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<!DOCTYPE html>
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<title>Analysis of single cell RNA-seq data</title>
<meta name="description" content="Analysis of single cell RNA-seq data">
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<meta name="twitter:title" content="Analysis of single cell RNA-seq data" />
<meta name="author" content="Vladimir Kiselev (wikiselev), Tallulah Andrews, Jennifer Westoby (Jenni_Westoby), Davis McCarthy (davisjmcc), Maren Büttner (marenbuettner) and Martin Hemberg (m_hemberg)">
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<li><a href="index.html">Table of Contents</a></li>
<li class="divider"></li>
<li class="chapter" data-level="1" data-path="index.html"><a href="index.html"><i class="fa fa-check"></i><b>1</b> About the course</a><ul>
<li class="chapter" data-level="1.1" data-path="index.html"><a href="index.html#video"><i class="fa fa-check"></i><b>1.1</b> Video</a></li>
<li class="chapter" data-level="1.2" data-path="index.html"><a href="index.html#registration"><i class="fa fa-check"></i><b>1.2</b> Registration</a></li>
<li class="chapter" data-level="1.3" data-path="index.html"><a href="index.html#github"><i class="fa fa-check"></i><b>1.3</b> GitHub</a></li>
<li class="chapter" data-level="1.4" data-path="index.html"><a href="index.html#docker-image-rstudio"><i class="fa fa-check"></i><b>1.4</b> Docker image (RStudio)</a></li>
<li class="chapter" data-level="1.5" data-path="index.html"><a href="index.html#manual-installation"><i class="fa fa-check"></i><b>1.5</b> Manual installation</a></li>
<li class="chapter" data-level="1.6" data-path="index.html"><a href="index.html#license"><i class="fa fa-check"></i><b>1.6</b> License</a></li>
<li class="chapter" data-level="1.7" data-path="index.html"><a href="index.html#prerequisites"><i class="fa fa-check"></i><b>1.7</b> Prerequisites</a></li>
<li class="chapter" data-level="1.8" data-path="index.html"><a href="index.html#contact"><i class="fa fa-check"></i><b>1.8</b> Contact</a></li>
</ul></li>
<li class="chapter" data-level="2" data-path="introduction-to-single-cell-rna-seq.html"><a href="introduction-to-single-cell-rna-seq.html"><i class="fa fa-check"></i><b>2</b> Introduction to single-cell RNA-seq</a><ul>
<li class="chapter" data-level="2.1" data-path="introduction-to-single-cell-rna-seq.html"><a href="introduction-to-single-cell-rna-seq.html#bulk-rna-seq"><i class="fa fa-check"></i><b>2.1</b> Bulk RNA-seq</a></li>
<li class="chapter" data-level="2.2" data-path="introduction-to-single-cell-rna-seq.html"><a href="introduction-to-single-cell-rna-seq.html#scrna-seq"><i class="fa fa-check"></i><b>2.2</b> scRNA-seq</a></li>
<li class="chapter" data-level="2.3" data-path="introduction-to-single-cell-rna-seq.html"><a href="introduction-to-single-cell-rna-seq.html#workflow"><i class="fa fa-check"></i><b>2.3</b> Workflow</a></li>
<li class="chapter" data-level="2.4" data-path="introduction-to-single-cell-rna-seq.html"><a href="introduction-to-single-cell-rna-seq.html#computational-analysis"><i class="fa fa-check"></i><b>2.4</b> Computational Analysis</a></li>
<li class="chapter" data-level="2.5" data-path="introduction-to-single-cell-rna-seq.html"><a href="introduction-to-single-cell-rna-seq.html#challenges"><i class="fa fa-check"></i><b>2.5</b> Challenges</a></li>
<li class="chapter" data-level="2.6" data-path="introduction-to-single-cell-rna-seq.html"><a href="introduction-to-single-cell-rna-seq.html#experimental-methods"><i class="fa fa-check"></i><b>2.6</b> Experimental methods</a></li>
<li class="chapter" data-level="2.7" data-path="introduction-to-single-cell-rna-seq.html"><a href="introduction-to-single-cell-rna-seq.html#what-platform-to-use-for-my-experiment"><i class="fa fa-check"></i><b>2.7</b> What platform to use for my experiment?</a></li>
</ul></li>
<li class="chapter" data-level="3" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html"><i class="fa fa-check"></i><b>3</b> Processing Raw scRNA-seq Data</a><ul>
<li class="chapter" data-level="3.1" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#fastqc"><i class="fa fa-check"></i><b>3.1</b> FastQC</a><ul>
<li class="chapter" data-level="3.1.1" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#solution-and-downloading-the-report"><i class="fa fa-check"></i><b>3.1.1</b> Solution and Downloading the Report</a></li>
</ul></li>
<li class="chapter" data-level="3.2" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#trimming-reads"><i class="fa fa-check"></i><b>3.2</b> Trimming Reads</a><ul>
<li class="chapter" data-level="3.2.1" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#solution"><i class="fa fa-check"></i><b>3.2.1</b> Solution</a></li>
</ul></li>
<li class="chapter" data-level="3.3" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#file-formats"><i class="fa fa-check"></i><b>3.3</b> File formats</a><ul>
<li class="chapter" data-level="3.3.1" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#fastq"><i class="fa fa-check"></i><b>3.3.1</b> FastQ</a></li>
<li class="chapter" data-level="3.3.2" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#bam"><i class="fa fa-check"></i><b>3.3.2</b> BAM</a></li>
<li class="chapter" data-level="3.3.3" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#cram"><i class="fa fa-check"></i><b>3.3.3</b> CRAM</a></li>
<li class="chapter" data-level="3.3.4" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#mannually-inspecting-files"><i class="fa fa-check"></i><b>3.3.4</b> Mannually Inspecting files</a></li>
<li class="chapter" data-level="3.3.5" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#genome-fasta-gtf"><i class="fa fa-check"></i><b>3.3.5</b> Genome (FASTA, GTF)</a></li>
</ul></li>
<li class="chapter" data-level="3.4" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#demultiplexing"><i class="fa fa-check"></i><b>3.4</b> Demultiplexing</a><ul>
<li class="chapter" data-level="3.4.1" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#identifying-cell-containing-dropletsmicrowells"><i class="fa fa-check"></i><b>3.4.1</b> Identifying cell-containing droplets/microwells</a></li>
</ul></li>
<li class="chapter" data-level="3.5" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#using-star-to-align-reads"><i class="fa fa-check"></i><b>3.5</b> Using STAR to Align Reads</a><ul>
