An open-source software analysis package for Microspheres with Ratiometric Barcode Lanthanide Encoding (MRBLEs)

RESEARCH ARTICLE An open-source software analysis package for Microspheres with Ratiometric Barcode Lanthanide Encoding (MRBLEs) Björn Harink ID1, Huy Nguyen1, Kurt Thorn2, Polly Fordyce ID1,3,4,5* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Genetics, Stanford University, Stanford, California, United States of America, 2 Department of Biochemistry & Biophysics, University of California, San Francisco, United States of America, 3 Department of Bioengineering, Stanford University, Stanford, California, United States of America, 4 ChEM-H Institute, Stanford University, Stanford, California, United States of America, 5 Chan Zuckerberg Biohub, San Francisco, California, United States of America * Abstract OPEN ACCESS Citation: Harink B, Nguyen H, Thorn K, Fordyce P (2019) An open-source software analysis package for Microspheres with Ratiometric Barcode Lanthanide Encoding (MRBLEs). PLoS ONE 14(3): e0203725. https://doi.org/10.1371/journal. pone.0203725 Editor: Vadim E. Degtyar, University of California Berkeley, UNITED STATES Received: August 20, 2018 Accepted: February 20, 2019 Published: March 22, 2019 Copyright: © 2019 Harink et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All images and input files used for this manuscript are available from the OSF database (accession number: 8kv35, link: https://osf.io/8kv35/). Source code is available through GitHub (link: https://github.com/ FordyceLab/MRBLEs). Funding: This work was supported by NIH/NIGMS grants DP2GM123641 and R01GM107132. In addition, P.M.F. is a Chan Zuckerberg Biohub Investigator and acknowledges support from the Beckman Foundation, and the Sloan Research Multiplexed bioassays, in which multiple analytes of interest are probed in parallel within a single small volume, have greatly accelerated the pace of biological discovery. Bead-based multiplexed bioassays have many technical advantages, including near solution-phase kinetics, small sample volume requirements, many within-assay replicates to reduce measurement error, and, for some bead materials, the ability to synthesize analytes directly on beads via solid-phase synthesis. To allow bead-based multiplexing, analytes can be synthesized on spectrally encoded beads with a 1:1 linkage between analyte identity and embedded codes. Bead-bound analyte libraries can then be pooled and incubated with a fluorescently-labeled macromolecule of interest, allowing downstream quantification of interactions between the macromolecule and all analytes simultaneously via imaging alone. Extracting quantitative binding data from these images poses several computational image processing challenges, requiring the ability to identify all beads in each image, quantify bound fluorescent material associated with each bead, and determine their embedded spectral code to reveal analyte identities. Here, we present a novel open-source Python software package (the mrbles analysis package) that provides the necessary tools to: (1) find encoded beads in a bright-field microscopy image; (2) quantify bound fluorescent material associated with bead perimeters; (3) identify embedded ratiometric spectral codes within beads; and (4) return data aggregated by embedded code and for each individual bead. We demonstrate the utility of this package by applying it towards analyzing data generated via multiplexed measurement of calcineurin protein binding to MRBLEs (Microspheres with Ratiometric Barcode Lanthanide Encoding) containing known and mutant binding peptide motifs. We anticipate that this flexible package should be applicable to a wide variety of assays, including simple bead or droplet finding analysis, quantification of binding to nonencoded beads, and analysis of multiplexed assays that use ratiometric, spectrally encoded beads. PLOS ONE | https://doi.org/10.1371/journal.pone.0203725 March 22, 2019 1 / 20 Open-source software analysis package for MRBLEs Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Introduction In traditional ‘single-plex’ assays, each set of potential interactions between 2 macromolecules (e.g. binding of a protein to DNA or another protein) is assessed in a single volume, with some readout to indicate whether or not a reaction has occurred. Multiplexing technologies enable the measurement of many analytes of interest within a single small volume, thereby saving precious reagents and reducing the cost and labor associated with each assay. Multiplexed beadbased assays, in particular, have been used for a wide range of applications in both research and diagnostics [1–8] In these assays, different analytes are tethered to beads with a one-toone linkage between analyte and beads. Bead-bound analyte libraries can then be pooled and tested in parallel for reaction with a probe molecule (e.g. testing protein binding or oligonucleotide hybridization to bead-bound peptides or oligos). Beads provide a support for solid-phase chemical synthesis (e.g. peptide or oligonucleotide synthesis), enabling rapid, precise, and lowcost library generation [9]. In addition, bead-based assays allow near-solution phase kinetics for measurement of up to hundreds of analytes simultaneously in volumes as low as 20 μL, with similar or better sensitivity and specificity relative to ELISA [1,10,11]. Finally, each assay can include many bead replicates for a particular analyte, reducing measurement error and enhancing the ability to detect subtle quantitative differences between analytes. Multiplexing bead-based assays requires the ability to reliably identify all bead-bound analytes. This can be accomplished via a ‘one-code-one-compound’ approach in which beads are encoded and each analyte is tethered to or synthesized on beads bearing a different code (Fig 1A) [9]. Various encoding strategies exist, including chemical encoding (in which beads are tagged with distinct chemical compounds) [1] and optical encoding (in which beads are produced with multiple shapes, embedded colors, or both) [12]. Optical encoding allows bead codes to be identified via imaging without a need for equipment-intensive downstream analysis (e.g. mass spectrometry or sequencing). In addition, optical encoding allows nondestructive code identification, facilitating kinetic monitoring of on-bead activity throughout an experiment. Finally, optically-encoded beads can be imaged with relatively cheap, or even mobile, equipment such as low-cost hyperspectral cameras, potentially enabling point-of-care applications [13]. We recently developed a novel technology for producing spectrally encoded beads, wh (...truncated)