Digital proteomics by DNA sequencing
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA291387 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:The simultaneous measurement of many different proteins presents a major challenge to the fields of surgical and clinical pathology, and there is a genuine need for the development of a more direct, quantitative approach to multiplex analysis of proteins in tissues and clinical assays. Here we present a novel, high-throughput immunoassay, driven by the hypothesis that highly sensitive, multiplex protein characterization can be obtained by coupling the unique specificity of antibodies with massively parallel sequencing to detect and quantitate antigens from formalin-fixed, paraffin-embedded tissue (FFPE) and in solution. The method involves conjugating antibodies with a universal anchor oligonucleotide that is hybridized with a second oligonucleotide specifying the sample, antibody, and a unique molecule identifier, akin to “make, model and serial” number. Recovery of the data-rich oligonucleotide from mixtures of antibodies from samples such as histological tissue sections, (herein breast carcinoma) or extracts in an ELISA-type format, enabling error free-amplification that increases the sensitivity and expands the range of analysis.
2025-07-01 | GSE71486 | GEO
Project description:We report a method for specific capture of an arbitrary subset of genomic targets for single molecule bisulfite sequencing, and for digital quantitation of DNA methylation at a single nucleotide resolution. We used targeted bisulfite sequencing to characterize the changes of DNA methylation during the de-differentiation of human fibroblasts into hybrid stem cells, and into induced pluripotent stem cells. We compared the methylation level of approximately 66,000 CpG sites within 2020 CpG islands on chromosome 12, chromosome 20, and 34 selected regions. A total of 288 differentially methylated regions were identified between fibroblasts and pluripotent cells. Methylation cluster analysis revealed distinct methylation patterns between fibroblasts and pluripotent cells. Furthermore iPS cells are globally more methylated than human embryonic stem cells, which could be due to the reprogramming process. This targeted bisulfite sequencing method is particularly useful for efficient and large-scale analysis of DNA methylation in organisms with large genomes.
2009-04-27 | GSE15007 | GEO
Project description:We introduce in vivo mRNA display, a technology that converts a variety of proteomics applications into a DNA sequencing problem.
2020-10-09 | GSE154643 | GEO
Project description:Single-cell DNA methylation sequencing is a powerful method for elucidating important physiological and pathological processes, identifying cell subpopulations, and constructing epigenetic regulatory networks. Existing methylome analyses typically require substantial starting materials, complex operations, and high cost and are susceptible to contamination. These problems have impeded the development of single-cell methylome technology for rare cell profiling. In this work, we report Digital Microfluidics-based single-cell Reduced Representation Bisulfite Sequencing (Digital-scRRBS), the first microfluidics-based single-cell methylome library construction platform, which is an automatic, efficient, reproducible, and reagent-economy approach to dissect the single-cell methylome. Taking advantage of our uniquely designed digital microfluidic chip, we realized efficient single-cell isolation in less than 15 seconds. Furthermore, with the advantages of a confined environment, superhydrophobic surface, and nano-scale reaction volume of our digital microfluidic chip, more amplifiable DNA is retained for library construction compared to other approaches. We have successfully constructed single-cell methylation sequencing libraries with a unique genome mapping rate of up to 53.6%, covering up to 2.26 million CpG sites. The application of Digital-scRRBS allows us to discriminate cell identity and dynamically monitor DNA methylation levels during drug administration. Digital-scRRBS provides the technology for widespread application of single-cell methylation methods as a versatile tool for epigenetic analysis in rare cells and highly heterogeneous populations.
2022-09-29 | GSE214106 | GEO
Project description:We analyzed levels of 5-methyl cytosine nnnn CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing. Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of SW48 colon cancer cell line genomic DNA. Genomic DNA spiked in with unmethylated, partially methylated and fully methylated standards was sequentially cut at CCCGGG sites with the methylation-sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5'CCGG overhangs), creating different end sequences that represented methylation status of the CCCGGG sites. These end sequences were analyzed by Illumina high throughput sequencing. Methylation status at individual CCCGGG sites across the genome was determined by counting the methylated reads with the CCGGG signature and unmethylated reads with the GGG signature at the beginnings of the sequencing reads after alignment to the human genome.