<li class="chapter" data-level="3.5.1" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#solution-for-star-alignment"><i class="fa fa-check"></i><b>3.5.1</b> Solution for STAR Alignment</a></li>
</ul></li>
<li class="chapter" data-level="3.6" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#kallisto-and-pseudo-alignment"><i class="fa fa-check"></i><b>3.6</b> Kallisto and Pseudo-Alignment</a><ul>
<li class="chapter" data-level="3.6.1" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#what-is-a-k-mer"><i class="fa fa-check"></i><b>3.6.1</b> What is a k-mer?</a></li>
<li class="chapter" data-level="3.6.2" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#why-map-k-mers-rather-than-reads"><i class="fa fa-check"></i><b>3.6.2</b> Why map k-mers rather than reads?</a></li>
<li class="chapter" data-level="3.6.3" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#kallistos-pseudo-mode"><i class="fa fa-check"></i><b>3.6.3</b> Kallisto’s pseudo mode</a></li>
<li class="chapter" data-level="3.6.4" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#solution-to-kallisto-pseudo-alignment"><i class="fa fa-check"></i><b>3.6.4</b> Solution to Kallisto Pseudo-Alignment</a></li>
<li class="chapter" data-level="3.6.5" data-path="processing-raw-scrna-seq-data.html"><a href="processing-raw-scrna-seq-data.html#understanding-the-output-of-kallisto-pseudo-alignment"><i class="fa fa-check"></i><b>3.6.5</b> Understanding the Output of Kallisto Pseudo-Alignment</a></li>
</ul></li>
</ul></li>
<li class="chapter" data-level="4" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html"><i class="fa fa-check"></i><b>4</b> Construction of expression matrix</a><ul>
<li class="chapter" data-level="4.1" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html#reads-qc"><i class="fa fa-check"></i><b>4.1</b> Reads QC</a></li>
<li class="chapter" data-level="4.2" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html#reads-alignment"><i class="fa fa-check"></i><b>4.2</b> Reads alignment</a></li>
<li class="chapter" data-level="4.3" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html#alignment-example"><i class="fa fa-check"></i><b>4.3</b> Alignment example</a></li>
<li class="chapter" data-level="4.4" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html#mapping-qc"><i class="fa fa-check"></i><b>4.4</b> Mapping QC</a></li>
<li class="chapter" data-level="4.5" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html#reads-quantification"><i class="fa fa-check"></i><b>4.5</b> Reads quantification</a></li>
<li class="chapter" data-level="4.6" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html#umichapter"><i class="fa fa-check"></i><b>4.6</b> Unique Molecular Identifiers (UMIs)</a><ul>
<li class="chapter" data-level="4.6.1" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html#introduction"><i class="fa fa-check"></i><b>4.6.1</b> Introduction</a></li>
<li class="chapter" data-level="4.6.2" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html#mapping-barcodes"><i class="fa fa-check"></i><b>4.6.2</b> Mapping Barcodes</a></li>
<li class="chapter" data-level="4.6.3" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html#counting-barcodes"><i class="fa fa-check"></i><b>4.6.3</b> Counting Barcodes</a></li>
<li class="chapter" data-level="4.6.4" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html#correcting-for-errors"><i class="fa fa-check"></i><b>4.6.4</b> Correcting for Errors</a></li>
<li class="chapter" data-level="4.6.5" data-path="construction-of-expression-matrix.html"><a href="construction-of-expression-matrix.html#downstream-analysis"><i class="fa fa-check"></i><b>4.6.5</b> Downstream Analysis</a></li>
</ul></li>
</ul></li>
<li class="chapter" data-level="5" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html"><i class="fa fa-check"></i><b>5</b> Introduction to R/Bioconductor</a><ul>
<li class="chapter" data-level="5.1" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#installing-packages"><i class="fa fa-check"></i><b>5.1</b> Installing packages</a><ul>
<li class="chapter" data-level="5.1.1" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#cran"><i class="fa fa-check"></i><b>5.1.1</b> CRAN</a></li>
<li class="chapter" data-level="5.1.2" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#github-1"><i class="fa fa-check"></i><b>5.1.2</b> Github</a></li>
<li class="chapter" data-level="5.1.3" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#bioconductor"><i class="fa fa-check"></i><b>5.1.3</b> Bioconductor</a></li>
<li class="chapter" data-level="5.1.4" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#source"><i class="fa fa-check"></i><b>5.1.4</b> Source</a></li>
</ul></li>
<li class="chapter" data-level="5.2" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#installation-instructions"><i class="fa fa-check"></i><b>5.2</b> Installation instructions:</a></li>
<li class="chapter" data-level="5.3" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#data-typesclasses"><i class="fa fa-check"></i><b>5.3</b> Data-types/classes</a><ul>
<li class="chapter" data-level="5.3.1" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#numeric"><i class="fa fa-check"></i><b>5.3.1</b> Numeric</a></li>
<li class="chapter" data-level="5.3.2" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#characterstring"><i class="fa fa-check"></i><b>5.3.2</b> Character/String</a></li>
<li class="chapter" data-level="5.3.3" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#logical"><i class="fa fa-check"></i><b>5.3.3</b> Logical</a></li>
<li class="chapter" data-level="5.3.4" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#factors"><i class="fa fa-check"></i><b>5.3.4</b> Factors</a></li>
<li class="chapter" data-level="5.3.5" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#checking-classtype"><i class="fa fa-check"></i><b>5.3.5</b> Checking class/type</a></li>
</ul></li>
<li class="chapter" data-level="5.4" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#basic-data-structures"><i class="fa fa-check"></i><b>5.4</b> Basic data structures</a></li>
<li class="chapter" data-level="5.5" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#more-information"><i class="fa fa-check"></i><b>5.5</b> More information</a></li>
<li class="chapter" data-level="5.6" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#data-types"><i class="fa fa-check"></i><b>5.6</b> Data Types</a><ul>
