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Sensitive and reproducible BCR clonotype detection

Sensitive and reproducible clonotype detection from a broad range of RNA amounts. Human BCR heavy chain (IgA/D/E/G/M) and light chain (IgK/L) libraries were generated from 10, 100, and 1,000 ng of total human PBMC RNA using SMART-Seq Human BCR (with UMIs). The sequence reads were processed using Cogent NGS Immune Profiler Software.

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Workflow

Fig. 1. SMART-Seq Human BCR (with UMIs) library preparation workflow leverages the SMART technology and semi-nested PCR approach. dT primed first-strand cDNA synthesis is performed by an MMLV-derived reverse transcriptase (RT). Upon reaching the 5’ end of each mRNA molecule, the RT adds non-templated nucleotides to the first-strand cDNA. The SMART UMI Oligo contains complementary sequences to the non-templated nucleotides added by the RT and hybridizes to the first-strand cDNA. In the template-switching step, the RT uses the remainder of the SMART UMI Oligo as a template for the incorporation of additional sequences at the end of the first-strand cDNA. Full-length variable regions of BCR cDNA are selectively amplified by PCR using primers that are complementary to the oligonucleotide-templated sequence (hBCR PCR1 Universal Forward) and the constant region(s) of BCR IgA/D/E/G/M/K/L isotypes (hBCR PCR1 Reverse). In a subsequent round of PCR, the variable regions of BCR-IgA/D/E/G/M and/or BCR- IgK/L subunits are further amplified and adapter sequences and indexes incorporated using hBCR PCR2 HC/LC Reverse primers and UDI primers, generating Illumina compatible BCR cDNA libraries for 300 bp (full-length V(D)J) or 150 bp (CDR3 only) paired-end sequencing. Easily analyze your data and obtain tabulated outputs and plots using our free Cogent NGS Immune Profiler Software and Cogent NGS Immune Viewer.

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Consistency

Fig. 2. SMART-Seq Human BCR (with UMIs) shows consistent performance across a variety of inputs. Bar plot showing clonotype counts for human BCR libraries from PBMC RNA (Panel A) and B-cell RNA (Panel B) at various inputs demonstrate a consistent increase in clonotype count with increase in RNA input. Sequencing outputs were down sampled to ~2 million, 6 million and 12 million reads for PBMC RNA and ~1 million, 5 million and 12 million reads for B-cell RNA. Data was processed using Takara Bio’s Immune profiling software (Cogent NGS Immune Profiler). HC=heavy chain, LC=light chain.

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Superior data quality

Fig. 3. Superior data quality with SMART-Seq Human BCR (with UMIs). Significantly higher clonotype counts are observed with the updated SMART-Seq Human BCR (with UMIs) (BCRv2) compared to both the previous version SMARTer Human BCR IgG IgM H/K/L Profiling Kit (BCRv1) and the competitor kit (Company N). Libraries were generated using 10 ng of human PBMC total RNA according to the manufacturer’s instructions and sequenced on the Illumina platform. Panel A. Sequencing outputs were down sampled to ~1 million for all the libraries. BCRv2 generated approximately 550% more clonotypes than previous version. Panel B. Sequencing outputs were down sampled to ~1 million for Company N libraries and 500,000 each for heavy and light chain libraries for BCRv2. In comparison to Company N, BCRv2 generated approximately 9.5K and 22.9K clonotypes for heavy chain (HC) and light chain (LC) isotypes respectively, representing a 127% increase against Company N.

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Sensitivity

Fig. 4. High sensitivity for the identification of low abundance clonotypes. 100pg, 10pg, 1pg and 0.1pg of RNA extracted from the TIB-190 cell line was spiked into 10 ng of PBMC RNA. Panel A. Clone counts at different spike-in levels are listed in the table. Libraries were normalized to 200,000 reads and all counts were measured after UMI-based consensus collapse. Panel B. Calculated correlation between spike-in RNA proportions and detected clonotype frequencies (both axes logarithmically transformed). Linear relationship between spike-in RNA sample (down to 0.001%) and clonotype frequencies observed with SMART-Seq Human BCR (with UMIs).

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Performance

Fig. 5. Highly reproducible BCR profiling with SMART-Seq Human BCR (with UMIs) between replicates and sequencing platforms. BCR profiling libraries from 10 ng of PBMC RNA and 1 ng B-cell RNA were prepared using SMART-Seq Human BCR (with UMIs). Panel A. Technical replicate libraries prepared with 10 ng PBMC RNA sequenced on the Illumina Miseq platform show high clonotype overlap (87%). Panel B. Libraries prepared using 1 ng B-cell RNA sequenced on the Miseq using a 600-cycle V3 cartridge or 300-cycle V2 cartridge as well as on the Miniseq platform show high clonotype overlap. For all libraries, data generated were downsampled to 1,000,000 reads and analyzed using our Cogent NGS Immune Profiler software.

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Comparison with previous version of BCR kit

Fig. 6. Comparison with previous version of BCR kit (SMARTer Human BCR IgG IgM H/K/L Profiling Kit)

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Reproducibility

Fig. 7. Correlation analysis for top 100 clonotypes between two biological replicates

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634776: SMART-Seq Human BCR (with UMIs)