High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire.

Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (VH and VL) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 104 capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion VH:VL linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of VH:VL pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG+ B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccinatio

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes

The molecular composition and binding epitopes of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following SARS-CoV-2 infection are unknown. Proteomic deconvolution of the IgG repertoire to the spike glycoprotein in convalescent subjects revealed that the response is directed predominantly (>80%) against epitopes residing outside the receptor-binding domain (RBD). In one subject, just four IgG lineages accounted for 93.5% of the response, including an N-terminal domain (NTD)-directed antibody that was protective against lethal viral challenge. Genetic, structural, and functional characterization of a multi-donor class of “public” antibodies revealed an NTD epitope that is recurrently mutated among emerging SARS-CoV-2 variants of concern. These data show that “public” NTD-directed and other non-RBD plasma antibodies are prevalent and have implications for SARS-CoV-2 protection and antibody escape

Systematic Characterization and Comparative Analysis of the Rabbit Immunoglobulin Repertoire

<div><p>Rabbits have been used extensively as a model system for the elucidation of the mechanism of immunoglobulin diversification and for the production of antibodies. We employed Next Generation Sequencing to analyze Ig germline V and J gene usage, CDR3 length and amino acid composition, and gene conversion frequencies within the functional (transcribed) IgG repertoire of the New Zealand white rabbit (<i>Oryctolagus cuniculus</i>). Several previously unannotated rabbit heavy chain variable (VH) and light chain variable (VL) germline elements were deduced bioinformatically using multidimensional scaling and k-means clustering methods. We estimated the gene conversion frequency in the rabbit at 23% of IgG sequences with a mean gene conversion tract length of 59±36 bp. Sequencing and gene conversion analysis of the chicken, human, and mouse repertoires revealed that gene conversion occurs much more extensively in the chicken (frequency 70%, tract length 79±57 bp), was observed to a small, yet statistically significant extent in humans, but was virtually absent in mice.</p></div

Light chain germline gene usage.

<p>(A) Vκ and (B) Jκ germline usage of unique kappa light chain sequences in rabbits. Sample sizes of unique sequences: rab1 Bone marrow PC, N = 10,446; rab2 Bone marrow PC, N = 7,481; rab3 Bone marrow PC, N = 12,580. Germline gene IDs are as listed in the IMGT database.</p

Gene conversion analysis: comparison of the frequencies, average scores, and average GC tract lengths in four different species.

<p>Gene conversion analysis: comparison of the frequencies, average scores, and average GC tract lengths in four different species.</p

Characterization of the CDRH3 and CDRL3 in rabbit as compared to other species.

<p>(A) CDRH3 lengths, (B) CDRL3 lengths, and (C) CDRH3 and CDRL3 amino acid composition. All data shown here is derived from unique heavy chain or light chain sequences. Sample sizes of unique IgG/IgY sequences: mouse heavy chain, N = 2,762; rabbit heavy chain, N = 29,439; rabbit light chain, N = 10,446; human heavy chain, N = 2,948; chicken heavy chain, N = 231,165.</p

MDS and k-means clustering of low scoring alignments for VH sequences.

<p>The first three principle components of the MDS are shown here, with k-means defined clusters colored differently. PC1 (principle component 1) v. PC2 (principle component 2) for (A) rab1, (B) rab2, and (C) rab3, and PC1 v. PC3 (principle component 3) for (D) rab1, (E) rab2, and (F) rab3. Each color represents a cluster of sequences as determined by k-means clustering of the Euclidean MDS-derived values.</p

Summary of 454 NGS data.

<p>Summary of 454 NGS data.</p

Diversification of the immunoglobulin repertoire – comparison of overall levels of nucleotide changes in the VH sequences across four species.

<p>The number of nucleotide differences from the nearest (assigned) VH germline derived from IgBLAST alignments as discussed in the text for rabbits.</p

Gene conversion analysis.

<p>(A) Examination of gene conversion donor VH germline usage for recipient query sequences. For the recipient query sequences examined here, the germline VH usage (i.e. the original recombined V gene) was either VH1-a3, VHs1, or VHn3. (B) Gene conversion tract lengths (i.e. lengths of recombined fragments). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101322#s2" target="_blank">Materials and Methods</a> for description of short (min) versus long (max) tract lengths. (C) The nucleotide positions along the VH gene sequence where the gene conversion recombination events start and stop. Start and end positions are based upon the short (min) tract as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0101322#s2" target="_blank">Materials and Methods</a>.</p