Significance analysis of xMap cytokine bead arrays.
Ontology highlight
ABSTRACT:
SUBMITTER: Won JH
PROVIDER: S-EPMC3286996 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Advancing technologies that quantify gene expression in space are transforming contemporary biology research. A class of spatial transcriptomics methods uses barcoded bead arrays that are optically decoded via microscopy and are later matched to sequenced data from the respective libraries. To obtain a detailed representation of the tissue in space, robust and efficient computational pipelines are required to process microscopy images and accurately basecall the bead barcodes. Optocoder is a computational framework that processes microscopy images to decode bead barcodes in space. It efficiently aligns images, detects beads, and corrects for confounding factors of the fluorescence signal, such as crosstalk and phasing. Furthermore, Optocoder employs supervised machine learning to strongly increase the number of matches between optically decoded and sequenced barcodes. We benchmark Optocoder using data from an in-house spatial transcriptomics platform, as well as from Slide-Seq(V2), and we show that it efficiently processes all datasets without modification. Optocoder is publicly available, open-source and provided as a stand-alone Python package on GitHub: https://github.com/rajewsky-lab/optocoder.
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Project description:Spatial transcriptomics technologies that can quantify gene expression in space are transforming contemporary biology research. Some of such methods use spatially barcoded bead arrays that are optically sequenced by a microscopy setup to detect bead barcodes in space which can be consecutively matched to cell barcodes from the respective single cell sequencing experiment. To have good quality barcodes and a high number of barcode matches in space, robust and efficient computational pipelines are needed to process raw microscopy images and call the bases of bead barcodes accurately. Here, we present Optocoder, a computational pipeline that takes raw optical sequencing microscopy images as input and outputs bead barcodes in space. Optocoder efficiently aligns images, detects beads, and corrects for confounding factors of the fluorescence signal such as crosstalk and phasing before base calling. Furthermore, we implement a machine learning pipeline that is trained using the signal from the beads that match to illumina barcodes in order to predict non-matching bead barcodes which can boost up the number of barcode matches. We benchmark Optocoder using data from an in-house spatial transcriptomics platform as well as data from the Slide-seq method and we show that it can efficiently process both datasets with minimal modification.
2022-06-24 | GSE193472 | GEO
Project description:BackgroundNo single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories.MethodsWe compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow-based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)]. All three analyzers quantify fluorescence signals generated by the Oncuria assay.ResultsAll three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically < 5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥ 1 analyte above the highest standard concentration, i.e., A1AT (n = 7/18), IL-8 (n = 5), and/or ANG (n = 2), while only one control sample registered an analyte (A1AT) above the highest standard concentration.ConclusionMultiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment responsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms.Trial registrationThis study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.
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Project description:BackgroundNo single marker of bladder cancer (BC) exists in urine samples with sufficient accuracy for disease diagnosis and treatment monitoring. The multiplex Oncuria BC assay noninvasively quantifies the concentration of 10 protein analytes in voided urine samples to quickly generate a unique molecular profile with proven BC diagnostic and treatment-tracking utility. Test adoption by diagnostic and research laboratories mandates reliably reproducible assay performance across a variety of instrumentation platforms used in different laboratories.MethodsWe compared the performance of the clinically validated Oncuria BC multiplex immunoassay when data output was generated on three different analyzer systems. Voided urine samples from 36 subjects (18 with BC and 18 Controls) were reacted with Oncuria test reagents in three 96-well microtiter plates on Day 1, and consecutively evaluated on the LED/image-based MagPix, and laser/flow based Luminex 200 and FlexMap 3D (all xMAP instruments from Luminex Corp., Austin, TX) on Day 2. The BC assay uses magnetic bead-based fluorescence technology (xMAP, Multi-analyte profiling; Luminex) to simultaneously quantify 10 protein