<li class="chapter" data-level="5.6.1" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#what-is-tidy-data"><i class="fa fa-check"></i><b>5.6.1</b> What is Tidy Data?</a></li>
<li class="chapter" data-level="5.6.2" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#what-is-rich-data"><i class="fa fa-check"></i><b>5.6.2</b> What is Rich Data?</a></li>
<li class="chapter" data-level="5.6.3" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#what-is-bioconductor"><i class="fa fa-check"></i><b>5.6.3</b> What is Bioconductor?</a></li>
<li class="chapter" data-level="5.6.4" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#singlecellexperiment-class"><i class="fa fa-check"></i><b>5.6.4</b> <code>SingleCellExperiment</code> class</a></li>
<li class="chapter" data-level="5.6.5" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#scater-package"><i class="fa fa-check"></i><b>5.6.5</b> <code>scater</code> package</a></li>
</ul></li>
<li class="chapter" data-level="5.7" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#bioconductor-singlecellexperiment-and-scater"><i class="fa fa-check"></i><b>5.7</b> Bioconductor, <code>SingleCellExperiment</code> and <code>scater</code></a><ul>
<li class="chapter" data-level="5.7.1" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#bioconductor-1"><i class="fa fa-check"></i><b>5.7.1</b> Bioconductor</a></li>
<li class="chapter" data-level="5.7.2" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#singlecellexperiment-class-1"><i class="fa fa-check"></i><b>5.7.2</b> <code>SingleCellExperiment</code> class</a></li>
<li class="chapter" data-level="5.7.3" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#scater-package-1"><i class="fa fa-check"></i><b>5.7.3</b> <code>scater</code> package</a></li>
</ul></li>
<li class="chapter" data-level="5.8" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#an-introduction-to-ggplot2"><i class="fa fa-check"></i><b>5.8</b> An Introduction to ggplot2</a><ul>
<li class="chapter" data-level="5.8.1" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#what-is-ggplot2"><i class="fa fa-check"></i><b>5.8.1</b> What is ggplot2?</a></li>
<li class="chapter" data-level="5.8.2" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#principles-of-ggplot2"><i class="fa fa-check"></i><b>5.8.2</b> Principles of ggplot2</a></li>
<li class="chapter" data-level="5.8.3" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#using-the-aes-mapping-function"><i class="fa fa-check"></i><b>5.8.3</b> Using the <code>aes</code> mapping function</a></li>
<li class="chapter" data-level="5.8.4" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#geoms"><i class="fa fa-check"></i><b>5.8.4</b> Geoms</a></li>
<li class="chapter" data-level="5.8.5" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#plotting-data-from-more-than-2-cells"><i class="fa fa-check"></i><b>5.8.5</b> Plotting data from more than 2 cells</a></li>
<li class="chapter" data-level="5.8.6" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#plotting-heatmaps"><i class="fa fa-check"></i><b>5.8.6</b> Plotting heatmaps</a></li>
<li class="chapter" data-level="5.8.7" data-path="introduction-to-rbioconductor.html"><a href="introduction-to-rbioconductor.html#principle-component-analysis"><i class="fa fa-check"></i><b>5.8.7</b> Principle Component Analysis</a></li>
</ul></li>
</ul></li>
<li class="chapter" data-level="6" data-path="tabula-muris.html"><a href="tabula-muris.html"><i class="fa fa-check"></i><b>6</b> Tabula Muris</a><ul>
<li class="chapter" data-level="6.1" data-path="tabula-muris.html"><a href="tabula-muris.html#introduction-1"><i class="fa fa-check"></i><b>6.1</b> Introduction</a></li>
<li class="chapter" data-level="6.2" data-path="tabula-muris.html"><a href="tabula-muris.html#downloading-the-data"><i class="fa fa-check"></i><b>6.2</b> Downloading the data</a></li>
<li class="chapter" data-level="6.3" data-path="tabula-muris.html"><a href="tabula-muris.html#reading-the-data-smartseq2"><i class="fa fa-check"></i><b>6.3</b> Reading the data (Smartseq2)</a></li>
<li class="chapter" data-level="6.4" data-path="tabula-muris.html"><a href="tabula-muris.html#building-a-scater-object"><i class="fa fa-check"></i><b>6.4</b> Building a scater object</a></li>
<li class="chapter" data-level="6.5" data-path="tabula-muris.html"><a href="tabula-muris.html#reading-the-data-10x"><i class="fa fa-check"></i><b>6.5</b> Reading the data (10X)</a></li>
<li class="chapter" data-level="6.6" data-path="tabula-muris.html"><a href="tabula-muris.html#building-a-scater-object-1"><i class="fa fa-check"></i><b>6.6</b> Building a scater object</a></li>
<li class="chapter" data-level="6.7" data-path="tabula-muris.html"><a href="tabula-muris.html#advanced-exercise"><i class="fa fa-check"></i><b>6.7</b> Advanced Exercise</a></li>
</ul></li>
<li class="chapter" data-level="7" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html"><i class="fa fa-check"></i><b>7</b> Cleaning the Expression Matrix</a><ul>
<li class="chapter" data-level="7.1" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#exprs-qc"><i class="fa fa-check"></i><b>7.1</b> Expression QC (UMI)</a><ul>
<li class="chapter" data-level="7.1.1" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#introduction-2"><i class="fa fa-check"></i><b>7.1.1</b> Introduction</a></li>
<li class="chapter" data-level="7.1.2" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#tung-dataset"><i class="fa fa-check"></i><b>7.1.2</b> Tung dataset</a></li>
<li class="chapter" data-level="7.1.3" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#cell-qc"><i class="fa fa-check"></i><b>7.1.3</b> Cell QC</a></li>
<li class="chapter" data-level="7.1.4" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#cell-filtering"><i class="fa fa-check"></i><b>7.1.4</b> Cell filtering</a></li>
<li class="chapter" data-level="7.1.5" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#compare-filterings"><i class="fa fa-check"></i><b>7.1.5</b> Compare filterings</a></li>
<li class="chapter" data-level="7.1.6" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#gene-analysis"><i class="fa fa-check"></i><b>7.1.6</b> Gene analysis</a></li>
<li class="chapter" data-level="7.1.7" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#save-the-data"><i class="fa fa-check"></i><b>7.1.7</b> Save the data</a></li>
