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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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Establishing a cut-off for reliable clonal detection. For each replicate (A: CDR3, B: VDJ), starting with the highest frequency clones, clones were tested for their simultaneous presence in the respective two other replicates. For each clone, presence or absence in the other two replicates were recorded. We regarded all those clones as “reliably detected” that belonged to the highest frequency set of clones with a ratio of presence to absence ≥99%. The percentage of clones passing the detection threshold is indicated by vertical red bars. Absolute numbers of reliably detected clones for CDR3-1M were (1427, 862, 791), and for CDR3-9M were (9414, 9485, 9460). For VDJ, 1M and 9M absolute numbers were (10741, 10731, 11055) and (18765, 18928, 18466), respectively. For a graphical overview of the method of reliable detection, please refer to Menzel et al. [53].
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Fig3: Establishing a cut-off for reliable clonal detection. For each replicate (A: CDR3, B: VDJ), starting with the highest frequency clones, clones were tested for their simultaneous presence in the respective two other replicates. For each clone, presence or absence in the other two replicates were recorded. We regarded all those clones as “reliably detected” that belonged to the highest frequency set of clones with a ratio of presence to absence ≥99%. The percentage of clones passing the detection threshold is indicated by vertical red bars. Absolute numbers of reliably detected clones for CDR3-1M were (1427, 862, 791), and for CDR3-9M were (9414, 9485, 9460). For VDJ, 1M and 9M absolute numbers were (10741, 10731, 11055) and (18765, 18928, 18466), respectively. For a graphical overview of the method of reliable detection, please refer to Menzel et al. [53].

Mentions: Leveraging the deep repertoire coverage, we proceeded by establishing a cutoff, which ensures that 99% of clones in a given replicate would be found in the two other replicates (Figure 3). Resulting from this reliable detection cutoff, for 1M triplicates, the top 1427, 862 and 791 CDR3s and for 9M triplicates, the top 9414, 9485 and 9460 CDR3s were found to be reliably detected (Figure 3, Additional file 6). Accordingly, the numbers of reliably detected VDJs for 1M were 10741, 10731 and 11055 and for 9M 18765, 18928 and 18466 (Figure 3, Additional files 3 and 6). The reliably detected CDR3/VDJ clones for each replicate were used in all analyses shown hereafter. Thus, replicate sequencing provided a powerful method to establish reliability of clonal detection.Figure 3


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)

Establishing a cut-off for reliable clonal detection. For each replicate (A: CDR3, B: VDJ), starting with the highest frequency clones, clones were tested for their simultaneous presence in the respective two other replicates. For each clone, presence or absence in the other two replicates were recorded. We regarded all those clones as “reliably detected” that belonged to the highest frequency set of clones with a ratio of presence to absence ≥99%. The percentage of clones passing the detection threshold is indicated by vertical red bars. Absolute numbers of reliably detected clones for CDR3-1M were (1427, 862, 791), and for CDR3-9M were (9414, 9485, 9460). For VDJ, 1M and 9M absolute numbers were (10741, 10731, 11055) and (18765, 18928, 18466), respectively. For a graphical overview of the method of reliable detection, please refer to Menzel et al. [53].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Establishing a cut-off for reliable clonal detection. For each replicate (A: CDR3, B: VDJ), starting with the highest frequency clones, clones were tested for their simultaneous presence in the respective two other replicates. For each clone, presence or absence in the other two replicates were recorded. We regarded all those clones as “reliably detected” that belonged to the highest frequency set of clones with a ratio of presence to absence ≥99%. The percentage of clones passing the detection threshold is indicated by vertical red bars. Absolute numbers of reliably detected clones for CDR3-1M were (1427, 862, 791), and for CDR3-9M were (9414, 9485, 9460). For VDJ, 1M and 9M absolute numbers were (10741, 10731, 11055) and (18765, 18928, 18466), respectively. For a graphical overview of the method of reliable detection, please refer to Menzel et al. [53].
Mentions: Leveraging the deep repertoire coverage, we proceeded by establishing a cutoff, which ensures that 99% of clones in a given replicate would be found in the two other replicates (Figure 3). Resulting from this reliable detection cutoff, for 1M triplicates, the top 1427, 862 and 791 CDR3s and for 9M triplicates, the top 9414, 9485 and 9460 CDR3s were found to be reliably detected (Figure 3, Additional file 6). Accordingly, the numbers of reliably detected VDJs for 1M were 10741, 10731 and 11055 and for 9M 18765, 18928 and 18466 (Figure 3, Additional files 3 and 6). The reliably detected CDR3/VDJ clones for each replicate were used in all analyses shown hereafter. Thus, replicate sequencing provided a powerful method to establish reliability of clonal detection.Figure 3

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