist in judging false positive and false negative in detection results.

Drawings

Fig. 1: in the quantification of D1-1 samples, the tri-tag fits the graph according to the amount incorporated and the detected proportion.

Fig. 2: in the quantification of D1-1 samples, the single tag fits the graph according to the amount incorporated and the detected ratio.

Fig. 3: in the quantification of D1-1 samples, the ditag fits the graph according to the amount incorporated and the detected proportion.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Partial term definition

Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the invention are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.

As used in this application, the indefinite or definite article when used in reference to a singular noun e.g. "a" or "an", "the" includes a plural of that noun.

As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

The term "about" in the present invention means a range of accuracy that one skilled in the art can understand while still guaranteeing the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

The above terms or definitions are provided solely to aid in the understanding of the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.

The absolute quantification method of the metagene sample generally comprises the following steps:

1) Taking a metagenome sample to be detected, labeling the sequence mixture, and uniformly mixing to obtain a mixed sample; 2) Mixing sample DNA extraction, library establishment and sequencing to obtain machine-on data; 3) Analyzing the detection proportion of pathogenic bacteria and tag sequences in the machine-down data; 4) Establishing a sample fitting linear relation, and fitting a sample linear relation formula by taking the detection proportion of each tag sequence as an abscissa and the known mass of the corresponding tag as an ordinate: y=kx; 5) Quantification of pathogenic bacteria: calculating the mass of the corresponding pathogenic bacteria according to a sample fitting linear relation y=kx and a pathogenic bacteria detection proportion x; defining a quality and copy number conversion formula, and calculating the copy number of pathogenic bacteria;

in some embodiments, the copy number=6.02×10≡23×10+ (-9) mass (ng)/(genome length bp×660 g/mol). In some embodiments, any one or more tags selected from the group consisting of SEQ ID nos. 1-10 are included in the tag sequence mixture; in some preferred embodiments, the tag sequence number is 3; in some more preferred embodiments, the tag sequence is the tag sequence shown in SEQ ID NO.1-3. In some further preferred embodiments, the tag sequence mixture has a tag concentration ratio of SEQ ID NO.1:2:3 of 50-100:5-10:0.5-1; in some further preferred embodiments, the SEQ ID NO.1:2:3 tag concentration ratio is 100:10:1.

In some embodiments, the tag sequence may be first prepared into a tag mixture for later use, for example, Q3 according to the present invention is prepared by preparing the tag sequence mixture according to the concentration ratio=100:10:1 of the tags shown in SEQ ID nos. 1, 2, and 3, and packaging the mixture into 30ul tubes for later use.

It will be appreciated that the sample sources described herein may be varied and in some embodiments, such as blood, sputum, alveolar lavage, etc., as referred to in the examples of the specification, are not limiting. In some preferred embodiments, the sample is a blood sample.

The invention is illustrated below in connection with specific embodiments.

Example 1 design, screening, specificity verification of quantitative tags

In the embodiment, 20 clinical samples are selected, including four sample types of plasma, alveolar lavage fluid, sputum and cerebrospinal fluid, 10 tags (corresponding sequences are shown in SEQ ID NO.1-10, the tag sequences are selected from plant-derived Arabidopsis specific sequences, 40 continuous identical bases are not arranged among the tags in the selection process, and 30 continuous similar bases are not arranged between the tags and human-derived and pathogenic microorganism sequences) which are designed and screened by self-research of the company are added into 20 samples. Each tag was added in an amount of 0.03ng (i.e., 1.00E+07 copy), and each tag was repeated twice.

And (5) completing extraction, library establishment and machine starting according to a metagenome detection flow, and calculating a non-specific index.

Non-specific index = number of non-specific/number of specific detection sequences, the lower the non-specific index, the stronger the tag specificity

The results are shown in Table 2 below, where the non-specific index is the lowest for tag A, B, C, and therefore A, B, C is preferred as a candidate tag for absolute quantification.

EXAMPLE 2 detection of background bacteria of quantitative tags

The quantitative thought of the patent is that three tag sequences are doped into a clinical sample, and absolute quantification is finally realized through metagenome detection. During the detection of pathogenic microorganisms, the introduction of environmental background bacteria from an external source can interfere with the interpretation of clinical results, and therefore the degree of cleanliness of the incorporated tag sequences and the pathogen carried must be known.

The reaction mixture was split into 0.2ml eight rows of transparent PCR thin-walled tubes for 10 reactions using gDNA sample from Nanjac, bai Biotechnology Co., ltd. And diluting the three sample tag sequences to 0.003ng (namely 1.00E+06 copy) by using Buffer EB, taking 10ul of the three tag sequences after fully mixing, and split charging the three tag sequences into 5 PCR tubes, namely Test groups, namely Test1, test2, test3, test4 and Test5, and the remaining 5 are control groups, namely CK1, CK2, CK3, CK4 and CK5. In a 10-ten thousand-level clean workshop environment, experiments are completed according to metagenomic library establishment and on-line processes, and data analysis results are as follows:

from the above detection results, it can be found that the detection level of the label test group is consistent with that of the control group, and it can be shown that the label of the invention has no exogenous introduction of excessive pathogenic bacteria.

Example 3 quantitative tag incorporation determination

In order to determine the reasonable doping amount of the three candidate tags, tags A/B/C with different concentration gradients are respectively doped in the simulated plasma sample, and the doping amount of the tags is set between 0.0003ng and 0.3ng, namely the corresponding copy number is 1.00E+05-1.00E+08copy. The extraction, library establishment and machine-on are completed according to the metagenome detection flow, and the label detection results are as follows:

by analyzing the data, the number of the detected sequences of the three sample labels and the gradient doping amount are strongly correlated, R ² Greater than 0.9, i.e., three tags meet the basic requirements for application in quantification.

And according to the number of detected sequences, three labels are finally selected to be doped in a gradient mode by 10 times, the highest doping amount is 0.03ng (1.00E+07 copy), thousands of sequence levels are detected under the corresponding 20M data amount, so that the remaining two doping amounts are 0.003ng (1.00E+06 copy) and 0.0003ng (1.00E+05opy) respectively, hundreds of sequence levels and dozens of sequence levels are detected under the corresponding 20M data amount, namely 3 doped gradients can meet the detection range of pathogenic bacteria in most clinical samples, and accurate quantification is convenient to realize. Thus, the above three labels were prepared in the following table proportions (a: B: C concentration ratio=100:10:1) and dispensed into 30ul tubes for use, designated Q3.

Example 4 quantitative method of the invention set up

The specific quantification method established according to the present invention, based on the label and the addition established in the above examples 1-3, is as follows:

1) Sample processing: taking a plasma sample with a known volume (according to the extraction kit), adding 30ul of the Q3 mixture, and uniformly mixing;

2) Wet experiment: completing the steps of extraction, library establishment and machine starting in metagenome detection according to the operation instruction;

3) Dry experiment: analyzing the detection proportion of the 3 tag sequences in the potential pathogenic bacteria and the Q3 mixture in the sample by using the off-machine data;

4) Q3 fits a linear relationship: and fitting a linear relation formula of the sample by taking the 3-tag sequence detection proportion as an abscissa and the known mass (ng) in Q3 as an ordinate: y=kx;

5) Quantification of potential pathogenic bacteria: from this sample fit linear relationship y=kx and the known pathogen ratio x, the corresponding mass (ng) can be calculated;

6) Quantification of potential pathogenic bacteria, namely defining a quality (ng) and copy number conversion formula, and calculating corresponding copy number; and the sample extraction volume is known, and the corresponding copy concentration can be calculated;

copy number=6.02×10≡23×10+ (-9) ×mass (ng)/(genome length bp×660 g/mol);

the concentration was calculated from the number of copies finally quantified divided by the volume of sample used for extraction.