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Project description:Replacement of high-value fish species with cheaper varieties or mislabelling of food unfit for human consumption is a global problem violating both consumers’ rights and safety. For distinguishing fish species in pure samples, DNA approaches are available; however, authentication and quantification of fish species in mixtures remains a challenge. In the present study, a novel high-throughput shotgun DNA sequencing approach applying masked reference libraries was developed and used for authentication and abundance calculations of fish species in mixed samples. Results demonstrate that the analytical protocol presented here can discriminate and predict relative abundances of different fish species in mixed samples with high accuracy. In addition to DNA analyses, shotgun proteomics tools based on direct spectra comparisons were employed on the same mixture. Similar to the DNA approach, the identification of individual fish species and the estimation of their respective relative abundances in a mixed sample also were feasible. Furthermore, the data obtained indicated that DNA sequencing using masked libraries predicted species-composition of the fish mixture with higher specificity, while at a taxonomic family level, relative abundances of the different species in the fish mixture were predicted with slightly higher accuracy using proteomics tools. Taken together, the results demonstrate that both DNA and protein-based approaches presented here can be used to efficiently tackle current challenges in feed and food authentication analyses.
2021-03-09 | MSV000087017 | MassIVE
Project description:<p>In order to explore opportunities for personalized and predictive health care, we collected serial clinical measurements, health surveys and multiomics profiles (genomics, proteomics, autoantibodies, metabolomics and gut microbiome) from 96 individuals. The participants underwent data-driven health coaching over a 16-month period with continuous digital monitoring of activity and sleep. Multiomics factor analysis resulted in an unsupervised, data-driven and integrated view of human health, revealing distinct and independent molecular factors linked to obesity, diabetes, liver function, cardiovascular disease, inflammation, immunity, exercise, diet and hormonal effects. The data revealed novel and previously uncovered associations between risk factors, molecular pathways, and quantitative lifestyle parameters. For example, ethinyl estradiol use had a distinct impact on metabolites, proteins and physiology. Multidimensional molecular and digital health signatures uncovered biological variability between people and quantitative effects of lifestyle changes, hence illustrating the value of the combined use of molecular and digital monitoring of human health.</p>
2021-09-02 | MTBLS2187 | MetaboLights
Project description:This clinical trial studies universal screening for deoxyribonucleic acid (DNA) mismatch repair deficiency in patients with endometrial cancer, mutations in the genes responsible for Lynch syndrome (inherited forms of endometrial cancers) and other DNA changes that could help guide treatment strategies. Universal tumor DNA sequencing may help doctors better understand how to personalize care, increase length of life, and increase quality of life in patients with endometrial cancer and their relatives.
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Project description:We established a protocol of the SuperSAGE technology combined with next-generation sequencing, coined “High-Throughput (HT-) SuperSAGE”. SuperSAGE is a method of digital gene expression profiling that allows isolation of 26-bp tag fragments from expressed transcripts. In the present protocol, index (barcode) sequences are employed to discriminate tags from different samples. Such barcodes permit to enable researchers to analyze digital tags from many transcriptomes of many samples in a single sequencing run by simply pooling the libraries. Here, we demonstrated that HT-SuperSAGE provided highly sensitive, reproducible and accurate digital gene expression data. By increasing throughput for analysis in HT-SuperSAGE, various applications were expected and several examples of its applications were introduced in the present study, including analyses of laser-microdissected cells, biological replicates or tag extraction using different anchoring enzymes. 27 different tissue samples from three different life organisms were analyzed. About 2 samples, three different anchoring enzymes were employed.
2010-09-30 | E-GEOD-20682 | biostudies-arrayexpress