CN114277091A

CN114277091A - Method for constructing high-quality immune repertoire library

Info

Publication number: CN114277091A
Application number: CN202111095473.0A
Authority: CN
Inventors: 张镇海; 余学清; 唐海培; 曾惠昆; 汪启龙; 朱燕; 陈青云; 蓝春红; 关俊杰; 谢文汐
Original assignee: Guangdong General Hospital
Current assignee: Guangdong General Hospital
Priority date: 2021-09-17
Filing date: 2021-09-17
Publication date: 2022-04-05
Anticipated expiration: 2041-09-17
Also published as: CN114277091B

Abstract

The invention provides a method for constructing a high-quality immune repertoire library with double-ended Barcode, double-ended UMI and double-ended Index. The method mainly comprises reverse transcription and three-round PCR amplification, wherein double-end Barcode is used for distinguishing different samples, double-end UMI is used for eliminating errors generated in the library construction process to the maximum extent, the molecular number and the gene expression quantity in an original sample are reduced, and double-end Index is used for eliminating errors generated in the sequencing process to the maximum extent.

Description

Method for constructing high-quality immune repertoire library

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for quickly and efficiently constructing a high-quality immune repertoire library.

Background

The immune repertoire (immune pretoire) is the sum of diversity of different B Cell Receptors (BCR) or T cell receptors (BCR) in the immune system of an organism, reflects the capability of the immune system of the organism to respond to external stimuli in a specific time period, and can accurately and comprehensively reflect the health condition of the immune system of the organism. The B Cell Receptor (BCR) consists of two heavy chains and two light chains, wherein the heavy chains are encoded by IGH genes and the light chains are encoded by IGL (Lamda chain) and IGK (Kappa chain) genes, respectively, and the composition of the BCR can be better reflected because the heavy chains have more complex internal compositions.

The TCR molecules, like BCRs, belong to the immunoglobulin superfamily, consisting of TCR α and TCR β chains or TCR γ and TCR δ chains. The complexity and diversity of BCRs and TCRs is unprecedented, arising from four mechanisms: diversification of different VDJ gene fragment combinations; random insertion and deletion during connection of different gene fragments cause connection diversification; diversification of different heavy and light chain combinations; and random high frequency somatic mutations specific to antibodies. It is estimated that these mechanisms can result in at least 10¹²Different antibodies are used.

Owing to the rapid development and wide application of the next-generation high-throughput sequencing technology, the DNA and RNA sequencing platform plays a very important role in various fields. Currently, this emerging technology has been widely applied to immune repertoire sequencing of BCRs and TCRs. Sequencing techniques for sequencing target sequences often require the construction of libraries, typically by adding sequencing adaptors by PCR amplification or by adding adaptors to both ends of the target fragment by ligation and transposase. However, because the similarity between BCR or TCR sequences is high, chimera is easily generated in a PCR amplification mode, and the phenomenon of low connection efficiency exists when a joint is added in a connection reaction mode. Therefore, a new fast and efficient database building method is needed to simplify the experimental steps and improve the database building efficiency.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for constructing a UMI (unique molecular identifier) with double ends Barcode (4bp fixed base) and double ends, wherein the UMI (unique molecular identifier) consists of random bases, the structure of the UMI designed by the invention is NNNNNNNNNNNN, the total length is 12bp, and the random number is 4¹²Excellent randomness) and double-ended Index (fixed bases of 8 bp), distinguishing different samples by using double-ended Barcode, eliminating errors generated in the library construction process to the maximum extent by using double-ended UMI, reducing the number of molecules and the gene expression amount in the original sample, and eliminating errors generated in the sequencing process to the maximum extent by using double-ended Index. Meanwhile, in the reverse transcription process, a plurality of Isotype primers are used for simultaneous operation, so that original molecular proportions of different Isotype can be obtained.

The invention provides a method for constructing a high-quality immune repertoire library with double-ended Barcode, double-ended UMI and double-ended Index, which comprises the following steps:

(1) extracting total RNA in a sample;

(2) performing reverse transcription on the total RNA in the step (1), synthesizing First-strand cDNA with single-ended Barcode and single-ended UMI, and purifying the First-strand cDNA;

(3) adding a PCR #1 reaction system into the First-strand cDNA to perform First round RCR amplification, and purifying to obtain a First round amplification product;

(4) adding a PCR #2 reaction system into the first round amplification product, carrying out second round RCR amplification, and purifying to obtain a second round amplification product;

(5) and adding a PCR #3 reaction system into the second round amplification product to amplify the adaptor sequence, and purifying the generated amplification product to obtain the immune repertoire library.

