Exploring DNA quality of single cells for genome analysis with simultaneous whole-genome amplification

Bäumer, Christiane; Fisch, Evelyn; Wedler, Holger; Reinecke, Frank; Korfhage, Christian

doi:10.1038/s41598-018-25895-7

Cited by 31 publications

(38 citation statements)

References 25 publications

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“…Even if the cells suffered from the same condition, the MTX effects and genetic status were different in each cell. This would significantly hinder technical analysis of the alignment and analysis of amplified patterns 19, 20 . Therefore, we had to optimize homogenously amplified patterns before sequencing by performing a serial clone selection and sub-culturing.…”

Section: Resultsmentioning

confidence: 99%

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells

Kim

Shin

Seo

2020

Preprint

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DHFR gene amplification is present in methotrexate (MTX)-resistant colon cancer cells and acute lymphoblastic leukemia. However, little is known about DHFR gene amplification due to difficulties in quantifying amplification size and recognizing the repetitive rearrangements involved in the process. In this study, we have proposed an integrative framework to characterize the amplified region by using a combination of single-molecule real time sequencing, next-generation optical mapping, and chromosome conformation capture (Hi-C). Amplification of the DHFR gene was optimized to generate homogenously amplified patterns. The amplification units of 11 genes, from the DHFR gene to the ATP6AP1L gene position on chromosome 5 (~2.2Mbp), and a twenty-fold tandemly amplified region were verified using longrange genome and RNA sequencing data. In doing so, a novel inversion at the start and end positions of the amplified region as well as frameshift insertions in most of the MSH and MLH genes were detected. These might stimulate chromosomal breakage and cause the dysregulation of mismatch repair pathways. Using Hi-C technology, high adjusted interaction frequencies were detected on the amplified unit and unsuspected position on 5q, which could have a complex network of spatial contacts to harbor gene amplification. Characterizing the tandem gene-amplified unit and genomic variants as well as chromosomal interactions on intra-chromosome 5 can be critical in identifying the mechanisms behind genomic rearrangements. These findings may give new insight into the mechanisms underlying the amplification process and evolution of drug resistance. Results Determination of MTX-resistant HT-29 cellsWhile generating MTX-resistant HT-29 cells (selected clone: C1-2) from singe-cell selection and MTX sensitization as previously described 15 , dramatic morphological changes in the cells themselves were detected in the form of rounded and circular shapes at the 1 st cycle of sensitization as well as rod and irregular shapes at the 2 nd cycle (Supplementary Fig. 1). The original shapes were again observed at the 3 rd cycle of sensitization, which might indicate that the HT-29 cells were becoming resistant to MTX and rapidly grew up under high MTX concentration.As expected, DHFR expression, which was normalized by the B2M housekeeping gene, was steadily increased from the 1 st to 3 rd cycle, as the HT-29 clones became resistant to MTX (Supplementary Fig. 2a). After confirming these morphological changes and increased DHFR expression, the DHFR copy number was measured in several clones.The clones' cycle quantification values (Ct) were used to compute the rate of copy number. These values dropped from 26.04 to 19.99, as the number of cycles increased ( Supplementary Table 1). Interestingly, there was a dramatic increase in the DHFR copy number between the 1 st (0.97 copies) and 2 nd (54.83 copies) cycles ( Supplementary Fig. 2b).Based on these results, we ascertained that a specific time period and set of conditions were required for clones to su...

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Section: Resultsmentioning

confidence: 99%

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells

Kim

Shin

Seo

2020

Preprint

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“…Despite this, our assays consumed only ~120,000 cells per patient for each drug condition tested, of which ~90,000 were used by the SMR mass assay. These sample requirements are comparable to next-generation sequencing, which is already a standard component of many clinical workflows and requires 10 ng-1 µg of DNA from typically 10 3 -10 5 tumor cells (34). However, the sample input requirements of the SMR mass assay could be reduced by several fold since sufficient statistical power can be obtained by weighing as few as ~2,000 cells per sample, which is considerably less than the 15,000 cells per sample used in the current protocol (Supplemental Note 1).…”

Section: Discussionmentioning

confidence: 99%

Functional drug susceptibility testing based on biophysical measurements predicts patient outcome in glioblastoma patient-derived neurosphere models

Stockslager

Malinowski

Touat

et al. 2020

Preprint

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Functional precision medicine aims to match each cancer patient to the most effective treatment by performing ex vivo drug susceptibility testing on the patient’s tumor cells. Despite promising feasibility studies, functional drug susceptibility testing is not yet used in clinical oncology practice to make treatment decisions. Often, functional testing approaches have measured ex vivo drug response using metabolic assays such as CellTiter-Glo, which measures ATP as a proxy for numbers of viable cells. As a complement to these existing metabolic drug response assays, we evaluated whether biophysical assays based on cell mass (the suspended microchannel resonator mass assay) or size as measured by microscopy (the IncuCyte assay) could be used as a readout for ex vivo drug response. Using these biophysical assays, we profiled the ex vivo temozolomide responses of a retrospective cohort of 70 glioblastoma patient-derived neurosphere models with matched clinical outcomes and found that both biophysical assays predicted patients’ overall survival with similar power to MGMT promoter methylation, the clinical gold standard biomarker for predicting temozolomide response in glioblastoma. These findings suggest that biophysical assays could be a useful complement to existing metabolic approaches as “universal biomarkers” to measure sensitivity or resistance to anti-cancer drugs with a wide variety of cytostatic or cytotoxic mechanisms.One-sentence summaryBy using biophysical assays to perform ex vivo drug susceptibility testing on 70 glioblastoma patient-derived neurosphere models, we find that functional testing predicts the duration that patients survive when treated with temozolomide, the standard of care chemotherapy.

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“…They are powerful tools for defining the characteristics of individual cells and identifying differences between them (Bäumer et al . ).…”

Section: Characteristics Of Sperm Dnamentioning

confidence: 97%

Potential biomarkers of DNA quality in cryopreserved fish sperm: impact on gene expression and embryonic development

Figueroa

Lee‐Estévez

Valdebenito

et al. 2018

Reviews in Aquaculture

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Sperm DNA quality and gene expression are crucial for early embryo development. Abnormal genomic processes can cause irreversible damage to totipotent cells, thereby altering the capacity for cell differentiation. Cryopreservation is a complex procedure that exposes cells to extreme conditions; it is therefore important to determine whether cryopreservation affects genomic stability. Despite this, few studies have focused on the effects of cryopreservation on the genomic stability of fish sperm. Thus, information is lacking to determine how the cryopreservation of fish sperm affects embryo quality and subsequently aquaculture. The aim of this review was to compile background information pertaining to the study of genomic stability in cryopreserved sperm. In this way, we hope to characterize more clearly the embryonic development of fish that are of biological and economic interest. We also discuss the potential use of antioxidants in cryopreservation to help preserve DNA integrity, gene expression and embryo quality. There is a current need to evaluate the use of potential biomarkers of genomic integrity to establish the effects of cryopreservation on the DNA quality of fish gametes and embryos.

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Exploring DNA quality of single cells for genome analysis with simultaneous whole-genome amplification

Cited by 31 publications

References 25 publications

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells

Functional drug susceptibility testing based on biophysical measurements predicts patient outcome in glioblastoma patient-derived neurosphere models

Potential biomarkers of DNA quality in cryopreserved fish sperm: impact on gene expression and embryonic development

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