BALDR – A Computational Pipeline for Immunoglobulin Reconstruction in Single-cell RNA-Seq data from B cells
Amit A. Upadhyay1, Alice Cho2, Amber Wolabaugh1, Robert Kauffman2, Gregory D. Tharp1, Nirav Patel1, F. Eun-Hyung Lee2, Jens Wrammert2, Steven E. Bosinger1,2*
1Yerkes National Primate Research Center, 2School of Medicine, Emory University, Atlanta, GA
B-cells play a critical role in the adaptive immune response by binding to pathogens through lineage specific B-cell receptors (BCR) and producing antibodies. BCR sequencing has applications in antibody discovery, vaccine design and cancer. Several pipelines have been described for T cell receptor reconstruction. However, similar methods for the longer BCR variable region are lacking. Our goal here was to develop a robust and efficient bioinformatic pipeline for accurate BCR reconstruction from sc-RNA-Seq data.
We generated Illumina single cell-RNA-Seq data for 86 human plasmablasts and 88 naïve B cells. Reconstruction was performed using de novo assembly, as well as assembly after different immunoglobulin transcript filtering strategies. Accuracy of clonotype-assignment was validated by comparing reconstructed plasmablast sequences to those obtained by conventional single-B cell cloning methodology.
Our methods resulted in reconstruction of productive IgH and IgL chains in all cells. When compared to single cell amplicon sequencing, the clonotype assignment accuracy was 100%. IG chains were identified unambiguously for 94% plasmablasts and 89% naive B cells. Considering the computational time and the accuracy of reconstructions, assembly using reads filtered by mapping to IG loci along with unmapped reads was found to give the best results. The pipeline used will be made as a software package (BALDR - BCR Assignment of Lineage using De novo Reconstruction).
The BALDR pipeline makes it possible to link the transcriptional programming of individual B cell clones at critical developmental stages with the eventual fate of the clonal lineage in vivo. This tool will be critical for dissection of molecular events that occur during vaccine induced humoral immunity in humans.