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1.
Elife ; 122023 01 24.
Article in English | MEDLINE | ID: mdl-36692004

ABSTRACT

High-throughput sequencing of adaptive immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful TCR/BCR repertoire reconstruction and analysis methods have been developed in the past decade. However, detecting and correcting the discrepancy between real and experimentally observed lymphocyte clone frequencies are still challenging. Here, we discovered a hallmark anomaly in the ratio between read count and clone count-based frequencies of non-functional clonotypes in multiplex PCR-based immune repertoires. Calculating this anomaly, we formulated a quantitative measure of V- and J-genes frequency bias driven by multiplex PCR during library preparation called Over Amplification Rate (OAR). Based on the OAR concept, we developed an original software for multiplex PCR-specific bias evaluation and correction named iROAR: immune Repertoire Over Amplification Removal (https://github.com/smiranast/iROAR). The iROAR algorithm was successfully tested on previously published TCR repertoires obtained using both 5' RACE (Rapid Amplification of cDNA Ends)-based and multiplex PCR-based approaches and compared with a biological spike-in-based method for PCR bias evaluation. The developed approach can increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.


Subject(s)
Adaptive Immunity , Software , DNA, Complementary , Clone Cells , High-Throughput Nucleotide Sequencing/methods , Receptors, Antigen, T-Cell/genetics
3.
Cancer Genet Cytogenet ; 167(1): 47-50, 2006 May.
Article in English | MEDLINE | ID: mdl-16682286

ABSTRACT

Deletions of Xq are extremely rare events in myelodysplastic syndromes (MDS) patients and were previously described in five patients, in two of them as a sole chromosome abnormality. We found isolated del(Xq) in 3 of 127 MDS patients with clonal chromosome changes. Detailed analysis of clinical and morphological data of presented and previously published cases indicates the following: (1) del(X)(q24) and del(X)(q13) are nonrandom chromosomal abnormalities in MDS; (2) MDS with deletions of Xq affect exclusively females ages 46-65; and (3) deletions of Xq are associated with refractory anemia with excess blasts (RAEB) and indicate an unfavorable prognosis.


Subject(s)
Chromosomes, Human, X , Cytogenetic Analysis , Gene Deletion , Myelodysplastic Syndromes/genetics , Sex Chromosome Aberrations , Adult , Aged , Bone Marrow Cells/cytology , Cells, Cultured , Fatal Outcome , Female , Humans , Middle Aged , Retrospective Studies
4.
Hematol J ; 4(1): 74-7, 2003.
Article in English | MEDLINE | ID: mdl-12692525

ABSTRACT

Total cell production and longevity of hematopoiesis in long-term bone marrow culture of tumor necrosis factor (TNF)-deficient mice (LTBM-TNFko) are increased. The rate of apoptosis is decreased during the first 40 weeks in culture, then the level of apoptosis reaches levels of wild-type cultures. Extended lifespan of primary cultures usually is the consequence of the neoplastic transformation. We set out to check this possibility in the LTBM-TNFko. Telomerase activity in suspension fraction (SF) of LTBM-TNFko increases with time and reaches maximum a year after culture initiation. Cytogenetic study reveals genome instability in SF and hyperploidy in the adhesion cell layer (ACL) of LTBM-TNFko. All of the above indicate the possibility of neoplastic transformation. However, histological study of cells and CFU-S-derived colonies of SF does not reveal a block of differentiation. Cells of SF are unable to grow without ACL. Although those cells could proliferate in the presence of exogenous growth factors, they are not able to be passaged. Attempts of passaging ACL cells failed as well. Neither healthy nor sublethally irradiated recipients injected intravenously or intraperitoneally with cells of SF develop tumors within 8 months of observation. In conclusion, abnormal dynamics of long-term bone marrow culture of TNF-deficient mice could not be explained by neoplastic transformation.


Subject(s)
Bone Marrow Cells/cytology , Hematopoiesis , Aneuploidy , Animals , Apoptosis , Bone Marrow Transplantation , Cell Adhesion , Cell Differentiation , Cell Division , Cell Transformation, Neoplastic , Cells, Cultured/cytology , Cells, Cultured/transplantation , Colony-Forming Units Assay , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Cells/cytology , Radiation Chimera , Telomerase/analysis , Time Factors , Tumor Necrosis Factor-alpha/deficiency , Tumor Necrosis Factor-alpha/genetics
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