<li class="chapter" data-level="7.1.8" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#big-exercise"><i class="fa fa-check"></i><b>7.1.8</b> Big Exercise</a></li>
<li class="chapter" data-level="7.1.9" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#sessioninfo"><i class="fa fa-check"></i><b>7.1.9</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="7.2" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#expression-qc-reads"><i class="fa fa-check"></i><b>7.2</b> Expression QC (Reads)</a></li>
<li class="chapter" data-level="7.3" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#data-visualization"><i class="fa fa-check"></i><b>7.3</b> Data visualization</a><ul>
<li class="chapter" data-level="7.3.1" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#introduction-3"><i class="fa fa-check"></i><b>7.3.1</b> Introduction</a></li>
<li class="chapter" data-level="7.3.2" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#visual-pca"><i class="fa fa-check"></i><b>7.3.2</b> PCA plot</a></li>
<li class="chapter" data-level="7.3.3" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#visual-tsne"><i class="fa fa-check"></i><b>7.3.3</b> tSNE map</a></li>
<li class="chapter" data-level="7.3.4" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#big-exercise-1"><i class="fa fa-check"></i><b>7.3.4</b> Big Exercise</a></li>
<li class="chapter" data-level="7.3.5" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#sessioninfo-1"><i class="fa fa-check"></i><b>7.3.5</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="7.4" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#data-visualization-reads"><i class="fa fa-check"></i><b>7.4</b> Data visualization (Reads)</a></li>
<li class="chapter" data-level="7.5" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#identifying-confounding-factors"><i class="fa fa-check"></i><b>7.5</b> Identifying confounding factors</a><ul>
<li class="chapter" data-level="7.5.1" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#introduction-4"><i class="fa fa-check"></i><b>7.5.1</b> Introduction</a></li>
<li class="chapter" data-level="7.5.2" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#correlations-with-pcs"><i class="fa fa-check"></i><b>7.5.2</b> Correlations with PCs</a></li>
<li class="chapter" data-level="7.5.3" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#explanatory-variables"><i class="fa fa-check"></i><b>7.5.3</b> Explanatory variables</a></li>
<li class="chapter" data-level="7.5.4" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#other-confounders"><i class="fa fa-check"></i><b>7.5.4</b> Other confounders</a></li>
<li class="chapter" data-level="7.5.5" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#exercise"><i class="fa fa-check"></i><b>7.5.5</b> Exercise</a></li>
<li class="chapter" data-level="7.5.6" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#sessioninfo-2"><i class="fa fa-check"></i><b>7.5.6</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="7.6" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#identifying-confounding-factors-reads"><i class="fa fa-check"></i><b>7.6</b> Identifying confounding factors (Reads)</a></li>
<li class="chapter" data-level="7.7" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#normalization-theory"><i class="fa fa-check"></i><b>7.7</b> Normalization theory</a><ul>
<li class="chapter" data-level="7.7.1" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#introduction-5"><i class="fa fa-check"></i><b>7.7.1</b> Introduction</a></li>
<li class="chapter" data-level="7.7.2" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#library-size-1"><i class="fa fa-check"></i><b>7.7.2</b> Library size</a></li>
<li class="chapter" data-level="7.7.3" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#normalisations"><i class="fa fa-check"></i><b>7.7.3</b> Normalisations</a></li>
<li class="chapter" data-level="7.7.4" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#effectiveness"><i class="fa fa-check"></i><b>7.7.4</b> Effectiveness</a></li>
</ul></li>
<li class="chapter" data-level="7.8" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#normalization-practice-umi"><i class="fa fa-check"></i><b>7.8</b> Normalization practice (UMI)</a><ul>
<li class="chapter" data-level="7.8.1" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#raw"><i class="fa fa-check"></i><b>7.8.1</b> Raw</a></li>
<li class="chapter" data-level="7.8.2" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#cpm-1"><i class="fa fa-check"></i><b>7.8.2</b> CPM</a></li>
<li class="chapter" data-level="7.8.3" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#size-factor-rle"><i class="fa fa-check"></i><b>7.8.3</b> Size-factor (RLE)</a></li>
<li class="chapter" data-level="7.8.4" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#upperquantile"><i class="fa fa-check"></i><b>7.8.4</b> Upperquantile</a></li>
<li class="chapter" data-level="7.8.5" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#tmm-1"><i class="fa fa-check"></i><b>7.8.5</b> TMM</a></li>
<li class="chapter" data-level="7.8.6" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#scran-1"><i class="fa fa-check"></i><b>7.8.6</b> scran</a></li>
<li class="chapter" data-level="7.8.7" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#downsampling-1"><i class="fa fa-check"></i><b>7.8.7</b> Downsampling</a></li>
<li class="chapter" data-level="7.8.8" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#normalisation-for-genetranscript-length"><i class="fa fa-check"></i><b>7.8.8</b> Normalisation for gene/transcript length</a></li>
<li class="chapter" data-level="7.8.9" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#exercise-1"><i class="fa fa-check"></i><b>7.8.9</b> Exercise</a></li>
<li class="chapter" data-level="7.8.10" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#sessioninfo-3"><i class="fa fa-check"></i><b>7.8.10</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="7.9" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#normalization-practice-reads"><i class="fa fa-check"></i><b>7.9</b> Normalization practice (Reads)</a></li>
<li class="chapter" data-level="7.10" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#dealing-with-confounders"><i class="fa fa-check"></i><b>7.10</b> Dealing with confounders</a><ul>