Example 5 quantitative detection of blood samples

20 clinical blood samples without staphylococcus aureus detection were selected, 500ul of plasma samples were taken, and staphylococcus aureus free nucleic acid fragments were added to a final concentration of 9.00E+04copy/ml (i.e., 1.47E+09fragment/ml). And finally, adding 30ul of Q3 prepared in advance into each sample to finish blood macro-gene detection (extraction, warehouse building and machine loading).

After 30ul of Q3 was added to the sample extraction library, the tag detection ratio was calculated for each library, as shown in the following Table, taking D1-1 as an example:

D1-1	Total DNA(ng)	detected proportion%
			A	0.033	6.9191
B	0.0033	0.6925
			C	0.00033	0.0519

Illustratively, FIG. 1 is a fitted linear curve of D1-1 according to the above tri-tag data.

According to the 3 label detection proportion and the corresponding quality in each sample, a fitting linear relation can be determined, and the slope K value of 20 sample fitting is calculated, wherein the specific values are as follows:

according to the known linear relationship, quantitative results of 20 samples incorporating pathogenic bacteria were obtained:

* The coefficient of variation cv= (standard deviation/average value) ×100%, and CV may reflect the absolute value of the degree of data dispersion, so the lower the CV value in this experiment, the more stable and accurate the quantitative method.

* The theoretical value of staphylococcus aureus is obtained by detecting the concentration (ng/ul) of the obtained DNA nucleic acid by using a Qubit quantitative instrument and then calculating by using a mass and copy number conversion formula. Because the pipettor and the quantifying instrument have deviation in the quantitative process of the Qubit, the theoretical value is only used as a comparison reference value in the quantitative process of the patent.

The quantitative concentration CV value of staphylococcus aureus of 20 clinical samples is 4.6% and the deviation from the theoretical value is about 1.57 times, thus illustrating the advantage of Q3 quantification in blood samples.

The Staphylococcus aureus copy number calculation procedure is described by taking D1-1 as an example:

species of species	Detected proportion%	K value	Total DNA(ng)	Genome Length (bp)	Copy
						A	6.9191	0.0048	0.033	2809	1.00E+07
B	0.6925	0.0048	0.0033	2800	1.00E+06
						C	0.0519	0.0048	0.00033	2848	1.00E+05
Staphylococcus aureus	42.14	0.0048	0.2022	2730000	6.76E+04

* Copy number=6.02×10≡23×10+ (-9)/(genome length bp×660 g/mol) mass-

* The concentration was further calculated from the number of copies finally quantified divided by the volume of sample used for extraction.

Example 5 quantitative result comparison of different tag count systems

This example uses single and double labels for quantification and compares the effect with the method of example 4 of the present invention.

According to the same quantitative concept as that of example 4, using D1-1 as an example, fig. 2 was fitted to each of the single and double tags according to the amount of incorporation and the ratio detected, and fig. 3 was a linear relationship (tag C was used for the single tag (tag C was tested to have a smaller CV than A, B, and therefore, tag C was selected), and tag b+c was used for the double tag).

Further, quantitative calculation is carried out on all samples by using the single label and the double label respectively, and the calculation results are as follows:

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as shown in the table, the single-label quantitative concentration CV value of staphylococcus aureus in 20 clinical samples is 9.8%, the double-label CV value is 6.4%, and the effect is weaker than that of the label CV value of example 3 by 4.6%. The result shows that the 3-label system three-point linear fitting accuracy is optimal, and the 3-label quantitative result is closer to a theoretical value and can be used for practical quantification.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Sequence listing

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gcctacgtct gcttttttct caagatattt ttttatagtt gcttgcttag caagaaactt 2520

tattaaaaag gtaataagaa agcagaatgg aaagaatttg aaattgggaa agattggatt 2580

tagatcatta tcgtatactt tgtggatatt ttcatggttt tgctgcttta tatataaact 2640

agctcaaaat tttatggaac cgaatatttt caatgaaaac aatatgaaat aacctaatct 2700

acacacatgg gttttctaac caatacatga ttcatattgt tggtctgcaa ttcattttac 2760

atagcattgc aaatatcacg atgggttaac caacaaatgt 2800

<210> 3

<211> 2848

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gcatacgcag cttcccttca tacttgtcag ttgaacactg ctctgcaagc tctatcactt 60

