scholarly journals EZH2 and CD79B mutational status over time in B‐cell non‐Hodgkin lymphomas detected by high‐throughput sequencing using minimal samples

2013 ◽  
Vol 121 (7) ◽  
pp. 377-386 ◽  
Author(s):  
Mauro Ajaj Saieg ◽  
William R. Geddie ◽  
Scott L. Boerner ◽  
Denis Bailey ◽  
Michael Crump ◽  
...  
Keyword(s):  
B Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Mutational Status ◽  
Over Time

scholarly journals A field-wide assessment of differential high throughput sequencing reveals widespread bias

2021 ◽  
Author(s):  
Taavi Päll ◽  
Hannes Luidalepp ◽  
Tanel Tenson ◽  
Ülo Maiväli
Keyword(s):  
High Throughput ◽  
Marked Improvement ◽  
High Throughput Sequencing ◽  
Experimental Treatment ◽  
Data Repository ◽  
P Value ◽  
P Values ◽  
Upper Limit ◽  
File Structures ◽  
Over Time

AbstractHere we assess reproducibility and inferential quality in the field of differential HT-seq, based on analysis of datasets submitted 2008-2019 to the NCBI GEO data repository. Analysis of GEO submission file structures places an overall 59% upper limit to reproducibility. We further show that only 23% of experiments resulted in theoretically expected p value histogram shapes, although both reproducibility and p value distributions show marked improvement over time. Uniform p value histogram shapes, indicative of <100 true effects, were extremely few. Our calculations of π0, the fraction of true nulls, showed that 36% of experiments have π0 <0.5, meaning that in over a third of experiments most RNA-s were estimated to change their expression level upon experimental treatment. Both the fraction of different p value histogram types and π0 values are strongly associated with the software used for calculating these p values by the original authors, indicating widespread bias.

scholarly journals An analysis of abnormalities in the B cell receptor repertoire in patients with systemic sclerosis using high-throughput sequencing

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8370
Author(s):  
Xiaodong Shi ◽  
Tihong Shao ◽  
Feifei Huo ◽  
Chenqing Zheng ◽  
Wanyu Li ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
B Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cell Receptor ◽  
Receptor Repertoire ◽  
Skin Sclerosis ◽  
Cdr3 Length ◽  
Gene Usage ◽  
Cell Receptor Repertoire

Systemic sclerosis is a chronic multisystem autoimmune disease that is associated with polyclonal B cell hyperreactivity. The CDR3 of BCRs is the major site of antigen recognition. Therefore, we analyzed the BCR repertoire of patients with SSc. The BCR repertoires in 12 subjects including eight SSc patients and four healthy controls were characterized by high-throughput sequencing, and bioinformatics analysis were studied. The average CDR3 length in the SSc group was significantly shorter. The SSc patient displayed more diverse BCR. Moreover, SSc patients with mild skin sclerosis, anti-Scl70, interstitial lung disease or female sex were more diversified. B cells from the SSc patients showed a differential V and J gene usage. SSc patients had distinct BCR repertoires.These findings reflected the differences of BCR repertoires between SSc patients and controls. The higher-usage genes for the BCR sequence might be potential biomarkers of B cell-targeted therapies or diagnosis for SSc.

scholarly journals Combined Loss of Tet1 and Tet2 Promotes B-Cell, but Not Myeloid Malignancies in Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3650-3650
Author(s):  
Zhigang Zhao ◽  
Lin Li ◽  
Meelad Dawlaty ◽  
Feng Pan ◽  
Zhe Li ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Cultures ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Hematological Malignancies ◽  
Cell Populations ◽  
Double Knockout ◽  
B Cell Malignancies ◽  
Myeloid Malignancies ◽  
Genome Wide

