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New Single Cell Tutorial in R #4141

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Camila-goclowski May 17, 2023
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski May 17, 2023
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Preamble edited
Camila-goclowski May 17, 2023
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski May 17, 2023
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Reformatted tutorial.md
Camila-goclowski May 17, 2023
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added cluster labelling might change filtering
Camila-goclowski May 17, 2023
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski May 17, 2023
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski Jun 13, 2023
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remainder of tutorial added
Camila-goclowski Jun 13, 2023
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reformatting
Camila-goclowski Jun 13, 2023
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski Jun 13, 2023
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Merge branch 'Filter,-Plot,-and-Explore_WIP' of https://github.com/Ca…
Camila-goclowski Jun 13, 2023
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removed tutorial.html per helena's rec
Camila-goclowski Jun 13, 2023
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Helena's comments fixed--pt2
Camila-goclowski Jun 13, 2023
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pt3
Camila-goclowski Jun 13, 2023
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski Jun 29, 2023
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formatting changes
Camila-goclowski Jun 29, 2023
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski Jul 10, 2023
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Merge branch 'Filter,-Plot,-and-Explore_WIP' of https://github.com/Ca…
Camila-goclowski Jul 10, 2023
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code formatting fix
Camila-goclowski Jul 10, 2023
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code formatting (equals) correction
Camila-goclowski Jul 10, 2023
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code block formatting
Camila-goclowski Jul 10, 2023
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authorship correction
Camila-goclowski Jul 11, 2023
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski Jul 11, 2023
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recorrected contributors
Camila-goclowski Jul 11, 2023
c8223f0
jekyll fix--hopefully
Camila-goclowski Jul 11, 2023
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fixed broken box?
Camila-goclowski Jul 11, 2023
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jekyll fix in preamble
Camila-goclowski Jul 11, 2023
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added first eight plots
Camila-goclowski Jul 11, 2023
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code format test
Camila-goclowski Jul 12, 2023
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski Jul 12, 2023
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fixed blocking issue at 141
Camila-goclowski Jul 12, 2023
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another block fix
Camila-goclowski Jul 12, 2023
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski Jul 13, 2023
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removed redundant library calling
Camila-goclowski Jul 13, 2023
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made adding metadata a little less clunky
Camila-goclowski Jul 13, 2023
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actually fixed block issue (extra space on 188)
Camila-goclowski Jul 13, 2023
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added plots 9-12
Camila-goclowski Jul 13, 2023
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added plots 13-17
Camila-goclowski Jul 13, 2023
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almost done formatting -- missing 2 plots
Camila-goclowski Aug 1, 2023
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added zonodo link
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corrected and added final plots
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line edits from Marisa
Camila-goclowski Aug 14, 2023
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added where data comes from
Camila-goclowski Aug 16, 2023
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added link to own history w/ data
Camila-goclowski Aug 16, 2023
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski Aug 16, 2023
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reformatted some tip/comment boxes
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Merge branch 'Filter,-Plot,-and-Explore_WIP' of https://github.com/Ca…
Camila-goclowski Aug 16, 2023
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DE box fix
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function fixes
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broken boxes ID'd by lint check
Camila-goclowski Aug 16, 2023
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two more potentisl broken boxes
Camila-goclowski Aug 16, 2023
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corrected broken links
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Update topics/single-cell/tutorials/scrna-case_FilterPlotandExploreRS…
Camila-goclowski Aug 23, 2023
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Update topics/single-cell/tutorials/scrna-case_FilterPlotandExploreRS…
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Update topics/single-cell/tutorials/scrna-case_FilterPlotandExploreRS…
Camila-goclowski Aug 23, 2023
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Camila-goclowski Aug 23, 2023
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Update topics/single-cell/tutorials/scrna-case_FilterPlotandExploreRS…
Camila-goclowski Aug 30, 2023
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Update topics/single-cell/tutorials/scrna-case_FilterPlotandExploreRS…
Camila-goclowski Aug 30, 2023
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Update topics/single-cell/tutorials/scrna-case_FilterPlotandExploreRS…
Camila-goclowski Aug 30, 2023
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Update topics/single-cell/tutorials/scrna-case_FilterPlotandExploreRS…
Camila-goclowski Aug 30, 2023
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Update topics/single-cell/tutorials/scrna-case_FilterPlotandExploreRS…
Camila-goclowski Aug 30, 2023
e073780
added zenodo and fixed hempty history
Camila-goclowski Aug 30, 2023
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added zenodo link and fixed empty history link
Camila-goclowski Aug 30, 2023
66cde2c
Merge branch 'Filter,-Plot,-and-Explore_WIP' of https://github.com/Ca…
Camila-goclowski Aug 30, 2023
9c56236
reorganized Get Data section
Camila-goclowski Sep 15, 2023
209cf23
reformatted first hands on box in preamble
Camila-goclowski Sep 15, 2023
6d820af
added plot 8 (genes x mito zoomed in)
Camila-goclowski Sep 15, 2023
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added saving object and plots to end
Camila-goclowski Sep 15, 2023
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linting error link removed
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Merge branch 'galaxyproject:main' into Filter,-Plot,-and-Explore_WIP
Camila-goclowski Sep 21, 2023
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Update topics/single-cell/tutorials/scrna-case_FilterPlotandExploreRS…
Camila-goclowski Sep 26, 2023
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added references for citations (tutorial.bib file)
Camila-goclowski Sep 26, 2023
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replaced author list commas with "and"s in bib
Camila-goclowski Sep 27, 2023
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Update topics/single-cell/tutorials/scrna-case_FilterPlotandExploreRS…
shiltemann Oct 2, 2023
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fix citations
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6 changes: 6 additions & 0 deletions CONTRIBUTORS.yaml
Original file line number Diff line number Diff line change
Expand Up @@ -319,6 +319,12 @@ cfusterbarcelo:
orcid: 0000-0002-4784-6957
bio: Post-doctoral researcher at UC3M