Further, reverse transcription was performed in the presence of specific primer GSP with Barcode, UMI and Read2, starting from the 5' end with the Read2 sequence, Barcode sequence, UMI sequence and BCR or TCR specific primer sequence GSP.

Furthermore, the Read2 sequence is 22bp long, Barcode is a fixed base of 4bp, UMI is composed of random bases and 12bp long, and the base sequence is NNNNNNNNNNNN.

Further, in step (2), after reverse transcription, RNase H treatment is performed before purification.

Further, in step (3), in the First round of RCR amplification, PCR extension is carried out by using specific primers with Barcode, UMI and Read1 by using the First-strand cDNA as a template, so as to form a First round of amplification products with double ends of Barcode and UMI.

Further, in the step (4), in the second round of RCR amplification, the first round of amplification products with double-ended Barcode and UMI are used as templates, and Read1 and Read2 are used as upstream and downstream primers for amplification enrichment to obtain second round of amplification products. The Read1 and Read2 sequences were 22bp in length.

Further, in the step (5), during the third round of RCR amplification, the enriched template is amplified by using the amplified and enriched second round amplification product as a template and the sequencing adapter containing double-ended Index as a primer, so as to obtain an amplification product with a sequencing adapter for on-machine sequencing.

Further, the primer sequences are SEQ ID No 1(TCR) and SEQ ID Nos 2-6(BCR) at the time of reverse transcription.

Further, in the first round of RCR amplification, the primer sequences are SEQ ID No 7-43(TCR) and SEQ ID No44-51 (BCR).

Further, in the second round of RCR amplification, the primer sequence is SEQ ID No 52-53.

Further, in the third round of RCR amplification, the primer sequence is SEQ ID No 54-55.

The invention also provides a group of primers for constructing a high-quality immune repertoire library with double-ended Barcode, double-ended UMI and double-ended Index, wherein the group of primers comprises SEQ ID No 1-55.

The invention also provides a kit for constructing a high-quality immune repertoire library with double-ended Barcode, double-ended UMI and double-ended Index, wherein the kit comprises a group of primers, and the group of primers comprises SEQ ID No 1-55.

The invention also provides a high-quality immune repertoire library, wherein two ends of the immune repertoire library contain Barcode, UMI and Index.

The invention also provides a group of oligonucleotides for constructing high-quality immune repertoire libraries, each oligonucleotide containing Barcode, UMI and Index at both ends.

Compared with the prior art, the invention has the characteristics and beneficial effects that:

(1) the invention is suitable for constructing immune repertoire sequencing libraries, comprises TCR and BCR, is suitable for mixed amplification of various Isotype, can accurately quantify the proportion of various Isotype in a sample under a real state, is used for constructing libraries containing double-ended Barcode, double-ended UMI and double-ended Index, and is directly used for on-machine sequencing of sequencing platforms such as Novaseq and the like.

(2) The invention provides a construction method of immune repertoire (including TCR and BCR) library, which directly adds sequencing joint into the library in the amplification process without further constructing the library, thereby saving time and material cost.

(3) In the invention, various types of BCRs can be amplified simultaneously by adding various Isotype primers in the reverse transcription process, and the proportion of the Isotype is obtained.

(4) The invention uses specific primers with Barcode, UMI and Read2 to carry out reverse transcription by a reverse transcription and 3-step PCR mode to obtain cDNA containing single-ended Barcode and UMI; the first round of PCR was performed with the cDNA generated as a template, and 1 cycle of PCR extension was performed with specific primers with Barcode, UMI and Read1, with the purpose of carrying Barcode and UMI on the other end, to form a molecule with both Barcode and UMI ends; in the second round of PCR, molecules with double ends Barcode and UMI are used as templates, and Read1 and Read2 are used as upstream and downstream primers to amplify and enrich the molecules for 4 cycles; and the third round of PCR takes the amplified and enriched product as a template and a sequencing joint containing double-ended Index as a primer to amplify the enriched template for 20 cycles, so as to obtain a PCR product with the sequencing joint, and construct the double-ended Barcode, the double-ended UMI and the double-ended Index into a library, so that the data analysis can be more accurate, and the analysis of the immune repertoire can be further optimized.

(5) The invention adopts 3 rounds of PCR amplification, so that the obtained library has high quality and more accurate data can be obtained.

(6) The library building method provided by the invention can be used for different sequencing platforms, is particularly suitable for a high-throughput Novaseq 6000 sequencing platform, and is wide in applicability and easy to popularize.

Drawings

FIG. 1 is a schematic diagram of construction of immune repertoire libraries with double ends Barcode, UMI and Index constructed in the present invention.

