1,721,324 research outputs found
Circulating fetal trophoblast enrichment and single cell genetic analysis in the context of cell-based non-invasive prenatal testing
No full textIn this dissertation, the main goal was to contribute to the development of a beginning-to-end cell-based non-invasive prenatal test (cbNIPT). For the enrichment of circulating fetal trophoblasts (CFTs) from maternal blood, the VTX-1 Liquid Biopsy System was assessed. This technology, based on inertial microfluidics and laminar microscale vortices, allows the successful enrichment of CFTs from maternal blood in seven out of ten cases with minimal contaminating maternal blood cells. After enrichment, putative fetal cells need to be isolated individually and the true fetal origin of each individual cell must be confirmed prior to the detection of chromosomal abnormalities. To allow multiple genetic analyses on a single cell, whole genome amplification (WGA) is required. Since WGA introduces errors, four WGA methods were compared regarding their suitability for downstream copy number variation (CNV) detection and short tandem repeat (STR) profiling for human identification. A different performance of the WGA methods is observed for unfixed and preserved cells, but overall, DOPlify™ WGA, Ampli1™ WGA Kit, and PicoPLEX® WGA Kit allow proper CNV detection and STR profiling, while REPLI-g Single Cell WGA Kit is not preferred. Finally, even with the most appropriate WGA method, incomplete STR profiling is expected after WGA on single cells. This challenges the reliable discrimination of single fetal and maternal cells in the context of cbNIPT, since mother and child share half of their DNA. Nevertheless, both length-based STR profiling and sequence-based SNP profiling proved to be capable of correctly discriminating single cells from a parent or an offspring after PicoPLEX WGA, with 100 % sensitivity and specificity, but a higher evidential value is obtained for SNP profiling
Cell-based non-invasive prenatal testing : circulating trophoblast enrichment and genetic analysis
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Exploring and optimizing variant detection and phasing methods in pharmacogenomics
No full textPharmacogenomics (PGx) studies the relationship between the patient genetic background and drug therapy. This dissertation aims to define which PGx tests are present on the market and which improvements are needed to these PGx tests to capture all essential variants and make them widely applicable in clinical practice. Therefore, in the first part, different PGx-specific tests, direct-to-customers assays, and exome sequencing were compared with the genetic variants in gene-drug interactions described in public databases. The studied tests could assay most of these genetic variants, mainly single nucleotide variants (SNVs). However, more complex and important haplotypes were missing. Therefore, in the second part of this dissertation, two new targeted sequencing technologies were tested to see to what extent they could call and phase the different variants into haplotypes for important pharmacogenes. First, the TLA technology was performed on four complex pharmacogenes and sequenced on the Illumina and ONT platforms. Both sequencing methods had a low on-target percentage, and not all positions in the genes were entirely covered. Nevertheless, CYP2D6, sequenced with the ONT platform, resulted in the correct genotype. However, less than half of the variants were phased correctly for the other studied genes. Second, we optimized the nanopore Cas9-targeted sequencing method to enrich the CYP2D6-CYP2D7 region. Although the low on-target percentage and low sequencing throughput, this method, combined with a newly developed analysis pipeline, resulted in the discovery of an inserted CYP2D6-CYP2D7 hybrid that goes undetected by other assays
STRide probes : single-labeled short tandem repeat identification probes
Full text linkThe demand for forensic DNA profiling at the crime scene or at police stations is increasing. DNA profiling is currently performed in specialized laboratories by PCR amplification of Short Tandem Repeats (STR) followed by amplicon sizing using capillary electrophoresis. The need for bulky equipment to identify alleles after PCR presents a challenge for shifting to a decentralized workflow. We devised a novel hybridization-based STRgenotyping method, using Short Tandem Repeat Identification (STRide) probes, which could help tackle this issue. STRide probes are fluorescently labeled oligonucleotides that rely on the quenching properties of guanine on fluorescein derivatives. Mismatches between STRide probes and amplicons can be detected by melting curve analysis after asymmetric PCR. The functionality of the STRide probes was demonstrated by analyzing synthetic DNA samples for the D16S539 locus. Next, STRide probes were developed for five different CODIS core loci (D16S539, TH01, TPOX, FGA, and D7S820). These probes were validated by analyzing 13 human DNA samples. Successful genotyping was obtained using inputs as low as 31 pg of DNA, demonstrating high sensitivity. The STRide probes are ideally suited to be implemented in a microarray and present an important step towards a portable device for fast on-site forensic DNA fingerprinting
