CN110241460A - A kind of immune group library method for screening the reaction of independent sample crossover - Google Patents
A kind of immune group library method for screening the reaction of independent sample crossover Download PDFInfo
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Abstract
The present invention relates to a kind of immune group library methods of examination independent sample crossover reaction, it is characterized by: using the RNA greater than 10ng as initial amount, RNA is made to carry out template switch when carrying out reverse transcription into cDNA using the principle of RACE, 2 pre- amplification experiments are carried out before PCR amplification, pre- amplification uses the upstream primer with UMI for the first time, so that the end 5` of original template has taken UMI, second of pre- amplification uses the downstream primer with UMI, so that the end 3` of template has taken UMI, PCR amplification is carried out using this product with both-end UMI as template again, finally obtained product obtains final sequencing library by purifying and connecting sequence measuring joints sequence, this method makes the both ends of every cDNA all mark different and unique UMI sequence Multiple samples can be merged one library of building after completing PCR amplification, it can solve the cross reaction problem that similar sequences in sample are generated in pcr amplification reaction, for the sample in different cell receptor sources, it is able to achieve in PCR amplification and only uses pair of primers progress PCR amplification, it realizes equivalent amplification, solves the problems, such as the primer Preference generated because multi-primers expand.
Description
Technical field
The invention belongs to biological medicine service fields, more particularly, to a kind of PCR amplification in people's immune group library, library construction
And the application in next-generation sequencing, especially a kind of immune group library method for screening the reaction of independent sample crossover.
Background technique
Immune system is mediated by immunized B cells and the surface receptor of T cell, with antigen derived from pathogen or pathogen
In conjunction with, and then play immune function and body is protected not encroached on.BCR heavy chain immunoglobulin (IGH) and TCR β chain (TCRB) are logical
V gene is crossed, the rearrangement of D gene and J constant gene segment C is to generate combination diversity.Junction between V-D and D-J lacks core
Thuja acid and at random addition nucleotide sequence produce receptor connection diversity.And light chain caused by antibody mediated immunity, ball is immunized
T cell receptor α (TCRA) during albumen λ or κ (IGL/K) and T cell are immune, which has, similarly to be reset, so as to form siberian crabapple
The diversity of system.It is in y-type structure by the antibody that immune group library B cell generates by taking antibody mediated immunity group library as an example, it is identical by two
Heavy chain and light chain composition.V (D) J recombinate when, it is V, D, J section multiple in respectively have a meeting be selected and recombinate, form a weight
The variable region of chain.And the upstream of the V section of front end, the sequence including ATG are known as the boot section of antibody.Each different V
Gene section corresponds to different guidance region sequences, target area (such as specification of the end 5` primer when which is the amplification of antibody group library
Shown in attached drawing 1).Similarly, we are by having found the guidance of TCR receptor sequence to the research in T cell surface receptor structure
Area, the target area as the end TCR sequence 5` design of primers.
Using high throughput sequencing technologies come to the analysis by the immune group library in blood or lymphoid organ, to be exceedingly fast
Speed develops and is increasing our cognitions to immune response.Such as immunoglobulin gene high throughput DNA sequencing is obtained
The information obtained can be used for detecting B cell malignant tumour in high sensitivity, finds the antibody special to target antigen, vaccine is instructed to open
Hair and understanding autoimmunity etc..Therefore, the research using high-flux sequence analysis method to immune group library can promote us right
Immunologic understanding and the solution method for not meeting clinical demand relevant to infectious disease, immune disorder and cancer.
Currently, there are mainly two types of existing people's immune group library PCR amplification and library construction techniques.1. based on multiplex PCR
Library constructing method: using DNA or cDNA as template, a plurality of upstream primer is designed for boot section, for different antibody phenotypes
It is expanded in constant region one downstream primer of design, amplification using the combination of many-to-one primer, then carries out library construction simultaneously
Sequencing.2. the library constructing method based on 5`RACE: using mRNA as template, being synthesized using the overall length that oligodT carries out cDNA, needle
To the special construction at the end 5` of mRNA, one section of universal sequence can be introduced at the end 5` simultaneously in the synthesis of cDNA, then using general
Sequence as upstream primer, and constant region design a downstream primer, expanded using pair of primers, finally carry out text
Library constructs and is sequenced.