cactaagcgt cttagccaca gattgcaact gctcgttgca gagcttgttc ttggagaagc 120

aagggtggct tgagaggtca gataaacaag ctggtatctg ttcgatcttc gagatgatcg 180

tcttccatct tcctgtaaac ttcttaaccg tttttgcctt gctgaggacg gatggtatta 240

gcgagttgac gcgcgatagc cattcttcta ctgattgttt atccatgatc atctggttaa 300

cttgctgctc ttctgccatg gctccaaatt ttcccatggt tcccaattct gccattgttc 360

ccattggaca aaaaagaaag aagaaaattc acagcttctt atgctatttg aacattcatc 420

ttctgtgttt ttgaaaacct gaagagaaaa atcaaaactt tacaacctta tatacatgtc 480

tgagccatga ttttccaagt ctatagggaa acaaaagaac tttacctcat ctgaaattga 540

attgtagcca gatttctcta gctcgatcaa tgattggtcg actccaacct gcaatgaaag 600

acgaagagaa gaataaaaac tttttgagct tttacatgtc tagtgagaaa cataaagaaa 660

agccaaaacc ttaccttgtc tccttgttcc ttttaacttc acttggagaa aaaatatcca 720

aaatgttccc cagttcttta tgttcctcac tgacaaattc agatcacaaa tgctgattaa 780

ctgtacttgg agattaataa agatcaaaac tttatggaag acactagtga gtagaagaca 840

aaaaaaaata agtatagatg aaaacaacat ctaactaata atacacacaa gccattgctt 900

aagaaaagaa ggaagtttcc acacgacata tcatttcgag tggtttctag atgcatatcc 960

attaaatgct tctccctaat atatcatatg aataacacat caaactcatt aacagccacg 1020

tattatactc accctccatg aacaatcatt gtcaatgcaa aaaacttttc aaaactcgta 1080

aaggttagtt tcgtgattgg tgaaagtttt cttaaaaagg ggaaacacaa gctgtaattt 1140

tctgatcaga atgattcaat gtgttgatag taacagaaag gaaaaatctg gctgcggata 1200

aaagttaact tgcagacttt tctttgaaga gaagtcaata ggaagtctct gtctctctat 1260

tcattagacc acaaacacaa gttgtaactt gtaagcacca atgtgaggaa gttagatact 1320

gagtgatcta ataaatattg aaattagccc aaaatttagt agtaatcttt agtggatgat 1380

ctaaagatag attttgtgta cagtttatac cttcaatcta gtagttgact tgagtgtgaa 1440

aatggttgca tccagccaaa atttgatctg tgaaattacg agagattcag taagtataca 1500

cacgtagagg ttaagcaaga ttttacagaa ttatgcagta tgatctttag gcgagtcact 1560

gagagaaaca cactcaaaac aaaaataaac ttgtgactat gaatctaatt caaaaacgga 1620

agatcggaaa ggagatatag gggaaagtat tgatcttcat aattaaacaa aagaatcaca 1680

gaagaaaaat cactcaattg tctctgtcga ctaaaaagca attataagtt agagatgaca 1740

actttagctt tatttttcta gtgaatacaa acatcaaccg taaagaacca accttttaag 1800

attcagaatc ttaaaataat tttcatcttt tgtttacgaa aatcaaaaag acaaaaaaaa 1860

aaaaaaaaaa aaaactgagg gatcggaaat ttgaactttt tattctgaat caaaaggaag 1920

tagaaaaatt acagtgatac aagagaagat aatttggatg gaatactcac acttacccag 1980

aaaccaagtt tgttgtgtaa tgagaagaga aactaaagcg gaggagctgt tgacaaagct 2040

gctgagtgct gacttctctc ttcctcaaac tcaaactgtt cctctcagaa taaatatttg 2100

aaaattttca aatatctcaa cacgatttag agagagaaag ggaatgagag agataactag 2160

gacagaggac aaaaactaaa gacaacttcg atcaaatccc gattctttaa tgataatagt 2220

attttttttt aaaatatccg tatttatgta ttagatgatt tactttctcg aaacagttga 2280

caaaattata taaaaaaaga agaagataca acagtgtcac tgagattcga aaaagaagcc 2340

ctacggtacg atctacgagt tttctcttaa aagtgctaac ttcccttggg atccgacggc 2400

tgcgatccat cttactacat tcaattttga ccgttagatc ttgttttgtt tcataacgtt 2460

acactctgtt actgacgtgt cttgttgtac gaccgtcgat ccggttaaac tcacaagatt 2520

tctttttacc ggcccattac gtggccactg tgccacgtgt tgtgtagaat tttgaatctg 2580

aatgaatact ttatgagttt attttacact attgtgcttc tactttagtg atattacaaa 2640

actgccaggc atagagagag cctgtgtgag tcgcgtgagc tatctatgcc tttggcccac 2700

ttctttacgt ccatttgctc tatttaataa attcattaaa ttgtttcttt tttctttctt 2760

tttttctgga ataggaataa actaaaactc ttacctaata ctataacaat ataattattt 2820

gaacatgttg cttcacatga caaatgat 2848

<210> 4

<211> 3157

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

tgagggcaag tagggatgga gagatatata gagtgcaaga aagataaaaa tattttaaag 60

attaataata gtaatgactt aacaaaaaaa aaaaaaagat ggaataatat tcgaatttaa 120

tgatgggtgg ttccgagata atactattag ctcaactact tatatgggac cttgatatta 180

aattaagtag tcagattatt tttcatgttt tgttcacacg aaatgcaagt tacaaaagtc 240

taacaaaacc tagctgaatc tcataagtca tatggtagat cataaatttg gtttatcttt 300

tttttcacac atatatttgg gcatcataac attctatttt ggtgtaacct aatttttttt 360

ttttttgaag tttttgataa acaacataac ttgttaatta taccactaca acacaacaaa 420

atatgacatg tttgttatat atttccttat aattaaactt aaacgtttta gacatccatg 480

atgtttttat tagcaagtgt atgtaggtta tccaaaattg agtgagggca agttataaaa 540

ttatatatca attgaatatt ataagcaggt tggctgcacg tagtgcaaat gcactgggtc 600

agcgacgggt aagttaatat aatatgttgt aaatagtaaa tttaaaggga gaacgtgttc 660

gagaaacgta cctctcattc atgagaagcg ttcactgtcc aaattgcaag aacaagggta 720

gacgaaagcg tgtcccaaat attcaaattt gttatttctt tgtataaatg tgtatcccac 780

atgcatacaa actccttaat atgggccatg gtaaattgat gataatgaat tactatgact 840

tcttaatagc agcacatttc gatttgtttc tccattatat accatcccta ttagctgttc 900

cagtattgta gtcattgtca tttataaatt tgctgtcttc gttaagacgt ttgttatata 960

agggaccctt tacataatga atttatttca tattataaat ctgataaaga agtaattagt 1020

tttggagttg catatctagt tagttttcga gtatttttct agttatttca tattgattgt 1080

gccaaggtgt ccaaaataga cctcggacgg gtagttgtag cttgtagctt tgtaggtaaa 1140

taaaatgttc ggatttgagg tcccaaagtg gacttattgt ccctgttttg ttttcattgg 1200

gataaagttg ttggtaaaat gtaatgcttt taatagttaa atcatttcta atatcgtgat 1260

tactgggaaa taatagagaa aaagaaaaca aagacaaaag gcagcgacgt cgtttccata 1320

atatcgtgat tactgcatga aacacatgtt tcaacagtac gcataccaac tctcaaactt 1380

caatattgga aagatccagt tcatcagatg ctaataattc atatatttag gtttatgtac 1440

agcataatga acaatagaga gaattaatca ataaacagat caaagtatta atttgcaaga 1500

taaatgaaat aatgatgata ataaatggta gactagtacc catgtgtaga cataaaccaa 1560

caattaatgt tggtcggctt aataccaact aactagaaac cagtccgtca tcaatgtatg 1620

aatgtacaca ctcacatctt gcaataattt taaaaaggac taacaattta aatggttgct 1680