Abstract Objective: Tet1/2/3 are methylcytosine dioxygenases regulating cytosine methylation in the genome. Tet1 and Tet2 are abundantly expressed in HSC/HPCs and implicated in hematological malignancies. Tet2 -deletion in mice causes myeloid malignancies, while Tet1 -null mice are overtly normal early in life. Here, we investigated the overlapping and non-redundant functions of Tet1/Tet2 in HSC maintenance and hematological malignancies using Tet1/2 double knockout (DKO) mice. Methods: 1) Kinetic analysis of the hematologicalparameters on WT, Tet1-/-, Tet2-/- and DKO mice; 2) Analysis of HSC, myeloid and lymphoid progenitors and various maturation stages of B-cell populations; 3) Competitive bone marrow reconstitution assay; 4) RAN-Seq on LK cells and B220+ cells from young/undiseased and diseased DKO mice respectively; 5) Chemical labeling and affinity purification method coupled with high-throughput sequencing (hMe-Seal) to profile the genome-wide distribution of 5hmC, and methylated DNA immunoprecipitation coupled with high-throughput sequencing (MeDIP-seq) to profile 5mC in BM LK cells from young WT, Tet2-/- and DKO mice; 6) q-PCR analysis of the mRNA expression levels of Tet1 and Tet2 on BM CD19+ cells from B-ALL patients and compared to that of CD19+ B-cells from healthy controls. Results: We found that T et 1 and T et 2 are often concomitantly down-regulatedin patients with B-ALL. Therefore, it is important to investigate the effects of combined loss of Tet1 and Tet2 on the hematopoietic phenotype and development of hematological malignancies in vivo. The LSK and CMP/GMP/MEP cell populations are comparable in yound WT, Tet1-/- and DKO mice, while were significantly increasedin Tet2-/- mice. When a replating assay was performed using LSK cells, Tet2-/- LSK cell cultures had a significant higher colony formation in each round of replating, while Tet1-/- and DKO LSK cell cultures only exhibited a moderate increase in the number of colonies at P2, but not P3 and P4. Furthermore, young DKO mice had an increased percentage of CLP, BLP and Pro-/Pre-/Immature-B cell populations in their BM as compared to WT, Tet1-/- and Tet2-/- mice. Consistent to the B-lineage phenotypic analysis, DKO BM cells contained higher pre-B cell colony forming cells than the three genotypes of control mice. Interestingly, DKO mice exhibited a strikingly decreased incidence and delayed onset of myeloid malignancies compared to Tet2-/- mice and in contrast developed lethal B-cell malignancies, most closely resembling B-ALL. The loss of Tet2 or DKO leads to genome-wide alterations of both 5mC and 5hmC. Significant overlaps between the differential hydroxymethylated regions (DhMRs) or differential methylated regions (DMRs) of two genotypes of LK cells were observed. However, intriguingly, the overlaps between DhMRs and DMRs within each genotype of LK cells were minimal, indicating that DhMRs and DMRs might represent distinct loci with altered epigenetic modifications under these conditions. When the expression of a pool of 654 genes that are known to be involved in regulating hematopoietic cell development and/or promoting leukemogenesis were overlap with DhMRs and DMRs identified above, we observed significant numbers of these genes with altered either 5hmC or 5mC modifications which however did not alter their gene expression. Furthermore, RNA-Seq on B-ALL DKO B220+ cells showed alteration of a set of genes involved in B-cell development and B-cell lymphoma/leukemogenesis. Conclusion: Using Tet1/2 double knockout mice, we found that Tet1 is required for Tet2 -deletion mediated HSC dysregulation, myeloid skewing and myeloid malignancy, indicating distinct roles of the two enzymes. Tet1 loss modulates the Tet2 -deletion mediated disease phenotype, not only decreasing the incidence and delaying the onset of myeloid malignancies, but also promoting the pathogenesis of B-cell malignancies. Furthermore, our observations highlight the roles of distinct cytosine modifications, particularly 5hmC, could play in marking the specific genes and enabling cells to fate decision change upon stimulation signals. These findings provide a pathological framework for further elucidating the molecular mechanisms and critical cross talks between Tet1 and Tet2 in the pathogenesis of hematological malignancies. Disclosures No relevant conflicts of interest to declare.

scholarly journals A multi-objective based clustering for identifying clonally-related sequences from high-throughput B cell repertoire data

2021 ◽  
Author(s):  
Nika Abdollahi ◽  
Anne Langlois De Septenville ◽  
Hugues Ripoche ◽  
Frederic Davi ◽  
Juliana Silva Bernardes
Keyword(s):  
B Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Somatic Hypermutation ◽  
Clustering Algorithms ◽  
Objective Functions ◽  
Cell Repertoire ◽  
Multi Objective ◽  
B Cell Repertoire ◽  
Attractive Option

The adaptive B cell response is driven by the expansion, somatic hypermutation, and selection of B cell clones. A high number of clones in a B cell population indicates a highly diverse repertoire, while clonal size distribution and sequence diversity within clones can be related to antigen's selective pressure. Identifying clones is fundamental to many repertoire studies, including repertoire comparisons, clonal tracking and statistical analysis. Several methods have been developed to group sequences from high-throughput B cell repertoire data. Current methods use clustering algorithms to group clonally-related sequences based on their similarities or distances. Such approaches create groups by optimizing a single objective that typically minimizes intra-clonal distances. However, optimizing several objective functions can be advantageous and boost the algorithm convergence rate. Here we propose a new method based on multi-objective clustering. Our approach requires V(D)J annotations to obtain the initial clones and iteratively applies two objective functions that optimize cohesion and separation within clones simultaneously. We show that under simulations with varied mutation rates, our method greatly improves clonal grouping as compared to other tools. When applied to experimental repertoires generated from high-throughput sequencing, its clustering results are comparable to the most performing tools. The method based on multi-objective clustering can accurately identify clone members, has fewer parameter settings and presents the lowest running time among existing tools. All these features constitute an attractive option for repertoire analysis, particularly in the clinical context to unravel the mechanisms involved in the development and evolution of B cell malignancies.

scholarly journals The underestimation of global microbial diversity

2016 ◽  
Author(s):  
Jay T Lennon ◽  
Kenneth J Locey
Keyword(s):  
Microbial Diversity ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequencing Technology ◽  
Recent Commentary ◽  
Quantum Leap ◽  
Over Time

In a recent commentary, Amann and Rosselló-Mórab summarize how the census of Bacteria and Archaea has changed over time (1). For decades, the number of recognized microbial taxa was underestimated owing to limitations associated with culture-based methods and the rules of nomenclature. The authors describe a "quantum leap" in the estimates of global microbial diversity following advances in high-throughput sequencing technology. Despite this, Amann and Rosselló-Mórab project that a complete census of microbial diversity will be reached within a few years culminating in the lower millions of taxa (1). While perhaps attractively optimistic to some, this presumption is misleading for the following reasons.

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