Although the quick high throughput of next generation's sequencing technologies energy carries out panorama type scanning to immune group library, at present
There are still more clearly disadvantageous for two methods.1. multiple PCR method has used a variety of primers, reaction system complex；It is more
Weight design of primers can only be designed according to known reference sequences, cannot be captured completely all etc. in the immune group library of full people
Position gene；More primer reactions can not accomplish equivalent amplification, and the amplification efficiency difference between different primers results in PCR Preference
In the presence of making result with truth, there are larger differences.2. although simple 5`RACE method can accomplish equivalent amplification,
It cannot distinguish between the cross reaction of the sequence in independent sample when building library.3. existing sequencing analysis method and experimental technique
There is no mitigation chimera sequence product as caused by the PCR later period, bring influences, and these products are two kinds of amplification sides
Can exist in method, and great interference is produced to the diversity quantitative analysis in sample.
The application is designed for the problem that current problem carries out completely new PCR method, be can solve and is currently encountered, and is reached true
The purpose of positive reduction immune group library truth.
Summary of the invention
It is similar in the sample generated during it is an object of the invention to screen immune group library PCR amplification and library construction
The cross reaction of sequence can be multiple samples after completing PCR amplification so as to really restore the truth in immune group library
Product merge one library of building, the cross reaction that similar sequences are generated in pcr amplification reaction in sample are solved the problems, such as, for not
With the sample in cell receptor source, it is able to achieve in PCR amplification and only uses pair of primers progress PCR amplification, realize equivalent expansion
Increase, solves the problems, such as the primer Preference generated because multi-primers expand.Unless otherwise defined, all skills used herein
Art and scientific term have and the normally understood identical meaning of general technical staff of the technical field of the invention.
Term:
UMI full name is unique molecular identifiers, is made of randomized bases, the UMI that the present invention designs
Structure be N (10) structure, length 10bp, random number 410It is a, there is fabulous randomness；
5 end rapid amplifying of 5`RACE:5`Rapid Amplification of cDNA Ends, cDNA.
The present invention is the principle of gene 5'RACE, the pre- amplification of a wheel twice is carried out before PCR amplification, so that every cDNA
Both ends all mark different and unique UMI sequence, after completing PCR amplification can multiple samples merge building one
Library.A kind of immune group library method for screening the reaction of independent sample crossover, it is characterised in that: the following steps are included:
The preparation of S1, cDNA template: using the RNA greater than 10ng as initial amount, make RNA inverse in progress using the principle of RACE
Template switch is carried out when being transcribed into cDNA into the cDNA of one section of anchor tip of band；
S2, then cDNA template carry out 2 pre- amplifications before PCR amplification, and pre- amplification is drawn using the upstream with UMI for the first time
Object forms product A so that the end 5` of original template has taken UMI；
S3, it is purified after, product A is carried out second pre- amplification, using the downstream primer with UMI, so that the 3` of template
End has taken UMI, forms product B；
S4, it is purified after, PCR amplification is carried out using the product B with both-end UMI in step S3 as template, finally
To product C；
Final product C obtained in S5, step S4 obtains final sequencing text by purifying and connecting sequence measuring joints sequence
Library.
In described step S2, S3, the S4, the upstream primer expanded in advance for the first time, the downstream primer that expands in advance for the second time and
The primer sequence of PCR amplification totally 38, as shown in sequence table.
The cDNA template preparation step of the step S1:
(1) cell RNA extracts: cell origin includes but is not limited to that the peripheral blood of people or mouse, rat and rabbit separates
The lymphocyte arrived, number of cells are that cell pyrolysis liquid is added and is cracked, is carried out using RNA extracts kit greater than 500
The extracting of RNA.
(2) tissue RNA is extracted: the tumor tissues and normal tissue or come that tissue-derived including but not limited to manpower art is got off
Derived from the various organization types of mouse, rat and rabbit, initial amount be greater than 1mg, using be added after liquid nitrogen grinding lysate into
Row cracking, the extracting of RNA is carried out using RNA extracts kit.
