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

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.

Eye on the B-ALL: B-cell receptor repertoires reveal persistence of numerous B-lymphoblastic leukemia subclones from diagnosis to relapse

“…Detection of very low-level B-ALL sequences in some qPCR-negative samples was reproducible in replicates of the same sample, with the exception of one sample (Supplementary Table S4, patient 1611, day 19 sample), from which clonotypic sequences were detected at low frequency (0.0656%) in only one of two repeats in keeping with stochastic ‘loss' of a rare variant. 8, 25, 26, 27 Likewise, the percentage of matching BCR sequences in DNA samples (UKALL XI) correlated with the % blasts in PB (Figure 1b). Notably, we detected clonotypic sequences in 6 of 10 patients at day 28, where blasts were not detected (<1%).…”

The mouse antibody heavy chain repertoire is germline-focused and highly variable between inbred strains

“…Although RFs were not equally used in the productive repertoires of C57BL/6 (RF1 15.4%, RF2 7.7%, RF3 76.9%) or BALB/c (RF1 15.6%, RF2 8.9%, RF3 75.5%), the usage frequencies were highly consistent between the two strains. Each IGHD The nomenclature of the VBASE2 database [26]. c The nomenclature of Johnstone et al [20].…”

Resident memory CD8 T cells persist for years in human small intestine

“…Correlation of the read counts for the clonotypes found within the three molecular replicates was high ( Figure S12C), indicating reproducibility of the TCR repertoire sequencing (Greiff et al, 2014). For downstream analysis, raw reads from molecular triplicates were cumulated, and only clonotypes with a minimal read count of 10 were used.…”

“…RNA was isolated and TCRα and TCRβ genes were amplified by semi-nested PCR including three cDNA replicates for each TCR chain ( Figure S12A). Correlation of the read counts for the clonotypes found within the three molecular replicates was high ( Figure S12C), indicating reproducibility of the TCR repertoire sequencing (Greiff et al, 2014). In order to determine whether different SI CD8 T cell subsets shared a common clonal origin we calculated the percentage of overlapping clones and the Morisita-Horn similarity index for all the pairwise comparisons of both TCRα and TCRβ clonotypes.…”