class: center, middle, inverse, title-slide # {flowDiv} workflow: reproducible cytometric diversity estimates ### María Victoria Quiroga, PhD --- class: left, middle .center[ <img src="img/bruno.png" style="border-radius: 80%;" width="100pxl" alt="Photo of BMS Wanderley" /> <img src="img/vi.jpg" style="border-radius: 80%;" width="100pxl" alt="Photo of MV Quiroga" /> <img src="img/Andre.jpeg" style="border-radius: 80%;" width="100pxl" alt="Photo of AM Amado" /> <img src="img/Fer.png" style="border-radius: 80%;" width="90pxl" alt="Photo of F Unrein" /> ] * __Bruno MS Wanderley__, Departamento de Oceanografia e Limnologia, Universidade Federal do Rio Grande do Norte, Brazil * __María Victoria Quiroga__, Instituto Tecnológico de Chascomús (INTECH, UNSAM-CONICET), Argentina * __André M Amado__, Departamento de Biologia, Universidade Federal de Juiz de Fora, Brazil * __Fernando Unrein__, Instituto Tecnológico de Chascomús (INTECH, UNSAM-CONICET), Argentina .right[.large[.bold[People behind _flowDiv_]]] --- class: chapter-slide # Background --- class: left, middle ### Basics of flow cytometry .pull-left[ .large[.bold[ * Single-cell multiple data (channels) * Scattered Light * Fluorescence * File type .fcs * Thousands of cells * High-throughput data ]]] .pull-right[ <img src="img/cytometer.png" width="420pxl" alt="Schematic functioning of a flow cytometer, showing laser, fluidic and optic systems and electronics" /> .right[.tiny[Figure source: Rowley (2012) [MATER METHODS 2:125](https://www.labome.com/method/Flow-Cytometry-A-Survey-and-the-Basics.html)]]] --- class: left, middle .pull-left[ ### Data visualization <img src="img/cito1.png" width="90%" alt="Cytogram of environmental bacteria, showing a biplot of channels SSC and FL1" /> Cytograms * Dots in the biplot = cells * 3D (colour) = abundance ] -- .pull-right[ ### Gating Strategy <img src="img/cito2.png" width="100%" alt="The same cytogram showing gating strategy" /> Gates: * Remove noise * Standard * Cytometric (sub)population ] --- class: left, middle ### Cytogram: Cytometric Fingerprint .large[.bold[ * Comparing cytograms is not straightforward * Environmental samples acquired with different: * Instrumental settings * Dilution factor ]] --- class: chapter-slide # Data processing --- ### Before _flowDiv_ .pull-left[.large[__1. [FlowJo Software__](https://docs.flowjo.com/flowjo/)] .full-width[.content-box-blue[**Sub**populations and Standard gates * Channel median values * Cell counts Export table into Excell]] .large[__3. Statistical Software__] .full-width[.content-box-red[ Multivariate analyzes]] ] .pull-right[.large[__2. Excel__] .full-width[.content-box-yellow[Data