<li class="chapter" data-level="7.10.1" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#introduction-6"><i class="fa fa-check"></i><b>7.10.1</b> Introduction</a></li>
<li class="chapter" data-level="7.10.2" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#remove-unwanted-variation"><i class="fa fa-check"></i><b>7.10.2</b> Remove Unwanted Variation</a></li>
<li class="chapter" data-level="7.10.3" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#combat"><i class="fa fa-check"></i><b>7.10.3</b> Combat</a></li>
<li class="chapter" data-level="7.10.4" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#mnncorrect"><i class="fa fa-check"></i><b>7.10.4</b> mnnCorrect</a></li>
<li class="chapter" data-level="7.10.5" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#glm"><i class="fa fa-check"></i><b>7.10.5</b> GLM</a></li>
<li class="chapter" data-level="7.10.6" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#how-to-evaluate-and-compare-confounder-removal-strategies"><i class="fa fa-check"></i><b>7.10.6</b> How to evaluate and compare confounder removal strategies</a></li>
<li class="chapter" data-level="7.10.7" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#big-exercise-2"><i class="fa fa-check"></i><b>7.10.7</b> Big Exercise</a></li>
<li class="chapter" data-level="7.10.8" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#sessioninfo-4"><i class="fa fa-check"></i><b>7.10.8</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="7.11" data-path="cleaning-the-expression-matrix.html"><a href="cleaning-the-expression-matrix.html#dealing-with-confounders-reads"><i class="fa fa-check"></i><b>7.11</b> Dealing with confounders (Reads)</a></li>
</ul></li>
<li class="chapter" data-level="8" data-path="biological-analysis.html"><a href="biological-analysis.html"><i class="fa fa-check"></i><b>8</b> Biological Analysis</a><ul>
<li class="chapter" data-level="8.1" data-path="biological-analysis.html"><a href="biological-analysis.html#clustering-introduction"><i class="fa fa-check"></i><b>8.1</b> Clustering Introduction</a><ul>
<li class="chapter" data-level="8.1.1" data-path="biological-analysis.html"><a href="biological-analysis.html#introduction-7"><i class="fa fa-check"></i><b>8.1.1</b> Introduction</a></li>
<li class="chapter" data-level="8.1.2" data-path="biological-analysis.html"><a href="biological-analysis.html#dimensionality-reductions"><i class="fa fa-check"></i><b>8.1.2</b> Dimensionality reductions</a></li>
<li class="chapter" data-level="8.1.3" data-path="biological-analysis.html"><a href="biological-analysis.html#clustering-methods"><i class="fa fa-check"></i><b>8.1.3</b> Clustering methods</a></li>
<li class="chapter" data-level="8.1.4" data-path="biological-analysis.html"><a href="biological-analysis.html#challenges-in-clustering"><i class="fa fa-check"></i><b>8.1.4</b> Challenges in clustering</a></li>
<li class="chapter" data-level="8.1.5" data-path="biological-analysis.html"><a href="biological-analysis.html#tools-for-scrna-seq-data"><i class="fa fa-check"></i><b>8.1.5</b> Tools for scRNA-seq data</a></li>
<li class="chapter" data-level="8.1.6" data-path="biological-analysis.html"><a href="biological-analysis.html#comparing-clustering"><i class="fa fa-check"></i><b>8.1.6</b> Comparing clustering</a></li>
</ul></li>
<li class="chapter" data-level="8.2" data-path="biological-analysis.html"><a href="biological-analysis.html#clust-methods"><i class="fa fa-check"></i><b>8.2</b> Clustering example</a><ul>
<li class="chapter" data-level="8.2.1" data-path="biological-analysis.html"><a href="biological-analysis.html#deng-dataset"><i class="fa fa-check"></i><b>8.2.1</b> Deng dataset</a></li>
<li class="chapter" data-level="8.2.2" data-path="biological-analysis.html"><a href="biological-analysis.html#sc3-1"><i class="fa fa-check"></i><b>8.2.2</b> SC3</a></li>
<li class="chapter" data-level="8.2.3" data-path="biological-analysis.html"><a href="biological-analysis.html#pcareduce-1"><i class="fa fa-check"></i><b>8.2.3</b> pcaReduce</a></li>
<li class="chapter" data-level="8.2.4" data-path="biological-analysis.html"><a href="biological-analysis.html#tsne-kmeans"><i class="fa fa-check"></i><b>8.2.4</b> tSNE + kmeans</a></li>
<li class="chapter" data-level="8.2.5" data-path="biological-analysis.html"><a href="biological-analysis.html#snn-cliq-1"><i class="fa fa-check"></i><b>8.2.5</b> SNN-Cliq</a></li>
<li class="chapter" data-level="8.2.6" data-path="biological-analysis.html"><a href="biological-analysis.html#sincera-1"><i class="fa fa-check"></i><b>8.2.6</b> SINCERA</a></li>
<li class="chapter" data-level="8.2.7" data-path="biological-analysis.html"><a href="biological-analysis.html#sessioninfo-5"><i class="fa fa-check"></i><b>8.2.7</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="8.3" data-path="biological-analysis.html"><a href="biological-analysis.html#feature-selection"><i class="fa fa-check"></i><b>8.3</b> Feature Selection</a><ul>
<li class="chapter" data-level="8.3.1" data-path="biological-analysis.html"><a href="biological-analysis.html#identifying-genes-vs-a-null-model"><i class="fa fa-check"></i><b>8.3.1</b> Identifying Genes vs a Null Model</a></li>
<li class="chapter" data-level="8.3.2" data-path="biological-analysis.html"><a href="biological-analysis.html#correlated-expression"><i class="fa fa-check"></i><b>8.3.2</b> Correlated Expression</a></li>
<li class="chapter" data-level="8.3.3" data-path="biological-analysis.html"><a href="biological-analysis.html#comparing-methods"><i class="fa fa-check"></i><b>8.3.3</b> Comparing Methods</a></li>
<li class="chapter" data-level="8.3.4" data-path="biological-analysis.html"><a href="biological-analysis.html#sessioninfo-6"><i class="fa fa-check"></i><b>8.3.4</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="8.4" data-path="biological-analysis.html"><a href="biological-analysis.html#pseudotime-analysis"><i class="fa fa-check"></i><b>8.4</b> Pseudotime analysis</a><ul>
<li class="chapter" data-level="8.4.1" data-path="biological-analysis.html"><a href="biological-analysis.html#first-look-at-deng-data"><i class="fa fa-check"></i><b>8.4.1</b> First look at Deng data</a></li>
<li class="chapter" data-level="8.4.2" data-path="biological-analysis.html"><a href="biological-analysis.html#tscan"><i class="fa fa-check"></i><b>8.4.2</b> TSCAN</a></li>