(3) Quality Identification and Concentration Testing are carried out to mRNA, it is desirable that the result of mRNA is complete, and RNA total amount is greater than 10ng.
(4) in order to improve the yield of cDNA, we are to have done optimization processing early period in synthesis cDNA, are configured according to the following table 1
Mixed system, and being reacted in PCR instrument, response procedures are as follows: 70 DEG C, 1min, immediately product is put after response procedures
In 3min on ice.Table 1
(5) mixed system for configuring the following table 2 is reacted with being placed in PCR instrument after the mixing of the product of step (4), is reacted
Program are as follows: 42 DEG C, 90min, 75 DEG C, 15min, 4 DEG C, ∞.Product after reaction is synthetic cDNA template, will be used for down
The experiment of one step.Table 2
The pre- amplification condition of cDNA template first of the S2 include the following:
(1) following PCR reaction system is configured
(2) PCR response procedures are set as follows:
(3) after the reaction was completed, reaction product is purified, is finally washed with 20ul Nuclease-free water
It is de-, obtain product A.
The product A of the S3 carries out the second pre- amplification condition include the following:
(1) following PCR reaction system is configured, product A is divided into 4 equal portions, while carrying out 4 reactions.
(2) PCR response procedures are as follows:
(3) after the reaction was completed, obtained product is reacted 4 and be mixed to get the product that a general pipeline adds up to 200ul, to this
Product is purified, and is finally eluted with 20ul Nuclease-free water, obtains product B.
The PCR amplification of the S4, include the following:
(1) PCR reaction is carried out by template of the purified product B of step 3, according to following preparation reaction system；
(2) PCR response procedures are as follows:
(3) after the reaction was completed, which is purified, is finally washed with 30ul Nuclease-free water
It is de-, obtain product C.
The primer sequence requires as follows:
The nucleotide sequence of RACE oligo includes or is made of SEQ ID NO:1, and wherein the 4 of end G is ribose core
Guanine in acid；The nucleotide sequence of Primer 1 in step 2 includes or is made of SEQ ID NO:2；
When receptor behaviour BCR IgA heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:3 composition；
When receptor behaviour BCR IgD heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:4 composition；
When receptor behaviour BCR IgE heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:5 composition；
When receptor behaviour BCR IgG heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:6 composition；
When receptor behaviour BCR IgM heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:7 composition；
When receptor behaviour BCR IgK light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:8 composition；
When receptor behaviour BCR IgL light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:9 composition；
When receptor behaviour TCR alpha chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:10 composition；
When receptor behaviour TCR beta chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:11 composition；
When receptor behaviour TCR delta chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:12 composition；
When receptor behaviour TCR gama chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:13 composition；
When receptor is mouse BCR IgA heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:14 composition；
When receptor is mouse BCR IgD heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:15 composition；
When receptor is mouse BCR IgE heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:16 composition；
When receptor is mouse BCR IgG heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:17 composition；
When receptor is mouse BCR IgM heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:18 composition；
When receptor is mouse BCR IgK light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:19 composition；
When receptor is mouse BCR IgL light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:20 composition；
When receptor is mouse TCR alpha chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:21 composition；
When receptor is mouse TCR beta chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:22 composition；
When receptor is mouse TCR delta chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:23 composition；
When receptor is rat BCR IgA heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:24 composition；
When receptor is rat BCR IgD heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:25 composition；
When receptor is rat BCR IgE heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:26 composition；
When receptor is rat BCR IgG heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:27 composition；
When receptor is rat BCR IgM heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:28 composition；
When receptor is rat BCR IgK light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:29 composition；
When receptor is rat BCR IgL light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:30 composition；
When receptor is rabbit BCR IgA heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:31 composition；
When receptor is rabbit BCR IgE heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:32 composition；
When receptor is rabbit BCR IgG heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:33 composition；
When receptor is rabbit BCR IgM heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:34 composition；
When receptor is rabbit BCR IgK light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:35 composition；
When receptor is rabbit BCR IgL light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID
NO:36 composition；
The nucleotide sequence of Primer 3 includes or is made of SEQ ID NO:37 in step 4；
The nucleotide sequence of Primer 4 includes or is made of SEQ ID NO:38 in step 4.
