Human B-cell Receptor Profiling service at MedGenome Inc. MedGenome Inc. offers unparalleled sequencing service to analyze B-cell receptor (BCR) repertoires from human or mouse species. MedGenome Inc. utilizes the Takara SMARTer BCR Profiling Kit that leverages SMART (Switching Mechanism at 5' End of RNA Template) technology coupled with 5' RACE approach, for unbiased gene and clonal amplification of BCR repertoire sequences. Repertoires sequenced on Illumina MiSeq platform are analyzed using Takara Bio Immune Profiler software for sensitive and accurate comprehensive BCR profiling. MedGenome Inc. will also support the development of new diagnostic experimental design and advanced bioinformatics analysis. Table 1: BCR - Sequencing Services Offered at MedGenome Inc. Source & Required Sequencing Analysis Information Technology Input Type Amount Method Method Obtained Bulk SMARTer Isolated Cells / RNA IgG/M BCR 10ng - 3μg RNA / Illumina Takara Bio or CDR3, V(D)J sequences Profiling Kit 50-10,000 cells MiSeq PE300 Immune Profiler PBMCs (Human/mouse) (Takara Bio USA) Cell isolation Template purification Molecule amplification CD10 CD19 CD24 B cell CD27 CD38 VDJ CH mRNA 5' RACE Chr14 5' AAA 3' 5' mRNA AAA 3' VJ CL Chr2 Data analysis Data processing Library construction (repertoire diversity (fitering, alignment and sequencing dynamics and clonality) and clustering) CDR-H3 V1 D1 J1 V2 D2 J2 V3 D3 J3 … … … Vn Dn Jn Figure 1: Schematics of steps for high-throughput BCR Repertoire Sequencing MEDGENOME INC. medgenome.com 1 What is B-Cell Receptor (BCR) Repertoire An adaptive immune system is fundamentally dependent upon the generation of diverse repertoires of B-lymphocytes antigen receptors (BCR). BCRs, a membrane bound cell surface receptors are assembled by genetic rearrangement and somatic recombination of vast immunoglobulin gene segments and are constantly diversified to specifically bind exogenous antigens and endogenous host responses. V(D)J recombination is a process of tandem arrangement of variable (V), diversity (D) and joining (J) gene segments, that often results in nucleotide insertion and/or deletion between gene segments at the junction (Figure 2). Human BCR undergo recombination at variable region of three gene segments of the immunoglobulin heavy chain locus (V, D, J) and two gene segments of immunoglobulin light chain locus (V, J). The collection of B-cells receptors genetically rearranged for different antigen specificity are known as BCR repertoire. V V V V D- J- C C C Stem cells Gene rearrangement C C C Naive B cells Somatic hypermutation and affinity maturation C C C Affinity-matured B cells Heavy chain Transcription Light chain Translation mRNA Protein (immunoglobulin) Figure 2. Gene rearrangement in the B cell receptor Immunoglobulin Heavy Chain Applications of BCR Repertoire Sequencing Tracking known Repertoire Disease Sequence Diagnostic Diagnosis and Marker Vaccination Discovery B-cell Repertoire Analysis Figure 3. BCR Repertoire Applications MEDGENOME INC. medgenome.com 2 Tracking known repertoire sequences Incorporating repertoire sequencing into antibody discovery projects will enable the identification of antibody sequences that are either novel or previously identified. Such