All Publications

  • VDJServer: A Cloud-Based Analysis Portal and Data Commons for Immune Repertoire Sequences and Rearrangements FRONTIERS IN IMMUNOLOGY Christley, S., Scarborough, W., Salinas, E., Rounds, W. H., Toby, I. T., Fonner, J. M., Levin, M. K., Kim, M., Mock, S. A., Jordan, C., Ostmeyer, J., Buntzman, A., Rubelt, F., Davila, M. L., Monson, N. L., Scheuermann, R. H., Cowell, L. G. 2018; 9: 976


    Recent technological advances in immune repertoire sequencing have created tremendous potential for advancing our understanding of adaptive immune response dynamics in various states of health and disease. Immune repertoire sequencing produces large, highly complex data sets, however, which require specialized methods and software tools for their effective analysis and interpretation.VDJServer is a cloud-based analysis portal for immune repertoire sequence data that provide access to a suite of tools for a complete analysis workflow, including modules for preprocessing and quality control of sequence reads, V(D)J gene segment assignment, repertoire characterization, and repertoire comparison. VDJServer also provides sophisticated visualizations for exploratory analysis. It is accessible through a standard web browser via a graphical user interface designed for use by immunologists, clinicians, and bioinformatics researchers. VDJServer provides a data commons for public sharing of repertoire sequencing data, as well as private sharing of data between users. We describe the main functionality and architecture of VDJServer and demonstrate its capabilities with use cases from cancer immunology and autoimmunity.VDJServer provides a complete analysis suite for human and mouse T-cell and B-cell receptor repertoire sequencing data. The combination of its user-friendly interface and high-performance computing allows large immune repertoire sequencing projects to be analyzed with no programming or software installation required. VDJServer is a web-accessible cloud platform that provides access through a graphical user interface to a data management infrastructure, a collection of analysis tools covering all steps in an analysis, and an infrastructure for sharing data along with workflows, results, and computational provenance. VDJServer is a free, publicly available, and open-source licensed resource.

    View details for DOI 10.3389/fimmu.2018.00976

    View details for Web of Science ID 000431682200001

    View details for PubMedID 29867956

    View details for PubMedCentralID PMC5953328

  • A Macrophage Colony-Stimulating-Factor-Producing γδ T Cell Subset Prevents Malarial Parasitemic Recurrence. Immunity Mamedov, M. R., Scholzen, A., Nair, R. V., Cumnock, K., Kenkel, J. A., Oliveira, J. H., Trujillo, D. L., Saligrama, N., Zhang, Y., Rubelt, F., Schneider, D. S., Chien, Y. H., Sauerwein, R. W., Davis, M. M. 2018; 48 (2): 350–63.e7


    Despite evidence that γδ T cells play an important role during malaria, their precise role remains unclear. During murine malaria induced by Plasmodium chabaudi infection and in human P. falciparum infection, we found that γδ T cells expanded rapidly after resolution of acute parasitemia, in contrast to αβ T cells that expanded at the acute stage and then declined. Single-cell sequencing showed that TRAV15N-1 (Vδ6.3) γδ T cells were clonally expanded in mice and had convergent complementarity-determining region 3 sequences. These γδ T cells expressed specific cytokines, M-CSF, CCL5, CCL3, which are known to act on myeloid cells, indicating that this γδ T cell subset might have distinct functions. Both γδ T cells and M-CSF were necessary for preventing parasitemic recurrence. These findings point to an M-CSF-producing γδ T cell subset that fulfills a specialized protective role in the later stage of malaria infection when αβ T cells have declined.

    View details for DOI 10.1016/j.immuni.2018.01.009

    View details for PubMedID 29426701

  • The Repertoire Dissimilarity Index as a method to compare lymphocyte receptor repertoires. BMC bioinformatics Bolen, C. R., Rubelt, F., Vander Heiden, J. A., Davis, M. M. 2017; 18 (1): 155-?


    The B and T cells of the human adaptive immune system leverage a highly diverse repertoire of antigen-specific receptors to protect the human body from pathogens. The sequencing and analysis of immune repertoires is emerging as an important tool to understand immune responses, whether beneficial or harmful (in the case of autoimmunity). However, methods for studying these repertoires, and for directly comparing different immune repertoires, are lacking.In this paper, we present a non-parametric method for directly comparing sequencing repertoires, with the goal of rigorously quantifying differences in V, D, and J gene segment utilization. This method, referred to as the Repertoire Dissimilarity Index (RDI), uses a bootstrapped subsampling approach to account for variance in sequencing depth, and, coupled with a data simulation approach, allows for direct quantification of the average variation between repertoires. We use the RDI method to recapitulate known differences in the formation of the CD4(+) and CD8(+) T cell repertoires, and further show that antigen-driven activation of naïve CD8(+) T cells is more selective than in the CD4(+) repertoire, resulting in a more specialized CD8(+) memory repertoire.We prove that the RDI method is an accurate and versatile method for comparisons of immune repertoires. The RDI method has been implemented as an R package, and is available for download through Bitbucket.

