Construction method of immune repertoire sequencing library and test kit
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A technology for sequencing libraries and immunological libraries, applied in chemical libraries, biochemical equipment and methods, combinatorial chemistry, etc., can solve the problems of inability to completely eliminate PCR amplification bias, prone to amplification bias, poor specificity, etc.
Pending Publication Date: 2020-08-25
北京全谱医学检验实验室有限公司
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Problems solved by technology
[0005] The multiple primer amplification method mainly amplifies the CDR3 region of TCR or BCR, and is prone to amplification bias and poor specificity, resulting in inaccurate detection results
[0006] The 5' RACE amplification method uses terminal transferase technology. After reverse transcription is completed, C is added to the end, polyG primer is combined and the second strand is synthesized, and a specific adapter is added to the end of cDNA. A pair of primers can be used to amplify TCR or BCR However, the shortcomings of PCR bias and poor specificity cannot be completely avoided. Although an inner primer and specific adapter are also used for amplification, this only solves the shortcomings of poor specificity and cannot completely eliminate PCR amplification bias.
Its disadvantages are the same as 5’ RACE, but the reverse transcription reaction and the addition of specific adapters can be completed in one step
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Embodiment 1
[0057] Example 1 Application of a Human TCR Diversity Detection Kit on the Illumina Sequencing Platform
[0058] 1. Extraction of total RNA.
[0059] 1. Add 5 times the volume of 1X red blood cell lysate H to 200uL of human whole blood.
[0060] 2. Incubate on ice for 10-15 min, and vortex to mix twice during the incubation.
[0061] 3. Centrifuge at 2,100 rpm (~400×g) for 10 min at 4°C to remove the supernatant completely.
[0062] 4. Add 1X Red Blood Cell Lysis Buffer H to the white blood cell pellet to resuspend the cells.
[0063] 5. Centrifuge at 2,100 rpm (~400×g) for 10 min at 4°C to remove the supernatant completely.
[0064] 6. Add 350uL Lysis Solution RLH (please add β-mercaptoethanol before use) to the white blood cell pellet, vortex or use a pipette to mix.
[0065] 7. Transfer the solution to the filter column CS (the filter column CS is placed in a collection tube), centrifuge at 12,000 rpm (~13,400×g) for 2 min, discard the filter column CS, and collect the ...
Embodiment 2
[0093] Example 2 Application of a Human BCR Diversity Detection Kit on the Illumina Sequencing Platform
[0094] 1. Extraction of total RNA.
[0095] 2. Reverse transcription.
[0096]1. Take 100ng total RNA, add IGHCo primer (SEQ ID No.11), IGKCo primer (SEQ ID No.12), IGLCo primer (SEQ ID No.13), dNTPs, reverse transcription Buffer, reverse transcriptase, ILTS linker (SEQ ID No.5), total system 20uL.
[0097] 2. React on PCR machine, 50°C for 30 minutes; 25°C for 5 minutes; 42°C for 25 minutes.
[0098] 3. Pre-amplification.
[0099] 1. Take 2uL reverse transcription product, add PCR master mix, ILT primer (SEQ ID No.6), IGHCi primer (SEQ ID No.14), IGKCi primer (SEQ ID No.15), IGLCi primer (SEQ ID No. 16), add water to 20uL system.
[0100] 2. React on a PCR machine, 95°C for 3 minutes; 95°C for 30 seconds, 65°C for 30s, 72°C for 30s, 20 cycles; 72°C for 5 minutes.
[0101] 3. Add 30uL kapa magnetic beads and mix well, let stand at room temperature for 5 minutes, sepa...
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Abstract
The invention discloses a construction method of an immune repertoire sequencing library and a test kit, belongs to the field of biological medicines, and relates to a detection method of immune repertoire diversity. One of the purposes of the invention is to provide the construction method of the immune repertoire sequencing library; the construction method comprises the following steps of (a) extracting sample RNA, (b) carrying out reverse transcription by using a reverse transcription primer to synthesize cDNA, and adding a specific linker containing a molecular tag sequence at the tail end; (c) by taking the product obtained in the step b as a template, carrying out amplification through a specific linker primer and a gene specific primer containing a molecular tag sequence; and (d) finally, adding a sequencing library linker to an amplification product, so as to complete library construction. When sequencing data is analyzed, original RNA molecules are identified through moleculartag sequences, and amplification bias is excluded. By using the method, all CDR regions of TCR and BCR can be covered, PCR amplification bias interference is effectively eliminated, the specificity is higher, and the analysis result is more accurate. The other purpose of the invention is to provide the test kit for immune repertoire sequencing.
Description
technical field [0001] The invention belongs to the field of biomedicine and relates to a detection method for the diversity of immune repertoire. Background technique [0002] The immune repertoire is the sum of all functionally diverse B and T cells in the circulation of an individual at any given time. Immune repertoire sequencing takes T / B lymphocytes as the research target, amplifies the complementarity determining region (CDR region) that determines the diversity of B cell receptor (BCR) or T cell receptor (TCR), combined with high-throughput sequencing technology , a comprehensive assessment of immune system diversity. [0003] Immunome sequencing enables observation and analysis of T and B cells with unprecedented depth and specificity. It is widely used in disease monitoring, antibody production, vaccine research, health examination and other fields. [0004] At present, the library construction methods widely used in immune repertoire sequencing mainly include m...
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