acataaataa acgatatagc cttacataaa actagcgaaa aaggttgata ttggctgtat 1740

aacatgcaca gtactaccag gctctaacat gcttcaaaat accgaatgat tcattaaatt 1800

gaaacattag agtaaacatt ttgtcgtggt cttgtatttg aatttcagta tataactgta 1860

tataatacaa tattattgat tttaatggtt tttcaaacaa tttttttttt taattactcg 1920

aattttttct taatctcgaa taagaaagat taatgctaat atatggaatt ataaatattt 1980

ggtcacgatt aatccgaatc caactacgaa ttcgatattg aaaatgtcaa tatcatcata 2040

tcaccagata aataaattct gtcaattgtt tctgttgcca aatcgtagtc atttgttttg 2100

aataaatcta tggattaaca aatagcaaca taatagcgac aaatgatcaa gtctttttat 2160

ccgccacaat ttcattgcta gttttgtcgt agacatggac catctatatc catattcttg 2220

gctatatatc cagcattatg tattttgtag taaaatcact acttgattgg caaattatta 2280

accaatccca tttataacaa aacaaaactt tatactcaga atatacttgg ttcgtagatt 2340

tatagctaac ataacttttt tgcttctctc tattattcta tcattgtgtg tttccaaatt 2400

gttgtcgata ttgtgtcact gtaacgcttt gccaaccgta gccataaagt gacaactttg 2460

tattcaacta tactcaaata tcgaacttcc cgagtatttt tgactaattg aattgtttga 2520

actttgactc ataggtctga ctaactcaaa gtaagccaac ccgagttttg aggatgatcg 2580

catatggcta ttggctaaca tatggatcta aatctgtagc aacttctagg tttggctttt 2640

tgtaaagaat agttctattt tgccaactcc agcatttttt gctattcatt atacattcaa 2700

ctttagttaa aaactttgaa ttgaacaact tgctattttt tttttttttt gcttttgttc 2760

ttcaacggct gcatcagtat tccagactac atcctccaat ccagatgttt ttgtagaact 2820

gcaaaagaga tttaactttt aagcatagaa aaccgaatta caacaaataa aattagaggt 2880

aactgaaccg gttggtaaag attaaaccga aaatatacct tgtgaaactc gagggtatca 2940

tcgccggttt tcctaagctc taccacgtgc aacgatggtg ccacttcaaa cacctattat 3000

aaccgataac caaaccaaaa taatcaggta ccggccgtat tcatgaaaat ctaaaaccgt 3060

actaaaccgg aatgttatca ttcctatctg tttttgttca taactctgta gctacagagt 3120

gctgaccttt acgagttacg accacttttg tctgcga 3157

<210> 5

<211> 2802

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ttgggttatc cacgagttcc actacgacct cttaccagaa catcaggttt tcttctattc 60

atatatatat atatatatat atgtggatat atatatatgt ggtttctgct gattcatagt 120

tagaatttga gttatgcaaa ttagaaacta tgtaatgtaa ctctatttag gttcagcagc 180

tattttaggc ttagcttact ctcaccaatg ttttatactg atgaacttat gtgcttacct 240

ccggaaattt tacagaggac atatgtcatc tgcagacttg agtacaaggg tgatgatgcg 300

gacattctat ctgcttatgc aatagatccc actcccgctt ttgtccccaa tatgactagt 360

agtgcaggtt ctgtggtgag tctttctcca tatacactta gctttgagta ggcagatcaa 420

aaaagagctt gtgtctactg atttgatgtt ttcctaaact gttgattcgt ttcaggtcaa 480

ccaatcacgt caacgaaatt caggatctta caacacttac tctgagtatg attcagcaaa 540

tcatggccag cagtttaatg aaaactctaa cattatgcag cagcaaccac ttcaaggatc 600

attcaaccct ctccttgagt atgattttgc aaatcacggc ggtcagtggc tgagtgacta 660

tatcgacctg caacagcaag ttccttactt ggcaccttat gaaaatgagt cggagatgat 720

ttggaagcat gtgattgaag aaaattttga gtttttggta gatgaaagga catctatgca 780

acagcattac agtgatcacc ggcccaaaaa acctgtgtct ggggttttgc ctgatgatag 840

cagtgatact gaaactggat caatggtaag ctttttttac tcatatataa tcacaaccta 900

tatcgcttct atatctcaca cgctgaattt tggcttttaa cagattttcg aagacacttc 960

gagctccact gatagtgttg gtagttcaga tgaaccgggc catactcgta tagatgatat 1020

tccatcattg aacattattg agcctttgca caattataag gcacaagagc aaccaaagca 1080

gcagagcaaa gaaaaggttt aacactctca ctgagaaaca tgactttgat acgaaatctg 1140

aatcaacatt tcatcaaaaa gatttagtca aatgacctct aaattatgag ctatgggtct 1200

gctttcaggt gataagttcg cagaaaagcg aatgcgagtg gaaaatggct gaagactcga 1260

tcaagatacc tccatccacc aacacggtga agcagagctg gattgttttg gagaatgcac 1320

agtggaacta tctcaagaac atgatcattg gtgtcttgtt gttcatctcc gtcattagtt 1380

ggatcattct tgttggttaa gaggtcaaat cggattcttg ctcaaaattt gtatttctta 1440

gaatgtgtgt ttttttttgt ttttttttct ttgctctgtt ttctcgctcc ggaaaagttt 1500

gaagttatat tttattagta tgtaaagaag agaaaaaggg ggaaagaaga gagaagaaaa 1560

atgcagaaaa tcatatatat gaattggaaa aaagtatatg taataataat tagtgcatcg 1620

ttttgtggtg tagtttatat aaataaagtg atatatagtc ttgtataaga aagggatttt 1680

acatgagacc caaatatgag taaagggtgt tggctcaaag attcatttag caaccaaagt 1740

tgcatttgca aggaaatgaa aaggtgttaa caatgtcact gcgtaacatg acttcgatac 1800

aaaatctcaa acaatacatc ttcaactgtg gattatgatg gacttggggt tgcaggtgat 1860

atgtctatag aaaaacgggt gggaatggaa aatggctgaa gaagaaagct ccaactaagc 1920

atcataactg gattgtttta gaggagatga gtcaaaggaa ttcacagtgg aactatctca 1980

agaacatgat cattggcttc ttattgttca tctccatcat tggctggatc attctggttc 2040

aagaggtcaa attatatata cataacggat ctaagaagta tagtgtagtc aattaaaaca 2100

aaacgagact tgaaaataag cataagtatt attaagttaa cccaatattc gtttcaatgc 2160

tttagttatc atcagaattc tcaacatttt cagatattta acttgccttt ggttgctttc 2220

tcgccatggt agtagcattc tccggataag aatcaagggg agcctcaact tcggcttcaa 2280

ccgtctcctc gtctttccta tgacattcac ttggtgttgc aacaatgtgt tgatgcccca 2340

cattcatagg agagtcccac atatctccaa cattcatagg agggttccac atatgcccac 2400

cattccaagg agggtaccac cacatatgcc cagcattcca agaaagatgg agcgacgata 2460

tcgtgaggaa caaggttttt gtagggcaaa taattgtagg tggttgagtt atatccgccc 2520

gcacagagta acagaccaac aattaacttt tgatatttta gtaaggtcta attcaatttt 2580

tggtggcgat aatatttggc ttagtcataa aatacagtat ggtataataa tgtaaaggtt 2640

tctcttatct tcaaaccaaa agactatact ggaagctgat gggatcatac gattctgaaa 2700

aaataagaca tatattgcaa cagagatcca atttgtatca aaaatattgt cggctcaaaa 2760

atctgaccca ccaagaatct aatcaagtgc gcgattaagc at 2802

<210> 6

<211> 2807

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cccttagtcg caatggcaaa agagatgata aatgcagcgt ttgcagataa gacaccaatc 60

