FAIR-SEQ™ VJ Multiplex Amplification Library Construction Kit

FAIR-SEQ ™ (Fast&Accurate Immune Repertoire Sequencing) This series of test kits adopts an experimental system optimized based on synthetic templates. After multiple rounds of optimization and adjustment of the primer system, the amplification deviation of the majority of primers in the adjusted experimental system is within one time, which can accurately reflect the true clone distribution of the sample. For specific content, please refer to the following detailed introduction.

FAIR-SEQ™ VJ Multiplex Amplification Library Construction Kit

FAIR-SEQ™ (Fast & Accurate Immune Repertoire Sequencing)

This series of kits adopts an optimized experimental system based on the synthetic template, and after multiple rounds of optimization and adjustment of the primer system, the amplification deviation of most of the primers in the adjusted experimental system is less than doubled, which can accurately reflect the true cloning distribution of the sample.

NEOIMMUNE product catalog

For Research Use Only

Item number

Product name

specification

Sample type

Scope of application

Sequencing recommended

Sequencing platform

Technical approach

R0011M

FAIR-SEQ™ Human IGH VJ  multiplex Kit (FR1)

16 Rxns

DNA/RNA

Human B cell surface heavy chain receptor (IGH, FR1 region) library construction

PE150

MGI

Multiplex PCR

R0012M

FAIR-SEQ™ Human IGH VJ  multiplex Kit (FR1)

96 Rxns

R0013M

FAIR-SEQ™ Human IGH VJ  multiplex Kit (FR3)

16 Rxns

Human B cell surface heavy chain receptor (IGH, FR3 region) library construction

R0014M

FAIR-SEQ™ Human IGH VJ  multiplex Kit (FR3)

96 Rxns

R0021M

FAIR-SEQ™ Human TRB VJ multiplex Kit

16 Rxns

Human T cell surface β chain receptor (TRB) library construction

R0022M

FAIR-SEQ™ Human TRB VJ multiplex Kit

96 Rxns

R0031M

FAIR-SEQ™ Human IGK/L VJ multiplex Kit

16 Rxns

Human B cell surface light chain receptor (IGK/L) library construction

R0032M

FAIR-SEQ™ Human IGK/L VJ multiplex Kit

96 Rxns

R0041M

FAIR-SEQ™ Human TRD VJ multiplex Kit

16 Rxns

Human T cell surface δ chain receptor (TRD) library construction

R0042M

FAIR-SEQ™ Human TRD VJ multiplex Kit

96 Rxns

R0051M

FAIR-SEQ™ Human TRG VJ multiplex Kit

16 Rxns

Human T cell surface γ chain receptor (TRG) library construction

R0052M

FAIR-SEQ™ Human TRG VJ multiplex Kit

96 Rxns

R0061M

FAIR-SEQ™ Human IGH VC multiplex Kit

16 Rxns

Construction of human B cell surface heavy chain receptor (IGH) VC region library

PE250

R0062M

FAIR-SEQ™ Human IGH VC multiplex Kit

96 Rxns

R0071M

FAIR-SEQ™ Human  BCR full-length Amplication Kit

16 Rxns

RNA

Human B-cell surface receptor full-length library construction

5'RACE

R0072M

FAIR-SEQ™ Human  BCR full-length Amplication Kit

96 Rxns

R0081M

FAIR-SEQ™ Human  TCR full-length Amplication Kit

16 Rxns

Human T cell surface receptor full-length library construction

R0082M

FAIR-SEQ™ Human  TCR full-length Amplication Kit

96 Rxns

R0091M

FAIR-SEQ™ Mouse  BCR full-length Amplication Kit

16 Rxns

Construction of murine B cell surface receptor full-length library

R0092M

FAIR-SEQ™ Mouse  BCR full-length Amplication Kit

96 Rxns

R0101M

FAIR-SEQ™ Mouse  TCR full-length Amplication Kit

16 Rxns

Construction of murine T cell surface receptor full-length library

R0102M

FAIR-SEQ™ Mouse  TCR full-length Amplication Kit

96 Rxns

R0011I

FAIR-SEQ™ Human IGH VJ  multiplex Kit (FR1)

16 Rxns

DNA/RNA

Human B cell surface heavy chain receptor (IGH, FR1 region) library construction

PE150

Illumina

Multiplex PCR

R0012I

FAIR-SEQ™ Human IGH VJ  multiplex Kit (FR1)

96 Rxns

R0013I

FAIR-SEQ™ Human IGH VJ  multiplex Kit (FR3)