Camila-goclowski:
name: Camila Goclowski
joined: 2023-01
email: [email protected]
linkedin: camila-goclowski

charitylaw:
name: Charity Law
joined: 2018-09
Expand Down
55 changes: 17 additions & 38 deletions Gemfile.lock
Original file line number Diff line number Diff line change
@@ -1,15 +1,8 @@
GEM
remote: https://rubygems.org/
specs:
Ascii85 (1.1.0)
addressable (2.8.4)
addressable (2.8.5)
public_suffix (>= 2.0.2, < 6.0)
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console (~> 1.10)
fiber-annotation
io-event (~> 1.1)
timers (~> 4.1)
awesome_bot (1.20.0)
parallel (= 1.20.1)
bibtex-ruby (6.0.0)
Expand All @@ -23,9 +16,6 @@ GEM
commander (4.6.0)
highline (~> 2.0.0)
concurrent-ruby (1.2.2)
console (1.17.2)
fiber-annotation
fiber-local
csl (2.0.0)
namae (~> 1.0)
rexml
Expand All @@ -38,26 +28,22 @@ GEM
ffi (>= 1.15.0)
eventmachine (1.2.7)
fastimage (2.2.7)
ffi (1.15.5)
fiber-annotation (0.2.0)
fiber-local (1.0.0)
ffi (1.16.2)
forwardable-extended (2.6.0)
google-protobuf (3.23.3-x86_64-linux)
hashery (2.1.2)
google-protobuf (3.24.3-x86_64-linux)
highline (2.0.3)
html-proofer (5.0.7)
html-proofer (4.4.3)
addressable (~> 2.3)
async (~> 2.1)
mercenary (~> 0.3)
nokogiri (~> 1.13)
pdf-reader (~> 2.11)
parallel (~> 1.10)
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typhoeus (~> 1.3)
yell (~> 2.0)
zeitwerk (~> 2.5)
http_parser.rb (0.8.0)
i18n (1.14.1)
concurrent-ruby (~> 1.0)
io-event (1.2.2)
jekyll (4.3.2)
addressable (~> 2.4)
colorator (~> 1.0)
Expand Down Expand Up @@ -94,40 +80,33 @@ GEM
rb-inotify (~> 0.9, >= 0.9.10)
mercenary (0.4.0)
namae (1.1.1)
nokogiri (1.15.2-x86_64-linux)
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parallel (1.20.1)
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forwardable-extended (~> 2.6)
pdf-reader (2.11.0)
Ascii85 (~> 1.0)
afm (~> 0.2.1)
hashery (~> 2.0)
ruby-rc4
ttfunk
pkg-config (1.5.2)
public_suffix (5.0.1)
pkg-config (1.5.5)
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racc (1.7.1)
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unicode-display_width (>= 1.1.1, < 3)
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zeitwerk (2.6.12)

