Due to their similarity to humans and easy genetic manipulation, mouse models have been widely used for understanding adaptive immune responses to facilitate disease research. B cells, or B lymphocytes, play a crucial role in adaptive immunity by secreting antibodies to neutralize invading pathogens or antigens. Besides functionality, B cells are also well known for their phenotypic and functional diversity. Hence, understanding B-cell diversity, formed by B-cell receptor (BCR) diversity and other molecular events, becomes essential in mouse models for various diseases, such as cancer, infectious diseases, and autoimmune disorders. Scientists who study B-cell development, activation, and response in mouse models will benefit from a sensitive and easy-to-use solution for mouse BCR profiling.
Our new SMART-Seq Mouse BCR (with UMIs) perfectly addresses the above needs. The new kit includes key improvements so customers can now capture BCR sequences from all isotypes (IgA, D, E, G, M for heavy chain; IgK, L for light chain) with unparalleled sensitivity. Accuracy and throughput have also been enhanced with the incorporation of UMIs (unique molecular identifiers) and UDIs (unique dual indexes), respectively.
Applications of SMART-Seq Mouse BCR (with UMIs) kits include but are not limited to:
Analyze B-cell clonal organization and diversity in tissues and circulation to understand how they affect B-cell differentiation pathway and functionalities.
Find BCRs with high affinity to inform better vaccination or therapy against infectious diseases.
Track changes and migration of antigen-specific B cells post adoptive transfer in mouse disease models for HIV and other diseases.
Check BCR sequences to study the development of autoantibodies against new epitopes over time and how that correlates with the severity of autoimmune diseases like lupus.
SMART technology empowers the detection of all BCR isotypes
The activation of B cells and their differentiation into antibody-secreting plasma cells are crucial components of humoral immune responses. All these activities depend on the diversity, specificity, and affinity of BCRs. BCRs go through a multistep development process. In the bone marrow, progenitor cells carry out V(D)J recombination to become naïve B cells, which migrate to secondary lymphoid tissues and undergo affinity maturation after encountering antigen stimulation. As a result, BCRs acquire increased affinity, avidity, and anti-pathogen activity, giving rise to antibody-secreting plasma cells, or memory B cells with long-lasting immunity to reinfection.
To help you understand B-cell diversity and responses in your mouse models, we leveraged the SMART (Switching Mechanism at the 5′ end of RNA Template) technology for sensitive and highly reproducible BCR detection. The SMART-Seq Mouse BCR (with UMIs) workflow (Figure 1) starts with first-strand cDNA synthesis is dT-primed and catalyzed by SMARTScribe Reverse Transcriptase (RT), which adds non-templated nucleotides upon reaching 5’ end of the mRNA template. The SMART UMI oligo contains sequences complementary to these non-templated nucleotides, so UMIs are incorporated during RT as well. Following reverse transcription, two rounds of PCR are performed to amplify the BCR locus. The first round of PCR amplifies the entire variable region and a considerable portion of the constant region from both heavy (HC) and light chains (LC). Using products from PCR1 as templates, the semi-nested PCR2 amplifies the entire variable region and a smaller part of the constant region.
Figure 1. SMART-Seq Mouse BCR (with UMIs) workflow. Input RNA or cells are oligo-dT primed using the dT Primer (dark blue). Following oligo dT-priming, SmartScribe Reverse Transcriptase performs first strand cDNA synthesis on input RNA or cells and adds non-templated nucleotides to the 5′ end of each cDNA molecule (XXXXX). Upon reaching the 5′ end of the RNA template, the SMART UMI oligo anneals to the non-templated nucleotides (XXXXX), incorporating the UMI (yellow) and partial Illumina adapter (light green) complementary to the mBCR PCR1 Universal Forward primer. Following reverse transcription, semi-nested PCR is performed to amplify BCR cDNAs. In PCR1, the mBCR PCR1 Universal Forward primer anneals to the complementary sequence carried by the SMART UMI oligo (light green), incorporating Illumina Read2 sequence (dark green). The mBCR PCR1 Reverse primer (orange) anneals to sequences in the constant regions of BCR cDNAs to amplify the entire BCR V(D)J region. During the PCR2, the nested mBCR LC (light chain) or mBCR HC (heavy chain) PCR2 Reverse primers anneal to sequences in BCR constant regions that are internal to the sequences bound by the mBCR PCR1 Reverse primer and add the Illumina Read 1 sequence (light purple). In the same PCR2 reaction, Unique Dual Index Kit primers anneal to sequence added by mBCR PCR1 Universal Forward primer or the mBCR LC or HC PCR2 Reverse primers to add Illumina P7-i7 and P5-i5 index sequences (dark blue). The result is a sequencing-ready library that contains the entire BCR light chain and/or heavy chain variable regions with a small portion of the constant region.
Results
Profile mouse BCR sequences with sensitivity
To evaluate the performance of SMART-Seq Mouse BCR (with UMIs), libraries were prepared from 10, 100, and 1,000 ng of mouse spleen RNA, generating 1.5 million sequencing reads. Clonotype counts from both heavy chain (IgA/D/E/G/M) and light chain (IgK/L) consistently increase with increasing amounts of input RNA (Figure 2).
Figure 2. SMART-Seq Mouse BCR (with UMIs) detected all BCR isotypes with outstanding sensitivity. Panel A. Detection of all BCR isotypes for both heavy chain (IgA/D/G/E/M) and light chain (IgK/L) by SMART-Seq Mouse BCR (with UMIs) in 10, 100, and 1,000 ng input of mouse spleen RNA. Comparison across various RNA inputs shows a consistent increase in clonotype counts with higher input. Panel B. Sensitivity of SMART-Seq Mouse BCR (with UMIs) ensures detection of more heavy-chain and light-chain clonotypes with more input RNA. Bar graphs illustrate both heavy-chain and light-chain clonotype counts consistently increase with a higher amount of input RNA.
Detect more BCR isotypes and clonotypes with SMART-Seq Mouse BCR (with UMIs)
With our initial mouse BCR kit (SMARTer Mouse BCR IgG H/K/L Profiling Kit), scientists can get BCR sequences for IgG (HC) and IgK/L (LC). Now with SMART-Seq Mouse BCR (with UMIs) kits, researchers can make more discoveries, spanning all BCR isotypes for both HC (IgA/D/E/G/M) and LC (IgK/L). Compared with SMARTer Mouse BCR IgG H/K/L Profiling Kit, SMART-Seq Mouse BCR (with UMIs) shows much higher sensitivity, manifested by 7x more clonotypes detected with SMART-Seq Mouse BCR (with UMIs) than with the SMARTer Mouse BCR IgG H/K/L Profiling Kit (Figure 3). Besides such expansion in the number and diversity of BCR sequences detected, ease-of-use also improved with the usage of pooled primers so all heavy-chain or light-chain libraries can be easily generated in one tube.
Figure 3. More isotypes and more clonotypes with a simpler workflow. Libraries were generated from 10 ng of mouse spleen RNA using the SMARTer Mouse BCR IgG H/K/L Profiling Kit (mBCRv1, blue) and SMART-Seq Mouse BCR (with UMIs) (mBCRv2; purple) workflow. The resulting libraries were sequenced on an Illumina NextSeq® sequencer. Sequencing data was downsampled to 1 million reads each for IgG, IgK, and IgL of SMARTer Mouse BCR IgG H/K/L Profiling Kit libraries and 1 million reads for each heavy and light chain of SMART-Seq Mouse BCR (with UMIs) libraries. Data was analyzed using Cogent Immune Profiler (CogentIP). Here, the bar graphs illustrate the extra BCR isotypes unraveled by SMART-Seq Mouse BCR (with UMIs) along with a higher number of BCR clonotypes detected with SMART-Seq Mouse BCR (with UMIs) for IgG, IgK, and IgL, exhibiting a 7 fold sensitivity increase.
SMART-Seq Mouse BCR (with UMIs) outperforms major competitors with unparalleled sensitivity
To compare the performance of SMART-Seq Mouse BCR (with UMIs) against other mouse BCR profiling solutions, we prepared BCR libraries using 10ng of mouse spleen RNA according to the manufacturer’s instructions. With sequencing reads downsampled to 1 million for NEB libraries and 500,000 each for heavy- and light-chain libraries for SMART-Seq Mouse BCR (with UMIs), we found SMART-Seq Mouse BCR (with UMIs) detects about 4 fold more BCR clonotypes than NEB kits at the same sequencing depth, demonstrating unmatched sensitivity with low input (Figure 4).
Takara Bio-1
Takara Bio-2
NEB-1
NEB-2
HC clonotypes
112,634
112,995
26,519
23,731
LC clonotypes
6,382
6,448
4,751
4,784
Figure 4. Unrivaled sensitivity of SMART-Seq Mouse BCR (with UMIs). Panel A. Sensitivity comparison of Takara Bio SMART-Seq Mouse BCR (with UMIs) vs NEB kit. Libraries were generated using 10ng of mouse spleen RNA in duplicates according to the manufacturer’s instructions. The resulting libraries were sequenced on Illumina MiSeq® with a 600-cycle cartridge. Sequencing outputs were downsampled to ~1 million for NEB libraries and 500,000 each for heavy and light chain libraries for SMART-Seq Mouse BCR (with UMIs). Data was analyzed using MiXCR v3. Here, the bar graphs illustrate the total BCR clonotype counts generated by SMART-Seq Mouse BCR (with UMIs) and the NEB kit for both heavy and light chains. Panel B. Total number of BCR clonotypes detected by Takara Bio SMART-Seq Mouse BCR (with UMIs) and NEB kit for HC and LC.
SMART-Seq Mouse BCR (with UMIs) ensures accurate and unbiased detection of rare clonotypes
Besides higher sensitivity and ability to detect more BCR isotypes, SMART-Seq Mouse BCR (with UMIs) also had accuracy guaranteed with UMI incorporation. To understand how UMI incorporation may affect the accuracy of BCR detection, we spiked 100, 10, and 1 pg of RNA extracted from the 10E8 hybridoma cell line into 10 ng of mouse spleen RNA. BCR libraries were prepared with SMART-Seq Mouse BCR (with UMIs) and sequenced on an Illumina NextSeq. During data analysis, libraries were normalized to 3 million reads and clonotype counts were measured after UMI-based consensus collapse in CogentIP. The ratio of detected 10E8 IgG and IgK reads showed great correlation with the corresponding spike-in concentration percentage (Figure 5).
10 ng spleen RNA+ spike-in RNA
% spike-in RNA
Detected reads (after UMI collapse) - HC
Detected reads (after UMI collapse) - LC
Total reads
100 pg spike-in
1
3,262
7,746
3E6
10 pg spike-in
0.10
310
321
3E6
1 pg spike-in
0.01
30
64
3E6
Figure 5. SMART-Seq Mouse BCR (with UMIs) guarantees accurate identification of rare BCR clonotypes. 100, 10, and 1 pg of RNA extracted from the 10E8 hybridoma cell line were spiked into 10 ng of mouse spleen RNA. Panel A. Calculated correlation between spike-in RNA ratio and detected 10E8 clonotype frequencies. Both axes are logarithmically transformed. Panel B. 10 million clonotype counts at different spike-in concentrations are listed in the table. Libraries were normalized to 3 million reads, and all counts were measured after UMI-based consensus collapse.
Sequence your BCR libraries on any Illumina sequencer and save money
We understand customers’ need for having one Illumina sequencer to satisfy different sequencing needs, so when developing the new SMART-Seq Mouse BCR (with UMIs) chemistry, we made sure customers can sequence the BCR libraries on any Illumina sequencer they have. Traditionally, BCR or TCR sequences, especially the full-length V(D)J information, are captured with 300 bp paired-end (300 PE) sequencing. With the emergence of new high-throughput Illumina sequencers, we saw more customers apply 150 bp paired-end (150 PE) sequencing to sequence the CDR3 region, which determines the antigen specificity of BCRs or TCRs. To ensure customers can enjoy such sequencing flexibility, we sequenced the BCR libraries using a 300-cycle (for 150 PE sequencing) and a 600-cycle (for 300 PE sequencing) cartridge, respectively. Both sequencing setups generated almost identical clonotype counts and there is over 75% overlap in the BCR clonotypes detected (Figure 6).