Above-mentioned immune group library amplification and library constructing method can summarize are as follows: using the RNA greater than 10ng as initial amount, benefit
Make RNA carry out template switch when carrying out reverse transcription into cDNA with the principle of RACE, 2 are then carried out before PCR amplification
Secondary pre- amplification experiment, pre- amplification uses the upstream primer with UMI for the first time, so that the end 5` of original template has taken UMI, the
Secondary pre- amplification uses the downstream primer with UMI, so that the end 3` of template has taken UMI, then the product of both-end UMI is had with this
PCR amplification is carried out as template, finally obtained product obtains final sequencing by purifying and connecting sequence measuring joints sequence
Library.
The invention has the benefit that
(1) while RNA reverse transcription is at cDNA, utilization expands in advance twice, adds simultaneously at the end 5` and the end 3` of cDNA
UMI realizes the independent marking to each sequence, can solve what similar sequences in sample were generated in pcr amplification reaction
Cross reaction problem.
(2) sample in different cell receptor sources is able to achieve in PCR amplification such as BCR or TCR and only uses one
PCR amplification is carried out to primer, realizes equivalent amplification, solves the problems, such as the primer Preference generated because multi-primers expand.
(3) present invention can meet the sample requirement for construction data base that RNA concentration is greater than 10ng.
Detailed description of the invention
Fig. 1 is heavy chain of antibody VDJ regrouping process schematic diagram；
Fig. 2 is cDNA preparation and PCR schematic diagram.
Specific embodiment
The explanation of following embodiment is merely used to help understand method and its core concept of the invention, it is noted that right
For those skilled in the art, without departing from the principle of the present invention, the present invention can also be carried out
Some improvements and modifications, such as design of primers or adjustment of the length in 18bp to 30bp of 200bp progress are originated in constant region domains
Base quantity of UMI etc., these improvements and modifications also fall within the scope of protection of the claims of the present invention.To disclosed implementation
The following the description of example, enables those skilled in the art to implement or use the present invention.Various modifications to these embodiments
It will be readily apparent to those skilled in the art, the general principles defined herein can not depart from this
In the case where the spirit or scope of invention, realize in other embodiments.Therefore, the present invention is not intended to be limited to illustrated herein
These embodiments in, but can be applied to meet broader model consistent with the principles and novel features disclosed in this article
It encloses.
1 1ml human peripheral sample immune group library high-throughput sequencing library construction method of embodiment
The preparation of 1.cDNA template
(1) human peripheral 1ml is acquired using EDTA anticoagulant tube, using single core in density gradient centrifugation method separation blood
Cell, is added 600ul cell pyrolysis liquid and carries out cell cracking, carries out RNA extracting using RNA extracts kit.(2) RNA is detected
Integrality, measure the concentration of RNA.(3) mixed system is configured according to the following table 9, and is reacted in PCR instrument, response procedures
Are as follows: 70 DEG C, 1min, product is immediately put in 3min on ice after response procedures.Table 9
(4) mixed system for configuring the following table 10 is reacted with being placed in PCR instrument after the mixing of the product of step (3), is reacted
Program are as follows: 42 DEG C, 90min, 75 DEG C, 15min, 4 DEG C, ∞.Product after reaction is synthetic cDNA template, will be used for down
The experiment of one step.Table 10
First time pre- amplified reaction before 2.PCR: (1) the PCR reaction system such as the following table 11 is configured.Table 11
The primer sequence of Primer 5 are as follows: CATGGACCTTACGACTAGCT (NNNNNNNNNN)4- 12AAGCAGTGGTATCAACGCAGAG
(2) PCR response procedures such as the following table 12: table 12
(3) after the reaction was completed, reaction product is purified, is finally washed with 20ul Nuclease-free water
It is de-, obtain product D.
