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

Greiff V, Menzel U, Haessler U, Cook SC, Friedensohn S, Khan TA, Pogson M, Hellmann I, Reddy ST - BMC Immunol. (2014)

Bottom Line: 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.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.Importantly, we show that both factors-(i) replicate sequencing and (ii) sequencing depth-are crucial for robust CDR3 and VDJ detection and ranking.

View Article: PubMed Central - PubMed

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.

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Diversity information was captured for all CDR3 and VDJ clones with an abundance of two or more regardless of ASC diversity (1M/9M). To address undersampling, we used a sequential sampling scheme (bootstrapping) in which CDR3 and VDJ datasets were divided into 1,000 read subsets, which were successively added to virtual samples until they had the same size as the original samples. We performed 200 simulation runs for one sample of each diversity scenario (1M/9M) having the highest ratio of different CDR3 clones to total numbers of CDR3s. Graphs show (A) the percentage of simulation runs with newly seen CDR3/VDJ clones in a given subset; (B) the normalized median CDR3/VDJ species richness; (C) normalized effective number of CDR3/VDJ species, where “normalized” signifies the division by the respective maximal value.
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Fig2: Diversity information was captured for all CDR3 and VDJ clones with an abundance of two or more regardless of ASC diversity (1M/9M). To address undersampling, we used a sequential sampling scheme (bootstrapping) in which CDR3 and VDJ datasets were divided into 1,000 read subsets, which were successively added to virtual samples until they had the same size as the original samples. We performed 200 simulation runs for one sample of each diversity scenario (1M/9M) having the highest ratio of different CDR3 clones to total numbers of CDR3s. Graphs show (A) the percentage of simulation runs with newly seen CDR3/VDJ clones in a given subset; (B) the normalized median CDR3/VDJ species richness; (C) normalized effective number of CDR3/VDJ species, where “normalized” signifies the division by the respective maximal value.

Mentions: Since comparison across replicates depends on their deep coverage, we first addressed the issue of undersampling. To do so, we relied on simulations that used a sequential sampling scheme (bootstrapping) for both CDR3 and VDJ clonal distributions. Simulations were performed for one replicate dataset from the 1M and 9M triplicates. The replicate that was chosen was based on having the unfavorably highest ratio of different CDR3s to total CDR3s. We performed a bootstrapping approach with 1,000 equally sized sampling steps, where in each step a random set of CDR3/VDJ sequences (from the replicate NGS dataset) was added to virtual samples. Sampling steps continued until virtual samples had accumulated the same number of sequences as the starting replicate (Figure 2).Figure 2


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

Greiff V, Menzel U, Haessler U, Cook SC, Friedensohn S, Khan TA, Pogson M, Hellmann I, Reddy ST - BMC Immunol. (2014)

Diversity information was captured for all CDR3 and VDJ clones with an abundance of two or more regardless of ASC diversity (1M/9M). To address undersampling, we used a sequential sampling scheme (bootstrapping) in which CDR3 and VDJ datasets were divided into 1,000 read subsets, which were successively added to virtual samples until they had the same size as the original samples. We performed 200 simulation runs for one sample of each diversity scenario (1M/9M) having the highest ratio of different CDR3 clones to total numbers of CDR3s. Graphs show (A) the percentage of simulation runs with newly seen CDR3/VDJ clones in a given subset; (B) the normalized median CDR3/VDJ species richness; (C) normalized effective number of CDR3/VDJ species, where “normalized” signifies the division by the respective maximal value.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4233042&req=5

Fig2: Diversity information was captured for all CDR3 and VDJ clones with an abundance of two or more regardless of ASC diversity (1M/9M). To address undersampling, we used a sequential sampling scheme (bootstrapping) in which CDR3 and VDJ datasets were divided into 1,000 read subsets, which were successively added to virtual samples until they had the same size as the original samples. We performed 200 simulation runs for one sample of each diversity scenario (1M/9M) having the highest ratio of different CDR3 clones to total numbers of CDR3s. Graphs show (A) the percentage of simulation runs with newly seen CDR3/VDJ clones in a given subset; (B) the normalized median CDR3/VDJ species richness; (C) normalized effective number of CDR3/VDJ species, where “normalized” signifies the division by the respective maximal value.
Mentions: Since comparison across replicates depends on their deep coverage, we first addressed the issue of undersampling. To do so, we relied on simulations that used a sequential sampling scheme (bootstrapping) for both CDR3 and VDJ clonal distributions. Simulations were performed for one replicate dataset from the 1M and 9M triplicates. The replicate that was chosen was based on having the unfavorably highest ratio of different CDR3s to total CDR3s. We performed a bootstrapping approach with 1,000 equally sized sampling steps, where in each step a random set of CDR3/VDJ sequences (from the replicate NGS dataset) was added to virtual samples. Sampling steps continued until virtual samples had accumulated the same number of sequences as the starting replicate (Figure 2).Figure 2

Bottom Line: 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.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.Importantly, we show that both factors-(i) replicate sequencing and (ii) sequencing depth-are crucial for robust CDR3 and VDJ detection and ranking.

View Article: PubMed Central - PubMed

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.

Show MeSH
Related in: MedlinePlus