Broadly neutralizing antibodies target a hemagglutinin anchor epitope, Guthmiller et al. 2021

This study was designed to analyze the B cell repertoire of cH5/1 binding B cells following vaccination with the cHA platform. Human B cells (CD19+IgM-CD27+cH5/1+) were sorted at day 113 and were used for downstream sequencing. This dataset is comprised of a collection of BCR sequences of 1952 antibodies with paired heavy chain and light chain, a collection of BCR sequences of 119 anchor antibodies, and a collection of BCR sequences of 365 VH1-69&kappa.

PROTOCOLS:
cH5/1+ memory B cells (CD19+CD27+HA+) were bulk sorted and partitioned into nanoliter-scale Gel Bead-In-Emulsions (GEMs) to achieve single cell resolution using the 10x Genomics Chromium Controller and according to the manufacturer’s instruction (10x Genomics). The sorted single cells were processed according to 5’ gene expression and B cell Immunoglobulin (Ig) enrichment instruction to prepare the libraries for sequencing. Libraries were sequenced using an Illumina HiSeq 4000 at Northwestern University or an Illumina NextSeq 500 at the University of Chicago.

Cellranger Single-Cell Software Suite (version 3.0) was used to perform sample de-multiplexing, barcode processing, and single-cell 5’ and V(D)J counting, and Cellranger mkfastq was used to de-multiplex raw base call (BCL) files into sample-specific fastq files. Subsequently, GRCh38-1.2.0 and cellranger-vdj-GRCh38-alta-ensembl-2.0.0 were used as references for the transcriptome and V(D)J assembly, respectively. Cellranger counts and Cellranger vdj package were used to identify gene expression and assemble V(D)J pairs of antibodies. BCR sequences were then processed by IgBlast and our in-house software VGenes.

Please see readme.xlsx file for more details.

本研究旨在分析经cHA平台免疫后结合cH5/1的B细胞受体库（B cell repertoire）。研究人员于第113天分选得到人B细胞（CD19+IgM-CD27+cH5/1+），并将其用于后续测序实验。本数据集包含三类B细胞受体（B cell receptor, BCR）序列集合：其一为1952条配对重链与轻链的抗体BCR序列，其二为119条锚定抗体的BCR序列，其三为365条VH1-69&kappa类型的BCR序列。

实验方案：
采用10x Genomics Chromium控制器，依照厂商说明书（10x Genomics），将cH5/1+记忆性B细胞（CD19+CD27+HA+）进行批量分选后，分配至纳升级凝胶微珠乳液（Gel Bead-In-Emulsions, GEMs）中以实现单细胞分辨率。分选得到的单细胞按照5'基因表达与B细胞免疫球蛋白（Ig）富集流程制备测序文库。文库分别在西北大学（Northwestern University）使用Illumina HiSeq 4000，或在芝加哥大学（University of Chicago）使用Illumina NextSeq 500进行测序。

使用Cellranger单细胞软件套件（版本3.0）完成样本拆分、条码处理以及单细胞5'转录组与V(D)J计数；使用Cellranger mkfastq将原始碱基识别（BCL）文件拆分为样本专属的fastq文件。随后分别以GRCh38-1.2.0与cellranger-vdj-GRCh38-alta-ensembl-2.0.0作为转录组与V(D)J组装的参考基因组。通过Cellranger counts与Cellranger vdj套件完成基因表达鉴定与抗体V(D)J配对组装。后续通过IgBlast与自研软件VGenes对BCR序列进行处理。

更多细节请参阅readme.xlsx文件。