Second of pre- amplified reaction before 3.PCR
(1) the PCR reaction system such as the following table 13 is configured, product D is divided into 4 equal portions, while carrying out 4 reactions.Table 13
The primer sequence of Primer 6 are as follows: ACTCGAAGTTCAGTCG (NNNNNNNNNN)4- 12GGGGAAGACCGATGGGCCCTTGGTGG
(2) PCR response procedures such as the following table 14: table 14
(3) after the reaction was completed, obtained product is reacted 4 and be mixed to get the product that a general pipeline adds up to 200ul, to this
Product is purified, and is finally eluted with 20ul Nuclease-free water, obtains product E.
4.PCR reaction.(1) PCR reaction is carried out by template of the purified product E of step (3), prepares and reacts according to table 15
System.Table 15
The primer sequence of Primer 7 are as follows: CATGGACCTTACGACTAGCT
The primer sequence of Primer 8 are as follows: GACTAGGAACTCGAAGTTCAGTCG
(2) PCR response procedures such as the following table 16: table 16
(3) after the reaction was completed, which is purified, is finally washed with 30ul Nuclease-free water
It is de-, obtain product F.
5. constructing sequencing library
(1) product F is subjected to connector connection using library construction Kit；
(2) library fragments screen and identify the quality in library using magnetic bead, as a result following table 17.
Table 17
The immune group library high-throughput sequencing library of embodiment 2:1mg human breast carcinoma tissue sample constructs
The preparation of 1.cDNA template
(1) human breast carcinoma tissue sample is taken out from liquid nitrogen or -80 DEG C of refrigerators, 1mg is weighed, using the method handle of liquid nitrogen grinding
Tissue grinder is added 600ul cell pyrolysis liquid and carries out cell cracking, RNA extracts kit is used to carry out RNA pumping at powdered
It mentions.(2) integrality for detecting RNA, measures the concentration of RNA.(3) mixed system is configured according to the following table 18, and is carried out in PCR instrument
Reaction, response procedures are as follows: 70 DEG C, 1min, product is immediately put in 3min on ice after response procedures.
Table 18
(4) mixed system for configuring the following table 19 is reacted with being placed in PCR instrument after the mixing of the product of step (3), is reacted
Program are as follows: 42 DEG C, 90min, 75 DEG C, 15min, 4 DEG C, ∞.Product after reaction is synthetic cDNA template, will be used for down
The experiment of one step.Table 19
First time pre- amplified reaction before 2.PCR
(1) the PCR reaction system such as the following table 20 is configured.Table 20
The primer sequence of Primer 9 are as follows: CATGGACCTTACGACTAGCT (NNNNNNNNNN)
4-12AAGCAGTGGTATCAACGCAGAG
(2) PCR response procedures such as the following table 21: table 21
(3) after the reaction was completed, reaction product is purified, is finally washed with 20ul Nuclease-free water
It is de-, obtain product G.
Second of pre- amplified reaction before 3.PCR
(1) the PCR reaction system such as the following table 22 is configured, product G is divided into 4 equal portions, while carrying out 4 reactions.Table 22
The primer sequence of Primer 10 are as follows: ACTCGAAGTTCAGTCG (NNNNNNNNNN)
4-12GGGGAAGACCGATGGGCCCTTGGTGG
(2) PCR response procedures such as the following table 25: table 25
(3) after the reaction was completed, obtained product is reacted 4 and be mixed to get the product that a general pipeline adds up to 200ul, to this
Product is purified, and is finally eluted with 20ul Nuclease-free water, obtains product H.
4.PCR reaction.
(1) PCR reaction is carried out by template of the purified product H of step (3), prepares reaction system according to table 26.Table 26
The primer sequence of Primer 11 are as follows: CATGGACCTTACGACTAGCT
The primer sequence of Primer 12 are as follows: GACTAGGAACTCGAAGTTCAGTCG
(2) PCR response procedures such as the following table 27: table 27
(3) after the reaction was completed, which is purified, is finally washed with 30ul Nuclease-free water
It is de-, obtain product I.
