阅读说明：本技术 一种单分子标签免疫组库高通量测序文库构建方法 (Construction method of high-throughput sequencing library of monomolecular label immune repertoire ) 是由 鞠巍 徐根明 潘艺 赵谦 于 2019-10-13 设计创作，主要内容包括：本发明属于医学检测领域,具体涉及一种单分子标签免疫组库高通量测序文库构建方法。本发明利用类似5’RACE的SMART技术和特异性引物,可特异性捕获并无偏好扩增人源RNA中所有低丰度TCRα、TCRβ、BCR重链H、BCR轻链L、BCR轻链K的整个CDR区域(可以单独捕获扩增其中一项,也可以同时捕获扩增所有项目),并采取一种简单高效的方式对其进行高通量测序文库的构建,从而进行TCR和BCR免疫组库分析。(The invention belongs to the field of medical detection, and particularly relates to a construction method of single molecule label immune repertoire high-throughput sequencing libraries, which utilizes a SMART technology similar to 5' RACE and specific primers, can specifically capture and preferentially amplify the whole CDR regions (can capture and amplify items independently or capture and amplify all items simultaneously) of all low-abundance TCR α, TCR β and BCR heavy chain H, BCR light chain L, BCR light chain K in human RNA, and adopts simple and efficient modes to construct a high-throughput sequencing library, so as to analyze the TCR and BCR immune repertoire.)

1, method for constructing high-throughput sequencing library of single-molecule tag immune repertoire, which comprises the following steps:

(1) extracting total RNA of the sample;

(2) taking the total RNA obtained in the step (1) as a template, adding an RT1 primer and a monomolecular label in sequence to perform reverse transcription and template conversion reaction to obtain cDNA with the monomolecular label;

(3) adding a TS-index primer and an RT2 primer into an strand composite serving as a template, performing semi-nested amplification, specifically amplifying a target region, and adding a sequencing joint to obtain DNA with a single-molecule label;

(4) after sorting and purifying the specific amplification product, adding a P7 joint primer and a P5 joint primer for PCR amplification to obtain a sequencing library.

2. The method for constructing the high-throughput sequencing library of the single-molecule tag immune repertoire according to claim 1, wherein in the step (2), the sequence of the single-molecule tag is shown as SQE ID No. 1.

3. The method for constructing the high-throughput sequencing library of the single-molecule tag immune repertoire according to claim 2, wherein in the step (2), the 3' end of the single-molecule tag is further connected with a plurality of degenerate bases, and the number of the degenerate bases is 1-20.

4. The method for constructing the high-throughput sequencing library of the single-molecule tag immune repertoire according to claim 3, wherein the degenerate base is an original base and/or a modified base, and the modified base comprises a thio modification, a methylation modification, an LNA modification and/or an hypoxanthine modification.

5. The method for constructing the high-throughput sequencing library of the single-molecule-tag immune repertoire according to claim 1, wherein in the step (2), the RT1 primer comprises a sequence shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and/or SEQ ID NO. 10.

6. The method for constructing the high-throughput sequencing library of the single-molecule-tag immune repertoire according to claim 1, wherein in the step (3), the RT2 primer comprises a sequence shown as SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 and/or SEQ ID NO. 17.

7. The method for constructing the high-throughput sequencing library of the single-molecule tag immune repertoire of claim 6, wherein the RT2 primer is further connected with an original base or a modified base at the 3' end, wherein the modified base comprises a thio modification, a methylation modification, an LNA modification and/or an hypoxanthine modification.

8. The method for constructing the high-throughput sequencing library of the single-molecule tag immune repertoire according to claim 1, wherein in the step (3), the sequence of the TS-index primer is shown as SEQ ID NO. 18.

9. The method for constructing the high-throughput sequencing library of the single-molecule-tag immune repertoire according to claim 1, wherein in the step (4), the sequence of the P7 adapter primer is shown as SEQ ID NO.19 or SEQ ID NO.18, and the sequence of the P5 adapter primer is shown as SEQ ID NO. 20.

10. The method for constructing the high-throughput sequencing library of the single-molecule-tagged immune repertoire of any one of claims 1-9, wherein in the step (2), the reaction temperature of the reverse transcription reaction is 25-50 ℃ and the reaction time is 30-90 min.

11. The method of constructing the high throughput sequencing library of any one of claims 1-9 to , wherein the sample comprises CDR regions of human TCR α, TCR β and BCR heavy chain H, BCR light chain L, BCR light chain K.

12. The method for constructing the high-throughput sequencing library of the single-molecule tag immune repertoire according to claim 1, wherein the technical platform for the method is a second-generation sequencing platform.

Technical Field

The invention belongs to the field of medical detection, and particularly relates to a construction method of a high-throughput sequencing library of single-molecule tag immune repertoires.