PLATFORMS
x86_64-linux
Expand All @@ -150,4 +129,4 @@ DEPENDENCIES
webrick

BUNDLED WITH
2.4.2
2.4.20
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---
layout: faq-page
---
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# Introduction

You’ve previously done all the work to make a single cell matrix. Now it’s time to fully process our data using Seurat: remove low quality cells, reduce the many dimensions of data that make it difficult to work with, and ultimately try to define clusters and find some biological meaning and insights! There are many packages for analysing single cell data - Seurat ({% cite Satija2015 %}), Scanpy ({% cite Wolf2018 %}), Monocle ({% cite Trapnell2014 %}), Scater ({% cite McCarthy2017 %}), and many more. We’re working with Seurat in RStudio because it is well updated, broadly used, and highly trusted within the field of bioinformatics.

> <comment-title></comment-title>
> This tutorial is significantly based on the Seurat documentation({% cite Satija2015 %}) as well as [Seurat's Guided Clustering Tutorial](https://satijalab.org/seurat/articles/pbmc3k_tutorial.html).
{: .comment}

> <agenda-title></agenda-title>
>
> In this tutorial, we will cover:
>
> 1. TOC
> {:toc}
>
{: .agenda}

We’ll provided you with experimental data to analyse from a mouse dataset of fetal growth restriction ({% cite Bacon2018 %}). This is the full dataset generated from [this tutorial]({% link topics/single-cell/tutorials/scrna-case_alevin/tutorial.md %}).

# Get Data onto Galaxy
To start, let's get our dataset loaded into Galaxy.

You can access the data for this tutorial in multiple ways:
1. **EBI Data Retrieval** - You may retrieve that files necessary to construct a Seurat Object in this way.Doing to will alleviate the necessity to convert AnnData (Python) objects into Seurat (R) objects:

> <hands-on-title>GetData</hands-on-title>
>
> Run{% tool [EBI SCXA Data Retrieval](toolshed.g2.bx.psu.edu/repos/ebi-gxa/retrieve_scxa/retrieve_scxa/v0.0.2+galaxy2) %} with the following parameters:
> - *"SC-Atlas experiment accession"*: `E-MTAB-6945`
> - *"Choose the type of matrix to download"*: `Raw filtered counts`
{: .hands_on}

2. **Importing from a history** - You can import [this history](https://usegalaxy.eu/u/camila-goclowski/h/fpe)

{% snippet faqs/galaxy/histories_import.md %}
This also alleviates the necessity to convert the AnnData object into a Seurat one.

3. **Uploading from Zenodo** (see below)

> <hands-on-title>Option 3: Uploading from Zenodo</hands-on-title>
>
> 1. Create a new history for this tutorial
> 2. Import the AnnData object from [Zenodo]({{ page.zenodo_link }})
>
> ```
> {{ page.zenodo_link }}/files/Mito-counted_AnnData
> ```
>
> {% snippet faqs/galaxy/datasets_import_via_link.md %}
>
> 3. **Rename** {% icon galaxy-pencil %} the datasets `Mito-counted AnnData`
> 4. Check that the datatype is `h5ad`
>
> {% snippet faqs/galaxy/datasets_change_datatype.md datatype="h5ad" %}
>
{: .hands_on}

# Important tips for easier analysis

{% snippet faqs/galaxy/tutorial_mode.md %}

> <comment-title></comment-title>
> - The Galaxy tool search panel sometimes doesn't find the tools we need from the thousands available.
> - You'll have a much easier time selecting tools from the panel (if you aren't using tutorial mode!) if you are on the [https://humancellatlas.usegalaxy.eu](https://humancellatlas.usegalaxy.eu)
{: .comment}

