scholarly journals Combined Molecular Genetic and Cytogenetic Analysis from Single Cells after Isothermal Whole-Genome Amplification

2011 ◽  
Vol 57 (7) ◽  
pp. 1032-1041 ◽  
Author(s):  
Thomas Kroneis ◽  
Jochen B Geigl ◽  
Amin El-Heliebi ◽  
Martina Auer ◽  
Peter Ulz ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Molecular Genetic ◽  
Target Cells ◽  
Whole Genome ◽  
Single Cell Level ◽  
Cell Level ◽  
Genome Amplification

BACKGROUND Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells' genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis. METHODS We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole-genome amplification (iWGA) of single and pooled cells. RESULTS DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells. CONCLUSIONS This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.

scholarly journals Evaluation of whole genome amplification and bioinformatic methods for the characterization of Leishmania genomes at a single cell level

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hideo Imamura ◽  
Pieter Monsieurs ◽  
Marlene Jara ◽  
Mandy Sanders ◽  
Ilse Maes ◽  
...  
Keyword(s):  
Single Cell ◽  
Leishmania Donovani ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Second Step ◽  
Whole Genome ◽  
Dna Testing ◽  
Cell Level ◽  
Genome Amplification

Abstract Here, we report a pilot study paving the way for further single cell genomics studies in Leishmania. First, the performances of two commercially available kits for Whole Genome Amplification (WGA), PicoPLEX and RepliG were compared on small amounts of Leishmania donovani DNA, testing their ability to preserve specific genetic variations, including aneuploidy levels and SNPs. We show here that the choice of WGA method should be determined by the planned downstream genetic analysis, PicoPLEX and RepliG performing better for aneuploidy and SNP calling, respectively. This comparison allowed us to evaluate and optimize corresponding bio-informatic methods. As PicoPLEX was shown to be the preferred method for studying single cell aneuploidy, this method was applied in a second step, on single cells of L. braziliensis, which were sorted by fluorescence activated cell sorting (FACS). Even sequencing depth was achieved in 28 single cells, allowing accurate somy estimation. A dominant karyotype with three aneuploid chromosomes was observed in 25 cells, while two different minor karyotypes were observed in the other cells. Our method thus allowed the detection of aneuploidy mosaicism, and provides a solid basis which can be further refined to concur with higher-throughput single cell genomic methods.

scholarly journals Comparison of seven single cell whole genome amplification commercial kits using targeted sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tamir Biezuner ◽  
Ofir Raz ◽  
Shiran Amir ◽  
Lilach Milo ◽  
Rivka Adar ◽  
...  
Keyword(s):  
Single Cell ◽  
Whole Genome Amplification ◽  
Genomic Sequence ◽  
Single Cells ◽  
Targeted Sequencing ◽  
Whole Genome ◽  
Cell Level ◽  
Genome Amplification ◽  
Commercial Kits ◽  
Commercial Kit

AbstractAdvances in whole genome amplification (WGA) techniques enable understanding of the genomic sequence at a single cell level. Demand for single cell dedicated WGA kits (scWGA) has led to the development of several commercial kit. To this point, no robust comparison of all available kits was performed. Here, we benchmark an economical assay, comparing all commercially available scWGA kits. Our comparison is based on targeted sequencing of thousands of genomic loci, including highly mutable regions, from a large cohort of human single cells. Using this approach we have demonstrated the superiority of Ampli1 in genome coverage and of RepliG in reduced error rate. In summary, we show that no single kit is optimal across all categories, highlighting the need for a dedicated kit selection in accordance with experimental requirements.

scholarly journals Evaluation of whole genome amplification and bioinformatic methods for the characterization of Leishmania genomes at a single cell level

2020 ◽  
Author(s):  
Hideo Imamura ◽  
Marlene Jara ◽  
Pieter Monsieurs ◽  
Mandy Sanders ◽  
Ilse Maes ◽  
...  
Keyword(s):  
Single Cell ◽  
Leishmania Donovani ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Second Step ◽  
Whole Genome ◽  
Dna Testing ◽  
Cell Level ◽  
Genome Amplification