FIG. 2 is a flow chart of construction of an immune repertoire library with double-ended Barcode, double-ended UMI and double-ended Index constructed according to the present invention.

Fig. 3BCR electrophoretic detection map.

FIG. 4TCR electrophoresis detection images.

FIG. 5 construction of quality control charts of the immune repertoire library.

FIG. 6BCR various Isotype proportion analysis chart.

FIG. 7 is a graph showing the hypermutation change of IgM somatic cells corrected by UMI.

Detailed Description

In order to achieve the above purpose, the invention takes BCR as an example, and provides the following technical scheme:

1. isolation of Peripheral Blood Mononuclear Cells (PBMC)

a, reversing and uniformly mixing a lymphocyte separation solution (TBD), and adding 5ml of lymphocyte separation solution into a 15ml centrifuge tube for later use;

b, adding 5ml of peripheral blood into a 15ml centrifuge tube filled with 1xPBS with the same amount, reversing, uniformly mixing, and diluting the blood; c, slowly dripping the diluted blood into a 15ml centrifuge tube filled with lymphocyte separation solution by using a disposable pasteur pipette at an oblique angle of 45 degrees;

d, centrifuging for 30min at the temperature of 25 ℃ of 750g and the acceleration of 3 and the deceleration of 0;

e, adding 9ml of 1xPBS into a 15ml centrifuge tube for later use;

f, carefully taking out the centrifuged sample, sucking 5ml of supernatant by using a vacuum pump, sucking the white membrane layer into the centrifuge tube prepared in the previous step by using a disposable pasteur pipette, and reversing and uniformly mixing;

g, 350g at 25 ℃, 9 at acceleration and 9 at deceleration, and centrifuging for 10 min;

h, (optional) if obvious red blood cell residues exist, fully resuspending the cell sediment by using 1ml of ACK (Lonza), standing for 2min, then adding 1xPBS to 15ml, reversing and uniformly mixing, at 25 ℃ of 160g, at the acceleration of 9, at the deceleration of 9, and centrifuging for 15 min;

i, resuspending the cell pellet with 1ml of 1xPBS and transferring to a 1.5ml centrifuge tube, centrifuging at 25 ℃ 8000Xg for 2min, removing the supernatant by aspiration, and extracting RNA from the pellet by direct cryopreservation at-80 ℃ or by resuspension with 350ul of RLT lysate (QIAGEN) containing 6ul of beta-ME.

2. Extraction of RNA

In the case of extracting RNA from a sample, an existing commercial kit may be used, or an RNA extraction reagent may be prepared by itself. The example of the RNeasy Mini Kit (250) Kit from QIAGEN is as follows:

a, taking out the sample, sequencing, counting the number of cells<＝5×10⁶Adding 350. mu.l RLT and 3.5. mu.l beta-ME, and thoroughly whipping or shaking to mix (if the number of cells is large)>5×10⁶And is<＝1×10⁷Adding 600 mul RLT and 6 mul beta-ME), pumping and uniformly mixing for 15-20 times by using a 1ml syringe until the precipitate or the cells are completely dissolved;

b, adding equal volume of 70% ethanol (which is used in the preparation), blowing, beating and uniformly mixing to obtain a uniformly mixed solution;

c, sucking 700 mul of the mixed solution into a red RNA extraction column, centrifuging at 12000rpm at room temperature for 30s, and repeating the operation if the mixed solution is larger than 700 ul;

d, discarding the flow-through liquid, adding 500 μ l RW1 into the red RNA extraction column, and centrifuging at 12000rpm for 30s at room temperature;

e, preparing DNase I solution (mixing 10ul DNase I stock solution with 70ul RDD buffer, reversing the mixture up and down, uniformly mixing, and centrifuging at low speed), adding 80ul DNase I solution into each red RNA extraction column, and standing at room temperature for 15 min;

f, adding 500 μ l RW1 into the red RNA extraction column, and centrifuging at 12000rpm at room temperature for 30 s;

g, discarding the flow-through liquid, adding 500 mul RPE into the red RNA extraction column, and centrifuging at 12000rpm for 30s at room temperature;

h, repeating the previous step;

i, replacing a new 2ml collecting pipe, and centrifuging for 2min at the room temperature of 12000 rpm;

j, changing a 1.5ml centrifuge tube, adding 25 mul of non-enzyme water into the center of the red RNA extraction column membrane for dissolving, standing for 5min, and centrifuging for 2min at the room temperature of 12000 rpm;

k, adding the flow-through liquid into the red RNA extraction column again, standing for 2min, and centrifuging for 2min at the room temperature of 12000 rpm;

l, preparing a mixture for measuring the Qubit RNA (1 ul of RNA BR Reagent in 199ul of RNA BR Buffer according to the proportion, shaking and mixing uniformly, and centrifuging at a low speed), and adding 190ul of the mixture for measuring the Qubit RNA into 10ul of RNA BR standard #1 and RNA BR standard #2 for correcting a Qubit instrument; additionally, 199ul of the Qubit RNA mixture was added to 1ul of the RNA sample for determination of RNA concentration;

m, storing the extracted RNA in a refrigerator at the temperature of minus 80 ℃.