handling * Subpopulation channel median relative to Standard * Dilution factors Reduced data dimension]] .right[.small[Publication: [Schiaffino _et al._ 2013](https://doi.org/10.3354/ame01612)]]] --- ### With _flowDiv_ .pull-left[.large[__1. [FlowJo Software__](https://docs.flowjo.com/flowjo/)] .full-width[.content-box-blue[Population and Standard gates * single cell data with channel values Delete subjective subgating ]]] .pull-right[.large[__2. R__] .full-width[.content-box-yellow[_**flowDiv**_ * Import gates into R * Channel normalization (Standard) * Volume correction * Diversity indexes Higher data dimension Multivariate analyzes]] .right[.small[Publication: [Quiroga _et al._ 2017](https://link.springer.com/article/10.1007/s10750-016-2969-2) [Wanderley _et al._ 2019](https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-019-2787-4)]] ] --- class: chapter-slide # _flowDiv_ arguments .right[<img src="https://media.springernature.com/lw685/springer-static/image/art%3A10.1186%2Fs12859-019-2787-4/MediaObjects/12859_2019_2787_Fig1_HTML.png?as=webp" width="55%" alt="Schematic view of the flowDiv workflow" /> <br /> <br /> <br /> <br /> <br /> <br /> <br /> .tiny[**Schematic view of the flowDiv workflow** Figure source: [Wanderley _et al._ 2019](https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-019-2787-4)]] --- class: left, middle ### Channel normalization .content-box-grey[.small[.code[flowDiv( ... , .blue[use.beads = TRUE, beads = "Standard"])]]] .bold[ <br />] <img src="img/normalizacionBeads.png" width="100%" alt="Schematic view of the normalization of two cytograms in relation to Standard mean" /> .bold[Centers all cytograms based on the Standard mean]<br /> .right[.tiny[Figure modified from: Wanderley 2019 [PhD Thesis](https://repositorio.ufrn.br/bitstream/123456789/28381/1/flowDivnova_Wanderley_2019.pdf)]] --- class: left, middle ### Binning resolution .bold[Interactive code: suggests number of binns] <img src="img/binns.png" width="100%" alt="Schematic view of channel binning and contingency table construction" /> Example: the channels were clustered into 5 binns .right[Contingency table: .small[cell count for the 5x5:25 cytometric categories]] .right[.tiny[Figure modified from: Wanderley 2019 PhD Defense]] --- class: left, middle ### Volume correction .content-box-grey[.small[.code[flowDiv( ... , .blue[dilutions = vector])]]] .bold[ <br /> Vector containing dilution factor for all samples] --- class: chapter-slide # Challenges and future goals --- class: left, middle ### Maintenance .large[.bold[ * Changes in [FlowJo Software](https://docs.flowjo.com/flowjo/) * Wait