EFB Dengue Literature Review

BCR Sequencing

11 min read

BCR Sequencing

Overview

BCR (B cell receptor) sequencing refers to the sequencing of rearranged immunoglobulin heavy and/or light chain variable region genes to assess somatic hypermutation (SHM), CDR3 diversity, clonal relationships, and selection pressure (replacement:silent mutation ratios in framework vs. CDR regions). In 2007-era studies, this was performed by PCR amplification of VH family genes from sorted B cell populations followed by Sanger sequencing of individual clones. Modern approaches use high-throughput next-generation sequencing (NGS) of bulk or single-cell BCR repertoires.

Key Points from Literature

  • Wei et al. used VH3 family-specific PCR primers with a Cγ constant region primer to amplify IgG⁺ sequences from sorted CD27⁺ and CD27⁻ (DN) B cell fractions; PCR products were cloned into pCR4Blunt-TOPO and individual colonies sequenced by BigDye Terminator cycle sequencing (see Wei2007 - DN Memory B Cells in SLE).

  • Analysis of ~28 sequences per group revealed that DN IgG⁺ cells carry ~3.2% nucleotide mutation rate (healthy) and ~2.6% (SLE), compared with ~5.4% and ~5.1% for CD27⁺ IgG⁺ memory cells — supporting antigen experience in DN cells but at a lower level than conventional memory (see Wei2007 - DN Memory B Cells in SLE).

  • Replacement:silent (R:S) mutation ratios in CDR1 were substantially higher in DN cells (13 in healthy, 3.2 in SLE) than CD27⁺ cells (3.9, 9.6), while FR region R:S ratios were broadly similar — broadly consistent with antigen-driven selection but with possible differences in selection stringency (see Wei2007 - DN Memory B Cells in SLE).

  • Limitation: analysis restricted to VH3 family sequences only; small sample size (n=28 per group); Sanger-based single-clone approach lacks the clonal diversity information available from modern NGS repertoire sequencing.

  • Tipton2015 NGS approach (Illumina MiSeq, VH1–7): RNA isolated from sorted B cell populations (naive, IgD⁻ memory, CD138⁻ ASC, CD138⁺ ASC, and acN cells); ~2 ng RNA converted to cDNA; PCR amplified with VH1-VH7 FR1 primers and constant-region primers (Cα, Cµ, Cγ) carrying Illumina Nextera tags. Sequenced on MiSeq (500-cycle v2 or 600-cycle v3). After paired-end joining, quality/length filtering, and IMGT/HighV-QUEST alignment, 50,000 sequences per dataset retained for downstream analysis. This approach covers all VH families (vs. VH3-only Sanger), enabling VH segment usage analysis (e.g., VH4-34 enrichment) (see Tipton2015 - ASC Diversity and Origin in SLE).

  • Clonality metrics (D20 and D50): Sequences are assigned to clones using shared VH/JH rearrangements + identical HCDR3 length + Hamming identity >85% in HCDR3. D20 = number of clones accounting for 20% of total sequences (lower = more oligoclonal); D50 = same for 50%. SLE CD138⁻ ASCs: D20 ~199, D50 ~1,722 vs. vaccination: D20 ~21, D50 ~159. acN cells have D20 index 28.7-fold higher than healthy naive B cells — consistent with antigen-driven clonal expansion (see Tipton2015 - ASC Diversity and Origin in SLE).

  • Clonal connectivity analysis between sorted populations: By applying the same clonal assignment metric across simultaneously sorted populations (naive, acN, memory, ASC), the fraction of sequences shared between populations can be quantified as a “connectivity” score. This approach revealed that acN cells contribute up to 32.5% of sequences clonally connected to co-circulating ASCs in SLE (see Tipton2015 - ASC Diversity and Origin in SLE).

  • Phylogenetic tree construction with IgTree: Clonal sequences aligned to germline with Clustal X; genealogical trees built with IgTree (distance-method algorithm). This allows identification of putative common ancestors and ordering of diversification steps within complex clones containing cells from multiple populations (see Tipton2015 - ASC Diversity and Origin in SLE).

  • Integration with serum proteomics: NGS databases from sorted populations serve as reference databases for LC-MS/MS identification of serum antibody sequences, enabling direct linkage of serum proteins to specific B cell clonotypes (see Tipton2015 - ASC Diversity and Origin in SLE; see also Serum Proteomics).

  • Jenks2018 clonal connectivity across aNAV, DN2, and PC: BCR sequencing of FACS-sorted aNAV, DN2, and PC populations demonstrated clonal sharing between all three — the in vivo repertoire evidence for the rNAV → aNAV → DN2 → plasmablast developmental pathway. IgG mutation rates in DN2 cells are similar to co-circulating PCs but lower than SWM, arguing against DN2 derivation from memory cells and supporting de novo EF differentiation from naive precursors (see Jenks2018 - DN2 B Cells and EF Pathway in SLE, BCR sequencing of sorted populations).