    View details for DOI 10.1186/s12859-017-1556-5

    View details for PubMedID 28264647

    View details for PubMedCentralID PMC5340033

  • VDJPipe: a pipelined tool for pre-processing immune repertoire sequencing data. BMC bioinformatics Christley, S., Levin, M. K., Toby, I. T., Fonner, J. M., Monson, N. L., Rounds, W. H., Rubelt, F., Scarborough, W., Scheuermann, R. H., Cowell, L. G. 2017; 18 (1): 448


    Pre-processing of high-throughput sequencing data for immune repertoire profiling is essential to insure high quality input for downstream analysis. VDJPipe is a flexible, high-performance tool that can perform multiple pre-processing tasks with just a single pass over the data files.Processing tasks provided by VDJPipe include base composition statistics calculation, read quality statistics calculation, quality filtering, homopolymer filtering, length and nucleotide filtering, paired-read merging, barcode demultiplexing, 5' and 3' PCR primer matching, and duplicate reads collapsing. VDJPipe utilizes a pipeline approach whereby multiple processing steps are performed in a sequential workflow, with the output of each step passed as input to the next step automatically. The workflow is flexible enough to handle the complex barcoding schemes used in many immunosequencing experiments. Because VDJPipe is designed for computational efficiency, we evaluated this by comparing execution times with those of pRESTO, a widely-used pre-processing tool for immune repertoire sequencing data. We found that VDJPipe requires <10% of the run time required by pRESTO.VDJPipe is a high-performance tool that is optimized for pre-processing large immune repertoire sequencing data sets.

    View details for DOI 10.1186/s12859-017-1853-z

    View details for PubMedID 29020925

    View details for PubMedCentralID PMC5637252

  • Adaptive Immune Receptor Repertoire Community recommendations for sharing immune-repertoire sequencing data. Nature immunology Rubelt, F., Busse, C. E., Bukhari, S. A., Bürckert, J. P., Mariotti-Ferrandiz, E., Cowell, L. G., Watson, C. T., Marthandan, N., Faison, W. J., Hershberg, U., Laserson, U., Corrie, B. D., Davis, M. M., Peters, B., Lefranc, M. P., Scott, J. K., Breden, F., Luning Prak, E. T., Kleinstein, S. H. 2017; 18 (12): 1274–78

    View details for DOI 10.1038/ni.3873

    View details for PubMedID 29144493

  • Reproducibility and Reuse of Adaptive Immune Receptor Repertoire Data. Frontiers in immunology Breden, F., Luning Prak, E. T., Peters, B., Rubelt, F., Schramm, C. A., Busse, C. E., Vander Heiden, J. A., Christley, S., Bukhari, S. A., Thorogood, A., Matsen Iv, F. A., Wine, Y., Laserson, U., Klatzmann, D., Douek, D. C., Lefranc, M. P., Collins, A. M., Bubela, T., Kleinstein, S. H., Watson, C. T., Cowell, L. G., Scott, J. K., Kepler, T. B. 2017; 8: 1418


    High-throughput sequencing (HTS) of immunoglobulin (B-cell receptor, antibody) and T-cell receptor repertoires has increased dramatically since the technique was introduced in 2009 (1-3). This experimental approach explores the maturation of the adaptive immune system and its response to antigens, pathogens, and disease conditions in exquisite detail. It holds significant promise for diagnostic and therapy-guiding applications. New technology often spreads rapidly, sometimes more rapidly than the understanding of how to make the products of that technology reliable, reproducible, or usable by others. As complex technologies have developed, scientific communities have come together to adopt common standards, protocols, and policies for generating and sharing data sets, such as the MIAME protocols developed for microarray experiments. The Adaptive Immune Receptor Repertoire (AIRR) Community formed in 2015 to address similar issues for HTS data of immune repertoires. The purpose of this perspective is to provide an overview of the AIRR Community's founding principles and present the progress that the AIRR Community has made in developing standards of practice and data sharing protocols. Finally, and most important, we invite all interested parties to join this effort to facilitate sharing and use of these powerful data sets (