agaacctgtt tcaaatgcga aattattacc ctttctaaac aatctcaatg acttaaatca 120

tttaaacctt aaaggaaaaa aaatctaatt aagtccatta aaaagaaacg atctaacctt 180

tatagataga agaacagggc tatcagaaaa gctcgattct tcatcacttt ttctcagtag 240

caagcttcta tctatgatga gttcagggct tttcaaataa gtttgccgat gaatctcacc 300

aactacacat ctgctagcta cactttgata gtaaaagatt ataaaacaaa aggatacaac 360

agtctagaag aagataggcg aagaccaact tccacaacag atgctgcaca cacacacaca 420

aaaaaaaaaa aagaacccaa caattcttat tggatcagag actactcaat atccccaaac 480

ttggaaatta gtttgttgct tgaggtctaa gatacttcta tatatggaaa aagattttca 540

aagccagata tttccacaag tttgtaatat caattcaaga taagagagct agaatcagac 600

aggaactagc aatgcttgaa atcaagaact tgaattgaaa tagtttttta cctgaatatt 660

gacagttgct ggattaattg cattgtagag gacgtgtcta tatacctttg gtctgtgaag 720

gattaaatcg atgaaaataa tctgccaaag aaaacaatta aagaaccaaa aaccaaaatt 780

ggaaagaaat agggaaacac ccaaaaaggg aaagaaagtg attaaaacag accatgcgtt 840

cacactcgat gtactcatct gctacttcct tgcaatttcc ctaaatataa caatatgatc 900

aaagatggaa actttgaaga aatttaatag agaatcttat aaaccctaat tgggtcaaag 960

aagatccatt aatacaaaaa tcttacgcat ttcatgagac gaatgttacc cggagagtat 1020

tgaatgaaca atgactttac cctaaaacca catcccacgc atctgtgttc actcgccgcc 1080

attgctctct ctctctctct ctctctctct ctctcaagag aagaagaata cggagcaatt 1140

agagtccggg tctgggctac tgttttaacc ctaaatgggc ttattcatgg gccaagtttt 1200

tgaagtctta actttaaatt tgttaggccc acttttgctc taagccgggg tatttgtacc 1260

ccaaaattta aaaatcatat acacgttgta atttataaat agttcaattt ggatcaaaat 1320

cttgtccata tgacatagca ttttaaaatg cgtaggttca tgaatgaaac atattatagg 1380

cctcagataa agatatacat attaagtcta aattatttag tcttcagaat ttaccacact 1440

tactgaaaag tctagtggtt cactaatatt attactgtcg tgttactttc tatatatagt 1500

tcatgacttg tgagttgtga tggataagtt tataagaaaa taaattattt attacaattc 1560

aacagtgaag aaatttattt agtttgatta aataagaaag gtaaataaat cttcgtttgc 1620

cacaccaaac aaaaaaaact ccttttgttg catgtaatta gcatatggct acaaaatgaa 1680

agatttgaca atgaggttta gataaaatag tttcctttca atataagtaa actcaacata 1740

aaaatgaaac ggtaagttaa aagtttaata tttgattatt actttattta gaaatgtatt 1800

agaaagggaa agaacccatt aacccccaat acgaagtttc cttatcatca ttaatcatgc 1860

ctatgaaaac cctaaccggc ctacatattt gcagtctctt ttaggtcagg aatctaatga 1920

gaatgaatta ttttatttta tttaacaata attggagcat gtcattaatt ctctttattc 1980

ttcttacaca actaatcatt tagatgtgtt acaatattat ttcctttagt cattttcata 2040

attttaatac ctccgtactt ttcactaata cctccccttt taattttcat tatttcttct 2100

tttctatcag tctatgcatg cattcttttg aatattaaaa tgcattttat attcttttga 2160

caactatgca caagcctttt gagacacatc tacacaatat aatagcacaa gcctttatga 2220

gacatatcta cacaatatca ttgctacttg tagactattt ggaatacgta tttacatatt 2280

ccattgtatc atccctttgc aaatgtttta tacatataac taacatatac attattcgta 2340

actttgttcc cctatatcga aaaatgtggg ctacacatat aactaacata tatatatata 2400

tgtatgttta tatgatatag tctccatgtc tatatatctt atatattaca tgtttcatgt 2460

ttacggtcaa gggagtattt ttatacgcat acacaatcat acacacttaa ccctacttat 2520

aatgatgtag gttcatatat ttatcttatt ttaggatcat tcgatcacaa attatacgga 2580

ccctcatact ctctaaagat atacaaaatc cgctatgtca tatccgatcc gaattagcag 2640

ctaaagaaaa acaaacacat gcatctactg aagatttgag tctcgagtgc ttagttacat 2700

gaactatcac aaaggatatg gataatataa ggtgtactga agtatgtcta tgcaatggga 2760

gggaaataca ttctgttaaa tgacttgtcg atttgatctt tcatgcc 2807

<210> 7

<211> 3093

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

aacaaaagcc cgagccgatt ggtttgctag gaaacttgag ttatacaaca tacgcatcat 60

aaaccaacta atagtccttt agccagatca aacaagtaat aataactcat acgacaaaat 120

atagtctcca atatccaact acaaagaaga aagacaagtg actgcgttta gtcaaacaaa 180

ccgaaaccca aatctccgtc actctcttct gctggctcat cctgcaatac caaaatgaga 240

tcaattttat atcacttatt agttagaaga atgatttcgt ctatcgttgc tatttcattt 300

accttcttct tctcctcagc agcaggagca gctgccgcac caccgccagc agctggagca 360

gcagctgcaa ccggagcacc acctccacca ccagcaccaa cgttcatgat gagatcagtc 420

acgttacgtt tctcagccat cttggcgaat agcattggcc agtatgactc aatactaaca 480

ccagcagctt tcaccaaggt cgcgattttg tcagcctatc aacgacataa acaaacacaa 540

gtaaagatct ttacccaatt caaaacacat ttagacagat acataaaata cttgttcaat 600

tcctgaccaa ataatactat accaaatcct tatcactcca taagaatcat tttcactaag 660

tttctaacta tcaagtcaaa agaaacggtt ccaaaaccct actgactaaa gcaaagagac 720

caatcaagat ttagaaatag acaagcagga gtcaaaggaa agataccgtg atagcgatac 780

cctcgtcctc gaggatcata acagcgtagc tgcaagcaag ctctccaact gtcgacattt 840

ttttatcctg aaacgattca aaaacagtaa caatagatta attgcttatt cacaaaaaaa 900

caaactttaa atccgtttta aaccaaaatt agagtaagtc gaacaaccca gacgatgatg 960

aaatgccaat aacaagtata aacggatcaa atggagaatc atagactacc taagagtaag 1020

atcgaaaatg cttttcgcaa ggcggcgaga gagacaaaat ttctagggtt tggatgcgga 1080

gacgggagat gcagagttta taaagctttt atccaattat ttgatgggcc gcataaatat 1140

tcgctaggcc cattttttaa ttttacatat atttgttttc agtattgggc cttcaaacct 1200

aatagccgtg aataaataaa agataagagc ccaaattaag agatttgact ttgaccagaa 1260

attgttggtt ggtttctagg gaacgaccac cgtggtccat catgcatgtg acagcacgag 1320