for dependency updates * [_CytoML_](https://bioconductor.org/packages/release/bioc/html/CytoML.html) * [_flowCore_](https://www.bioconductor.org/packages/release/bioc/html/flowCore.html) * [_flowWorkspace_](https://www.bioconductor.org/packages/release/bioc/html/flowWorkspace.html) * Update _flowDiv_]] --- class: left, middle ### The path forward .large[.bold[ Develop * Suitable gating approach in R<br /> * _flowDiv_ tutorial (learnr <svg viewBox="0 0 640 512" style="height:1em;position:relative;display:inline-block;top:.1em;" xmlns="http://www.w3.org/2000/svg"> <path d="M425.7 256c-16.9 0-32.8-9-41.4-23.4L320 126l-64.2 106.6c-8.7 14.5-24.6 23.5-41.5 23.5-4.5 0-9-.6-13.3-1.9L64 215v178c0 14.7 10 27.5 24.2 31l216.2 54.1c10.2 2.5 20.9 2.5 31 0L551.8 424c14.2-3.6 24.2-16.4 24.2-31V215l-137 39.1c-4.3 1.3-8.8 1.9-13.3 1.9zm212.6-112.2L586.8 41c-3.1-6.2-9.8-9.8-16.7-8.9L320 64l91.7 152.1c3.8 6.3 11.4 9.3 18.5 7.3l197.9-56.5c9.9-2.9 14.7-13.9 10.2-23.1zM53.2 41L1.7 143.8c-4.6 9.2.3 20.2 10.1 23l197.9 56.5c7.1 2 14.7-1 18.5-7.3L320 64 69.8 32.1c-6.9-.8-13.5 2.7-16.6 8.9z"></path></svg>)]] --- class: left, middle ### Questions/Feedback .large[ .bold[Bruno MS Wanderley]<br/> bruno.mattoswanderley@gmail.com <svg viewBox="0 0 496 512" style="height:1em;position:relative;display:inline-block;top:.1em;" xmlns="http://www.w3.org/2000/svg"> <path d="M165.9 397.4c0 2-2.3 3.6-5.2 3.6-3.3.3-5.6-1.3-5.6-3.6 0-2 2.3-3.6 5.2-3.6 3-.3 5.6 1.3 5.6 3.6zm-31.1-4.5c-.7 2 1.3 4.3 4.3 4.9 2.6 1 5.6 0 6.2-2s-1.3-4.3-4.3-5.2c-2.6-.7-5.5.3-6.2 2.3zm44.2-1.7c-2.9.7-4.9 2.6-4.6 4.9.3 2 2.9 3.3 5.9 2.6 2.9-.7 4.9-2.6 4.6-4.6-.3-1.9-3-3.2-5.9-2.9zM244.8 8C106.1 8 0 113.3 0 252c0 110.9 69.8 205.8 169.5 239.2 12.8 2.3 17.3-5.6 17.3-12.1 0-6.2-.3-40.4-.3-61.4 0 0-70 15-84.7-29.8 0 0-11.4-29.1-27.8-36.6 0 0-22.9-15.7 1.6-15.4 0 0 24.9 2 38.6 25.8 21.9 38.6 58.6 27.5 72.9 20.9 2.3-16 8.8-27.1 16-33.7-55.9-6.2-112.3-14.3-112.3-110.5 0-27.5 7.6-41.3 23.6-58.9-2.6-6.5-11.1-33.3 2.6-67.9 20.9-6.5 69 27 69 27 20-5.6 41.5-8.5 62.8-8.5s42.8 2.9 62.8 8.5c0 0 48.1-33.6 69-27 13.7 34.7 5.2 61.4 2.6 67.9 16 17.7 25.8 31.5 25.8 58.9 0 96.5-58.9 104.2-114.8 110.5 9.2 7.9 17 22.9 17 46.4 0 33.7-.3 75.4-.3 83.6 0 6.5 4.6 14.4 17.3 12.1C428.2 457.8 496 362.9 496 252 496 113.3 383.5 8 244.8 8zM97.2 352.9c-1.3 1-1 3.3.7 5.2 1.6 1.6 3.9 2.3 5.2 1 1.3-1 1-3.3-.7-5.2-1.6-1.6-3.9-2.3-5.2-1zm-10.8-8.1c-.7 1.3.3 2.9 2.3 3.9 1.6 1 3.6.7 4.3-.7.7-1.3-.3-2.9-2.3-3.9-2-.6-3.6-.3-4.3.7zm32.4 35.6c-1.6 1.3-1 4.3 1.3 6.2 2.3 2.3 5.2 2.6 6.5 1 1.3-1.3.7-4.3-1.3-6.2-2.2-2.3-5.2-2.6-6.5-1zm-11.4-14.7c-1.6 1-1.6 3.6 0 5.9 1.6 2.3 4.3 3.3 5.6 2.3 