Antibody repertoire sequencing analysis

Abstract

High-throughput sequencing for B cell receptor (BCR) repertoire provides useful insights for the adaptive immune system. With the continuous development of the BCR-seq technology, many efforts have been made to develop methods for analyzing the ever-increasing BCR repertoire data. In this review, we comprehensively outline different BCR repertoire library preparation protocols and summarize three major steps of BCR-seq data analysis, i. e., V(D)J sequence annotation, clonal phylogenetic inference, and BCR repertoire profiling and mining. Different from other reviews in this field, we emphasize background intuition and the statistical principle of each method to help biologists better understand it. Finally, we discuss data mining problems for BCR-seq data and with a highlight on recently emerging multiple-sample analysis.

Keywords: BCR repertoire analysis; high-throughput sequencing; profiling and data mining; statistical method.

Figure 1

Methods for BCR repertoire preparation Multiplex PCR: regardless of the template types, degenerated primer sets targeting the V gene segments are adopted. Different colors of arrows symbolizes different primer sets targeting various V classes. When mRNA (from bulk or single cell) is used as input, reverse primers annealing to C segments or poly-A sequence are commonly used to amplify the V(D)J. While when genomic DNA is subject to library preparation, the reverse primers usually anneal to the J intron region. One-side PCR methods: In the context of using bulk mRNA as template, after reverse transcription, several cytidines are over-hanged to the 3’ end of synthesized cDNA. Then a “template switch” primer with poly-G sequence will add adaptor on this side. In bulk mRNA samples, 5’ RACE is a popular example. The biotinylated primers annealing to J intron segment are used to select the target ssDNA sequence with streptavidin. In the next step, a dsDNA “bridge primer” is used to mediate the following ligation and synthesize of the other strand. The one-side PCR strategy of single-cell samples, e.g., 5’ seq, is similar to that of bulk mRNA, and the “template switch” primers are attached to a barcoded bead for discrimination between different cell samples.

Figure 2

A reference analysis pipeline for BCR-seq data