5. constructing sequencing library
(1) product I is subjected to connector connection using library construction Kit；(2) library fragments are sieved using magnetic bead
It selects and the quality in library is identified, as a result following 28. table 28 of table
Embodiment 3
By comparing the type number of UMI in sample sequence, after UMI sequence self-correcting error correction, according to both-end UMI's
Correction, screens sequence, improves the accuracy of sample sequence, deviation shadow caused by reducing because of PCR error and sequencing mistake
It rings.The data cases in test 14 libraries used are as shown in table 29 and Fig. 2.Table 29:
Table Fig. 2:
It can be seen that the both-end UMI sequence that the present invention is added in the sample by the result of table 29 and table Fig. 2 to be distributed more
Uniformly, technical feasibility.By the application of both-end UMI, there is most UMI type to be corrected, passes through the both-end after correction
UMI sequence and considerable UMI sequence number of the same race are greater than 2 combination, we can carry out more accurately sample sequence
Screening considerably reduces the influence of PCR and sequencing mistake to sample sequence.
7.cDNA preparation and PCR schematic diagram, as shown in Figure 2.
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Sequence table
<110>Hospital of Southern Medical University
<120>a kind of immune group library method for screening the reaction of independent sample crossover
<141> 2019-05-31
<160> 38
<170> SIPOSequenceListing 1.0
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<211> 26
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<211> 52
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<213>artificial sequence (Homo sapiens)
<220>
<221> misc_feature
<222> (20)..(30)
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<213>artificial sequence (Homo sapiens)
<220>
<221> misc_feature
<222> (17)..(26)
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actcgaagtt cagtcgnnnn nnnnnnggct cagcgggaag acct 44
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<213>artificial sequence (Homo sapiens)
<220>
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actcgaagtt cagtcgnnnn nnnnnngggg aagaccgatg ggcccttggt gg 52
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<211> 44
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<213>artificial sequence (Homo sapiens)
<220>
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actcgaagtt cagtcgnnnn nnnnnnttgg ggcggatgca ctcc 44
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<211> 46
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<213>artificial sequence (Homo sapiens)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 8
actcgaagtt cagtcgnnnn nnnnnnagat ggtgcagcca cagttc 46
<210> 9
<211> 56
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<213>artificial sequence (Homo sapiens)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 9
actcgaagtt cagtcgnnnn nnnnnncctt gttggcttgr agctcctcag aggagg 56
<210> 10
<211> 46
<212> DNA
<213>artificial sequence (Homo sapiens)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 10
actcgaagtt cagtcgnnnn nnnnnncagg gtcagggttc tggata 46
<210> 11
<211> 46
<212> DNA
<213>artificial sequence (Homo sapiens)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 11
actcgaagtt cagtcgnnnn nnnnnntctg atggctcaaa cacagc 46
<210> 12
<211> 46
<212> DNA
<213>artificial sequence (Homo sapiens)
<220>
<221> misc_feature
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<223> n is a, c, g or t
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actcgaagtt cagtcgnnnn nnnnnngatg gtttggtatg aggctg 46
<210> 13
<211> 46
<212> DNA
<213>artificial sequence (Homo sapiens)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 13
actcgaagtt cagtcgnnnn nnnnnntatg ttccagcctt ctggag 46
<210> 14
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 14
actcgaagtt cagtcgnnnn nnnnnnaggt cacattcatc gtgccg 46
<210> 15
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 15
actcgaagtt cagtcgnnnn nnnnnngcac tctgagagga ggaaca 46
<210> 16
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 16
actcgaagtt cagtcgnnnn nnnnnnggtc atggaagcag tgcctt 46
<210> 17
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 17
actcgaagtt cagtcgnnnn nnnnnngaca gggatccaga gttcca 46
<210> 18
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 18
actcgaagtt cagtcgnnnn nnnnnngcac cagattctta tcagac 46
<210> 19
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 19
actcgaagtt cagtcgnnnn nnnnnngayt gaggcacctc cagatg 46
<210> 20
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 20
