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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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Motivation and experimental setup. (A) The problem of undersampling in NGS antibody repertoire sequencing is most easily explained by the marble analogy. Assuming an urn is filled with k numbers of marbles of different species in varying frequencies–urn and marbles represent the original antibody mixture. The problem is clear: if only a sample of size n (n < k) is drawn, then three qualitatively different sampling outcomes can arise: (1) If n is too small, species richness (number of different colors in the urn) is not accurately determined and consequently neither are species frequencies. (2) In the case that n is larger, species richness is accurately represented but species frequencies can be off. (3) Only if n is large enough, both species richness and frequency are accurately reflected in the sample. This study set out to answer, which outcome best describes antibody repertoire NGS data from ASCs of immunized mice. (B) To address undersampling concerns, we explored two different scenarios of ASC diversity: 10 female BALB/c mice were immunized with NP-CGG and sacrificed 14 days post-injection. Subsequently, bone marrow plasma cells were isolated as described previously, as were CD138-positive splenocytes [4]. ASCs of 1 mouse (1M) were pooled as were those of 9 mice (9M). RNA was isolated, followed by RT-PCR and Illumina MiSeq sequencing of triplicates (see Methods).
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Fig1: Motivation and experimental setup. (A) The problem of undersampling in NGS antibody repertoire sequencing is most easily explained by the marble analogy. Assuming an urn is filled with k numbers of marbles of different species in varying frequencies–urn and marbles represent the original antibody mixture. The problem is clear: if only a sample of size n (n < k) is drawn, then three qualitatively different sampling outcomes can arise: (1) If n is too small, species richness (number of different colors in the urn) is not accurately determined and consequently neither are species frequencies. (2) In the case that n is larger, species richness is accurately represented but species frequencies can be off. (3) Only if n is large enough, both species richness and frequency are accurately reflected in the sample. This study set out to answer, which outcome best describes antibody repertoire NGS data from ASCs of immunized mice. (B) To address undersampling concerns, we explored two different scenarios of ASC diversity: 10 female BALB/c mice were immunized with NP-CGG and sacrificed 14 days post-injection. Subsequently, bone marrow plasma cells were isolated as described previously, as were CD138-positive splenocytes [4]. ASCs of 1 mouse (1M) were pooled as were those of 9 mice (9M). RNA was isolated, followed by RT-PCR and Illumina MiSeq sequencing of triplicates (see Methods).

Mentions: Reproducible sequencing of antibody repertoires is of paramount importance for the development of diagnostic approaches [40,41]. In light of recent findings that antibody clonal abundance is correlated to antigen specificity [4,42,43], reliable capture of ranking information is necessary for monoclonal antibody discovery and profiling of antibody responses to vaccines and infections. The concerns of undersampling the antibody repertoire naturally lead to the following questions [17,40]: (i) To what extent are antibody clones within one sample being detected by NGS? (ii) What percentage of detected clones can be reliably and reproducibly identified? (iii) To what extent do reliably detected clones bear accurate frequency information (Figure 1A)?Figure 1


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)

Motivation and experimental setup. (A) The problem of undersampling in NGS antibody repertoire sequencing is most easily explained by the marble analogy. Assuming an urn is filled with k numbers of marbles of different species in varying frequencies–urn and marbles represent the original antibody mixture. The problem is clear: if only a sample of size n (n < k) is drawn, then three qualitatively different sampling outcomes can arise: (1) If n is too small, species richness (number of different colors in the urn) is not accurately determined and consequently neither are species frequencies. (2) In the case that n is larger, species richness is accurately represented but species frequencies can be off. (3) Only if n is large enough, both species richness and frequency are accurately reflected in the sample. This study set out to answer, which outcome best describes antibody repertoire NGS data from ASCs of immunized mice. (B) To address undersampling concerns, we explored two different scenarios of ASC diversity: 10 female BALB/c mice were immunized with NP-CGG and sacrificed 14 days post-injection. Subsequently, bone marrow plasma cells were isolated as described previously, as were CD138-positive splenocytes [4]. ASCs of 1 mouse (1M) were pooled as were those of 9 mice (9M). RNA was isolated, followed by RT-PCR and Illumina MiSeq sequencing of triplicates (see Methods).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Motivation and experimental setup. (A) The problem of undersampling in NGS antibody repertoire sequencing is most easily explained by the marble analogy. Assuming an urn is filled with k numbers of marbles of different species in varying frequencies–urn and marbles represent the original antibody mixture. The problem is clear: if only a sample of size n (n < k) is drawn, then three qualitatively different sampling outcomes can arise: (1) If n is too small, species richness (number of different colors in the urn) is not accurately determined and consequently neither are species frequencies. (2) In the case that n is larger, species richness is accurately represented but species frequencies can be off. (3) Only if n is large enough, both species richness and frequency are accurately reflected in the sample. This study set out to answer, which outcome best describes antibody repertoire NGS data from ASCs of immunized mice. (B) To address undersampling concerns, we explored two different scenarios of ASC diversity: 10 female BALB/c mice were immunized with NP-CGG and sacrificed 14 days post-injection. Subsequently, bone marrow plasma cells were isolated as described previously, as were CD138-positive splenocytes [4]. ASCs of 1 mouse (1M) were pooled as were those of 9 mice (9M). RNA was isolated, followed by RT-PCR and Illumina MiSeq sequencing of triplicates (see Methods).
Mentions: Reproducible sequencing of antibody repertoires is of paramount importance for the development of diagnostic approaches [40,41]. In light of recent findings that antibody clonal abundance is correlated to antigen specificity [4,42,43], reliable capture of ranking information is necessary for monoclonal antibody discovery and profiling of antibody responses to vaccines and infections. The concerns of undersampling the antibody repertoire naturally lead to the following questions [17,40]: (i) To what extent are antibody clones within one sample being detected by NGS? (ii) What percentage of detected clones can be reliably and reproducibly identified? (iii) To what extent do reliably detected clones bear accurate frequency information (Figure 1A)?Figure 1

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