cttggtcaag caatacttga tggatgtcct tttcgcacta gtaacataat tcttcatttg 1380

acatatttat agtcttcaat aaacatatca tatgcgatcc aatatatttt cattttttct 1440

tgtacattcg tcttggtcca aaaatcacga gtttgattag ttctttaacc ccaaacttca 1500

caaaatcatg tagttgtaga caccaagttt gattatattt gtcaattaat aaaaaaaatg 1560

atttatacaa tttgtttaga atctcgataa atacttgttc atcctactac cattccaacc 1620

aactttatat aatagtagta aaaattacgc atgcgctaag ccaaatattg gcatttagta 1680

gaataacaga aaaaaaataa ataaaaatgt agattatgga gctaaaacaa cgtgcaatct 1740

ctctccttat ctagtctacg tatattgtcc cgcacggtat ataatcaata tggacccaaa 1800

aacgaatata ttcatgttaa tatcgatgcc aacaaaataa attaataata ctatgtattt 1860

ggtgaaaaga taaaatttat taaagagaaa gaaacacagt tattggtgtc gtggttgcag 1920

taaaagaggt ggtcagactc agagcaactt cttacaaagc aaccaaaggt tatctgtact 1980

ttctgtttct ctctcactcc ttatttctga aaaaaaaatc tacagctaaa tacctaaatc 2040

tcaacttcac ggaacaccga tacataaaga caatagagtg gtaataattt gttgagacca 2100

aaagtaatta aacacattac acatggggaa aaagaacggc tcttcttctt ggctcaccgc 2160

tgtaaagcga gctttccgat ctccgacgaa gaaagatcat agtaatgacg tcgaagaaga 2220

tgaagaaaag gtttcttatt cttagatctt attatttgct tactatattc aaagtatgat 2280

tattggtttt catttttctc tgttttttta aaaaaagaag agagagaaga gacggtggtt 2340

tagaaaaccg gcgactcaag aatctccggt gaagtcttcc ggtatctctc caccagcacc 2400

tcaggaagat tcacttaacg taaactcaaa accctctccg gagacagcac cgagttacgc 2460

aactacgacg ccgccatcca acgccggtaa acccccgtcc gccgtagtcc ccatcgccac 2520

gtcagcatct aagacactcg caccgaggcg gatttattac gcaagagaaa attacgctgc 2580

tgttgtcatc cagacttctt tcagaggata tttggtgagc aattattaat gacctatatt 2640

tttactttca tttttttaac caacttgact tctagaccac tttttagtga caatgatgtg 2700

tgtctatgtc tataaaatta acattgctta tacaagatct accatttaaa agattgggat 2760

ttcatttaga taattttttt ttgggtcttg atataagttt tgttctttct aatttggtaa 2820

ggcaagaaga gcattaagag cattaaaagg gttagtgaag ctacaagcat tggtgagggg 2880

acataatgtg agaaagcaag ctaaaatgac attaaggtgt atgcaagctc tggttcgagt 2940

ccagtctcgt gtgcttgacc aacgcaaacg cttgtctcat gacggtagtc gcaaatccgc 3000

gttcagtgac tctcacgctg tttttgaatc tcgctatctt caagatttgt cagatcgaca 3060

atccatggtt agtatagcct cctaggcctt gcc 3093

<210> 8

<211> 3115

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

aaccgaactg aaccggagta tttttttctt tcttacctct gtagctacgg agagctgacc 60

tttacgacca cttttgtctc ctttcatttt tatctgcgaa gtttacttaa acaatgttta 120

acgaagctac aaagacatta gttgacacaa acaaaatcaa gaagaagaag aagaagaaga 180

acagaccttg tagttgtctt tacggacatt aaagcctaat ggctttgcgg tttcttccat 240

tttcgacatt atttcactcg cagatcgttg agaagtaaac cgcgtttctt tcttcacaag 300

ttgctgcaaa caacaacaaa tcagatttag ttagataaaa cctatgcatg attttagaaa 360

atcaagacaa actgtaagta gaaaaaagtt cttaccgctt gcttctcgaa caagttttca 420

agactgaact cgcttgagct agagataagt tcgaaagcgt tcatggatac aggtttctcc 480

ttcttctcag ttacaagaca ttcctagagt agcagcacaa aaagagtgat attagttggt 540

aacaatacat tggtttactt aaaccgaact atgatataca caaccagatg gagaatttgg 600

aaatggcggt gtttaccttg gagttactaa aagcagcatc aacatcatct atggttatgt 660

cctcgtcatc ttggtcaaat gatggcggct tgtacccttt cttgaaccat tcatcttcga 720

gcaactctgc aatacttatt ctctgcattt cagaaataag tacatactta agctcttgca 780

caatctatgg ttggcagcat gtcatgttca cattttcaca actattactt catgtaagag 840

gcacttacgg taatagggtt gggttcgaga atacgcttga tgactctctt ggcaccttgc 900

gagaaccatg gtgggcagct aaactcagcc ttgcatatct aaagtatata ttaaacacca 960

aagatgagac tagaaaagta ctgcagaaga tcaagaagca gagggagaga taatttacac 1020

gtacacgttt gtataatgtc atgagattcg gctcatcaaa aggcaagtaa ccagccataa 1080

gcacaaagag aatgacacca caagaccaga catctgctgc tgcaccgtca tagcctttgt 1140

ccgacaaaac ctgccagcaa atccaacaca atgcagtaga caacattaaa ggcatatctc 1200

ttatatacat cataaataaa gaagattctg caaatgaagt ctatatatac gtatagcatt 1260

tgttagattt ccttcctttt taaaaacgta ttagcagttc ttgaattcct taaaatctta 1320

ttttccaaag aggatctaca gaacatttca tattctttgc ctttttaagt gctgaaagtt 1380

agtacataat acctcagggg caacgtagtt tggcgttcca caagctgtat gaagcaaacc 1440

atcttcctgc attcaaaaga acaatgctta gaacacttca ttcaaattga tatcttggtt 1500

atatatatca gaaattaatt gccaagtgaa aaatataatg gcattacccg aacttgtcgt 1560

gagaaggcgc ttaacccaaa atcagagact ttcaaaaccc catttgcgtc aaggatcaga 1620

ttttcaggct gcaacaagga acagatgata acaaaagaaa cttgaataag aactaagaag 1680

aatgaatatc gtaatcacag gaaacaagaa gagtgcacaa aaacaaaaga aagaccttga 1740

gatctctgtg gtagacacct cgactgtggc agtaatccac agcattgatg agctgctgaa 1800

aatatctccg agcttcatcc tccttaagcc tcccttgttg cgcctatgta tatgatttaa 1860

atccaagaat taacataaga gtaagaagag agaaagacag agaacatatg tcaatatttg 1920

tgaagagaaa agagaggaag attctctaag ttaggttact tacgatttta tcaaagagtt 1980

cacctccatt gacaagctca agaacgatat agatcttcgt tttgctcgcc ataacctacc 2040

acagagagat gttttacacg ctaagattca aaacttaatt taaaaaatat ctaaggaatg 2100

ataactaatt agtttcgtac ctcaatgatt tcaaccacat ttggatgttt aatcagtttc 2160

attgtagata tttctctttt aagctgcaca acaacaaaaa aataaaactt tatgatcatt 2220

atatcttcga gatcttgctc ataacttcca atcatacata tatagcatac aaaactaaat 2280