actcgaagtt cagtcgnnnn nnnnnnagct cttcagagga aggtgg 46
<210> 21
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 21
actcgaagtt cagtcgnnnn nnnnnnaact ggtacacagc aggttc 46
<210> 22
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 22
actcgaagtt cagtcgnnnn nnnnnnggtg gagtcacatt tctcag 46
<210> 23
<211> 47
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 23
actcgaagtt cagtcgnnnn nnnnnncaat cttcttggat gatctga 47
<210> 24
<211> 45
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 24
actcgaagtt cagtcgnnnn nnnnnngtct cagtgggtag atggt 45
<210> 25
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 25
actcgaagtt cagtcgnnnn nnnnnngagg aacaagtcag gttcct 46
<210> 26
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 26
actcgaagtt cagtcgnnnn nnnnnntttc gctgctacag ggcttc 46
<210> 27
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 27
actcgaagtt cagtcgnnnn nnnnnnagac agatggggct gttgtt 46
<210> 28
<211> 49
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 28
actcgaagtt cagtcgnnnn nnnnnnccac caaattctca tcagacagg 49
<210> 29
<211> 46
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 29
actcgaagtt cagtcgnnnn nnnnnngata cagttggtgc agcatc 46
<210> 30
<211> 47
<212> DNA
<213>artificial sequence (Mouse)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 30
actcgaagtt cagtcgnnnn nnnnnntggg agtggacttg ggctgac 47
<210> 31
<211> 46
<212> DNA
<213>artificial sequence (Rabbit)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 31
actcgaagtt cagtcgnnnn nnnnnngatc aggcagccga ygacca 46
<210> 32
<211> 45
<212> DNA
<213>artificial sequence (Rabbit)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 32
actcgaagtt cagtcgnnnn nnnnnnctct gcagcaggag gccaa 45
<210> 33
<211> 46
<212> DNA
<213>artificial sequence (Rabbit)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 33
actcgaagtt cagtcgnnnn nnnnnncggt cttgtccact ttggtg 46
<210> 34
<211> 46
<212> DNA
<213>artificial sequence (Rabbit)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 34
actcgaagtt cagtcgnnnn nnnnnngtac agagttggag atgaca 46
<210> 35
<211> 46
<212> DNA
<213>artificial sequence (Rabbit)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 35
actcgaagtt cagtcgnnnn nnnnnntggt gggaagakga ggacag 46
<210> 36
<211> 48
<212> DNA
<213>artificial sequence (Rabbit)
<220>
<221> misc_feature
<222> (17)..(26)
<223> n is a, c, g or t
<400> 36
actcgaagtt cagtcgnnnn nnnnnngggt agaagtcayt gatcagac 48
<210> 37
<211> 20
<212> DNA
<213>artificial sequence (Rabbit)
<400> 37
catggacctt acgactagct 20
<210> 38
<211> 24
<212> DNA
<213>artificial sequence (Rabbit)
<400> 38
gactaggaac tcgaagttca gtcg 24
Claims (6)
1. a kind of immune group library method for screening the reaction of independent sample crossover, it is characterised in that: the following steps are included:
The preparation of S1, cDNA template: using the RNA greater than 10ng as initial amount, RNA is made to carry out reverse transcription using the principle of RACE
At progress template switch when cDNA at the cDNA of one section of anchor tip of band；
S2, then cDNA template carry out 2 pre- amplifications before PCR amplification, and pre- amplification uses the upstream primer with UMI for the first time,
So that the end 5` of original template takes UMI, product A is formed；
S3, it is purified after, product A is carried out second pre- amplification, using the downstream primer with UMI, so that the end the 3` band of template
UMI has been gone up, product B is formed；
S4, it is purified after, using the product B with both-end UMI in step S3 as template carry out PCR amplification, finally obtain production
Object C；
Final product C obtained in S5, step S4 obtains final sequencing library by purifying and connecting sequence measuring joints sequence.
2. a kind of a kind of examination independent sample crossover reaction described in claim 1 is prepared according to claim 1
Immune group library method, it is characterised in that: in described step S2, S3, the S4, the upstream primer that expands in advance for the first time, second
The primer sequence of the downstream primer and PCR amplification that expand in advance totally 38, as shown in sequence table.
3. a kind of a kind of examination independent sample crossover reaction described in claim 1 is prepared according to claim 2
Immune group library method, it is characterised in that: the pre- amplification condition of cDNA template first of the S2 include the following:
(1) following PCR reaction system is configured
(2) PCR response procedures are set as follows:
(3) after the reaction was completed, reaction product is purified, is finally eluted with 20ul Nuclease-free water,
Obtain product A.
