title: Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  In 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.
authors.forename: Victor
authors.initials: V
authors.lastname: Greiff
authors.forename: Ulrike
authors.initials: U
authors.lastname: Menzel
authors.forename: Ulrike
authors.initials: U
authors.lastname: Haessler
authors.forename: Skylar C
authors.initials: SC
authors.lastname: Cook
authors.forename: Simon
authors.initials: S
authors.lastname: Friedensohn
authors.forename: Tarik A
authors.initials: TA
authors.lastname: Khan
authors.forename: Mark
authors.initials: M
authors.lastname: Pogson
authors.forename: Ina
authors.initials: I
authors.lastname: Hellmann
authors.forename: Sai T
authors.initials: ST
authors.lastname: Reddy
mesh_terms: D000818:Animals; D000906:Antibodies / Q000235:genetics; D002999:Clone Cells; D022801:Complementarity Determining Regions / Q000235:genetics; D005260:Female; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D007114:Immunization; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008807:Mice, Inbred BALB C; D015203:Reproducibility of Results; D060152:V(D)J Recombination / Q000235:genetics
chemical_list: D000906:Antibodies; D022801:Complementarity Determining Regions; D007135:Immunoglobulin Variable Region