B cells are an essential part of the adaptive immune response, functioning via B-cell receptors (BCRs) expressed on their surface. Each B cell expresses a different BCR that allows it to recognize molecular patterns in pathogens. Development of BCRs (Figure 1) is a multistep process in which a progenitor cell undergoes V(D)J recombination in the germline followed by somatic hypermutation (SHM), resulting in a final product with a specific CDR3 (complementarity determining region 3) sequence in the hypervariable region of the immunoglobulin (Ig). These molecular events facilitate receptor diversity and the generation of heavy chain isotypes. Upon exposure to a stimulus or stimuli, the lambda and kappa light chain genes of the BCR undergo rearrangements to generate different light chain isotypes from the same B-cell clone.
Understanding the profiles of BCRs, (i.e., sequencing the full-length CDR3 regions to determine the diversity of receptors and the clonotypes, defined by expression of specific heavy and light chain gene segments) can not only aid in gaining insights into the adaptive immune response in healthy individuals, but also in those with a wide range of conditions, including infectious diseases, allergies, autoimmune disorders, cancers, and aging (Yaari & Kleinstein, 2015).
Next-generation sequencing (NGS) approaches for profiling B-cell repertoires have provided valuable insights into the adaptive immune response and antibody engineering. There are two major NGS approaches used in profiling B-cell repertoires—multiplex PCR or 5' RACE. While the multiplex PCR approach allows for amplification of multiple BCR genes within one reaction, challenges with sensitivity, specificity, and biases in amplification of certain sequences can lead to difficulties in accurate identification of isotypes. On the other hand, the 5'-RACE method reduces variability and allows for priming from the constant regions of the BCR heavy or light chains. However, the burden of designing optimized primers falls onto the individual researcher.
The SMARTer Mouse BCR IgG H/K/L Profiling Kit (SMARTer mouse BCR kit) solves these problems by combining the benefits of 5' RACE with gene-specific amplification (Figure 2) to provide a highly sensitive and reproducible method for profiling B-cell repertoires. The high sensitivity of the kit accurately identifies top B-cell Ig clonotypes and reliably assigns isotype in a majority of cases, based on sequencing of the gamma (G) heavy chain and the kappa (K) and lambda (L) light chains.
The kit leverages SMART technology (Switching Mechanism at 5' End of RNA Template) and employs a 5'-RACE-like approach to capture complete V(D)J variable regions of BCR transcripts. First-strand cDNA synthesis is dT-primed, and the template-switching activity ensures that only sequences from full-length cDNAs undergo PCR amplification (Figure 2). Two rounds of PCR are then performed in succession to amplify cDNA sequences corresponding to the variable regions of the BCR chain transcripts (Figure 2, Panel B). The first PCR reaction uses first-strand cDNA as a template to amplify each BCR Ig transcript (G, K, or L) in a separate reaction. Then, the first PCR product is used as the template with semi-nested primers to amplify the entire variable region and a portion of the constant region. Following post-PCR purification, size selection, and quality analysis, the library is ready for sequencing on an Illumina MiSeq® sequencer using the 600-cycle MiSeq® Reagent Kit v3.
Specific amplification of Ig heavy (G) and light (K and L) chains
To determine if the BCR-specific primers amplified the expected regions of the BCR loci, RNA was isolated from three mouse hybridoma samples (10E8, HB-8117, and TIB-127). Then, 10 ng of RNA from each sample was used as input for the SMARTer mouse BCR kit, and the resulting libraries were analyzed for quality and sequenced. Bioanalyzer traces for each hybridoma sample show the expected peaks for the G, K, and L chains, demonstrating correct amplification (Figure 3, Panel A).
Analysis of sequencing data showed that alignment of total reads to Ig sequences were over 90% for these samples (Figure 3B). We consistently see over 75% alignment to Ig, indicating that the SMARTer mouse BCR kit produces BCR-specific libraries. The top two clones identified in our sequencing data, representing an H chain transcript and a K light chain transcript, make up >70% of the clonotype fraction combined. We also detected a small number of clones with a clone fraction of ≥0.0001. These data indicate a highly clonal BCR population, which is expected for hybridoma cell line samples. In addition, the percentage of overlapping reads was over 80%, which indicates good sequencing depth—an attribute that is potentially helpful in identifying base pair changes resulting from somatic hypermutation.
Figure 3. Highly specific amplification of Ig chains. Libraries containing BCR G and K chain transcripts were generated using the SMARTer Mouse BCR IgG H/K/L Profiling kit, starting with 10 ng of RNA isolated from a hybridoma developed in-house (10E8) and two manufactured ATCC hybridoma samples (HB-8117 and TIB-127). Panel A. Bioanalyzer traces showing gene-specific amplification of G, K and L chains for each hybridoma. Peaks labeled "LM" and "UM" correspond to DNA reference markers included in each analysis. Panel B. Mapping metrics were determined using MiXCR software (version 188.8.131.52; software not provided with kit) and aligned against all Ig reference sequences, with a clone fraction threshold of 0.01%. V, D, and J IMGT allele outputs, alignment scores and consensus CDR3 amino acid CDR3 for the top heavy (G) and light (K) chain clone for each hybridoma are displayed. For cases in which the MiXCR software determined the presence of more than one V, D, or J allele, all determined alleles with alignment scores are shown.
