Dataset Information

Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour dna Mutations.

ABSTRACT: : It has now been established that in biological fluids such as blood, it is possible to detect cancer causing genomic alterations by analysing circulating tumour DNA (ctDNA). Information derived from ctDNA offers a unique opportunity to enrich our understanding of cancer biology, tumour evolution and therapeutic efficacy and resistance. Here, we propose a workflow to identify targeted mutations by a customized microarray-based assay for the simultaneous detection of single point mutations in different oncogenes (KRAS, NRAS and BRAF) followed by droplet digital PCR (ddPCR) to determine the fractional abundance of the mutated allele. Genetic variants were determined in the plasma of 20 metastatic colorectal cancer (mCRC) patients previously genotyped on tissue biopsy at the diagnosis for medication planning (T0) and following the tumour genetic evolution during treatment phase (T1 and T2) with the objective of allowing therapy response prediction and monitoring. Our preliminary results show that this combined approach is suitable for routine clinical practice. The microarray platform enables for a rapid, specific and sensitive detection of the most common mutations suitable for high-throughput analysis without costly instrumentation while, the ddPCR, consents an absolute quantification of the mutated allele in a longitudinal observational study on patients undergoing targeted therapy.

SUBMITTER: Galbiati S

PROVIDER: S-EPMC6721623 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour dna Mutations.

Galbiati Silvia S Damin Francesco F Ferraro Lucia L Soriani Nadia N Burgio Valentina V Ronzoni Monica M Gianni Luca L Ferrari Maurizio M Chiari Marcella M

Cells 20190724 8

<b>:</b> It has now been established that in biological fluids such as blood, it is possible to detect cancer causing genomic alterations by analysing circulating tumour DNA (ctDNA). Information derived from ctDNA offers a unique opportunity to enrich our understanding of cancer biology, tumour evolution and therapeutic efficacy and resistance. Here, we propose a workflow to identify targeted mutations by a customized microarray-based assay for the simultaneous detection of single point mutation ...[more]

PMID: 31344983

Similar Datasets

Project description:BackgroundMicroarrays depend on appropriate probe design to deliver the promise of accurate genome-wide measurement. Probe design, ideally, produces a unique probe-target match with homogeneous duplex stability over the complete set of probes. Much of microarray pre-processing is concerned with adjusting for non-ideal probes that do not report target concentration accurately. Cross-hybridizing probes (non-unique), probe composition and structure, as well as platform effects such as instrument limitations, have been shown to affect the interpretation of signal. Data cleansing pipelines seldom filter specifically for these constraints, relying instead on general statistical tests to remove the most variable probes from the samples in a study. This adjusts probes contributing to ProbeSet (gene) values in a study-specific manner. We refer to the complete set of factors as biologically applied filter levels (BaFL) and have assembled an analysis pipeline for managing them consistently. The pipeline and associated experiments reported here examine the outcome of comprehensively excluding probes affected by known factors on inter-experiment target behavior consistency.ResultsWe present here a 'white box' probe filtering and intensity transformation protocol that incorporates currently understood factors affecting probe and target interactions; the method has been tested on data from the Affymetrix human GeneChip HG-U95Av2, using two independent datasets from studies of a complex lung adenocarcinoma phenotype. The protocol incorporates probe-specific effects from SNPs, cross-hybridization and low heteroduplex affinity, as well as effects from scanner sensitivity, sample batches, and includes simple statistical tests for identifying unresolved biological factors leading to sample variability. Subsequent to filtering for these factors, the consistency and reliability of the remaining measurements is shown to be markedly improved.ConclusionsThe data cleansing protocol yields reproducible estimates of a given probe or ProbeSet's (gene's) relative expression that translates across datasets, allowing for credible cross-experiment comparisons. We provide supporting evidence for the validity of removing several large classes of probes, and for our approaches for removing outlying samples. The resulting expression profiles demonstrate consistency across the two independent datasets. Finally, we demonstrate that, given an appropriate sampling pool, the method enhances the t-test's statistical power to discriminate significantly different means over sample classes.

Project description:Monkeypox virus (MPXV) poses a global health threat. Droplet digital PCR (ddPCR) holds potential as an accurate diagnostic tool for clinical microbiology. However, there is limited literature on the applicability of ddPCR in clinical settings. In this study, the clinical features of patients with MPXV during the initial outbreak in China in June 2023 were reviewed, and an optimized ddPCR method with dilution and/or inhibitor removal was developed to enhance MPXV detection efficiency. Eighty-two MPXV samples were tested from nine different clinical specimen types, including feces, urine, pharyngeal swabs, anal swabs, saliva, herpes fluid, crust, and semen, and the viral load of each specimen was quantified. A comparative analysis was performed with qPCR to assess sensitivity and specificity and to investigate the characteristics of MPXV infection by analyzing viral loads in different clinical specimens. Consequently, common pharyngeal and gastrointestinal symptoms were observed in patients with MPXV. The optimized ddPCR method demonstrated relatively high sensitivity for MPXV quantification in the clinical materials, with a limit of detection of 0.1 copies/μL. This was particularly evident in low-concentration samples like whole blood, semen, and urine. The optimized ddPCR demonstrated greater detection accuracy compared with normal ddPCR and qPCR, with an area under the curve (AUC) of 0.939. Except for crust samples, viral loads in the specimens gradually decreased as the disease progressed. Virus levels in feces and anal swabs kept a high detection rate at each stage of post-symptom onset, and feces and anal swabs samples may be suitable for clinical diagnosis and continuous monitoring of MPXV.ImportanceThe ddPCR technique proved to be a sensitive and valuable tool for accurately quantifying MPXV viral loads in various clinical specimen types. The findings provided valuable insights into the necessary pre-treatment protocols for MPXV diagnosis in ddPCR detection and the potentially suitable sample types for collection. Therefore, such results can aid in comprehending the potential characteristics of MPXV infection and the usage of ddPCR in clinical settings.

Dataset Information

Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour dna Mutations.

Publications

Microarray Approach Combined with ddPCR: An Useful Pipeline for the Detection and Quantification of Circulating Tumour dna Mutations.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets