Basics of the B-cell repertoire

B cells are vital members of the adaptive humoral immune response. They express receptors (BCRs) on their surface. BCRs recognize the unique molecular patterns of invading pathogens and block the spread of infection by secreting neutralizing antibodies. A developing B cell undergoes recombination; afterward, the BCR has two identical heavy chains (generated by the recombination of V, D, and J gene segments) and two identical light chains (generated by the recombination of V and J gene segments). 

The diverse collection of clonotypes—the BCR repertoire—is key to shaping immunity. Profiling this repertoire with NGS has provided valuable insights, such as informing the design of therapeutic interventions against SARS-CoV-2. Current technologies are limited in their ability to generate data accurately and reproducibly for all human BCR heavy and light chains. There is an urgent need for both academic and clinical researchers to get a precise readout of BCR sequences for a good understanding of B cell responses in normal and disease states.

SMART-Seq Human BCR (with UMIs) kit chemistry

We developed a refined kit for profiling the BCR repertoire to satisfy this need. It leverages SMART technology (Switching Mechanism at 5′ End of RNA Template) and pairs NGS with a 5′ RACE approach. This optimized chemistry for BCR profiling delivers sensitivity, accuracy, and reliability. The 5′-RACE method used in hBCRv2 does not require any prior knowledge of the BCR sequences, reduces variability, and allows for priming from the constant region of all heavy and light chains (Figure 1).

When used with CogentIP, PCR duplicates and sequencing errors can be detected and removed from the data, leading to more accurate and reliable clonotype calling and quantification. The addition of the UDIs lets researchers pool multiple samples—currently up to 384—while providing greater confidence in sample integrity when sequencing on a patterned Illumina flow cell. We have improved our chemistry from our initial human BCR kit (hBCRv1) to make the BCR libraries compatible with any Illumina sequencer, allowing researchers to save on sequencing costs and increase sample multiplexing on high-throughput sequencers such as the NovaSeq™ system. Full-length V(D)J transcript information can be obtained when libraries are sequenced on the MiSeq® or NextSeq® system, or the CDR3 region using shorter sequencing read lengths on any sequencer. hBCRv2 is designed to generate a consistent library yield from 10 ng–1 ug of PBMC RNA, 1 ng–100 ng of B cell RNA, or up to 100 ng of whole blood RNA, ensuring a sufficient yield for any sequencing configurations. We have also deployed pooled primers to greatly simplify the workflow and allow researchers to study heavy chains found in all isotypes (IgG, IgM, IgD, IgA, and IgE) and immunoglobulin light chain (IgK and IgL) in a much more seamless way.

More sensitive and comprehensive BCR profiling with hBCRv2

To evaluate the sensitivity of hBCRv2, libraries were prepared from 10, 100, and 1,000 ng of RNA extracted from PBMCs, generating approximately 2 million, 6 million, and 12 million sequencing reads, respectively. Clonotype counts from both heavy chain (IgG/M/D/A/E) and light chain (IgK and IgL) consistently increase as the amount of RNA input increases (Figure 2, Panel A). Chord diagrams for both heavy-chain (HC) and light-chain (LC) libraries prepared from 10 ng input showed confident detection of clonotypes with varying abundance, as indicated by chord of various thickness (Figure 2, Panel B).

To demonstrate how SMART technology combined with 5′ RACE is the best-in-class approach for BCR profiling, we prepared BCR libraries using 10 ng of human PBMC total RNA according to the manufacturer’s instructions. With sequencing reads downsampled to ~1 million for Company N libraries and 500,000 each for heavy- and light-chain libraries for hBCRv2, we found a 127% increase in clonotype counts against Company N, manifesting superior data quality (Figure 3).