<li class="chapter" data-level="8.4.3" data-path="biological-analysis.html"><a href="biological-analysis.html#monocle"><i class="fa fa-check"></i><b>8.4.3</b> monocle</a></li>
<li class="chapter" data-level="8.4.4" data-path="biological-analysis.html"><a href="biological-analysis.html#diffusion-maps"><i class="fa fa-check"></i><b>8.4.4</b> Diffusion maps</a></li>
<li class="chapter" data-level="8.4.5" data-path="biological-analysis.html"><a href="biological-analysis.html#slicer"><i class="fa fa-check"></i><b>8.4.5</b> SLICER</a></li>
<li class="chapter" data-level="8.4.6" data-path="biological-analysis.html"><a href="biological-analysis.html#ouija"><i class="fa fa-check"></i><b>8.4.6</b> Ouija</a></li>
<li class="chapter" data-level="8.4.7" data-path="biological-analysis.html"><a href="biological-analysis.html#comparison-of-the-methods"><i class="fa fa-check"></i><b>8.4.7</b> Comparison of the methods</a></li>
<li class="chapter" data-level="8.4.8" data-path="biological-analysis.html"><a href="biological-analysis.html#expression-of-genes-through-time"><i class="fa fa-check"></i><b>8.4.8</b> Expression of genes through time</a></li>
<li class="chapter" data-level="8.4.9" data-path="biological-analysis.html"><a href="biological-analysis.html#sessioninfo-7"><i class="fa fa-check"></i><b>8.4.9</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="8.5" data-path="biological-analysis.html"><a href="biological-analysis.html#imputation"><i class="fa fa-check"></i><b>8.5</b> Imputation</a><ul>
<li class="chapter" data-level="8.5.1" data-path="biological-analysis.html"><a href="biological-analysis.html#scimpute"><i class="fa fa-check"></i><b>8.5.1</b> scImpute</a></li>
<li class="chapter" data-level="8.5.2" data-path="biological-analysis.html"><a href="biological-analysis.html#magic"><i class="fa fa-check"></i><b>8.5.2</b> MAGIC</a></li>
<li class="chapter" data-level="8.5.3" data-path="biological-analysis.html"><a href="biological-analysis.html#sessioninfo-8"><i class="fa fa-check"></i><b>8.5.3</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="8.6" data-path="biological-analysis.html"><a href="biological-analysis.html#dechapter"><i class="fa fa-check"></i><b>8.6</b> Differential Expression (DE) analysis</a><ul>
<li class="chapter" data-level="8.6.1" data-path="biological-analysis.html"><a href="biological-analysis.html#bulk-rna-seq-1"><i class="fa fa-check"></i><b>8.6.1</b> Bulk RNA-seq</a></li>
<li class="chapter" data-level="8.6.2" data-path="biological-analysis.html"><a href="biological-analysis.html#single-cell-rna-seq"><i class="fa fa-check"></i><b>8.6.2</b> Single cell RNA-seq</a></li>
<li class="chapter" data-level="8.6.3" data-path="biological-analysis.html"><a href="biological-analysis.html#differences-in-distribution"><i class="fa fa-check"></i><b>8.6.3</b> Differences in Distribution</a></li>
<li class="chapter" data-level="8.6.4" data-path="biological-analysis.html"><a href="biological-analysis.html#models-of-single-cell-rnaseq-data"><i class="fa fa-check"></i><b>8.6.4</b> Models of single-cell RNASeq data</a></li>
</ul></li>
<li class="chapter" data-level="8.7" data-path="biological-analysis.html"><a href="biological-analysis.html#de-in-a-real-dataset"><i class="fa fa-check"></i><b>8.7</b> DE in a real dataset</a><ul>
<li class="chapter" data-level="8.7.1" data-path="biological-analysis.html"><a href="biological-analysis.html#introduction-8"><i class="fa fa-check"></i><b>8.7.1</b> Introduction</a></li>
<li class="chapter" data-level="8.7.2" data-path="biological-analysis.html"><a href="biological-analysis.html#kolmogorov-smirnov-test"><i class="fa fa-check"></i><b>8.7.2</b> Kolmogorov-Smirnov test</a></li>
<li class="chapter" data-level="8.7.3" data-path="biological-analysis.html"><a href="biological-analysis.html#wilcoxmann-whitney-u-test"><i class="fa fa-check"></i><b>8.7.3</b> Wilcox/Mann-Whitney-U Test</a></li>
<li class="chapter" data-level="8.7.4" data-path="biological-analysis.html"><a href="biological-analysis.html#edger"><i class="fa fa-check"></i><b>8.7.4</b> edgeR</a></li>
<li class="chapter" data-level="8.7.5" data-path="biological-analysis.html"><a href="biological-analysis.html#monocle-1"><i class="fa fa-check"></i><b>8.7.5</b> Monocle</a></li>
<li class="chapter" data-level="8.7.6" data-path="biological-analysis.html"><a href="biological-analysis.html#mast"><i class="fa fa-check"></i><b>8.7.6</b> MAST</a></li>
<li class="chapter" data-level="8.7.7" data-path="biological-analysis.html"><a href="biological-analysis.html#slow-methods-1h-to-run"><i class="fa fa-check"></i><b>8.7.7</b> Slow Methods (>1h to run)</a></li>
<li class="chapter" data-level="8.7.8" data-path="biological-analysis.html"><a href="biological-analysis.html#bpsc"><i class="fa fa-check"></i><b>8.7.8</b> BPSC</a></li>
<li class="chapter" data-level="8.7.9" data-path="biological-analysis.html"><a href="biological-analysis.html#scde"><i class="fa fa-check"></i><b>8.7.9</b> SCDE</a></li>
<li class="chapter" data-level="8.7.10" data-path="biological-analysis.html"><a href="biological-analysis.html#sessioninfo-9"><i class="fa fa-check"></i><b>8.7.10</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="8.8" data-path="biological-analysis.html"><a href="biological-analysis.html#comparingcombining-scrnaseq-datasets"><i class="fa fa-check"></i><b>8.8</b> Comparing/Combining scRNASeq datasets</a><ul>
<li class="chapter" data-level="8.8.1" data-path="biological-analysis.html"><a href="biological-analysis.html#introduction-9"><i class="fa fa-check"></i><b>8.8.1</b> Introduction</a></li>
<li class="chapter" data-level="8.8.2" data-path="biological-analysis.html"><a href="biological-analysis.html#datasets"><i class="fa fa-check"></i><b>8.8.2</b> Datasets</a></li>
<li class="chapter" data-level="8.8.3" data-path="biological-analysis.html"><a href="biological-analysis.html#projecting-cells-onto-annotated-cell-types-scmap"><i class="fa fa-check"></i><b>8.8.3</b> Projecting cells onto annotated cell-types (scmap)</a></li>
<li class="chapter" data-level="8.8.4" data-path="biological-analysis.html"><a href="biological-analysis.html#cell-to-cell-mapping"><i class="fa fa-check"></i><b>8.8.4</b> Cell-to-Cell mapping</a></li>
<li class="chapter" data-level="8.8.5" data-path="biological-analysis.html"><a href="biological-analysis.html#metaneighbour"><i class="fa fa-check"></i><b>8.8.5</b> Metaneighbour</a></li>