Figure 6. Sequencing flexibility of SMART-Seq Mouse BCR (with UMIs) chemistry. Panel A. Comparison of clonotype counts between 2x300 sequencing for full-length V(D)J and 2x150 sequencing for CDR3 only. BCR libraries were prepared with 10 ng of mouse spleen RNA following the SMART-Seq Mouse BCR (with UMIs) protocol. The resulting libraries were sequenced on the Illumina MiSeq® platform with a 300-cycle and a 600-cycle cartridge, respectively. Data were then analyzed using Cogent IP. The bar graphs show a similar number of total clonotype counts identified with two different sequencing setups. Panel B. Evaluation of BCR data consistency between different sequencing setups. Venn diagram shows over 75% clonotype overlap between the SMART-Seq Mouse BCR (with UMIs) data sequenced with different parameters.
With sensitivity, reproducibility is never left behind
When performing mouse studies, scientists may have several biological or technical replicates, so reproducibility is extremely important. We also took this into account when developing the new SMART-Seq Mouse BCR (with UMIs) chemistry. Here, 10 ng mouse spleen RNA was used to prepare BCR libraries in duplicates, and the resulting data showed excellent reproducibility, as demonstrated by a Pearson correlation (r) of 0.8536 for the heavy chain and 0.999 for the light chain.
Figure 7. SMART-Seq Mouse BCR (with UMIs) libraries show a high level of reproducibility. HC and LC libraries were generated in duplicate with 10 ng of mouse spleen RNA. Plots show the abundance of each HC (Panel A) or LC (Panel B) clonotype from the technical replicates.
Speed up your large-scale project with an automation-friendly protocol
Clinical studies involving large patient cohorts and demand for easily adaptable protocols for automation are increasing. Accordingly, we conducted tests on our SMART-Seq Mouse BCR (with UMIs) protocol using the Mantis liquid handler, both at full-volume and half-volume reaction settings. Remarkably, the half-volume setup on the Mantis liquid handler yielded clonotype counts equal to those obtained with full-volume, affirming the seamless compatibility of our SMART-Seq Mouse BCR (with UMIs) kits with automated solutions. Combining our mouse BCR profiling solution with automation empowers you to efficiently process a high volume of samples, delivering cost-effectiveness and productivity.
Figure 8. SMART-Seq Mouse BCR (with UMIs) embraces an automation-friendly protocol. Heavy chain (Panel A) and light chain (Panel B) libraries were generated with full-volume (FV) or half-volume (HV) reactions on the MANTIS liquid handler or manually with 10 ng of mouse spleen RNA. The resulting libraries were sequenced on a NextSeq 550 sequencer with 2 million reads each. Bar graphs show the equal sensitivity of FV and HV reactions as indicated by the number of clonotype counts.
Conclusion
SMART-Seq Mouse BCR (with UMIs) is a long overdue research tool for scientists who study B cells using mouse models. It leverages SMART technology and gene-specific amplification to capture the BCR sequences in an unbiased manner. The design of the chemistry also guarantees the detection of all BCR isotypes, satisfying different research needs. Meanwhile, UDIs and UMIs are incorporated into the workflow to increase multiplexing capability and accuracy. Sequencing can also be done very economically as you have the flexibility to sequence either the full-length V(D)J or just the CDR3 region. Once sequencing is completed, we have the Cogent NGS Immune Profiler and the web-based Cogent NGS Immune Viewer for seamless data analysis and visualization.
Materials and Methods
BCR libraries were prepared from mouse spleen RNA. For the spike-in test, total RNA from the 10E8 hybridoma cell line was spiked into 10 ng of mouse spleen RNA. Total RNA from 10E8 cells was extracted using the NucleoSpin RNA Plus kit (Takara Bio, Cat. # 740984.50).
All BCR libraries were generated using SMART-Seq Mouse BCR (with UMIs), as per the user manual. Following purification and size selection, libraries were quantified using the Qubit and Agilent Bioanalyzer 2100. Sequencing was performed on a MiSeq sequencer with 600-cycle V3 cartridges (Illumina, Cat. # MS-102-3003), a MiSeq sequencer with 300-cycle V2 cartridges (Illumina, Cat. # MS-102-2002), and a NextSeq sequencer with 300-cycle cartridges. Data analysis was completed using the Cogent NGS Immune Profiler.