3. Reverse transcription

a, taking out a sample, sequencing, using a 0.2ml centrifuge tube, absorbing RNA (1ug), supplementing RNase free water to 10.8 mu l, adding 0.2ul 10uM GSP primer (the GSP primer is formed by equivalently mixing IgG, IgM, IgA, IgD and IgE primers, the sequence is SEQ ID No 2-6, the final concentration is 2pmol) and 1 mu l dNTP (10mM), mixing uniformly, reacting for 5min at 65 ℃ in a PCR instrument, and immediately placing on ice for standing for 3min after time;

b, putting the sample back to a refrigerator at the temperature of minus 80 ℃;

c, adding the components according to the following reaction system:

d, after blowing, beating and uniformly mixing, placing on a PCR instrument to execute the following procedures: hold at 42 ℃ for 2min and 4 ℃;

e, adding 1 μ l SuperScript II Reverse Transcriptase into the reaction system, blowing, mixing uniformly, and placing on a PCR instrument to execute the following programs: hold at 42 ℃ for 50min, 70 ℃ for 15min and 4 ℃;

f, respectively adding 0.2ul of RNase H (10U/ul) into each reaction system, and reacting at 37 ℃ for 20min, wherein the hold is 4 ℃;

g, the reverse transcribed sample is placed in a refrigerator at-20 ℃ for storage or further operation is carried out.

4. Magnetic bead purification

a, vortexing for 30s to fully resuspend the AMPure XP magnetic beads, and adding the AMPure XP magnetic beads according to the following volume according to a PCR reaction system:

b, blowing and beating the mixture for 20 times by using a pipettor, and fully mixing the mixture, and incubating the mixture for 5min at room temperature;

c, after a short centrifugation (1s), placing the PCR tube in a magnetic rack for 2min, and removing the supernatant;

d, keep the PCR tube in the magnetic rack, add 200ul 70% ethanol (now ready for use), incubate at room temperature for 30s, remove the supernatant. Repeating the rinsing for 2 times, and sucking clean ethanol for the last time;

e, uncovering the cover, drying the cover with air until no ethanol residue exists (no liquid drops exist at the bottom and the pipe wall of the PCR pipe), and observing the surfaces of the magnetic beads such as a frosted sample to avoid drying cracks;

f, adding 24ul (suitable for a subsequent 50ul PCR reaction system) of enzyme-free water for elution, blowing and beating for 20 times by using a pipette, fully and uniformly mixing, standing at room temperature for 2min, and transferring to a magnetic frame for standing for 2min after short centrifugation (3 s);

and g, transferring the supernatant to a new PCR tube for subsequent PCR, and taking up the magnetic beads without touching.

5, first round PCR reaction

The first round of PCR is carried out, and a PCR reaction system is configured according to the following volumes:

wherein the Forward primer is formed by mixing 8 specific primers of V region in equal amount, and the sequence is SEQ ID No 44-51.

The PCR instrument was set with the following conditions:

6. magnetic bead purification

Reference step 4

7. Second round PCR reaction

And (3) carrying out second round PCR, and configuring a PCR reaction system according to the following volumes:

(a) the PCR reaction system is configured according to the following volume:

wherein the Read1 primer and the Read2 primer are primers containing 22bp and have the sequences of SEQ ID Nos. 52-53.

The PCR instrument was set with the following conditions:

8. magnetic bead purification

Reference step 4

7. Second round PCR reaction

(a) the PCR reaction system is configured according to the following volume:

the PCR instrument was set with the following conditions:

and (3) detecting the PCR product by using 1.5% agarose gel electrophoresis, selecting a BCR fragment with the fragment length of about 560bp, cutting and recovering the gel to obtain a purified BCR fragment, wherein the gel recovery and purification step adopts a gel purification kit of MN company and is carried out according to the conventional laboratory operation.