<li class="chapter" data-level="8.8.6" data-path="biological-analysis.html"><a href="biological-analysis.html#mnncorrect-1"><i class="fa fa-check"></i><b>8.8.6</b> mnnCorrect</a></li>
<li class="chapter" data-level="8.8.7" data-path="biological-analysis.html"><a href="biological-analysis.html#cannonical-correlation-analysis-seurat"><i class="fa fa-check"></i><b>8.8.7</b> Cannonical Correlation Analysis (Seurat)</a></li>
<li class="chapter" data-level="8.8.8" data-path="biological-analysis.html"><a href="biological-analysis.html#sessioninfo-10"><i class="fa fa-check"></i><b>8.8.8</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="8.9" data-path="biological-analysis.html"><a href="biological-analysis.html#search-scrna-seq-data"><i class="fa fa-check"></i><b>8.9</b> Search scRNA-Seq data</a><ul>
<li class="chapter" data-level="8.9.1" data-path="biological-analysis.html"><a href="biological-analysis.html#about"><i class="fa fa-check"></i><b>8.9.1</b> About</a></li>
<li class="chapter" data-level="8.9.2" data-path="biological-analysis.html"><a href="biological-analysis.html#dataset"><i class="fa fa-check"></i><b>8.9.2</b> Dataset</a></li>
<li class="chapter" data-level="8.9.3" data-path="biological-analysis.html"><a href="biological-analysis.html#gene-index"><i class="fa fa-check"></i><b>8.9.3</b> Gene Index</a></li>
<li class="chapter" data-level="8.9.4" data-path="biological-analysis.html"><a href="biological-analysis.html#marker-genes"><i class="fa fa-check"></i><b>8.9.4</b> Marker genes</a></li>
<li class="chapter" data-level="8.9.5" data-path="biological-analysis.html"><a href="biological-analysis.html#search-cells-by-a-gene-list"><i class="fa fa-check"></i><b>8.9.5</b> Search cells by a gene list</a></li>
<li class="chapter" data-level="8.9.6" data-path="biological-analysis.html"><a href="biological-analysis.html#sessioninfo-11"><i class="fa fa-check"></i><b>8.9.6</b> sessionInfo()</a></li>
</ul></li>
</ul></li>
<li class="chapter" data-level="9" data-path="seurat-chapter.html"><a href="seurat-chapter.html"><i class="fa fa-check"></i><b>9</b> Seurat</a><ul>
<li class="chapter" data-level="9.1" data-path="seurat-chapter.html"><a href="seurat-chapter.html#seurat-object-class"><i class="fa fa-check"></i><b>9.1</b> <code>Seurat</code> object class</a></li>
<li class="chapter" data-level="9.2" data-path="seurat-chapter.html"><a href="seurat-chapter.html#expression-qc"><i class="fa fa-check"></i><b>9.2</b> Expression QC</a></li>
<li class="chapter" data-level="9.3" data-path="seurat-chapter.html"><a href="seurat-chapter.html#normalization"><i class="fa fa-check"></i><b>9.3</b> Normalization</a></li>
<li class="chapter" data-level="9.4" data-path="seurat-chapter.html"><a href="seurat-chapter.html#highly-variable-genes-1"><i class="fa fa-check"></i><b>9.4</b> Highly variable genes</a></li>
<li class="chapter" data-level="9.5" data-path="seurat-chapter.html"><a href="seurat-chapter.html#dealing-with-confounders-1"><i class="fa fa-check"></i><b>9.5</b> Dealing with confounders</a></li>
<li class="chapter" data-level="9.6" data-path="seurat-chapter.html"><a href="seurat-chapter.html#linear-dimensionality-reduction"><i class="fa fa-check"></i><b>9.6</b> Linear dimensionality reduction</a></li>
<li class="chapter" data-level="9.7" data-path="seurat-chapter.html"><a href="seurat-chapter.html#significant-pcs"><i class="fa fa-check"></i><b>9.7</b> Significant PCs</a></li>
<li class="chapter" data-level="9.8" data-path="seurat-chapter.html"><a href="seurat-chapter.html#clustering-cells"><i class="fa fa-check"></i><b>9.8</b> Clustering cells</a></li>
<li class="chapter" data-level="9.9" data-path="seurat-chapter.html"><a href="seurat-chapter.html#marker-genes-1"><i class="fa fa-check"></i><b>9.9</b> Marker genes</a></li>
<li class="chapter" data-level="9.10" data-path="seurat-chapter.html"><a href="seurat-chapter.html#sessioninfo-12"><i class="fa fa-check"></i><b>9.10</b> sessionInfo()</a></li>
</ul></li>
<li class="chapter" data-level="10" data-path="ideal-scrnaseq-pipeline-as-of-oct-2017.html"><a href="ideal-scrnaseq-pipeline-as-of-oct-2017.html"><i class="fa fa-check"></i><b>10</b> “Ideal” scRNAseq pipeline (as of Oct 2017)</a><ul>
<li class="chapter" data-level="10.1" data-path="ideal-scrnaseq-pipeline-as-of-oct-2017.html"><a href="ideal-scrnaseq-pipeline-as-of-oct-2017.html#experimental-design"><i class="fa fa-check"></i><b>10.1</b> Experimental Design</a></li>
<li class="chapter" data-level="10.2" data-path="ideal-scrnaseq-pipeline-as-of-oct-2017.html"><a href="ideal-scrnaseq-pipeline-as-of-oct-2017.html#processing-reads"><i class="fa fa-check"></i><b>10.2</b> Processing Reads</a></li>
<li class="chapter" data-level="10.3" data-path="ideal-scrnaseq-pipeline-as-of-oct-2017.html"><a href="ideal-scrnaseq-pipeline-as-of-oct-2017.html#preparing-expression-matrix"><i class="fa fa-check"></i><b>10.3</b> Preparing Expression Matrix</a></li>
<li class="chapter" data-level="10.4" data-path="ideal-scrnaseq-pipeline-as-of-oct-2017.html"><a href="ideal-scrnaseq-pipeline-as-of-oct-2017.html#biological-interpretation"><i class="fa fa-check"></i><b>10.4</b> Biological Interpretation</a></li>
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<li class="chapter" data-level="11" data-path="advanced-exercises.html"><a href="advanced-exercises.html"><i class="fa fa-check"></i><b>11</b> Advanced exercises</a></li>
<li class="chapter" data-level="12" data-path="resources.html"><a href="resources.html"><i class="fa fa-check"></i><b>12</b> Resources</a><ul>
<li class="chapter" data-level="12.1" data-path="resources.html"><a href="resources.html#scrna-seq-protocols"><i class="fa fa-check"></i><b>12.1</b> scRNA-seq protocols</a></li>
<li class="chapter" data-level="12.2" data-path="resources.html"><a href="resources.html#external-rna-control-consortium-ercc"><i class="fa fa-check"></i><b>12.2</b> External RNA Control Consortium (ERCC)</a></li>
<li class="chapter" data-level="12.3" data-path="resources.html"><a href="resources.html#scrna-seq-analysis-tools"><i class="fa fa-check"></i><b>12.3</b> scRNA-seq analysis tools</a></li>
<li class="chapter" data-level="12.4" data-path="resources.html"><a href="resources.html#scrna-seq-public-datasets"><i class="fa fa-check"></i><b>12.4</b> scRNA-seq public datasets</a></li>
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<li class="chapter" data-level="13" data-path="references.html"><a href="references.html"><i class="fa fa-check"></i><b>13</b> References</a></li>