AbstractHere, we report a pilot study paving the way for further single cell genomics studies in Leishmania. First, the performances of two commercially available kits for Whole Genome Amplification (WGA), PicoPlex and RepliG was compared on small amounts of Leishmania donovani DNA, testing their ability to preserve specific genetic variations, including aneuploidy levels and SNPs. We show here that the choice of WGA method should be determined by the planned downstream genetic analysis, Picoplex and RepliG performing better for aneuploidy and SNP calling, respectively. This comparison allowed us to evaluate and optimize corresponding bio-informatic methods. As PicoPlex was shown to be the preferred method for studying single cell aneuploidy, this method was applied in a second step, on single cells of L. braziliensis, which were sorted by fluorescence activated cell sorting (FACS). Even sequencing depth was achieved in 28 single cells, allowing accurate somy estimation. A dominant karyotype with three aneuploid chromosomes was observed in 25 cells, while two different minor karyotypes were observed in the other cells. Our method thus allowed the detection of aneuploidy mosaicism, and provides a solid basis which can be further refined to concur with higher-throughput single cell genomic methods.

scholarly journals Comparison of seven single cell Whole Genome Amplification commercial kits using targeted sequencing

2017 ◽  
Author(s):  
Tamir Biezuner ◽  
Ofir Raz ◽  
Shiran Amir ◽  
Lilach Milo ◽  
Rivka Adar ◽  
...  
Keyword(s):  
Single Cell ◽  
Whole Genome Amplification ◽  
Single Cells ◽  
Targeted Sequencing ◽  
Whole Genome ◽  
Genome Coverage ◽  
Genome Amplification ◽  
Cell Experiment ◽  
Commercial Kits ◽  
Genomic Regions

AbstractAdvances in biochemical technologies have led to a boost in the field of single cell genomics. Observation of the genome at a single cell resolution is currently achieved by pre-amplification using whole genome amplification (WGA) techniques that differ by their biochemical aspects and as a result by biased amplification of the original molecule. Several comparisons between commercially available single cell dedicated WGA kits (scWGA) were performed, however, these comparisons are costly, were only performed on selected scWGA kit and more notably, are limited by the number of analyzed cells, making them limited for reproducibility analysis. We benchmarked an economical assay to compare all commercially available scWGA kits that is based on targeted sequencing of thousands of genomic regions, including highly mutable genomic regions (microsatellites), from a large cohort of human single cells (125 cells in total). Using this approach, we could analyze the genome coverage, the reproducibility of genome coverage and the error rate of each kit. Our experimental design provides an affordable and reliable comparative assay that simulates a real single cell experiment. Results demonstrate the needfor a dedicated kit selection depending on the desired single cell assay.

scholarly journals Characteristics and requirements of the interaction between human monocytes and tumor cells on the single cell level

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 390-396 ◽  
Author(s):  
MG Golightly ◽  
DG Fischer ◽  
C Ohlander ◽  
HS Koren
Keyword(s):  
Tumor Cell ◽  
Single Cell ◽  
Tumor Cells ◽  
Autologous Serum ◽  
Target Cells ◽  
Single Cell Level ◽  
Human Monocytes ◽  
Blood Monocytes ◽  
Cell Level ◽  
Effector Cells

Abstract Highly purified (97%-99%) and viable (99%) peripheral blood monocytes obtained by EDTA-reversible adherence to autologous-serum-precoated plastic surfaces could rapidly lyse a variety of tumor cells in a 3–4 hr 51Cr release assay. Using these monocytes as effectors, a short-term agarose/conjugate assay was utilized, permitting us to examine the interaction between fresh human monocytes and neoplastic target cells on a single cell level. That the tumor-bound effector cells were indeed monocytes was confirmed by employing the monocyte-specific monoclonal antibody 61D3, which stained 95%-99% of the mononuclear cells bound to conjugated and killed K562 tumor targets. The binding of monocytes to target cells appeared to be temperature dependent and was extremely rapid, reaching a plateau after 5 min at 30 degrees C. Our findings demonstrated for the first time that only a proportion of human blood monocytes can bind to a particular target cell and that only a fraction of the binding cells have the intrinsic potential to kill those neoplastic targets. The proportion of monocytes capable of binding and killing varies between individuals and also depends on the tumor cell used, indicating heterogeneity in the monocyte and tumor cell populations. The highest proportion of monocytes bind to the human erythromyeloid leukemia K562 cell line (13%-50%). The frequency of monocytes capable of killing K562 tumor cells is relatively low (7%- 13%). The system described here should be useful to study the heterogeneity of mononuclear phagocytes and to analyze the molecular basis of the interaction between those effector cells and neoplastic target cells.

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