Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice.

Victor Greiff,Sai T Reddy,Simon Friedensohn,Tarik A Khan,Mark Pogson,Ina Hellmann,Skylar C Cook,Ulrike Menzel,Ulrike Haessler

doi:10.1186/s12865-014-0040-5

Abstract

BackgroundNext-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies: thus, it is essential to establish the reliability of antibody NGS data.ResultsWe isolated RNA from antibody-secreting cells (ASCs) from either 1 mouse or a pool of 9 immunized mice in order to simulate both normal and high diversity populations. Next, we prepared three technical replicates of antibody libraries by RT-PCR from each diversity scenario, which were sequenced using the Illumina MiSeq platform resulting in >106 250 bp paired-end reads per replicate. We then assessed the robustness of antibody repertoire data based on clonal identification defined by amino acid sequence of either full-length VDJ region or the complementarity determining region 3 (CDR3). Leveraging modeling approaches adapted from mathematical ecology, we found that in either diversity scenario both CDR3 and VDJ detection nears completeness indicating deep coverage of ASC repertoires. Additionally, we defined reliability thresholds for accurate quantification and ranking of CDR3s and VDJs. Importantly, we show that both factors–(i) replicate sequencing and (ii) sequencing depth–are crucial for robust CDR3 and VDJ detection and ranking.ConclusionsIn summary, we established widely applicable experimental and computational guidelines for robust antibody NGS and analysis, which will help advance systems immunology studies related to the quantitative profiling of antibody responses following infection and vaccination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-014-0040-5) contains supplementary material, which is available to authorized users.

Highlights

Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses
Antibody repertoire NGS has become a powerful tool to quantitatively address fundamental questions in immunology related to lymphocyte development and differentiation [20], discovery of clinical diagnostics based on antibody sequence biomarkers [14,21,22], and antibody repertoire diversity [20,23,24,25]
High quality Illumina triplicate sequencing of antibody-secreting cells from pooled and individual mouse samples Two scenarios of immunological relevance were assessed for NGS reproducibility

Summary

Introduction

Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. An emerging systems immunology method to quantitatively assess the antibody repertoire’s immense diversity is high-throughput immune repertoire analysis, which combines next-generation sequencing (NGS), bioinformatics, and statistical analysis of variable regions [17,18,19]. One of the principal advantages of antibody NGS is the quantitative determination of clonal diversity and distribution, which provides valuable insight into clonal selection and expansion during humoral responses [26] This assessment of clonal diversity and distribution offers new approaches for vaccine profiling and monoclonal antibody discovery and engineering [1,4,13,27,28,29,30,31]

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Results

Discussion

Conclusion