Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Method Study

ABSTRACT: High-throughput sequencing (HTS) of antibody amplicon libraries has become a powerful method in the emerging field of systems immunology. However, numerous sources of bias in HTS workflows may affect antibody repertoire data. A crucial step in antibody amplicon library preparation is the addition of adapter sequences, which are platform-specific short nucleotide sequences. In addition to conventional ligation-based adapter addition, it is also possible to use PCR-based methods such as Direct Addition (one-step adapter addition, DA) and Primer Extension (two-step adapter addition, PE), which have led to the lack of a consensus method and uncertainty regarding the impact of adapter addition on repertoire HTS data. Therefore, we compared all three methods by performing HTS with the Illumina MiSeq platform using total RNA originating from mouse antibody-secreting cells. We used technical replicate-based validation and clonal overlap and rank statistics to demonstrate that the two PCR-based methods produced HTS repertoires equivalent to ligation. Specifically: Nine BALB/c mice (8-10 weeks old, Charles River, specific-pathogen free) were immunized with 50 ug alum-precipitated chicken gamma globulin (CGG) conjugated to 4-hydroxy-3-nitrophenylacetyl (NP, NP-CGG, BioCat). Mice were sacrificed 14 days post-immunization (dpi) and their spleens and bone marrow (from femurs and tibia) were harvested. Antibody-secreting CD138-positive cells were enriched from spleen and bone marrow 14 dpi as previously described (Reddy, 2010, Nat Biotech).

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Mus musculus

SUBMITTER: Victor Greiff

PROVIDER: E-MTAB-2226 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody sequencing.

Menzel Ulrike U Greiff Victor V Khan Tarik A TA Haessler Ulrike U Hellmann Ina I Friedensohn Simon S Cook Skylar C SC Pogson Mark M Reddy Sai T ST

PloS one 20140508 5

High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not ...[more]

PMID: 24809667

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Project description: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. Therefore, two conditions of immunological relevance were assessed for NGS reproducibility. First, we pooled spleen plasmablasts and plasma cells (CDR138-enriched) and bone marrow plasma cells (CD45R-depleted and CD138-enriched 4) of 1 mouse (1M) immunized with NP-CGG (chicken gamma globulin (CGG) conjugated to 4-hydroxy-3-nitrophenylacetyl) and sacrificed 14 days post-immunization (dpi). The thus created cell pool contained approximately 3 x 106 viable ASCs. Second, in order to model extreme antibody diversity, we repeated the same cell isolation procedure from 9 immunized mice (9M) resulting in an ASC pool of approximately 2.5 x 10^7 viable cells. From isolated cells, we recovered total RNA and used RT-PCR to amplify expressed rearranged IgG variable heavy VDJ genes; PCR was performed using a well-characterized and utilized primer set based on variable framework 1 region forward primers and one IgG constant region 1 reverse primer (covering all IgG isotypes) 43. Similarly to previously published methods (Vollmers et al., 2013, PNAS), Illumina adaptors were added during the PCR step by using a direct addition approach where adaptors are added at the 5’ end of the gene-specific portion of the primer set, thus circumventing the need for ligation of adaptors following PCR. For each of the two conditions (1M/9M), triplicates were prepared, where a triplicate signifies three separately indexed samples prepared from the same starting cDNA pool; thus variable region PCR was independently performed in each of the triplicates. All six samples (triplicates of 1M and 9M) were sequenced using the Illumina MiSeq platform with 250bp paired-end reads.

Dataset Information

Method Study

Publications

Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody sequencing.

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