A streamlined workflow to profile all heavy chains

One of the most important improvements we made in hBCRv2 as compared to hBCRv1 was a much more simplified workflow enabling detection of all BCR heavy chains. While the previous kit only allowed researchers to sequence IgG and IgM for the heavy chains, hBCRv2 can profile all BCR heavy chains (i.e., IgG/M/D/A/E) with great confidence. Compared with hBCRv1, hBCRv2 also displays much higher sensitivity, as indicated by more clonotypes detected for both heavy and light chains at the same sequencing depth (Figure 4). Besides revamped chemistry to support ultra-sensitive readout of more BCR clonotypes, we also redesigned the workflow with pooled primers, allowing researchers to generate all BCR heavy-chain libraries in one reaction.

hBCRv2 provides accurate and unbiased amplification of low-abundance clonotypes

Another key benefit of hBCRv2 is its superior accuracy powered by UMIs. To evaluate how UMIs guaranteed accurate profiling of BCR clonotypes, we spiked 100, 10, 1, and 0.1 pg of RNA extracted from the TIB-190 cell line into 10 ng of PBMC RNA. BCR libraries were prepared with hBCRv2 and sequenced on an Illumina MiSeq sequencer. During data analysis, libraries were normalized to 200,000 reads and clonotype counts were measured after UMI-based consensus collapse in CogentIP. The ratio of detected TIB-190 reads versus total reads showed good correlation with the spike-in percentage of TIB-190 RNA (Figure 5). This result indicates that UMI-powered analysis with hBCRv2 enables researchers to identify low-abundance BCR clonotypes with great confidence and accuracy.

Best-in-class reproducibility is needed for biomarker discoveries in clinical research

Immune repertoire sequencing, such as BCR profiling, has been widely used for biomarker discoveries in clinical research, making it extremely important that the underlying workflow can generate reproducible and high-quality data each time. To evaluate the reproducibility of hBCRv2, we prepared BCR libraries from 10 ng of PBMC RNA and 1 ng of B-cell RNA using the hBCRv2 protocol. Technical replicates prepared with 10 ng of PBMC RNA were sequenced on a MiSeq sequencer with 300-cycle and 600-cycle kits. The Venn diagram illustrates 87% clonotype overlap (Figure 6, Panel A). Libraries prepared using B-cell RNA also displayed great reproducibility, as indicated by ≥85% clonotype overlap between the libraries sequenced on different platforms (Figure 6, Panel B).

SMART-Seq Human BCR (with UMIs) is an innovative and powerful tool for profiling human B-cell receptors. By leveraging SMART technology and combining a 5′-RACE approach with gene-specific amplification, this workflow captures complete V(D)J variable regions of BCRs and is optimized for sensitive and reproducible clonotype detection. Incorporation of UMIs during reverse transcription also enhances the accuracy of this workflow by removing sequencing reads derived from PCR amplification bias. Furthermore, we have also modified the workflow with pooled primers so researchers can enjoy a much easier protocol with all BCR chains profiled in one reaction. Incorporating UDIs into the libraries allows for both pooling of up to 384 samples and sequencing on patterned flow cells without worrying about index hopping.

BCR libraries were prepared from human PBMC total RNA (Takara Bio, Cat. # 636592) and human B-Cell (CD19⁺) total RNA (Miltenyi Biotech, Cat. # 130-0930169). For the sensitivity test, total RNA from the TIB-190 B-cell carcinoma cell line (ATCC) was spiked into 10 ng of PBMC RNA at the listed concentrations. Total RNA from TIB-190 cells was extracted using the NucleoSpin RNA plus kit (Takara Bio, Cat. # 740984.50).

All BCR libraries were generated using SMART-Seq Human 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 V3 cartridges (Illumina, Cat. # MS-102-3001), and a MiniSeq sequencer with 300-cycle cartridges (Illumina, Cat. # FC-420-1002). Data analysis was completed using Cogent NGS Immune Profiler. The report of heavy-chain and light-chain clonotypes from 10 ng of PBMC RNA generated by CogentIP was uploaded to VDJviz browser for chord diagram visualization.