Constructing an accurate antibody repertoire

What is an antibody repertoire?

The immune repertoire is the collection of unique immunoglobulin and T-cell receptor sequences present in an individual at a particular time. At Digital Proteomics we focus primarily on the immunoglobulin, or B-cell receptor repertoires of humans and other mammals. The total number of B cells and plasma cells that encode full-length immunoglobulins in an adult human is estimated to be 1010-1011, which puts an upper bound on the total size of the B-cell receptor, or antibody, repertoire. The antibody repertoire is dynamic, with sequence diversity and composition of the repertoire changing dramatically over time. In this post, we discuss the approach to immune repertoire construction that we employ in our Reptor and Alicanto services. Before repertoire construction and analysis can begin, the B-cell receptor transcripts must be sequenced. For a bit about that process, read our previous post on immunosequencing.

Due to high sequence diversity, antibody repertoire sequencing and assembly of an individual repertoire is complex and requires specialized tools. It’s important to recognize the unique challenges of antibody repertoire sequence and analysis, which make them ill-suited for standard RNA-seq or target-enrichment workflows.

Antibody repertoire construction overview

Read quality filtering

As in any bioinformatic pipeline, for immune repertoire analysis ‘garbage in = garbage out’. Read filtering is an important part of the process, and the best practices for any RNA/DNA sequencing analysis apply. This includes removing reads with low quality, removing reads that are generated from a spiked-in library (such as PhiX), and trimming primer and sequencing adapter sequences. Looking at overall statistics for your run using a free tool such as FastQC is also a great way to spot problems in your sequencing run.

Helpful resources for further reading

The Adaptive Immune Receptor Repertoire (AIRR) Community holds regular meetings and publishes standards for repertoire sequencing and analysis.

Practical guidelines for B-cell receptor repertoire sequencing analysis.