  • Single-cell V(D)J via 10x Genomics Chromium in COVID-19: ASCs from an ICU patient were captured at 10,000 cells per sample on a 10x Chromium device (Next GEM Single Cell V(D)J Reagent Kits v1.1). Sequencing on Illumina NovaSeq (2×150 bp, >70% saturation at 5,000 reads/cell) yielded 5,338 cells and 2,017 clonotypes. Combined with bulk V(D)J from CD138-enriched ASCs of 2 additional patients, this revealed germline-dominant repertoire (>50% unmutated VH), balanced IgM/IgG1/IgA1, ongoing CSR, and oligoclonal expansions — the first single-cell ASC repertoire analysis in an acute viral infection through the EF pathway lens (see Woodruff2020 - EF B Cell Responses in COVID-19, 10x Chromium + Illumina NovaSeq and MiSEQ).

  • Lineage analysis tools: GLaMST (Grow Lineages along Minimum Spanning Tree) for lineage tree construction from single-cell V(D)J data. Circos plots for visualising contemporaneous IgM↔IgG1/IgA1 class switching connections. Both approaches complement the IgTree phylogenetics used in Tipton2015 (see Woodruff2020 - EF B Cell Responses in COVID-19).

  • FIRST DENGUE BCR REPERTOIRE DATA — 454 pyrosequencing of VH from unsorted PBMC gDNA: Parameswaran2013 amplified VH rearrangements from 100 ng PBMC genomic DNA using BIOMED-2 barcoded multiplex primers in 6 independent PCR reactions per sample. Sequencing on GS FLX (454/Roche) with dual runs; iHMMune-align for V/D/J assignment; median ~2,000 reads per sample (range 450–9,000). The use of gDNA ensures each B cell contributes a single copy regardless of Ig expression level, unlike RNA-based approaches where plasmablasts are overrepresented. This makes clonality estimates unbiased by expression level but precludes isotype resolution (see Parameswaran2013 - Convergent Antibody Signatures in Dengue, n=60 dengue + 16 controls, 454 pyrosequencing).

  • Replicate library sequencing for clonality estimation (P(collision)): By sequencing 6 independent PCR libraries from separate DNA aliquots of the same sample, sequences observed in >1 replicate are inferred to derive from sufficiently expanded clones. P(collision) — the probability that two randomly chosen B cells share clonal origin — is estimated from inter-replicate coincidences. This metric is independent of sequencing depth (validated by subsampling) and captures global B cell expansion without requiring antigen-specific pre-selection. P(collision) was significantly higher in acute vs. convalescent (p=0.0004) and acute vs. post-convalescent (p<0.0001) dengue, and higher in secondary vs. primary acute dengue (p=0.0409) (see Parameswaran2013 - Convergent Antibody Signatures in Dengue).

  • Convergent CDR3 identification by cross-validation: The 44-patient dataset was split into training and test sets; CDR3 amino acid sequences and their one-mismatch derivatives were screened for high prevalence in acute samples and low prevalence in post-convalescent/healthy samples. Six CDR3s (10-mers and 13-mers) were validated across training set, test set, an independent 16-patient cohort, and confirmed absent in >1,000 datasets from 640 non-dengue individuals. Four independent analytical approaches (mismatch cross-validation, BLOSUM62 similarity, association testing with FDR correction, L1-regularized logistic regression) yielded overlapping CDR3 candidates, with the cluster-based logistic regression achieving AUC 0.834 for dengue classification (see Parameswaran2013 - Convergent Antibody Signatures in Dengue).

  • Convergent CDR3s encoded by multiple V gene families — evidence of true convergent evolution: The most prevalent CDR3 (ARLD(Y)₅GMDL) was encoded by 6 distinct V genes from V1, V3, and V5 families across individuals. Nucleotide sequences underlying the same CDR3 amino acid sequence differed across individuals, with synonymous codon variation and diverse V/D/J usage. This rules out PCR contamination or shared germline alleles and establishes independent derivation through convergent evolution under antigen selection (see Parameswaran2013 - Convergent Antibody Signatures in Dengue).

  • Combined Sanger single-cell and 454 pool sequencing of sorted B cells in dengue: Appanna2016 used two complementary BCR sequencing approaches on sorted populations: (1) Sanger sequencing of single-cell VH/VL from plasmablasts and MBCs (RT-PCR from 96-well plates), enabling paired heavy/light chain cloning and mAb expression; (2) 454 GS-FLX Titanium pyrosequencing (250,000 reads/sample) of pool-sorted populations using iRepertoire primers covering V and C regions. CDR3 clones were defined by same V/D/J usage, same length, and ≥85% sequence identity. Clonal overlap analysis (Venn diagrams) revealed very few shared CDR3s between PBs and DENV-binding MBCs — the first direct clonal comparison of these two compartments in dengue. MiXCR v1.8.2 was used for V/D/J assignment and isotype extraction from 454 data (see Appanna2016 - Plasmablasts as Subset of Memory B Cell Pool, n=12 dengue, FACSAria sorting → Sanger + 454).