    View details for DOI 10.3389/fimmu.2017.01418

    View details for PubMedID 29163494

    View details for PubMedCentralID PMC5671925

  • Identifying specificity groups in the T cell receptor repertoire. Nature Glanville, J., Huang, H., Nau, A., Hatton, O., Wagar, L. E., Rubelt, F., Ji, X., Han, A., Krams, S. M., Pettus, C., Haas, N., Arlehamn, C. S., Sette, A., Boyd, S. D., Scriba, T. J., Martinez, O. M., Davis, M. M. 2017


    T cell receptor (TCR) sequences are very diverse, with many more possible sequence combinations than T cells in any one individual. Here we define the minimal requirements for TCR antigen specificity, through an analysis of TCR sequences using a panel of peptide and major histocompatibility complex (pMHC)-tetramer-sorted cells and structural data. From this analysis we developed an algorithm that we term GLIPH (grouping of lymphocyte interactions by paratope hotspots) to cluster TCRs with a high probability of sharing specificity owing to both conserved motifs and global similarity of complementarity-determining region 3 (CDR3) sequences. We show that GLIPH can reliably group TCRs of common specificity from different donors, and that conserved CDR3 motifs help to define the TCR clusters that are often contact points with the antigenic peptides. As an independent validation, we analysed 5,711 TCRβ chain sequences from reactive CD4 T cells from 22 individuals with latent Mycobacterium tuberculosis infection. We found 141 TCR specificity groups, including 16 distinct groups containing TCRs from multiple individuals. These TCR groups typically shared HLA alleles, allowing prediction of the likely HLA restriction, and a large number of M. tuberculosis T cell epitopes enabled us to identify pMHC ligands for all five of the groups tested. Mutagenesis and de novo TCR design confirmed that the GLIPH-identified motifs were critical and sufficient for shared-antigen recognition. Thus the GLIPH algorithm can analyse large numbers of TCR sequences and define TCR specificity groups shared by TCRs and individuals, which should greatly accelerate the analysis of T cell responses and expedite the identification of specific ligands.

    View details for DOI 10.1038/nature22976

    View details for PubMedID 28636589

  • VDJML: a file format with tools for capturing the results of inferring immune receptor rearrangements. BMC bioinformatics Toby, I. T., Levin, M. K., Salinas, E. A., Christley, S., Bhattacharya, S., Breden, F., Buntzman, A., Corrie, B., Fonner, J., Gupta, N. T., Hershberg, U., Marthandan, N., Rosenfeld, A., Rounds, W., Rubelt, F., Scarborough, W., Scott, J. K., Uduman, M., Vander Heiden, J. A., Scheuermann, R. H., Monson, N., Kleinstein, S. H., Cowell, L. G. 2016; 17: 333-?


    The genes that produce antibodies and the immune receptors expressed on lymphocytes are not germline encoded; rather, they are somatically generated in each developing lymphocyte by a process called V(D)J recombination, which assembles specific, independent gene segments into mature composite genes. The full set of composite genes in an individual at a single point in time is referred to as the immune repertoire. V(D)J recombination is the distinguishing feature of adaptive immunity and enables effective immune responses against an essentially infinite array of antigens. Characterization of immune repertoires is critical in both basic research and clinical contexts. Recent technological advances in repertoire profiling via high-throughput sequencing have resulted in an explosion of research activity in the field. This has been accompanied by a proliferation of software tools for analysis of repertoire sequencing data. Despite the widespread use of immune repertoire profiling and analysis software, there is currently no standardized format for output files from V(D)J analysis. Researchers utilize software such as IgBLAST and IMGT/High V-QUEST to perform V(D)J analysis and infer the structure of germline rearrangements. However, each of these software tools produces results in a different file format, and can annotate the same result using different labels. These differences make it challenging for users to perform additional downstream analyses.To help address this problem, we propose a standardized file format for representing V(D)J analysis results. The proposed format, VDJML, provides a common standardized format for different V(D)J analysis applications to facilitate downstream processing of the results in an application-agnostic manner. The VDJML file format specification is accompanied by a support library, written in C++ and Python, for reading and writing the VDJML file format.The VDJML suite will allow users to streamline their V(D)J analysis and facilitate the sharing of scientific knowledge within the community. The VDJML suite and documentation are available from . We welcome participation from the community in developing the file format standard, as well as code contributions.