knowledge can aid in the development of new therapeutic studies. Diagnostic Marker Discovery against Infectious Diseases High throughput repertoire sequencing can provide broad information of disease-specific BCR clones and their dynamic changes in clonality during an infectious state. Sequencing data from antigen-specific clones with stereotyped features in the post-infection repertoires, can provide greater insights and contribute to diagnostic marker discovery against infectious diseases such as H1N1, malaria and COVID-19. Disease Diagnosis and Vaccination Repertoire sequencing can provide insight into disease associated antibody repertoire information such as measure of diversity and rate of abundance of different antibody clone sequences. This repertoire sequence data can significantly aid in understanding immunological mechanisms in vaccine development and improve our understanding in correlating repertoire sequence data to immunological assay functional measures. Major Technologies Gene Amplification Technology Takara SMARTer Human BCR Profiling kit leverages SMART technology (Switching Mechanism at 5’ End of RNA Template) and pairs NGS with a 5’ RACE approach (Figure 4A). cDNA synthesis is dT-primed and full-length cDNA is achieved using MMLV-derived SMARTScribe Reverse Transcriptase (RT) enzyme, that adds SMART UMI oligo’s annealed to non-templated nucleotides at 5’end of each mRNA template. The first strand cDNA is then subjected to two rounds of gene-specific PCR amplification are performed using semi-nested PCR. The nested PCR reduces variability and allows for priming from the constant region of heavy or light chains (Figure 4B). This method generates highly sensitive and reproducible B-cell repertoire profiling, and allow to capture complete V(D)J variable regions of BCR transcripts. Figure 4. Takara SMARTer Human BCR Profiling kit workflow MEDGENOME INC. medgenome.com 3 Next Generation Sequencer (Illumina MiSeq) Nucleotide sequences of B-cell Receptor (BCR) genes require long read sequencing (400-600 bp) to read and assign the V, D, J and C regions of the BCR transcripts. For this purpose, BCR repertoire is sequenced using Illumina MiSeq (Figure 5). Figure 5. Illumina MiSeq BCR Repertoire Analysis MedGenome Inc. utilized Takara Bio Immune Profiler Software to analyze V, D, J and C regions of the BCR transcripts. The software incorporates two third-party software packages, MIGEC and MiXCR for accurate and reliable clonotype calling and quantification. A validation study at MedGenome Inc. was performed using total RNA from human spleen as well as B-cell expressing cell lines and PBMCs using the human Takara SMARTer BCR Profiling Kit. Sequencing data was analyzed using Takara Bio Immune Profiler software following recommended guidelines. We provide a representative BCR repertoire report using human spleen total RNA. A B 5000 5000 Lower 691 Upper Lower 741 Upper 4000 4000 3000 3000 2000 2000 1000 1000 Sample Intensity (Normalized FU) Sample Intensity (Normalized FU) 0 0 Size (nt) Size (bp) 25 50 25 50 100 200 300 400 500 700 Size (bp) 100 200 300 400 500 700 1000 1500 1000 1500 C D 5000 5000 Lower 691 Upper Lower 1000 Upper 4000 4000 3000 3000 2000 2000 1000 1000 Sample Intensity (Normalized