High-throughput sequencing of the IGH repertoire in human peripheral blood and bone marrow
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE181689 | GEO | 2021/10/27
REPOSITORIES: GEO
Similar Datasets
Project description:Purpose: IGH sequencing has revolutionized analysis of structural and functional signatures of B cell clonality. The goals of this study are to compare IGH repertoire of splenic B cells before and after knockdown of long isoform of PRLR. Methods: After amplifying the IGH repertoire of splenic B cells using PCR primers for VHJ558 variable and Cμ constant regions. For next generation sequencing (NGS), a TrueSeq library was prepared by adapter ligation to full-length products and sequenced on the MiSeq Illumina 2x150 bp platform. Results: In this project, Unique nucleotide sequences were identified, their abundances were calculated, and their complementary determining region 3 (CDR3) length, CDR3 spectrum, VDJ usage, and diversity were analyzed using the International Immunogenetics Information System (IMGT) HighV-QUEST software. Controls are included C3,C4,C5,C7,C8 and C9. LFPRLR SMO treated mice samples are: A3,A4,A5,A6,A7 and A8
2023-03-06 | GSE207186 | GEO
Project description:To study longitudinal dynamics of IGH BCR repertoires and clonal lineages evolution of memory B-cells, plasmablasts and plasma cells from peripheral blood of healthy donors, which were sampled three times within a year
2022-09-15 | E-MTAB-11193 | biostudies-arrayexpress
Project description:Developing B lymphocytes undergo V(D)J recombination to assemble germline V, D, and J gene segments into exons that encode the antigen-binding variable region of immunoglobulin (Ig) heavy (H) and light (L) chains. IgH and IgL chains associate to form the B cell receptor (BCR), which upon antigen binding activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. Ability of V(D)J recombination to generate vast primary B cell repertoires results from combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as HTGTS repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or non-productive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions and also for following antibody affinity maturation processes. We employed high-throughput genome-wide translocation sequencing adapted repertoire sequencing (HTGTS-Rep-seq) to study antibody repertoires. For HTGTS-Rep-seq libraries, we utilize bait coding ends of J segments to identify, in unbiased fashion, mouse IgH DJH repertoires [processed tlx files] along with both productive and non-productive IgH V(D)J repertoires from both pro-B and peripheral B cells [processed xls files of samples 1-18, 21-51]. Similarly, we also identify mouse productive and non-productive Igk repertoires from peripheral B cells [processed xls files of samples 19,20,52-57].
2016-07-22 | E-GEOD-82126 | biostudies-arrayexpress
Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5M-bM-^@M-^Y-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus. For the ChIP-seq, input and immunoprecipitated DNA was given to The Scripps DNA Array Facility, where it was prepared for massively parallel sequencing on Illumina HiSeq2000.
2013-08-05 | E-GEOD-47766 | biostudies-arrayexpress
Project description:A diverse antibody repertoire is formed through the rearrangement of V, D, and J segments at the immunoglobulin heavy chain (Igh) loci. The C57BL/6 murine Igh locus has over 100 functional VH gene segments that can recombine to a rearranged DJH. While the non-random usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question we conducted deep sequencing of 5M-bM-^@M-^Y-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. ChIP-seq results for several histone modifications and RNA polymerase II binding, RNA-seq for sense and antisense non-coding germline transcripts, and proximity to CTCF and Rad21 sites were compared to the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and non-coding RNA in general, but having a CTCF site near their RSS is critical, suggesting that position near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have high levels of histone marks and non-coding RNA, which may compensate for their poorer RSS and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for of each the four domains that comprise this locus. RNA was extracted from C57BL/6 RAG-/- pro-B cells using TrizolM-BM-. (Life Technologies Corp., Carlsbad CA) and genomic DNA was eliminated using the genomic DNA wipeout buffer in the QuantiTect Reverse transcription kit (QIAGEN). A final purification of the RNA was performed with the RNeasy kit from QIAGEN. For each sample, 100 ng of total RNA was used to make RNASeq libraries using the NuGEN Encore Complete DR kits following manufacturer's recommended protocols. Sequencing libraries were gel purified to ensure insert sizes were larger than 100 bp in length and sequenced on an Ilumina HiSeq2000 for 100 bases plus 7 bases for indexing.