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<li><a href="http://www.sanger.ac.uk/science/groups/hemberg-group" target="blank">Hemberg Lab</a></li>
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<h1>
<i class="fa fa-circle-o-notch fa-spin"></i><a href="./">Analysis of single cell RNA-seq data</a>
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<h1><span class="header-section-number">11</span> Advanced exercises</h1>
<p>For the final part of the course we would like you to work on more open ended problems. The goal is to carry out the type of analyses that you would be doing for an actual research project.</p>
<p>Participants who have their own dataset that they are interested in should feel free to work with them.</p>
<p>For other participants we recommend downloading a dataset from the <a href="http://imlspenticton.uzh.ch:3838/conquer/">conquer</a> resource (<b>co</b>nsistent <b>qu</b>antification of <b>e</b>xternal <b>r</b>na-seq data). <a href="http://imlspenticton.uzh.ch:3838/conquer/">conquer</a> uses <a href="http://salmon.readthedocs.io/en/latest/salmon.html">Salmon</a> to quantify the transcript abundances in a given sample. For a given organism, the fasta files containing cDNA and ncRNA sequences from Ensembl are complemented with ERCC spike-in sequences, and a Salmon quasi-mapping index is built for the entire catalog. Then <a href="http://salmon.readthedocs.io/en/latest/salmon.html">Salmon</a> is run to estimate the abundance of each transcript. The abundances estimated by <a href="http://salmon.readthedocs.io/en/latest/salmon.html">Salmon</a> are summarised and provided to the user in the form of a <code>MultiAssayExperiment</code> object. This object can be downloaded via the buttons in the <em>MultiAssayExperiment</em> column. The provided <code>MultiAssayExperiment</code> object contains two “experiments”, corresponding to the gene-level and transcript-level values.</p>
<p>The gene-level experiment contains four “assays”:</p>
<ul>
<li>TPM</li>
<li>count</li>
<li>count_lstpm (count-scale length-scaled TPMs)</li>
<li>avetxlength (the average transcript length, which can be used as offsets in count models based on the count assay, see <a href="http://f1000research.com/articles/4-1521/">here</a>).</li>
</ul>
<p>The transcript-level experiment contains three “assays”:</p>
<ul>
<li>TPM</li>
<li>count</li>
<li>efflength (the effective length estimated by <a href="http://salmon.readthedocs.io/en/latest/salmon.html">Salmon</a>)</li>
</ul>
<p>The <code>MultiAssayExperiment</code> also contains the phenotypic data (in the <code>colData</code> slot), as well as some metadata for the data set (the genome, the organism and the Salmon index that was used for the quantification).</p>
<p>Here we will show you how to create an <code>SCE</code> from a <code>MultiAssayExperiment</code> object. For example, if you download <code>Shalek2013</code> dataset you will be able to create an <code>SCE</code> using the following code:</p>
<div class="sourceCode"><pre class="sourceCode r"><code class="sourceCode r"><span class="kw">library</span>(MultiAssayExperiment)
<span class="kw">library</span>(SummarizedExperiment)
<span class="kw">library</span>(scater)
d <-<span class="st"> </span><span class="kw">readRDS</span>(<span class="st">"~/Desktop/GSE41265.rds"</span>)
cts <-<span class="st"> </span><span class="kw">assays</span>(<span class="kw">experiments</span>(d)[[<span class="st">"gene"</span>]])[[<span class="st">"count_lstpm"</span>]]
tpms <-<span class="st"> </span><span class="kw">assays</span>(<span class="kw">experiments</span>(d)[[<span class="st">"gene"</span>]])[[<span class="st">"TPM"</span>]]
phn <-<span class="st"> </span><span class="kw">colData</span>(d)
sce <-<span class="st"> </span><span class="kw">SingleCellExperiment</span>(
<span class="dt">assays =</span> <span class="kw">list</span>(
<span class="dt">countData =</span> cts,
<span class="dt">tpmData =</span> tpms
),
<span class="dt">colData =</span> phn
)</code></pre></div>
<p>You can also see that several different QC metrics have already been pre-calculated on the <a href="http://imlspenticton.uzh.ch:3838/conquer/">conquer</a> website.</p>
<p>Here are some suggestions for questions that you can explore:</p>
<ul>
<li><p>There are two mESC datasets from different labs (i.e. <code>Xue</code> and <code>Kumar</code>). Can you merge them and remove the batch effects?</p></li>
<li><p>Clustering and pseudotime analysis look for different patterns among cells. How might you tell which is more appropriate for your dataset?</p></li>
<li><p>One of the main challenging in hard clustering is to identify the appropriate value for <code>k</code>. Can you use one or more of the clustering tools to explore the different hierarchies available? What are good mathematical and/or biological criteria for determining <code>k</code>?</p></li>
<li><p>The choice of normalization strategy matters, but how do you determine which is the best method? Explore the effect of different normalizations on downstream analyses.</p></li>
<li><p>scRNA-seq datasets are high-dimensional and since most dimensions (ie genes) are not informative. Consequently, dimensionality reduction and feature selection are important when analyzing and visualizing the data. Consider the effect of different feature selection methods and dimensionality reduction on clustering and/or pseudotime inference.</p></li>
<li><p>One of the main challenges after clustering cells is to interpret the biological relevance of the subpopulations. One approach is to identify <a href="http://geneontology.org/">gene ontology</a> terms that are enriched for the set of marker genes. Identify marker genes (e.g. using <code>SC3</code> or <code>M3Drop</code>) and explore the ontology terms using <a href="http://biit.cs.ut.ee/gprofiler/">gProfiler</a>, <a href="http://www.webgestalt.org/">WebGestalt</a> or <a href="https://david.ncifcrf.gov/">DAVID</a>.</p></li>
<li><p>Similarly, when ordering cells according to pseudotime we would like to understand what underlying cellular processes are changing over time. Identify a set of changing genes from the aligned cells and use ontology terms to characterize them.</p></li>
</ul>
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