SMART-Seq Human BCR (with UMIs)

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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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Schematic of technology and workflow for SMART-Seq Human BCR (with UMIs)

Schematic of technology and workflow for SMART-Seq Human BCR (with UMIs). 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 hBCR PCR1 Universal Forward primer. Following reverse transcription, semi-nested PCR is performed to amplify BCR cDNAs. In PCR1, the hBCR PCR1 Universal Forward primer anneals to the complementary sequence carried by the SMART UMI Oligo (light green), incorporating the Illumina Read2 sequence (dark green). The hBCR 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 hBCR LC (light chain) or hBCR HC (heavy chain) PCR2 Reverse primers anneal to sequences in BCR constant regions that are internal to the sequences bound by the hBCR PCR1 Reverse primer and add the Illumina Read 1 sequence (light purple). In the same PCR2 reaction, Unique Dual Index Kit primers anneal to the sequence added by hBCR PCR1 Universal Forward primer or the hBCR 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.

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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).

Back

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.

Back

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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634777: SMART-Seq Human BCR (with UMIs)

Back

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.

Back

Schematic of technology and workflow for SMART-Seq Human BCR (with UMIs)

Schematic of technology and workflow for SMART-Seq Human BCR (with UMIs). 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 hBCR PCR1 Universal Forward primer. Following reverse transcription, semi-nested PCR is performed to amplify BCR cDNAs. In PCR1, the hBCR PCR1 Universal Forward primer anneals to the complementary sequence carried by the SMART UMI Oligo (light green), incorporating the Illumina Read2 sequence (dark green). The hBCR 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 hBCR LC (light chain) or hBCR HC (heavy chain) PCR2 Reverse primers anneal to sequences in BCR constant regions that are internal to the sequences bound by the hBCR PCR1 Reverse primer and add the Illumina Read 1 sequence (light purple). In the same PCR2 reaction, Unique Dual Index Kit primers anneal to the sequence added by hBCR PCR1 Universal Forward primer or the hBCR 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.

Back

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.

Back

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.

Back

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).

Back

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.

Back

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)

Back

Reproducibility

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

Back

634778: SMART-Seq Human BCR (with UMIs)

Back

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.

Back

Schematic of technology and workflow for SMART-Seq Human BCR (with UMIs)

Schematic of technology and workflow for SMART-Seq Human BCR (with UMIs). 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 hBCR PCR1 Universal Forward primer. Following reverse transcription, semi-nested PCR is performed to amplify BCR cDNAs. In PCR1, the hBCR PCR1 Universal Forward primer anneals to the complementary sequence carried by the SMART UMI Oligo (light green), incorporating the Illumina Read2 sequence (dark green). The hBCR 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 hBCR LC (light chain) or hBCR HC (heavy chain) PCR2 Reverse primers anneal to sequences in BCR constant regions that are internal to the sequences bound by the hBCR PCR1 Reverse primer and add the Illumina Read 1 sequence (light purple). In the same PCR2 reaction, Unique Dual Index Kit primers anneal to the sequence added by hBCR PCR1 Universal Forward primer or the hBCR 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.

Back

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.

Back

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.

Back

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).

Back

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.

Back

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)

Back

Reproducibility

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

Back

634776: SMART-Seq Human BCR (with UMIs)