1.6-1.3 1.6-3.9 0-6.2-1.4-2.3-4-3.3-5.6-2z"></path></svg>[bmsw](https://github.com/bmsw)<br/> .bold[María Victoria Quiroga]<br/> mvquiroga@iib.unsam.edu.ar <svg viewBox="0 0 512 512" style="height:1em;position:relative;display:inline-block;top:.1em;" xmlns="http://www.w3.org/2000/svg"> <path d="M459.37 151.716c.325 4.548.325 9.097.325 13.645 0 138.72-105.583 298.558-298.558 298.558-59.452 0-114.68-17.219-161.137-47.106 8.447.974 16.568 1.299 25.34 1.299 49.055 0 94.213-16.568 130.274-44.832-46.132-.975-84.792-31.188-98.112-72.772 6.498.974 12.995 1.624 19.818 1.624 9.421 0 18.843-1.3 27.614-3.573-48.081-9.747-84.143-51.98-84.143-102.985v-1.299c13.969 7.797 30.214 12.67 47.431 13.319-28.264-18.843-46.781-51.005-46.781-87.391 0-19.492 5.197-37.36 14.294-52.954 51.655 63.675 129.3 105.258 216.365 109.807-1.624-7.797-2.599-15.918-2.599-24.04 0-57.828 46.782-104.934 104.934-104.934 30.213 0 57.502 12.67 76.67 33.137 23.715-4.548 46.456-13.32 66.599-25.34-7.798 24.366-24.366 44.833-46.132 57.827 21.117-2.273 41.584-8.122 60.426-16.243-14.292 20.791-32.161 39.308-52.628 54.253z"></path></svg>[@ViQu20](https://twitter.com/ViQu20) <svg viewBox="0 0 496 512" style="height:1em;position:relative;display:inline-block;top:.1em;" xmlns="http://www.w3.org/2000/svg"> <path d="M165.9 397.4c0 2-2.3 3.6-5.2 3.6-3.3.3-5.6-1.3-5.6-3.6 0-2 2.3-3.6 5.2-3.6 3-.3 5.6 1.3 5.6 3.6zm-31.1-4.5c-.7 2 1.3 4.3 4.3 4.9 2.6 1 5.6 0 6.2-2s-1.3-4.3-4.3-5.2c-2.6-.7-5.5.3-6.2 2.3zm44.2-1.7c-2.9.7-4.9 2.6-4.6 4.9.3 2 2.9 3.3 5.9 2.6 2.9-.7 4.9-2.6 4.6-4.6-.3-1.9-3-3.2-5.9-2.9zM244.8 8C106.1 8 0 113.3 0 252c0 110.9 69.8 205.8 169.5 239.2 12.8 2.3 17.3-5.6 17.3-12.1 0-6.2-.3-40.4-.3-61.4 0 0-70 15-84.7-29.8 0 0-11.4-29.1-27.8-36.6 0 0-22.9-15.7 1.6-15.4 0 0 24.9 2 38.6 25.8 21.9 38.6 58.6 27.5 72.9 20.9 2.3-16 8.8-27.1 16-33.7-55.9-6.2-112.3-14.3-112.3-110.5 0-27.5 7.6-41.3 23.6-58.9-2.6-6.5-11.1-33.3 2.6-67.9 20.9-6.5 69 27 69 27 20-5.6 41.5-8.5 62.8-8.5s42.8 2.9 62.8 8.5c0 0 48.1-33.6 69-27 13.7 34.7 5.2 61.4 2.6 67.9 16 17.7 25.8 31.5 25.8 58.9 0 96.5-58.9 104.2-114.8 110.5 9.2 7.9 17 22.9 17 46.4 0 33.7-.3 75.4-.3 83.6 0 6.5 4.6 14.4 17.3 12.1C428.2 457.8 496 362.9 496 252 496 113.3 383.5 8 244.8 8zM97.2 352.9c-1.3 1-1 3.3.7 5.2 1.6 1.6 3.9 2.3 5.2 1 1.3-1 1-3.3-.7-5.2-1.6-1.6-3.9-2.3-5.2-1zm-10.8-8.1c-.7 1.3.3 2.9 2.3 3.9 1.6 1 3.6.7 4.3-.7.7-1.3-.3-2.9-2.3-3.9-2-.6-3.6-.3-4.3.7zm32.4 35.6c-1.6 1.3-1 4.3 1.3 6.2 2.3 2.3 5.2 2.6 6.5 1 1.3-1.3.7-4.3-1.3-6.2-2.2-2.3-5.2-2.6-6.5-1zm-11.4-14.7c-1.6 1-1.6 3.6 0 5.9 1.6 2.3 4.3 3.3 5.6 2.3 1.6-1.3 1.6-3.9 0-6.2-1.4-2.3-4-3.3-5.6-2z"></path></svg>[mvquiroga](https://github.com/mvquiroga)]