Journal: bioRxiv

Article Title: Benchmarking and integrating human B-cell receptor genomic and antibody proteomic profiling

doi: 10.1101/2023.11.01.565093

Figure Lengend Snippet: (a) The sequence information of the Ig repertoire can be examined on the nucleotide level by bulk BCR sequencing (bulkBCR-seq) and single-cell BCR sequencing (scBCR-seq) or on the protein level by bottom-up antibody mass spectrometry (Ab-seq). Still, there is a lack of joint analysis integrating and comparing the overlap between methods, and few studies where Ab-seq data augment BCR data. (b) To address this problem, we developed a workflow that extracts repertoire information by combining high-throughput bulkBCR-seq with natively paired receptor information in scBCR-seq, then leveraging this sequence information to examine the composition of the serum antibody repertoire using Ab-seq. Our findings indicate that: (c) VH-gene usage is conserved within individuals despite differences in sequencing methods, (d) the gap in sampling depth between bulkBCR-seq and scBCR-seq resulted in low clonal sequence overlap, contributing to lower biological coverage of the Ig repertoire, and (e) it is possible to recover clonal sequences from peptide sequences characterized by Ab-seq, with paired chain BCR sequencing contributing to paired chain V(D)J sequence reconstruction.

Article Snippet: This allowed efficient full-length BCR sequencing while keeping the amplicon length under 600 bp for Illumina MiSeq , .

Techniques: Sequencing, Mass Spectrometry, High Throughput Screening Assay, Sampling

Journal: bioRxiv

Article Title: Benchmarking and integrating human B-cell receptor genomic and antibody proteomic profiling

doi: 10.1101/2023.11.01.565093

Figure Lengend Snippet: Peptides from digestion of serum Abs were analyzed by LC-MS/MS, and their sequences were aligned to references made from bulkBCR-seq and scBCR-seq data of the same individual. (a) Sample setup for Ab-seq. (b) Number of peptides identified by LC-MS/MS (All), aligned to reference BCR sequences (Ab-specific), and overlapping at least 3 aa with the reference sequence’s CDR3 (CDR3-overlapping). (c) Ab-seq peptide length in regard to the length of overlap with its reference’s CDRH/L3. (d) Number of CDR3-overlapping peptides by the sequencing method of the reference match: bulkBCR-seq, scBCR-seq, or both bulkBCR-seq and scBCR-seq (both). (e) Number of CDR3-overlapping peptides by protease treatment: AspN, Chymotrypsin (Ct), Chymotrypsin followed by Trypsin (Ct+Tryp), and Trypsin (Tryp). (f) Number of CDR3-overlapping peptides that mapped to only one reference clonotype or multiple clonotypes . (g) Number of uniquely mapped CDR3-overlappping peptides by clonal size ranking in descending order of the BCR-seq reference match in log10 scale.

Article Snippet: This allowed efficient full-length BCR sequencing while keeping the amplicon length under 600 bp for Illumina MiSeq , .

Techniques: Liquid Chromatography with Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: Benchmarking and integrating human B-cell receptor genomic and antibody proteomic profiling

doi: 10.1101/2023.11.01.565093

Figure Lengend Snippet: Uniquely mapped CDR3-overlapping peptides (quantified in ) were utilized to recover the clonotype information provided by BCR-seq, with overlapping segment in bold. (a) Example of Ab-seq peptides mapped to a bulkBCR-seq used to recover the single chain V(D)J sequence. (b) Example of Ab-seq peptides mapped to a scBCR-seq used to recover the paired chain V(D)J sequence by utilizing the cell barcode unique to each single-cell droplet. See Supplementary file 1 for the full table of all V(D)J sequences recovered by Ab-seq.

Article Snippet: This allowed efficient full-length BCR sequencing while keeping the amplicon length under 600 bp for Illumina MiSeq , .

Techniques: Sequencing