  • 454-Roche IgG VH cDNA sequencing with ImmunediveRsity pipeline in dengue: GodoyLozano2016 used 5ʹ RACE-PCR with an IgHG CH1 exon-specific antisense primer to generate VH libraries from total peripheral blood RNA (unsorted IgG⁺ B cells). Sequencing on GS FLX Titanium (454-Roche, ~450–500 bp reads, 3ʹ→5ʹ direction to prioritise CDRH3 quality). 2.36 million raw reads yielded 2.04 million pass-filter sequences, reconstructed into 146,565 clonotypes and 385,206 lineages across 30 libraries (19 acute + 11 post-convalescent). The custom ImmunediveRsity pipeline performs IgBLAST V(D)J assignment, CDRH3-based clonotype clustering (97% identity), lineage consensus (99.5% identity), Shannon entropy/rarefaction diversity estimation, and SHM quantification via IMGT/HighVQuest. IGHV usage was expressed at three levels: raw read proportion (relative transcription), clonotype frequency, and lineage frequency — enabling discrimination of transcription-level bias (high Ig expression per cell) from clonal expansion. Hierarchical clustering of ΔA−Pc IGHV usage identified patient groups correlated with clinical (DWS−/DWS+) and immune (primary/secondary) status (see GodoyLozano2016 - Lower IgG SHM Rates in Acute Dengue, n=19 acute + n=11 post-convalescent, 454-Roche).

  • Monte Carlo simulation for B cell subpopulation deconvolution: A second-order Monte Carlo simulation estimated the probability of sampling memory B cells vs. ASCs in unsorted IgG⁺ repertoire data, modelling individual variation in mB:ASC ratios and differential Ig transcription (ASCs ~12-fold higher than mB cells, gamma-distributed). At the expected acute dengue plasmablast proportion (56% of IgG⁺ per Wrammert2012), the probability of sampling a single read from a memory B cell was 0.015. During post-convalescence (mB ~95.8%), lineages ≥30 reads were estimated to derive exclusively from ASCs (p = 2.06E-06 for mB origin). This approach enables in silico sorting of repertoire data by Ig expression level, partially compensating for the lack of physical cell sorting (see GodoyLozano2016 - Lower IgG SHM Rates in Acute Dengue).

  • De novo convergent CDRH3 identification: Beyond testing against previously published signatures (Parameswaran2013), GodoyLozano2016 identified de novo convergent CDRH3s by screening for identical amino acid sequences shared in ≥3 individuals. Found 1,365 clonotypes representing 269 identical CDRH3s; 70% had a predominant 10-residue length. Two CDRH3s (ARQFGNWFDS, ARQWGNWFDL) were shared in 10/19 (52%) of individuals. Convergent clones carried significantly lower SHM than non-convergent clones (p<0.001) (see GodoyLozano2016 - Lower IgG SHM Rates in Acute Dengue).

  • Smart-seq2 full-length BCR from scRNA-seq of alternative lineage B cells: Sutton2021 obtained BCR sequences from 163 FACS-sorted antigen-specific B cells (PfCSP- and HA-tetramer⁺) processed by Smart-seq2 plate-based scRNA-seq. The full-length mRNA capture enabled V gene usage analysis and SHM quantification alongside transcriptomic cluster assignment. All non-naive clusters (including atBC1, atBC2, atBC3, MBC1) showed significant somatic hypermutation, consistent with post-GC origin for the alternative lineage. The low throughput (163 cells) limits statistical power for rare clonotype detection but provides paired transcriptome + BCR data at single-cell resolution (see Sutton2021 - Alternative Lineage B Cells in Vaccination and Infection, Smart-seq2, n=11 donors).

  • Microdissection + Vκ PCR sequencing — the original EF SHM method. William2002 pioneered the use of microdissection to isolate B cells from defined anatomical sites for BCR sequencing. Id⁺ B cell clusters at the T zone–red pulp border were captured with a glass micropipette (Eppendorf Transferman micromanipulator), and Vκ8/Jκ4 rearranged sequences were amplified by nested PCR using Pfu Turbo (Stratagene) from picks of 5–50 cells. Amplified DNA was cloned into bacterial vectors and colonies sequenced with T3 primers. From 8 mice, 45 independent PCR libraries yielded 305 total sequences and 125 unique sequences. Sequences were assigned to genealogical trees based on shared/unique mutations and VJ junctions. This approach — physical isolation of cells from a defined tissue site followed by clonal sequencing — provides spatial resolution that no other BCR sequencing method in the wiki achieves, and was essential for proving that SHM was occurring in situ at EF sites rather than in GCs followed by migration (see William2002 - Extrafollicular Somatic Hypermutation in Autoimmune Mice, Pfu-based nested PCR from microdissected frozen sections).

Contradictions & Debates

None documented in current wiki sources.

FACS Sorting, Somatic Hypermutation, Double-Negative B Cell, DN2 B Cell, Activated Naive B Cell, Memory B Cell, Plasmablast, Immunohistochemistry, Extrafollicular Response

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