2013-08-05 | E-GEOD-47885 | biostudies-arrayexpress
Project description:Over 80% of patients with diffuse large B-cell lymphoma (DLBCL) treated with R-CHOP achieve complete remission with variable 5-year overall survival (OS) rates of 60 to 70% depending on the preexisting risk factors. We herein attempted to stratify DLBCL by the patterns of immunoglobulin heavy chain gene rearrangement (IgH-R) by using a PCR-based clonality detection method to predict prognosis of the patients with DLBCL. We retrospectively analyzed 81 patients with DLBCL who were treated at our hospital from 2006 to 2013 and had data on IgH-R of lymphoma cells at diagnosis. IgH-R analyses identified a uniform pattern of IgH-R harboring all frame regions (FR)1-3 not completely involving DH1-7 in 15 out of 26 patients, who failed to obtain complete remission (CR). Moreover, CR rate was quite low ((7/22 (32%)) in these patients with this IgH-R pattern. We evaluated prognostic parameters of these patients with special reference to patterns of IgH-R by univariate and multivariate proportional-hazard model. Multivariate analysis revealed that IgH-R in all FR1-3 without entire DH1-7 was significantly associated with adverse overall survival. Then, gene expression profiling of mRNA elicited the candidate genes associated with poorer prognosis stratified by IgH-R patterns. Among them, CDC25A mRNA was significantly amplified by real-time PCR and then further analysis of its protein with immunostaining confirmed that CDC25A could be a responsible gene for prognosis of patients with DLBCL. These results may shed light on individualized therapeutic modalities by the stratification of patients in accordance with patterns of IgH-R.
2018-02-24 | GSE111069 | GEO
Project description:The immunoglobulin heavy-chain (Igh) locus undergoes large-scale contraction in pro-B cells, which facilitates VH-DJH recombination by juxtaposing distal VH genes next to the DJH- rearranged gene segment in the proximal Igh domain. By high-resolution mapping of long-range interactions, we now demonstrate that an array of local interaction domains establishes the three- dimensional structure of the extended Igh locus in lymphoid progenitors and thymocytes. In pro- B cells, these local domains engage in long-range interactions across the entire Igh locus, which depend on the transcription factors Pax5, YY1 and CTCF. The large VH gene cluster thereby undergoes flexible long-range interactions with the more rigidly structured 3M-bM-^@M-^Y proximal domain, which ensures that all VH genes can participate with similar probability in VH-DJH recombination to generate a diverse antibody repertoire. Notably, these long-range interactions appear to be an intrinsic feature of the VH gene cluster, as they are still generated upon mutation of the EM-NM-< enhancer, IGCR1 insulator or 3M-bM-^@M-^Y regulatory region present in the 3M-bM-^@M-^Y proximal Igh domain. 4C sequencing from mutliple celltypes with multiple viewpoints; uneven number of replicates ChIP-Seq
2013-09-30 | E-GEOD-43008 | biostudies-arrayexpress
Project description:High-throughput sequencing of the IGH repertoire in human peripheral blood and bone marrow
| PRJNA753048 | ENA
Project description:We report the application of 4C-seq for analysis of the 3’ end of he Igh locus during B cell development. We generated genome-wide 4C maps for BM derived Rag2-/- pro-B cells, resting splenic B cells, LPS+IL4 activated B cells and ConA stimulated splenic T cells using two viewpoints, Em and hs4 located in 3’Ea. We find that long range interactions are present only on B lineage cells reflecting enhancer activation status.
2021-11-23 | GSE183006 | GEO