4. a kind of a kind of examination independent sample crossover reaction described in claim 1 is prepared according to claim 2
Immune group library method, it is characterised in that: the product A of the S3 carries out the second pre- amplification condition include the following:
(1) following PCR reaction system is configured, product A is divided into 4 equal portions, while carrying out 4 reactions.
(2) PCR response procedures are as follows:
(3) after the reaction was completed, obtained product is reacted 4 and be mixed to get the product that a general pipeline adds up to 200ul, to the product
It is purified, is finally eluted with 20ul Nuclease-free water, obtain product B.
5. a kind of a kind of examination independent sample crossover reaction described in claim 1 is prepared according to claim 2
Immune group library method, it is characterised in that: the PCR amplification of the S4, include the following:
(1) PCR reaction is carried out by template of the purified product B of step 3, according to following preparation reaction system；
(2) PCR response procedures are as follows:
(3) after the reaction was completed, which is purified, is finally eluted, is obtained with 30ul Nuclease-free water
To product C.
6. a kind of according to claim 3,4 or 5 prepare a kind of examination independent sample crossover described in claim 1
The immune group library method of reaction, it is characterised in that: the primer sequence requires as follows:
The nucleotide sequence of RACE oligo includes or is made of SEQ ID NO:1, and wherein the 4 of end G is in ribonucleic acid
Guanine；
The nucleotide sequence of Primer 1 in step 2 includes or is made of SEQ ID NO:2；
When receptor behaviour BCR IgA heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:3
Composition；
When receptor behaviour BCR IgD heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:4
Composition；
When receptor behaviour BCR IgE heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:5
Composition；
When receptor behaviour BCR IgG heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:6
Composition；
When receptor behaviour BCR IgM heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:7
Composition；
When receptor behaviour BCR IgK light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:8
Composition；
When receptor behaviour BCR IgL light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:9
Composition；
When receptor behaviour TCR alpha chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:10
Composition；
When receptor behaviour TCR beta chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:11
Composition；
When receptor behaviour TCR delta chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:12
Composition；
When receptor behaviour TCR gama chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:13
Composition；
When receptor is mouse BCR IgA heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
14 compositions；
When receptor is mouse BCR IgD heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
15 compositions；
When receptor is mouse BCR IgE heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
16 compositions；
When receptor is mouse BCR IgG heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
17 compositions；
When receptor is mouse BCR IgM heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
18 compositions；
When receptor is mouse BCR IgK light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
19 compositions；
When receptor is mouse BCR IgL light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
20 compositions；
When receptor is mouse TCR alpha chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
21 compositions；
When receptor is mouse TCR beta chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
22 compositions；
When receptor is mouse TCR delta chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
23 compositions；
When receptor is rat BCR IgA heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
24 compositions；
When receptor is rat BCR IgD heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
25 compositions；
When receptor is rat BCR IgE heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
26 compositions；
When receptor is rat BCR IgG heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
27 compositions；
When receptor is rat BCR IgM heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
28 compositions；
When receptor is rat BCR IgK light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
29 compositions；
When receptor is rat BCR IgL light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
30 compositions；
When receptor is rabbit BCR IgA heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
31 compositions；
When receptor is rabbit BCR IgE heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
32 compositions；
When receptor is rabbit BCR IgG heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
33 compositions；
When receptor is rabbit BCR IgM heavy chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
34 compositions；
When receptor is rabbit BCR IgK light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
35 compositions；
When receptor is rabbit BCR IgL light chain: the nucleotide sequence of the Primer 2 in step 3 includes or by SEQ ID NO:
36 compositions；
The nucleotide sequence of Primer 3 includes or is made of SEQ ID NO:37 in step 4；
The nucleotide sequence of Primer 4 includes or is made of SEQ ID NO:38 in step 4.
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| CN113621609A (en) * | 2021-09-15 | 2021-11-09 | 深圳泛因医学有限公司 | Library construction primer group and application thereof in high-throughput detection |
| CN113999891A (en) * | 2021-09-17 | 2022-02-01 | 广东省人民医院 | Method for constructing immune repertoire high-throughput sequencing library for removing chimera sequences in sample, and group of primers and kit |
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