Background

The sequencing of the immune repertoire is techniques for researching the diversity of TCR or BCR coding genes by using a high-throughput sequencing technology, and can reflect the relation between T/B cell clone change and diseases, and the method is widely applied to fields of tumors, autoimmune diseases, infectious diseases, transplantation and the like at present.

However, the use of DNA as the sequencing template for the immunohistochemical library has the disadvantages that ① amplification process uses a large number of primer pairs, but perfect match amplification between primers is impossible, and non-authentic recombinant sequences are easily generated, that ② J-C regions have a large number of introns, so that the downstream primers must be located in J regions, that ③ primers are designed from known reference sequences, and unknown sequences cannot be captured.

Chinese patent 201410442470.3 uses whole blood mRNA as template, and performs library construction based on 5' RACE method, but only can detect TCR- β immune repertoire, but not TCR- α and BCR immune repertoire, and mismatch is introduced in PCR process.

Chinese patent 201510488029.3 uses cfDNA as a template and adopts multiplex PCR amplification technology to realize the detection of immune repertoire of BCR H chain and TCR β chain, but because of the technical defects of multiplex PCR itself in , a certain region is preferentially amplified and non-specific amplification exists, and in , the patent does not detect TCR- α and BCR light chain and cannot say that the detection is complete immune repertoire.

In addition, there are also methods of performing library construction using 5 'RACE or similar 5' RACE methods, but most of them use dTprimer for RT, followed by two PCRs, and finally ligation of sequencing adapters for library construction. These methods, which can only use RNA with a Poly A tail for RT, have limitations on low abundance transcripts.

Disclosure of Invention

The invention aims to provide a method for constructing a high-throughput sequencing library of a single-molecule tag immune repertoire.

The method for constructing the high-throughput sequencing library of the single-molecule tag immune repertoire comprises the following steps:

(1) extracting total RNA of the sample;

(2) taking the total RNA obtained in the step (1) as a template, adding an RT1 primer and a monomolecular label in sequence to perform reverse transcription and template conversion reaction to obtain cDNA with the monomolecular label;

(3) adding a TS-index primer and an RT2 primer into an strand composite serving as a template, performing semi-nested amplification, specifically amplifying a target region, and adding a sequencing joint to obtain DNA with a single-molecule label;

(4) after sorting and purifying the specific amplification product, adding a P7 joint primer and a P5 joint primer for PCR amplification to obtain a sequencing library.

Preferably, in the step (2), the sequence of the single molecule tag is shown as SQE ID NO. 1.

Preferably, in step (2), the single molecule tag further comprises a plurality of degenerate bases connected to the 3' end thereof, wherein the number of the degenerate bases is 1-20.

Preferably, the degenerate base is an original base and/or a modified base, the modified base comprising a thio modification, a methylation modification, an LNA modification and/or a hypoxanthine modification.

Preferably, the RT1 primer comprises a sequence as shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and/or SEQ ID NO. 10.

Preferably, the RT2 primer comprises a sequence as shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 and/or SEQ ID NO. 17.

Preferably, the RT2 primer further has an original base or a modified base attached to its 3' end, wherein the modified base comprises a thio modification, a methylation modification, an LNA modification and/or an hypoxanthine modification.

Preferably, in the step (3), the sequence of the TS-index primer is shown as SEQ ID NO. 18.

Preferably, the base at the 3 'end of the TS-Index primer can be complementary to the adaptor after conversion of the TS-3G primer template with the single molecule label, and the 5' end comprises a P7 binding sequence, an Index sequence and a Read 2 sequencing sequence.

Preferably, in step (4), the sequence of the P7 adapter primer is shown as SEQ ID NO.19 or SEQ ID NO.18, and the sequence of the P5 adapter primer is shown as SEQ ID NO. 20.

Preferably, in the step (2), the reaction temperature of the reverse transcription reaction is 25-50 ℃ and the reaction time is 30-90 min.

Preferably, the sample comprises CDR regions of a human TCR α, TCR β, BCR heavy chain H, BCR light chain L, BCR light chain K.

Preferably, the technology platform for which the method is applicable is a second generation sequencing platform.

Preferably, the primer sequences used in the present invention are as follows:

note: wherein Index-I7 is prior art disclosed in illumina.

The invention has the beneficial effects that:

the invention can specifically capture and preferentially amplify the whole CDR region of all low abundance TCR α, TCR β and BCR heavy chain H, BCR light chain L, BCR light chain K in human RNA ( items can be captured and amplified independently or all items can be captured and amplified simultaneously), and simple and efficient modes are adopted to construct a high-throughput sequencing library, so that the analysis of TCR and BCR immune repertoire is carried out.