cttcaacaga ttcaaatcga accaaatcaa aaacaaagct actcgaactt caaaaccata 2340

tccatatcat agttaatagt tatcttgatc aatgcgtact aagatcagca tcgtgtagat 2400

atatacagaa acaaaaacat tcatacaaaa ttcgatattt taaaatatta ttgttaacac 2460

tttaatcaaa cattaacctt aataaccgaa caccaaccta ggaggacatg tgggtcccta 2520

atcacgaaat taaactgttc tttatagtac ttaaaaacaa acaaatccat aatcatttta 2580

cgtgatcaaa ccaaacacaa ttaacgaacc tgttcgacca ttttgtgacg gaagaccttt 2640

tctcggtcga ggattttgat agcggcttga tctccggtga cggtgttctt ggcgtatttc 2700

accttagcga agcttccttc tccgagagtt cgtcccatct cgtaattccc tactcgcgtc 2760

ctactcgccg gcgtcgcctt ccttctgctt ccactcattt tctttttccg attaagaaaa 2820

tcaacggctc tagatcggcg gcgaatctat caatgtgatt cttctccctc aatatctctc 2880

cttcttctag tggctgcctg gttttaaggc tgacgagaga aagtggacga gcgagagatt 2940

taataggcgc gactgggagt cacgcgcgta ttgtgggtcc cgtatgccac gtaatgtcaa 3000

aaatcattat tttgctctgt tccgcgtcag tcccgtgtta tttgtacttg tgtacgttcg 3060

taacgttcat tcttcattca tataaaaaaa ttccataatt ttgttcttgt gtggt 3115

<210> 9

<211> 3065

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

acgactattc tcctgcttgc ttaccatttg actttttcta ttgttagtca acgacctttc 60

cttttaatca tctacttttt tatatacttt ccaacttaca tatgtatgcg ctttttcatt 120

ccaaacaata taaatagatt aattagaaga aaatttataa catgtaaatt tcgttgtaaa 180

taacagatag ctactaatct gtaactatca aggctaaata taatttttat agctatctcc 240

tgcaaaaatt gtcacctctt tagtaaaata tatagatcgt cgaagtgttt aagacaccag 300

caaaatttat cataacattg tggccctgaa tccaaatttc ttcttatagt atttgtttaa 360

ttcagttaat acatatactt atcttaactc gatcctatcg tttattgaaa atatataaaa 420

gttcaaagga gtaatttaca ttgttcttat ataagaatct ttcttttgct aatatacata 480

aactaattag taactttaca cataatacag aggtatcagc atagaaaact aagtagtcac 540

aaagcacttg gtggccgatt aatcatatgt gcttagatac aatgtaaata ttaatgtgtg 600

atgttttata tatctgatta tataatatac tctccttttt aggtcataag aggaaaagcg 660

agtttgtgat tggagcactt caaatgattc ggttgcatgc aaaaataaat agaatcatat 720

cttccattag atttggatta tattccagat tctaacgtga cactcacaat tgatgtcacg 780

aaatatctac ttaatgaact tattgtggtt ctgatcattt cactatccta atctttgaaa 840

acaaatctcc atcatttcac taccatatag tttagacttt ttttttaaaa aattccttgc 900

tagaaatatt gataaaactc tagtttttac ttaacatttt gattgaatag aagaaactcc 960

ttggaacttg acagaataaa agaattgttt tttctttaca tatacaagaa tgtcaaagag 1020

attacgagtg acacatgaat agctttttga ttttactata agcaccacct ctaattctct 1080

aaacatgaaa aacccaaaca aagatccgag tcatatatgg gcttaaagaa aggcccaata 1140

attggctttt gattttatgg cctatcactc aaaatttcaa cacgagcacg aagcttgttc 1200

cttgatatta ctagtctcaa gatccgaacc agagatgaca tcaattctcg aaccgtcaag 1260

tttcacggtg gctccaccgt cggattccat agcttttctc gaaacaacaa cgcggctgaa 1320

aatctcctcg agtaaacgga aaaaagcttc gtcgacgttt ccgccgctta gagccgagac 1380

ttccgagaaa aaaagccgtt gagtctcggc gaattcgact gcatcctccg taggaacggc 1440

acgtttacca acggagagat cggctttgtt tccgacgagc atgatcacgg cggaatcatc 1500

ggcgtgagca cgtaattcct cgacccaacg tgcaacgtgg tcgaatgaga ggcgtttggt 1560

aatgtcgtaa accaccatcg cacctaacgc acctctgtag taggcgctag tcaccgctct 1620

gtatctgacc gccgaaacag ggtaagcaaa caaatatttt ttaattgaat atattataac 1680

aaaatctgac attgcagacc aaatcaagaa agaccaataa ttattcaaaa tcccataaat 1740

ttaaatatcg tgatcctaga taatataacg aaattctact ctagtttatt ttaacattta 1800

atttgtatac aaatctgaga ttcagctata aagaattatc aatgtaagac tgcaatttct 1860

tcgctcttta attaatgcaa aacaaaaata tacgcaacat ttatacatat tttatcaaga 1920

aaaataatat gaaaaaacga tattaaaaga aacttttgca tcggtaagaa aaatataaag 1980

ttttacttat gcaagtatgc aacaaagtat tcggtgagtt attgcatttg accttttgat 2040

ctataagtgg ttgcaaaaag acaaaaacaa aatatgcaga aaagaaatct gacattgtat 2100

taaccaaatc aagaacacca atggttattc aaatccatac cacaattctt tacccaacaa 2160

aaatattcca tattgtatta gaaatatatt tagttacaaa atcaaatcaa accatatcta 2220

aattaccgtg atccaagtga aatgaagtga tagtctaaac aaatgatttg aatccaaaag 2280

tgagagctta ataaaatata taaagaatta tcaatgtgtt tttggatata gatgatataa 2340

cctctcttga ccggcggtgt cccagatctg agctttgacg agtttgcccc gaagagtgat 2400

ggtccgtgtc tgaaactcga caccaatggt tgattttgaa tcataacaga actcgttgtg 2460

tgtaaaacgt gagaggagct gcgttttccc gacagcagaa tctccaatga ccaccacctt 2520

gaacacgtaa tcgatctttt ccggcatcgt cggtttcttt acgtgtttgt tattctccgg 2580

cgactcaccg ctcatctctt cgttcattat tgcgctgatg aagaaaacaa ctataaagtt 2640

taagtctctt ttgagtaaag tgggagtgag tgaaacagag aaaagagaag aaaggagaag 2700

aaatttggtt ttatatggtt cagtacgaat ataccgaatt aacaaaaatt ctgaaccact 2760

tcagtgagat tggttggatt cactcataac cattcgcaac catatttaat aaaactaaaa 2820

ctgctacaaa ccaaaatttc gtaaatttgg tttggtttaa gtcgttgaaa taacccaaac 2880

taaaattaaa tattgagtat cctaacctaa aagtgttgat atatgaagac agcaacaatg 2940

ctctgtgcat tattttaatt agctcttctt ttatatataa ttttaattag ctttttatat 3000

atgattcaaa atgaatatac aatgtttaaa tatagctgtg gcttgtgggc atggaccgac 3060

ataat 3065

<210> 10

<211> 2981

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tcctgtcaac gaaccaaccg gtcaagtacg gatttcttta attgtaaagt tgggtacgtg 60