    View details for PubMedID 27766961

  • Individual heritable differences result in unique cell lymphocyte receptor repertoires of naive and antigen-experienced cells NATURE COMMUNICATIONS Rubelt, F., Bolen, C. R., McGuire, H. M., Vander Heiden, J. A., Gadala-Maria, D., Levin, M., Euskirchen, G. M., Mamedov, M. R., Swan, G. E., Dekker, C. L., Cowell, L. G., Kleinstein, S. H., Davis, M. M. 2016; 7
  • Onset of Immune Senescence Defined by Unbiased Pyrosequencing of Human Immunoglobulin mRNA Repertoires PLOS ONE Rubelt, F., Sievert, V., Knaust, F., Diener, C., Lim, T. S., Skriner, K., Klipp, E., Reinhardt, R., Lehrach, H., Konthur, Z. 2012; 7 (11)


    The immune system protects us from foreign substances or pathogens by generating specific antibodies. The variety of immunoglobulin (Ig) paratopes for antigen recognition is a result of the V(D)J rearrangement mechanism, while a fast and efficient immune response is mediated by specific immunoglobulin isotypes obtained through class switch recombination (CSR). To get a better understanding on how antibody-based immune protection works and how it changes with age, the interdependency between these two parameters need to be addressed. Here, we have performed an in depth analysis of antibody repertoires of 14 healthy donors representing different gender and age groups. For this task, we developed a unique pyrosequencing approach, which is able to monitor the expression levels of all immunoglobulin V(D)J recombinations of all isotypes including subtypes in an unbiased and quantitative manner. Our results show that donors have individual immunoglobulin repertoires and cannot be clustered according to V(D)J recombination patterns, neither by age nor gender. However, after incorporating isotype-specific analysis and considering CSR information into hierarchical clustering the situation changes. For the first time the donors cluster according to age and separate into young adults and elderly donors (>50). As a direct consequence, this clustering defines the onset of immune senescence at the age of fifty and beyond. The observed age-dependent reduction of CSR ability proposes a feasible explanation why reduced efficacy of vaccination is seen in the elderly and implies that novel vaccine strategies for the elderly should include the "Golden Agers".

    View details for DOI 10.1371/journal.pone.0049774

    View details for Web of Science ID 000312376100028

    View details for PubMedID 23226220

  • A streamlined protocol for emulsion polymerase chain reaction and subsequent purification ANALYTICAL BIOCHEMISTRY Schuetze, T., Rubelt, F., Repkow, J., Greiner, N., Erdmann, V. A., Lehrach, H., Konthur, Z., Gloekler, J. 2011; 410 (1): 155-157


    Compartmentalization of polymerase chain reaction (PCR) reduces artifacts, especially when complex libraries are amplified. It allows clonal amplification of templates from complex mixtures in a bias-free manner. Here we describe a rapid, straightforward, and easy protocol for PCR in a water-in-oil emulsion (ePCR) including sample recovery by DNA purification. Furthermore, no special laboratory equipment is needed and inexpensive components are used. Therefore, our flexible protocol allows ePCR to be readily implemented in daily routine experiments for a broad range of applications.

    View details for DOI 10.1016/j.ab.2010.11.029

    View details for Web of Science ID 000286711300023

    View details for PubMedID 21111698

  • V-gene amplification revisited - An optimised procedure for amplification of rearranged human antibody genes of different isotypes NEW BIOTECHNOLOGY Lim, T. S., Mollova, S., Rubelt, F., Sievert, V., Duebel, S., Lehrach, H., Konthur, Z. 2010; 27 (2): 108-117


    For studying human antibody variable (V)-gene usage in any group of individuals or for the generation of recombinant human antibody libraries for phage display, quality and yield of the amplified V-gene repertoire is of utmost importance. Key parameters affecting the amplification of full antibody repertoires are V-gene specific primer design, complementary DNA (cDNA) synthesis from total RNA extracts of peripheral blood mononuclear cells (PBMCs) and ultimately the polymerase chain reaction (PCR). In this work we analysed all these factors; we performed a detailed bioinformatic analysis of V-gene specific primers based on VBASE2 and evaluated the influence of different commercially available reverse transcriptases on cDNA synthesis and polymerases on PCR efficiency. The primers presented cover near to 100% of all functional and putatively functional V-genes in VBASE2 and the final protocol presents an optimised combination of commercial enzymes and reaction additives for cDNA synthesis and PCR conditions for V-gene amplification. Finally, applying this protocol in combination with different immunoglobulin (Ig) chain specific reverse primers we were able to amplify rearranged antibody genes of different isotypes under investigation.

    View details for DOI 10.1016/j.nbt.2010.01.001

    View details for Web of Science ID 000279133600006

    View details for PubMedID 20083243