analytes in urine specimens [i.e., angiogenin (ANG), apolipoprotein E (ApoE), carbonic anhydrase IX (CA9), CXCL8/interleukin-8 (IL-8), matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-10 (MMP-10), serpin A1/alpha-1 anti-trypsin (A1AT), serpin E1/plasminogen activator inhibitor-1 (PAI-1), CD138/syndecan-1 (SDC1), and vascular endothelial growth factor-A (VEGF-A)].ResultsAll three platforms categorized all 10 analytes in identical samples at nearly identical concentrations, with variance across systems typically <5%. While the most contemporary instrument, the FlexMap 3D, output higher raw fluorescence values than the two comparator systems, standard curve slopes and analyte concentrations determined in urine samples were concordant across all three units. Forty-four percent of BC samples registered ≥1 analyte above the highest standard concentration, i.e., A1AT (n=7/18), IL-8 (n=5), and/or ANG (n=2). In Controls, A1AT was higher in one sample.ConclusionMultiplex BC assays generate detailed molecular signatures useful for identifying BC, predicting treatment esponsiveness, and tracking disease progression and recurrence. The similar performance of the Oncuria assay across three different analyzer systems supports test adaptation by clinical and research laboratories using existing xMAP platforms.Trial registrationThis study was registered at ClinicalTrials.gov as NCT04564781, NCT03193528, NCT03193541, and NCT03193515.
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Project description:Single nucleotide polymorphisms (SNPs) of genes that affect cytokine production and function are known to influence the susceptibility and progression of immune-related conditions such as infection, autoimmune diseases, transplantation, and cancer. We established a multiplex genotyping method to analyze the SNPs of cytokine genes by combining the multiplex PCR and bead array platform. Thirteen cytokine gene regions, including 20 SNPs, were amplified, and allele-specific primer extension was performed in a single tube. High-quality allele-specific primers were selected for signals greater than 1000 median fluorescence intensity (MFI) for positive alleles, and less than 500 MFI for negative alleles. To select and improve the extension primers, modifications for the reverse direction, length or refractory were performed. 24 primers in the forward or reverse direction step and 12 primers in length or refractory modifications were selected and showed high concordance with results by nucleotide sequencing. Among the 13 candidate cytokine genes, the SNPs of 12 cytokine genes, including IL-1?, IL-1R, IL-1RA, IL-1?, IL-2, IL-4, IL-4R?, IL-6, IL-10, IL-12, TGF-?1, and TNF-?, were successfully defined with the selected allele-specific primers in healthy Korean subjects. Our genotyping system provides a fast and accurate detection for SNPs of multiple cytokine genes to investigate their association with immune-related diseases and transplantation outcomes.
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Project description:The assembly of a highly parallel force spectroscopy tool requires careful placement of single-molecule targets on the substrate and the deliberate manipulation of a multitude of force probes. Since the probe must approach the target biomolecule for covalent attachment, while avoiding irreversible adhesion to the substrate, the use of polymer microspheres as force probes to create the tethered bead array poses a problem. Therefore, the interactions between the force probe and the surface must be repulsive at very short distances (<5 nm) and attractive at long distances. To achieve this balance, the chemistry of the substrate, force probe, and solution must be tailored to control the probe-surface interactions. In addition to an appropriately designed chemistry, it is necessary to control the surface density of the target molecule in order to ensure that only one molecule is interrogated by a single force probe. We used gold-thiol chemistry to control both the substrate's surface chemistry and the spacing of the studied molecules, through binding of the thiol-terminated DNA and an inert thiol forming a blocking layer. For our single molecule array, we modeled the forces between the probe and the substrate using DLVO theory and measured their magnitude and direction with colloidal probe microscopy. The practicality of each system was tested using a probe binding assay to evaluate the proportion of the beads remaining adhered to the surface after application of force. We have translated the results specific for our system to general guiding principles for preparation of tethered bead arrays and demonstrated the ability of this system to produce a high yield of active force spectroscopy probes in a microwell substrate. This study outlines the characteristics of the chemistry needed to create such a force spectroscopy array.
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