16 Rxns

Human B cell surface heavy chain receptor (IGH, FR3 region) library construction

R0014I

FAIR-SEQ™ Human IGH VJ  multiplex Kit (FR3)

96 Rxns

R0021I

FAIR-SEQ™ Human TRB VJ multiplex Kit

16 Rxns

Human T cell surface β chain receptor (TRB) library construction

R0022I

FAIR-SEQ™ Human TRB VJ multiplex Kit

96 Rxns

R0031I

FAIR-SEQ™ Human IGK/L VJ multiplex Kit

16 Rxns

Human B cell surface light chain receptor (IGK/L) library construction

R0032I

FAIR-SEQ™ Human IGK/L VJ multiplex Kit

96 Rxns

R0041I

FAIR-SEQ™ Human TRD VJ multiplex Kit

16 Rxns

Human T cell surface δ chain receptor (TRD) library construction

R0042I

FAIR-SEQ™ Human TRD VJ multiplex Kit

96 Rxns

R0051I

FAIR-SEQ™ Human TRG VJ multiplex Kit

16 Rxns

Human T cell surface γ chain receptor (TRG) library construction

R0052I

FAIR-SEQ™ Human TRG VJ multiplex Kit

96 Rxns

R0061I

FAIR-SEQ™ Human IGH VC multiplex Kit

16 Rxns

Construction of human B cell surface heavy chain receptor (IGH) VC region library

PE250

R0062I

FAIR-SEQ™ Human IGH VC multiplex Kit

96 Rxns

R0071I

FAIR-SEQ™ Human  BCR full-length Amplication Kit

16 Rxns

RNA

Human B-cell surface receptor full-length library construction

5'RACE

R0072I

FAIR-SEQ™ Human  BCR full-length Amplication Kit

96 Rxns

R0081I

FAIR-SEQ™ Human  TCR full-length Amplication Kit

16 Rxns

Human T cell surface receptor full-length library construction

R0082I

FAIR-SEQ™ Human  TCR full-length Amplication Kit

96 Rxns

R0091I

FAIR-SEQ™ Mouse  BCR full-length Amplication Kit

16 Rxns

Construction of murine B cell surface receptor full-length library

R0092I

FAIR-SEQ™ Mouse  BCR full-length Amplication Kit

96 Rxns

R0101I

FAIR-SEQ™ Mouse  TCR full-length Amplication Kit

16 Rxns

Construction of murine T cell surface receptor full-length library

R0102I

FAIR-SEQ™ Mouse  TCR full-length Amplication Kit

96 Rxns

 

FAIR-SEQ™ Human TRB VJ multiplex amplification library construction kit

Human TRB VJ multiplex kit based on FAIR-SEQ™ technology has a low-preference amplification system, library construction time is as low as 5h, no need to cut rubber recovery, easy to operate, the success rate of healthy human whole blood DNA sample bank construction is 95%, and the data efficiency can reach 90%.

Product specifications

Item number

Product name

specification

R002-1

FAIR-SEQ™ Human TRB VJ multiplex Kit(beads excluded)

16 Rxns

R002-2

FAIR-SEQ™ Human TRB VJ multiplex Kit(beads included)

16 Rxns

R002-3

FAIR-SEQ™ Human TRB VJ multiplex Kit(beads excluded)

96 Rxns

R002-4

FAIR-SEQ™ Human TRB VJ multiplex Kit(beads included)

96 Rxns

Product introduction

Immune Repertoire sequencing technology takes T/B lymphocyte surface receptors as the research target, deeply evaluates the immune status of the body, and explores the relationship between human immune status and diseases. At present, the main methods for studying the immune panel are 5'RACE and multiplex PCR, which is suitable not only for RNA but also DNA, but also for a wider range of sample sources, and DNA samples are relatively easier to obtain and easier to store and transport than 5'RACE is only for RNA samples.

T cell receptor (TCR) is a heterodimer that specifically recognizes and binds to the molecular structure of the antigen peptide-MHC molecule by T cells, and is composed of two different subunits. 95%~99% of T cell receptors are composed of α subunits and β subunits, and 1%~5% are composed of γ and δ subunits, which will change with the development of the individual and the health of the body. This kit takes the peptide chain β the T lymphocyte surface receptor as the research target, uses the unbiased primer amplification system optimized by the synthetic template, and the distribution of each clone in the reaction sample is more realistic, and the TRB library constructed with this product can be used on the Illumina platform (Novaseq6000 is recommended), and the sequencing type is PE150.

Low bias amplification system

The primer system was optimized for multiple rounds using synthetic template sequences, and most of the optimized system amplification bias was within doubling.

Easy to operate, no need for glue recycling, can complete the warehouse within 5 hours

Good repeatability and stable system

The Pearson correlation coefficient of 5.3 for the CDR0 amino acid sequence was 9618.5 for two experimental replicates of the synthetic template sequence (Figure 3 left) and 0.9954 for the sample two experimental replicates (Figure <>, right).

High enrichment efficiency and high effective data rate

After filtering the data after low-quality and junction contamination, the V gene alignment rate, the J gene alignment rate, and the sequences of V and J were found to reach more than 95%.

Good specificity, VJ pairing covers the whole range

For synthetic templates mixed with equal or unequal ratios (including all Functional V and J genes), the coverage of VJ pairing combinations can reach 100%; However, due to the uncontrollable factors of the real sample, the detection rate of VJ pairing of healthy whole blood DNA samples is currently more than 95%.

DEMO data display

The following figures (from top to bottom, from left to right) are: CDR3 length distribution, CDR3 frequency distribution, Top10 CDR3 frequency distribution, CDR3 abundance distribution and V-J pairing three-dimensional distribution, the complete results also include: Reads insert distribution, saturation curve, CDR3 V(D)J gene length distribution, V(D) J gene length distribution, V(D) J gene deletion length distribution, V(D)J gene insrtion length distribution, V(D)J gene high-frequency mutation rate (base) distribution, J gene usage distribution, V gene usage distribution.

References

[1]. Wu J, Wang X, Lin L, Li X, Liu S, Zhang W, et al. Developing an Unbiased Multiplex PCR System to Enrich the TRB Repertoire Toward Accurate Detection in Leukemia. Frontiers in Immunology. (2020) 11:1631. doi:10.3389/fimmu.2020.01631

[2]. Cao, K., et al., T-cell receptor repertoire data provides new evidence for hygiene hypothesis of allergic diseases. Allergy, 2020. 75(3): p. 681-683.

[3].  Wang T, Wang C, Wu J, He C, Zhang W, Liu J, Zhang R, et al. The different T-cell receptor repertoires in breast cancer tumors, draining lymph nodes, and adjacent tissues. Cancer immunology research. (2017)5(2):148-56.doi: 10.1158/2326-

[4].  Zhang W, Du Y, Su Z, Wang C, Zeng X, Zhang R, et al. IMonitor: a robust pipeline for TCR and BCR repertoire analysis. Genetics. (2015) 201:459–72. doi: 10.1534/genetics.115.176735

[5]. Liu, X., et al., T cell receptor beta repertoires as novel diagnostic markers for systemic lupus erythematosus and rheumatoid arthritis. Ann Rheum Dis, 2019. 78(8): p. 1070-1078.

FAIR-SEQ™ Human IGH VJ multiplex amplification library construction kit

FAIR-SEQ™ (Fast & Accurate Immune Repertoire Sequencing)
This series of kits adopts an optimized experimental system based on synthetic templates, and after multiple rounds of optimization and adjustment of the primer system, the amplification deviation of most primers in the adjusted experimental system is less than doubled, which can accurately reflect the true clonal distribution of the sample.

FAIR-SEQ Human IGH VJ 
Multiplex Amplification Library Kit Based on FAIR-SEQ™ technology human IGH VJ multiplex kit, with a preference-free amplification system, library construction time as low as 5h, no need to cut rubber recovery, easy to operate, the success rate of bank construction of whole blood DNA samples in healthy people is
currently 95% , most of the data efficiency is 95%.
 

Product specifications

Item number

Product name

specification

R001-1

FAIR-SEQ™ Human IGH VJ multiplex Kit(beads excluded)

16 Rxns

R001-2

FAIR-SEQ™ Human IGH VJ multiplex Kit(beads included)

16 Rxns

R001-3

FAIR-SEQ™ Human IGH VJ multiplex Kit(beads excluded)

96 Rxns

R001-4

FAIR-SEQ™ Human IGH VJ multiplex Kit(beads included)

96 Rxns

Product introduction

Immune Repertoire sequencing technology takes T/B lymphocyte surface receptors as the research target, deeply evaluates the immune status of the body, and explores the relationship between human immune status and diseases. At present, the main methods for studying immunorepertoire are 5'RACE and multiplex PCR, which is suitable not only for RNA but also for DNA, but also for a wider range of sample sources, and DNA samples are relatively easier to obtain and easier to store and transport than 5'RACE is only for RNA samples.

B cell receptor (BCR) is a composite dimer structure, which is formed by the pairing of two heavy light chains of IGH/KL, heavy chain IGH is composed of V, D, J, C, and light chain IGK/L is composed of V, J and C. BCR is an immunoglobulin expressed on the surface of B cells, through which B cells recognize antigens, receive antigen stimulation signals, and initiate humoral immune responses. This kit aims to study BCR heavy chain IGH with a higher diversity, using an unbiased primer amplification system optimized by synthetic templates to provide a more realistic distribution of each clone in the reaction sample.

IGH libraries built with this product are available on the Illumina platform (Novaseq6000 recommended) and the sequencing type is PE150/PE250. Different sequencing types can be selected according to different research purposes, such as PE3 sequencing is recommended for only the study of CDR150 region, and PE3 sequencing is recommended for SHM (Somatic Hyper Mutation) and CDR250 region information if you need to study IGH full-length sequences.

Product advantages

Unbiased amplification system

Multiple rounds of primer optimization were performed using synthetic template sequences, and most of the amplification bias of the optimized system was within doubling. 

Easy to operate, no need for glue recycling, can complete the warehouse within 5 hours

It is suitable for a wide range of starting volumes, as low as 300ng whole blood gDNA can be created

Good repeatability and stable system

The Pearson correlation coefficient of CDR7 amino acid sequence was 3.0 for two experimental replicates of the synthetic template sequence (Figure 9935 left), and the Pearson correlation coefficient for CDR7 amino acid sequence for two experimental replicates of the sample (Figure 3 right) was 0.8445.

The Pearson correlation coefficient of VJ pairing for two experimental replicates of the synthetic template sequence (Figure 8 left) was 0.9935, and the Pearson correlation coefficient of the VJ pairing sequence for the sample two experimental replicates (Figure 8 right) was 0.9870.

High enrichment efficiency and high effective data rate
After filtering the data after low-quality and connector contamination, the V gene alignment rate and J gene alignment rate, and the sequences of V and J were found to be above 95%.

 

Good specificity, VJ pairing covers the whole range

For synthetic templates mixed with equal or unequal ratios (including all Functional V and J genes), the coverage of VJ pairings can reach 100%; However, due to the uncontrollable factors of the real sample, the detection rate of VJ pairing combination of healthy whole blood DNA samples is currently more than 95%.

DEMO data display

The results of some chart results after standard data analysis using IMonitor were shown, and the results were: Reads insert distribution, saturation curve, CDR3 length distribution, CDR3 abundance distribution, CDR3 frequency distribution, Top10 CDR3 frequency distribution, CDR3 V(D)J gene length distribution, V(D)J gene deletion length distribution, V(D)J gene insrtion length distribution, V(D)J gene high-frequency mutation rate (base) distribution, J gene usage distribution, V gene usage distribution, V-J pairing three-dimensional distribution.

References

 

[1].    Wu J, Wang X, Lin L, Li X, Liu S, Zhang W, et al. Developing an Unbiased Multiplex PCR System to Enrich the TRB Repertoire Toward Accurate Detection in Leukemia. Frontiers in Immunology. (2020) 11:1631. doi:10.3389/fimmu.2020.01631

[2]. Huang C, Li X, Wu J, Zhang W, Sun S, Lin L, Wang X, Li H, Wu X, Zhang P, Xu G. The landscape and diagnostic potential of T and B cell repertoire in Immunoglobulin A Nephropathy. Journal of autoimmunity. ( 2019 )97: 100-107.doi10.1016/j.jaut.2018.10.018

[3]. Zhang W, Feng Q, Wang C, Zeng X, Du Y, Lin L, Wu J, et al. Characterization of the B Cell Receptor Repertoire in the Intestinal Mucosa and of Tumor-Infiltrating Lymphocytes in Colorectal Adenoma and Carcinoma.The Journal of Immunology. (2017) 198 (9) 3719-3728; DOI: 10.4049/jimmunol.1602039

[4].    Wu J, Jia S, Wang C, Zhang W, Liu S, Zeng X, Mai H, et al. Minimal residual disease detection and evolved IGH clones analysis in acute B lymphoblastic leukemia using IGH deep sequencing. Frontiers in immunology. (2016)7:403.doi: 10.3389/fimmu.2016.00403

[5].    Zhang W, Du Y, Su Z, Wang C, Zeng X, Zhang R, et al. IMonitor: a robust pipeline for TCR and BCR repertoire analysis. Genetics. (2015) 201:459–72. doi: 10.1534/genetics.115.176735

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