tttatgaagc gaacgtgttt gatagaaaaa aaatgaatgc ttgcaactgt gaagactgaa 120

aagtcatgat ttagtgttga gacttattca aattattatg tccacattct ttaaacagtg 180

ggaagagaga gattggtgac agcattaaat tggtggtcac acataccaat atcacaaaat 240

ttagtacttg taccccataa agaagagggt cactatattc aattattcat gttttatgat 300

gtaatatact atagtagatg ttaacatatc gaagcattga atatgaaagt tccaaatctt 360

gatggtcatt atttcgaatt cttgtgggcg aataaatgaa aagagcgttt gatgatatcg 420

acattctaac aagatcgtga gatctaaagg tcgtcgtgat gatttagtaa taagataaaa 480

aagtcgtagc aacgtttttt tggtttaaaa tgtaaaaaga agaccgtgat gcattgtttg 540

taatgtatac atgaataaaa taagtactta tttaacaacg tagtgtatta gattatcttt 600

cgtcatgtat ttgcatgtat attttccccc atgtggaatg tttcagtttg tgttatctca 660

agagcgaaaa tgtgccacca attgtgggga aattattggg catcattaat taactactag 720

tagtatataa aacgagcttt tcacttttca caaaataatt atagttaaaa taagcaaatt 780

atttcatgac gtaaaaaaag aaagaaaaag aagctaccac accacttgca tatttcttaa 840

gtgtctgtta tccaccgaat ctaaaatgtc gcttttgcat tcccacaatc ttattagata 900

tttgtattcc cagagtaaaa gtataattca aacatttatt attattatta taggaattga 960

aagaaaatta aattttcaaa aagaccgctt acttgtttat tatatacaca gagaagagaa 1020

cagtctcact cgccactttc ctcgagaaaa cgaaaaaaaa agatggtagg aagaagaaaa 1080

gctttactct tttctctctg tttcttcttc ctctctcttc cttccttctc ttcccttcca 1140

tccttccaaa ccctattccc caattctcat tcccttcctt gtgcttctcc cgtctcattc 1200

caacccgact ccgactccga gtcactgcta gaatccgaat ttgaatccgg atccgactcc 1260

gagtcttctt cttccattac tctaaacctc gaccacatcg atgctctctc ctccaacaaa 1320

acacccgacg agctattcag ctcccgtctc caacgtgact cccggcgcgt gaaatctatc 1380

gccacactcg ccgctcaaat ccccggaaga aacgtgactc acgcgccacg acccggtgga 1440

ttcagcagct cagtcgtctc tggtctctct caaggaagcg gtgaatactt cacgcgcctc 1500

ggtgtcggca ctccggcgag atatgtttac atggtgctcg acaccggcag cgacatcgtc 1560

tggctacaat gcgctccttg tcggagatgc tactctcaat ccgacccgat attcgacccg 1620

agaaaatcca agacctatgc cacaatcccc tgttcttcac ctcactgccg ccgattagac 1680

tccgccggat gcaacacccg tcgtaagact tgtctctacc aagtctctta cggagatggt 1740

tctttcaccg tcggcgattt ctccaccgaa acgctaactt tccggcgaaa tcgcgttaaa 1800

ggcgttgctc tcggatgtgg ccacgacaac gaaggtctct tcgtcggagc cgccggttta 1860

ttaggactcg gtaaaggtaa attatcgttc cccggtcaaa ccggtcaccg gtttaatcag 1920

aaattctctt actgtttagt cgacagatcc gcttcttcga aaccttcctc ggtcgtcttc 1980

ggaaacgccg ccgtatcacg aatcgcgaga ttcacgccac ttttgtcaaa cccaaaactc 2040

gacactttct actacgtcgg gcttctggga attagcgtcg gagggacacg tgtccccggc 2100

gtaacggctt ctctttttaa acttgaccag atcggtaacg gtggagttat tatcgattcg 2160

ggtacgtctg tgacccggtt gatccgacca gcttatattg ctatgagaga cgcgttccgg 2220

gtcggagcta agacattgaa aagagctccg gatttctcac tcttcgacac gtgtttcgat 2280

ctctccaata tgaacgaggt taaagtaccg acggtggttt tacatttccg tggagctgac 2340

gtatcacttc cggcgacgaa ttacctgatc cctgtggata ccaacggcaa gttctgtttt 2400

gcgtttgccg gtacgatggg cggactatcc ataattggga atatccagca acagggtttc 2460

cgggttgtat acgacttagc gagttcccgg gtcgggtttg ctccaggagg atgcgcttaa 2520

taattagatc tgaccggagc cggtacggat aatgtacttt tgttttctca atttaatgct 2580

tagacttgtt tttttttttt ttataaatcg gacaagtgaa tctttaagcg gtggagttga 2640

atacgtgttg ataaaatctt cacgtgttgt cggctgtatt attagtatcg tctttatttt 2700

acatctcgga ccaaagagag cgtgtatgat tttctaaaaa attggcaatt ttacttttag 2760

ttatccaaca aatgacaact gcggaaatta gttagatcaa agattattca atataatttc 2820

caagtcagac aaatctgatt atctgaggat ggtagagtac gtctataata gtataaatta 2880

agatagattt tggtaaatta ctgtttacgt aatacatagg gcagatgaaa aattgtgttg 2940

gagttgttaa tcagacaaaa acgaagattc agcgcgaaga t 2981

Claims (9)

1. A method for absolute quantification of a metagenomic sample, the method comprising the steps of:

1) Taking a metagenome sample to be detected, labeling the sequence mixture, and uniformly mixing to obtain a mixed sample;

2) Mixing sample DNA extraction, library establishment and sequencing to obtain machine-on data;

3) Analyzing respective detection proportions of pathogenic bacteria and tag sequences in the machine-down data;

4) Establishing a sample fitting linear relation, and fitting a sample linear relation formula by taking the detection proportion of each tag sequence as an abscissa and the known mass of the corresponding tag as an ordinate: y=kx;

5) Quantification of pathogenic bacteria: calculating the mass of the corresponding pathogenic bacteria according to a sample fitting linear relation y=kx and a pathogenic bacteria detection proportion x; defining a quality and copy number conversion formula, and calculating the copy number of pathogenic bacteria;

the copy number=6.02 x 10 x 23 x 10 (-9) x mass (ng)/(genome length bp x 660 g/mol);

the tag sequence mixture comprises 3 tags;

the tag sequence is shown as SEQ ID NO.1-3.

2. The absolute quantification method of claim 1, wherein:

the concentration ratio of SEQ ID NO.1, 2 and 3 tags in the tag sequence mixture is 50-100:5-10:0.5-1.

3. The method of absolute quantification of claim 2, wherein the tag sequence mixture is prepared by a method comprising: preparing a mixture of tag sequences shown in SEQ ID NO.1, SEQ ID NO. 2 and SEQ ID NO. 3 according to a concentration ratio of 100:10:1, and subpackaging the mixture into a 30ul system for later use.

4. A method of absolute quantification according to any of claims 1-3, wherein the sample is a blood sample.

5. A tag sequence for absolute quantification of a metagenomic blood sample, wherein the tag sequence is shown in SEQ ID No.1-3.

6. The tag sequence according to claim 5, wherein the concentration ratio of SEQ ID NOS.1, 2, 3 is 50 to 100:5 to 10:0.5 to 1.

7. A product for absolute quantification of a metagenomic blood sample, characterized in that it comprises a tag sequence according to any one of claims 5-6.

8. The product of claim 7, wherein the final concentration of the tags SEQ ID NO.1, 2, 3 in the product is 0.0011ng/ul, 0.00011ng/ul and 0.000011ng/ul, respectively.

9. Any one of the following uses of the tag sequences of any one of claims 5-6:

1) The application in preparing reagents for detecting blood sample infection;

2) The application in absolute quantification of metagenomic blood samples.