Optimized workflow for clonotype detection
The optimized workflow of the SMARTer mouse BCR kit workflow allows for flexibility in sequencing and accurate clonotype identification. Following reverse transcription, the reaction is split to allow for amplification of the G, K, and L chains in separate reactions. The second PCR adds the same sequencing indexes to each amplified Ig chain transcript for a given sample, but distinct indexes for each individual sample (e.g. G, K, L of sample 1 would all receive index A, but G, K, L of sample 2 would all receive index B). After sample validation, libraries are pooled for sequencing. This key strategy allows highly sensitive sequencing of different chains from the same sample even with a very small amount of starting material.
We demonstrated that PCR pooling strategy affects percentage of sequencing read aligning to Ig (Figure 5). Clonal B-cell populations or hybridomas primarily express a K or L light chain —not both. The bioanalyzer traces for the K chains in our hybridoma samples contained a sharp peak representing amplification of this transcript, while the bioanalyzer traces for the L chains lacked this peak, suggesting the K light chain is the light chain expressed within our hybridoma samples (Figure 3, Panel A). Since we observed no amplification of the L chain, we omitted the L chain and pooled only the G and K chains together for sequencing (“GK” in the table below). Over 70% of sequencing reads aligned to Ig when the GK pooling strategy was used. When the L chain products were included in the pool ("GKL" in the table below), the percentage of reads aligning to Ig dropped significantly. Additionally, the proportion of the identified CDR3 sequence for the G and K chains were the same for both pooling strategies. These data demonstrate that the SMARTer mouse BCR kit allows for flexibility in sequencing approach in cases where high Ig alignment rates are required without compromising accuracy of clonotype identification.
% successful alignment
Chains sequenced together
% aligned to IG
Gamma (G) chain CDR3
CARAYDY DRAWFG YW 50%
CARAYDY DRAWFG YW 51%
CARKSSY YGSTYVY FDVW 45%
CARKSSY YGSTYVY FDVW 44%
Kappa (K) chain CDR3
CQQSND NPLTF 46%
CQQSND NPLTF 44%
CQQSRK VPSTF 27%
CQQSRK VPSTF 28%
CGQGYS YPYTF 50%
CGQGYS YPYTF 51%
Figure 5. Effect of PCR pooling strategy on Ig alignment rate and clonotype identification. G and K chain PCR products (GK) or G, K and L chain PCR products (GLK) were pooled and sequenced for each hybridoma sample. Panel A. The percentage of reads aligning to Ig reference sequences for GK pooling, GKL pooling, and L only sequencing strategies as determined by MiXCR software. Panel B. The identified CDR3 amino acid consensus sequence and percent distribution for the top heavy (G) and light (K) chain clone for GK pooling or GKL pooling strategies as determined by the MiXCR software. The clone fraction threshold was set to 0.01%.
The SMARTer Mouse BCR IgG H/K/L Profiling Kit is a powerful tool for profiling mouse B-cell receptors. By leveraging SMART technology and combining a 5' RACE-like approach with gene-specific primer amplification, this workflow captures complete V(D)J variable regions of BCRs and is optimized for highly sensitive and specific clonotype detection. With primers that incorporate Illumina-specific adaptor sequences during cDNA amplification, the protocol generates indexed libraries ready for sequencing on the Illumina MiSeq platform. An additional advantage is the unique PCR cycling and pooling workflow which reduces sequencing cost while enabling accurate clonotype identification. By avoiding multiplex PCR, this kit also avoids the pitfalls of biases in amplification of certain sequences, helping to provide a complete and accurate view of mouse BCR repertoires.
Libraries containing BCR heavy and light chain sequences were generated using the SMARTer Mouse BCR IgG H/K/L Profiling Kit as per the protocol given in the user manual. 10 ng of RNA was obtained as starting material from the indicated hybridomas (10E8, HB-8117, and TIB- 127) with different Ig subtypes. 10E8 is an in-house hybridoma; the others are ATCC lines. ATCC determined the expected isotype for their lines; 10E8 was determined in-house. Hybridomas were cultured according to established methods. Libraries were produced using the first-strand cDNA as a template in three different PCRs for the gamma heavy chain and the kappa and lambda light chains. The product of these PCRs was used as template in a set of nested PCRs, one for each chain. Following purification and size selection, libraries were validated using the Agilent 2100 Bioanalyzer. Libraries were then sequenced on an Illuminia MiSeq sequencer and analyzed using MiXCR software (version 184.108.40.206, software not provided).
Bolotin, D.A. et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat. Methods12, 380–381 (2015).
Yaari, G. and Kleinstein, S.H. Practical guidelines for B-cell receptor repertoire sequencing analysis. Genome Med.7:121 (2015).