RESUMO
Liquid biopsies as a minimally invasive approach have the potential to revolutionize molecular diagnostics. Yet, although protocols for sample handling and the isolation of circulating tumor DNA (ctDNA) are numerous, comprehensive guidelines for diagnostics and research considering all aspects of real-life multicenter clinical studies are currently not available. These include limitations in sample volume, transport, and blood collection tubes. We tested the impact of commonly used (EDTA and heparin) and specialized blood collection tubes and storage conditions on the yield and purity of cell-free DNA for the application in down-stream analysis. Moreover, we evaluated the feasibility of a combined workflow for ctDNA and tumor cell genomic testing and parallel flow cytometric analysis of leukocytes. For genomic analyses, EDTA tubes showed good results if stored for a maximum of 4 hours at room temperature or for up to 24 hours when stored at 4°C. Spike-in experiments revealed that EDTA tubes in combination with density gradient centrifugation allowed the parallel isolation of ctDNA, leukocytes, and low amounts of tumor cells (0.1%) and their immunophenotyping by flow cytometry and down-stream genomic analysis by whole genome sequencing. In conclusion, adhering to time and temperature limits allows the use of routine EDTA blood samples for liquid biopsy analyses. We further provide a workflow enabling the parallel analysis of cell-free and cellular features for disease monitoring and for clonal evolution studies.
Assuntos
Coleta de Amostras Sanguíneas/métodos , DNA Tumoral Circulante/genética , Testes Diagnósticos de Rotina/métodos , Citometria de Fluxo/métodos , Testes Genéticos/métodos , Leucócitos , Sequenciamento Completo do Genoma/métodos , Adolescente , Adulto , Idoso , Doadores de Sangue , Ácido Edético/química , Estudos de Viabilidade , Feminino , Heparina/química , Humanos , Biópsia Líquida/métodos , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/métodos , Fenótipo , Temperatura , Fatores de Tempo , Adulto JovemRESUMO
Clinical decision-making in cancer and other diseases relies on timely and cost-effective genome-wide testing. Classical bioinformatic algorithms, such as Rawcopy, can support genomic analysis by calling genomic breakpoints and copy-number variations (CNVs), but often require manual data curation, which is error prone, time-consuming, and thus substantially increasing costs of genomic testing and hampering timely delivery of test results to the treating physician. We aimed to investigate whether deep learning algorithms can be used to learn from genome-wide single-nucleotide polymorphism array (SNPa) data and improve state-of-the-art algorithms. We developed, applied, and validated a novel deep neural network (DNN), DeepSNP. A manually curated data set of 50 SNPa analyses was used as truth-set. We show that DeepSNP can learn from SNPa data and classify the presence or absence of genomic breakpoints within large genomic windows with high precision and recall. DeepSNP was compared with well-known neural network models as well as with Rawcopy. Moreover, the use of a localization unit indicates the ability to pinpoint genomic breakpoints despite their exact location not being provided while training. DeepSNP results demonstrate the potential of DNN architectures to learn from genomic SNPa data and encourage further adaptation for CNV detection in SNPa and other genomic data types.
Assuntos
Genômica/métodos , Polimorfismo de Nucleotídeo Único/genética , Algoritmos , Hibridização Genômica Comparativa/métodos , Biologia Computacional/métodos , Variações do Número de Cópias de DNA/genética , Aprendizado Profundo , Genoma Humano/genética , Humanos , Redes Neurais de Computação , Análise de Sequência com Séries de Oligonucleotídeos/métodosRESUMO
Research regarding warning compliance has often emphasized the physical aspects of the warning itself. Here, we examine the role of the perceiver in sensation seeking and health orientation as individual difference variables that affect behavioral compliance to a health warning. The experiment used a laboratory-based simulation of a chemistry demonstration that has been used in previous warnings research. In addition, however, individual difference effects of sensation seeking and health orientation were investigated. Among the significant findings were a significant interaction between condition assignment and sensation seeking on compliance outcome and a significant interaction between condition and health orientation. These results indicate that individual difference variables represent significant influences on the degree to which persons comply with warnings.