Changes in DNA methylation in Arabidopsis thaliana plants exposed over multiple generations to gamma radiation
No full textPrevious studies have found indications that exposure to ionising radiation (IR) results in DNA methylation changes in plants. However, this phenomenon is yet to be studied across multiple generations. Furthermore, the exact role of these changes in the IR-induced plant response is still far from understood. Here, we study the effect of gamma radiation on DNA methylation and its effect across generations in young Arabidopsis plants. A multigenerational set-up was used in which three generations (Parent, generation 1, and generation 2) of 7-day old Arabidopsis thaliana plants were exposed to either of the different radiation treatments (30, 60, 110, or 430 mGy/h) or to natural background radiation (control condition) for 14 days. The parental generation consisted of previously non-exposed plants, whereas generation 1 and generation 2 plants had already received a similar irradiation in the previous one or two generations, respectively. Directly after exposure the entire methylomes were analysed with UPLC-MS/MS to measure whole genome methylation levels. Whole genome bisulfite sequencing was used to identify differentially methylated regions (DMRs), including their methylation context in the three generations and this for three different radiation conditions (control, 30 mGy/h, and 110 mGy/h). Both intra- and intergenerational comparisons of the genes and transposable elements associated with the DMRs were made. Taking the methylation context into account, the highest number of changes were found for cytosines followed directly by guanine (CG methylation), whereas only limited changes in CHG methylation occurred and no changes in CHH methylation were observed. A clear increase in IR-induced DMRs was seen over the three generations that were exposed to the lowest dose rate, where generation 2 had a markedly higher number of DMRs than the previous two generations (Parent and generation 1). Counterintuitively, we did not see significant differences in the plants exposed to the highest dose rate. A large number of DMRs associated with transposable elements were found, the majority of them being hypermethylated, likely leading to more genetic stability. Next to that, a significant number of DMRs were associated with genes (either in their promoter-associated region or gene body). A functional analysis of these genes showed an enrichment for genes related to development as well as various stress responses, including DNA repair, RNA splicing, and (a)biotic stress responses. These observations indicate a role of DNA methylation in the regulation of these genes in response to IR exposure and shows a possible role for epigenetics in plant adaptation to IR over multiple generations
Various Evolutionary Trajectories Lead to Loss of the Tobramycin-Potentiating Activity of the Quorum-Sensing Inhibitor Baicalin Hydrate in Burkholderia cenocepacia Biofilms
Full text linkCombining antibiotics with potentiators that increase their activity is a promising strategy to tackle infections caused by antibiotic-resistant bacteria. As potentiators do not interfere with essential processes, it has been hypothesized that they are less likely to induce resistance. However, evidence supporting this hypothesis is lacking. In the present study, we investigated whether Burkholderia cenocepacia J2315 biofilms develop reduced susceptibility toward one such adjuvant, baicalin hydrate (BH). Biofilms were repeatedly and intermittently treated with tobramycin (TOB) alone or in combination with BH for 24 h. After treatment, the remaining cells were quantified using plate counting. After 15 cycles, biofilm cells were less susceptible to TOB and TOB + BH compared to the start population, and the potentiating effect of BH toward TOB was lost. Whole-genome sequencing was performed to probe which changes were involved in the reduced effect of BH, and mutations in 14 protein-coding genes were identified (including mutations in genes involved in central metabolism and in BCAL0296, encoding an ABC transporter). No changes in the MIC or MBC of TOB or changes in the number of persister cells were observed. However, basal intracellular levels of reactive oxygen species (ROS) and ROS levels found after treatment with TOB were markedly decreased in the evolved populations. In addition, in evolved cultures with mutations in BCAL0296, a significantly reduced uptake of TOB was observed. Our results indicate that B. cenocepacia J2315 biofilms rapidly lose susceptibility toward the antibiotic-potentiating activity of BH and point to changes in central metabolism, reduced ROS production, and reduced TOB uptake as mechanisms
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