# Open RStudio in Galaxy
You now should have imported the matrix.mtx, genes.tsv, barcodes.tsv, and exp_design.tsv files into your Galaxy history. For the rest of the workflow, let's move onto RStudio and get coding!
> <hands-on-title>Open RStudio in Galaxy</hands-on-title>
> Run {% tool [RStudio](interactive_tool_rstudio)%}
{: .hands_on}


><comment-title>Next Step</comment-title>
> The interactive RStudio tool should begin to load now. Make your way over to your Active Interactive Tools page (User (in the top bar of the usegalaxy page)> Active Interactive Tools > RStudio)
>
>Alternatively, you may use the view (eye) button in your Galaxy History to open the interactive RStudio environment.
{: .comment}
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# This is the bibliography file for your tutorial.
#
# To add bibliography (bibtex) entries here, follow these steps:
# 1) Find the DOI for the article you want to cite
# 2) Go to https://doi2bib.org and fill in the DOI
# 3) Copy the resulting bibtex entry into this file
#
# To cite the example below, in your tutorial.md file
# use {% cite Batut2018 %}
#
# If you want to cite an online resourse (website etc)
# you can use the 'online' format (see below)
#
# You can remove the examples below

@article{Bacon2018,
doi = {10.3389/fimmu.2018.02523},
url = {https://doi.org/10.3389/fimmu.2018.02523},
year = {2018},
month = nov,
publisher = {Frontiers Media {SA}},
volume = {9},
author = {Wendi A. Bacon and Russell S. Hamilton and Ziyi Yu and Jens Kieckbusch and Delia Hawkes and Ada M. Krzak and Chris Abell and Francesco Colucci and D. Stephen Charnock-Jones},
title = {Single-Cell Analysis Identifies Thymic Maturation Delay in Growth-Restricted Neonatal Mice},
journal = {Frontiers in Immunology}
}

@article{Wolf2018,
doi = {10.1186/s13059-017-1382-0},
url = {https://doi.org/10.1186/s13059-017-1382-0},
year = {2018},
month = feb,
publisher = {Springer Science and Business Media {LLC}},
volume = {19},
number = {1},
author = {F. Alexander Wolf and Philipp Angerer and Fabian J. Theis},
title = {{SCANPY}: large-scale single-cell gene expression data analysis},
journal = {Genome Biology}
}

@article{Satija2015,
doi = {10.1038/nbt.3192},
url = {https://doi.org/10.1038/nbt.3192},
year = {2015},
month = May,
publisher = {Nature Anerica Inc. All rights reserved},
volume = {33},
number = {5},
author = {Rahul Satija and Jeffrey A. Farrell and David Gennert and Alexander F Schier and Aviv Regev},
title = {Spatial reconstruction of single-cell gene expression data},
journal = {Nature Biotechnology}
}

@article{Trapnell2014,
doi = {10.1038/nbt.2859},
url = {https://doi.org/10.1038/nbt.2859},
year = {2014},
month = April,
publisher = {Nature Anerica Inc. All rights reserved},
volume = {32},
number = {4},
author = {Cole Trapnell and Davide Cacchiarelli and Jonna Grimsby and Prapti Pokharel and Shuqiang Li and Michael Morse and Niall J Lennon and Kenneth J Livak and Tarjei S Mikkelsen and John L Rinn},
title = {Pseudo-temporal ordering of individual cells reveals dynamics and regulators of cell fate decisions},
journal = {Nature Biotechnology}
}

@article{McCarthy2017,
doi = {10.1093/bioinformatics/btw777},
url = {https://doi.org/10.1093/bioinformatics/btw777},
year = {2017},
month = January,
publisher = {Advanced Access Publication},
volume = {33},
number = {8},
author = {Davis J McCarthy and Kieran R Campbell and Aaron T L Lun and Quin F Wills},
title = {Scater: pre-processing, quality control, normalization and visualization of single-cell RNA-seq data in R},
journal = {Bioinformatics}
}
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