Sequencing returns data and by analysis, the proportion of each Isotype in the sample can be specified (as shown in figure 6). At the same time, with paired-end UMI, the source of each molecule can be determined, based on which re-analysis of IgM Somatic Hypermutation (SHM) ratios revealed that SHM after alignment of Consensus (Consensus, C) sequences was significantly lower than Total IgM (Total, T), suggesting that the quality of the data obtained from sequencing can be significantly improved after UMI correction (fig. 7).

110, Guangdong province people hospital

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ctacacgacg ctcttccgat ctactgnnnn nnnnnnnnga ggtgcagctg gtggagtct 59

<210> 49

<211> 60

<212> DNA

<213> Artificial sequence

<400> 49

ctacacgacg ctcttccgat ctactgnnnn nnnnnnnnca ggtgcagcta cagcagtggg 60

<210> 50

<211> 60

<212> DNA

<213> Artificial sequence

<400> 50

ctacacgacg ctcttccgat ctactgnnnn nnnnnnnnca ggtgcagctg caggagtcgg 60

<210> 51

<211> 22

<212> DNA

<213> Artificial sequence

<400> 51

ctacacgacg ctcttccgat ct 22

<210> 52

<211> 22

<212> DNA

<213> Artificial sequence

<400> 53

cagacgtgtg ctcttccgat ct 22

<210> 53

<211> 62

<212> DNA

<213> Artificial sequence

<223> the number of bases not incorporated into the Index sequence was counted by inserting the Index sequence between base C at position 29 and base A at position 30, and the Index sequence varied depending on the sequencing platform.

<400> 53

aatgatacgg cgaccaccga gatctacac-Index-a cactctttcc ctacacgacg ctcttccgat 60

ct 62

<210> 54

<211> 58

<212> DNA

<213> Artificial sequence

<223> the number of bases not incorporated into the Index sequence was counted by inserting the Index sequence between base T at position 24 and base G at position 25, and the Index sequence varied depending on the sequencing platform.

<400> 54

caagcagaag acggcatacg agat-Index-gtgact ggagttcaga cgtgtgctct tccgatct 58

Claims

1. A method of constructing a high quality immune repertoire library with paired ends Barcode, paired ends UMI and paired ends Index, the method comprising the steps of:

(1) extracting total RNA in a sample;

2. The method of claim 1, wherein reverse transcription is carried out in the presence of GSP specific primers with Barcode, UMI and Read2, the GSP starting from the 5' end being the Read2 sequence, the Barcode sequence, the UMI sequence and the BCR or TCR subtype specific sequence, the Read2 sequence being 22bp long, the Barcode being a 12bp variable sequence and the fixed base ACTG being 4bp, the UMI consisting of random bases, being 12bp long and the base sequence being NNNNNNNNNNNN.

3. The method according to claim 1, wherein in the step (2), after the reverse transcription, RNase H treatment is performed before purification.

4. The method of claim 1, wherein in step (3), in the First round of RCR amplification, PCR extension is performed using specific primers with Barcode, UMI and Read1 using the First-strand cDNA as a template, thereby forming a First round of amplification products with double-ended Barcode and UMI.

5. The method of claim 1, wherein in step (4), in the second round of RCR amplification, a first round of amplification products with double-ended Barcode and UMI are used as templates, and Read1 and Read2 are used as upstream and downstream primers for amplification enrichment to obtain a second round of amplification products.

6. The method according to claim 1, wherein in the step (5), in the third round of RCR amplification, the enriched second round amplification product is amplified by using the amplified enriched second round amplification product as a template and the sequencing adaptor containing double-ended Index as a primer, so as to obtain the amplification product with the sequencing adaptor for on-machine sequencing.

7. The method of claim 2, wherein the primer sequences are SEQ ID No 1(TCR) and SEQ ID nos 2-6(BCR) at the time of reverse transcription.

8. The method of claim 4, wherein the primer sequences are SEQ ID Nos 7-43(TCR) and SEQ ID Nos 44-51(BCR) during the first round of RCR amplification.

9. The method of claim 5, wherein the primer sequence is SEQ ID Nos 52-53 during the second round of RCR amplification.

10. The method of claim 6, wherein the primer sequence is SEQ ID nos 54-55 during the third round of RCR amplification.

11. A set of primers for constructing a library of high quality immune repertoires with double ends Barcode, double ends UMI and double ends Index, wherein said set of primers comprises SEQ ID nos 1-55.

12. A kit for constructing a library of high quality immune repertoires with double-ended Barcode, double-ended UMI and double-ended Index, comprising a set of primers comprising SEQ ID nos 1-55.

13. A high quality immune repertoire library comprising Barcode, UMI and Index at each end of the library.