FU) Sample Intensity (Normalized FU) 0 0 Size (nt) 25 50 Size (bp) 25 50 100 200 300 400 500 700 Size (bp) 100 200 300 400 500 700 1000 1500 1000 1500 Figure 6. TapeStation traces show representative profiles of B-cell receptor repertoire libraries generated from 10 ng of total RNA from human spleen to specifically amplify the IgG heavy chain (A), IgM heavy chain (B) Kappa (C) and Lambda (D) chains respectively. The libraries were generated using the Takara SMARTer Human BCR IgG IgM H/K/L Profiling Kit and following manufacturers’ instructions. Sequencing was performed using the Illumina MiSeqV3 600 cycle kit, and analysis was performed using the immune profiler pipeline provided by Takara. MEDGENOME INC. medgenome.com 4 Table 2. Mapping statistics shows specificity of amplification of heavy and light chains in BCR libraries of RNA from human spleen total RNA. Sample undeter- IGG IGM IGK IGL short flc total Name mined Human Spleen - 139813(97.0%) 53(0.0%) 93(0.1%) 263(0.2%) 0(0.0%) 3953(2.7%) 0(0.0%) 144175(100.0%) 10ng (IgG) Human Spleen - 55(0.1%) 51847(97.1%) 45(0.1%) 75(0.1%) 0(0.0%) 1393(2.6%) 0(0.0%) 53415(100.0%) 10ng (IgM) Human Spleen - 109(0.1%) 44(0.0%) 152162(97.1%) 93(0.1%) 0(0.0%) 4257(2.7%) 0(0.0%) 156665(100.0%) 10ng (IgK) Human Spleen - 144(0.1%) 80(0.1%) 261(0.2%) 101928(96.0%) 0(0.0%) 3783(3.6%) 0(0.0%) 106196(100.0%) 10ng (IgL) Human Spleen - 2235787(97.3%) 238(0.0%) 540(0.0%) 1988(0.1%) 0(0.0%) 59298(2.6%) 0(0.0%) 2297851(100.0%) 100ng (IgG) Human Spleen - 274(0.0%) 644372(97.6%) 582(0.1%) 291(0.0%) 0(0.0%) 14750(2.2%) 0(0.0%) 660269(100.0%) 100ng (IgM) Human Spleen - 453(0.0%) 186(0.0%) 1155708(97.4%) 334(0.0%) 0(0.0%) 30286(2.6%) 0(0.0%) 1186967(100.0%) 100ng (IgK) Human Spleen - 674(0.0%) 183(0.0%) 293(0.0%) 1546070(96.7%) 0(0.0%) 51057(3.2%) 0(0.0%) 1598277(100.0%) 100ng (IgL) Table 3. Representative table of final clonotype counts from BCR libraries generated using 10 ng of total RNA human spleen (Top 5 clonotypes) each amplifying the IgG heavy and light chains. Clone Clone All VHits All DHits All JHits All CHits Sample ID CloneId AA Seq CDR3 Count Fraction With Score With Score With Score With Score S3034351_IGL_ IGLV2-14*00(2125), 0 2 1 IGLJ1*00(351) IGLC1*00(84.5) CSSYTTSSTYIF mig_cdr3 IGLV2-18*00(1874) S3034351_IGM_ 0 1 1 IGHV3-7*00 (1462) IGHD3-3*00(40) IGHJ4*00(401) IGHM*00(266) CARSFWRFDYW mig_cdr3 IGHJ4*00(372), IGHG1*00(82.7), S3034352_IGG_ IGHD3-16*00(25), 0 206 0.009528655 IGHV3-48*00 (3179.4) IGHJ5*00(350), IGHG2*00(81.8), CTRGLFENW mig_cdr3 IGHD5-12*00(25) IGHJ1*00(331) IGHGP*00(81) IGHG1*00(79.8), S3034352_IGG_ IGHD3-16*00(25), IGHJ4*00(293.7), 1 113 0.005226884 IGHV3-7*00 (3466.7) IGHG2*00(79), CEGGGPKADHW mig_cdr3 IGHD3-3*00(25) IGHJ5*00(283.7) IGHGP*00(78.8) IGHG1*00(88.3), CARDPSGIGVGEL- S3034352_IGG_ IGHD3-10*00(42), 2 68 0.003145381 IGHV3-74*00(3681) IGHJ6*00(332.9) IGHG2*00(86.9), RWGPEWNHLRN- mig_cdr3 IGHD1-14*00(41) IGHGP*00(85.1) KKYGMDVW CARDHI- S3034352_IGG_ IGHG3*00(84.1), 3 68 0.003145381 IGHV1-18*00(2714.6) IGHD6-6*00(56) IGHJ6*00(347) ATRPQYNYGM- mig_cdr3 IGHG4*00(84.1) DVW IGHG1*00(81.3), S3034352_IGG_ IGHD3-10*00(30), 4 45 0.002081502 IGHV3-7*00(3946.1) IGHJ4*00(413) IGHG2*00(80.2), CAGETYYYDHW mig_cdr3 IGHD3-16*00(30) IGHGP*00(78.2) MEDGENOME INC.