The method for constructing the immune repertoire library can be used for high-throughput sequencing platforms such as Roche, Illumina, ThermoFisher, Pacific Biosciences, Huada genes, Oxford Nanopore Technologies, Huanyang and Hanhai genes.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows a schematic diagram of construction of an immune repertoire library;

FIG. 2 shows a flow chart of an experiment for constructing an immune repertoire library;

FIG. 3 shows the results of 1.5% agarose gel electrophoresis library detection in example 1 of the present invention;

FIG. 4 shows the results of 1.5% agarose gel electrophoresis library detection in example 2 of the present invention;

FIG. 5 shows the results of the library detection by 1.5% agarose gel electrophoresis in example 3 of the present invention.

Detailed Description

The technical solution of the present invention will be described in detail below to make the objects, technical solutions and advantages of the present invention clearer.

The present invention is further illustrated in by reference to the example of the Illumina platform.

The schematic diagram and the experimental flow chart of the construction method of the immune repertoire library are respectively shown in the figure 1 and the figure 2:

firstly, obtaining total RNA, then taking quantitative total RNA, adding RT1 primer group, monomolecular label primer TS-3G-N and corresponding reaction reagent in sequence, and reacting and synthesizing chain product with monomolecular label under constant temperature condition.

And adding a specific amplification primer RT2 and a P7 adaptor primer into the obtained chain product, adding a reagent for PCR1 reaction, and amplifying a specific complete immune repertoire according to corresponding reaction conditions.

Finally, adding a linker to the obtained complete immune repertoire by PCR2 to obtain an on-machine immune repertoire library with two ends respectively containing P5 and P7 sequences.

The primers used in the invention are as follows:

1. the sequences of the immunohistochemical library specific PCR primer set RT1 are shown in table 1:

TABLE 1 RT1 primer set sequences

Primer types	Primer name	Primer sequence (5' ->3’)
			TCRα	TCR-RT1-A	GTCTAGCACAGTTTTGTC
TCRβ	TCR-RT1-B	GTATCTGGAGTCATTGA
			BCR H	BCR-RT1-IGHA	AGGCAGGCGATGACCACGTT
BCR H	BCR-RT1-IGHG	CTTGACCAGGCAGCCCAGGGCCGCTGTG
			BCR H	BCR-RT1-IGHE	AGTCACGGAGGTGGCATTGGAGGGAATGT
BCR H	BCR-RT1-IGHM	AGGGGGAAAAGGGTTGGGGCGGATGCACT
			BCR H	BCR-RT1-IGHD	TGGGGAACACATCCGGAGCCTTGGTGGGT
BCR K	BCR-RT1-IGK	GCTCATCAGATGGCGGGAAGATGAAGA
			BCR L	BCR-RT1-IGL	TCACGGCTCCCGGGTAGAAGTCACTTATG

or a combination of these primer sets RT1 can be used.

Adopting a semi-nested strategy, carrying out steps of specific synthesis and amplification of a target fragment, using RT2 and RT1 , carrying out double proofreading, and greatly improving the specificity, wherein the sequence of an immune repertoire specific PCR primer group RT2 is shown in a table 2:

TABLE 2RT 2 primer set sequences

The primer set RT2 was used selectively and in combination with either or several of the same types of the primer set RT 1.

The combination of the RT1 primer and RT2 primer can be used to construct different libraries, as specified in the following table:

serial number	Constructed library types	Selected RT1 primer	Selected RT2 primer
				1	TCRα	TCR-RT1-A	TCR-RT2-A
2	TCRβ	TCR-RT1-B	TCR-RT2-B
				3	BCR H	BCR-RT1-IGHA/D/E/G/M	BCR-RT2-IGHJ
4	BCR K	BCR-RT1-IGK	BCR-RT2-IGKJ 1/2/3
				5	BCR L	BCR-RT1-IGL	BCR-RT2-IGLJ

If a TCR α library and a TCR β library are to be constructed simultaneously, only primers with sequence numbers 1 and 2 need to be mixed with , for example, a mixture of TCR-RT1-A and TCR-RT1-B is used as an RT1 primer, a mixture of TCR-RT2-A and TCR-RT2-B is used as an RT2 primer, and the like is repeated in other cases.

TS-3G-N primer sequence with single molecular label:

5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNNX-3'。

wherein "N" represents degenerate bases, the single molecule tag may be composed of 1-20 degenerate bases, and not only 12 "N" s listed herein, the number of "N" s being any natural number from 1-20. Meanwhile, "N" may be not only an original base but also a modified base, and the base modification includes thio, methylation, LNA, hypoxanthine, and the like.

TS-index sequence:

5'-CAAGCAGAAGACGGCATACGAGAT[Index-I7]GTGACTGGAGTTCAGACGTGT-3'；

p7 sequence: 5'-CAAGCAGAAGACGGCATACGAGAT-3', respectively;

p5 adapter Primer (TS-Primer1) sequence:

5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGAC-3'。