rnaseq deseq2 tutorial

Next, get results for the HoxA1 knockdown versus control siRNA, and reorder them by p-value. 3.1.0). Here, for demonstration, let us select the 35 genes with the highest variance across samples: The heatmap becomes more interesting if we do not look at absolute expression strength but rather at the amount by which each gene deviates in a specific sample from the genes average across all samples. Figure 1 explains the basic structure of the SummarizedExperiment class. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. We subset the results table to these genes and then sort it by the log2 fold change estimate to get the significant genes with the strongest down-regulation: A so-called MA plot provides a useful overview for an experiment with a two-group comparison: The MA-plot represents each gene with a dot. I will visualize the DGE using Volcano plot using Python, If you want to create a heatmap, check this article. This tutorial will walk you through installing salmon, building an index on a transcriptome, and then quantifying some RNA-seq samples for downstream processing. To avoid that the distance measure is dominated by a few highly variable genes, and have a roughly equal contribution from all genes, we use it on the rlog-transformed data: Note the use of the function t to transpose the data matrix. We look forward to seeing you in class and hope you find these . To install this package, start the R console and enter: The R code below is long and slightly complicated, but I will highlight major points. To view the purposes they believe they have legitimate interest for, or to object to this data processing use the vendor list link below. Good afternoon, I am working with a dataset containing 50 libraries of small RNAs. # plot to show effect of transformation It is essential to have the name of the columns in the count matrix in the same order as that in name of the samples of RNA sequencing technology. If you would like to change your settings or withdraw consent at any time, the link to do so is in our privacy policy accessible from our home page.. There is a script file located in, /common/RNASeq_Workshop/Soybean/STAR_HTSEQ_mapping/bam_files called bam_index.sh that will accomplish this. such as condition should go at the end of the formula. The workflow for the RNA-Seq data is: The dataset used in the tutorial is from the published Hammer et al 2010 study. Converting IDs with the native functions from the AnnotationDbi package is currently a bit cumbersome, so we provide the following convenience function (without explaining how exactly it works): To convert the Ensembl IDs in the rownames of res to gene symbols and add them as a new column, we use: DESeq2 uses the so-called Benjamini-Hochberg (BH) adjustment for multiple testing problem; in brief, this method calculates for each gene an adjusted p value which answers the following question: if one called significant all genes with a p value less than or equal to this genes p value threshold, what would be the fraction of false positives (the false discovery rate, FDR) among them (in the sense of the calculation outlined above)? controlling additional factors (other than the variable of interest) in the model such as batch effects, type of # "trimmed mean" approach. This function also normalises for library size. goal here is to identify the differentially expressed genes under infected condition. PLoS Comp Biol. We get a merged .csv file with our original output from DESeq2 and the Biomart data: Visualizing Differential Expression with IGV: To visualize how genes are differently expressed between treatments, we can use the Broad Institutes Interactive Genomics Viewer (IGV), which can be downloaded from here: IGV, We will be using the .bam files we created previously, as well as the reference genome file in order to view the genes in IGV. This script was adapted from hereand here, and much credit goes to those authors. Well use these KEGG pathway IDs downstream for plotting. The package DESeq2 provides methods to test for differential expression analysis. It is important to know if the sequencing experiment was single-end or paired-end, as the alignment software will require the user to specify both FASTQ files for a paired-end experiment. How many such genes are there? Much of Galaxy-related features described in this section have been . While NB-based methods generally have a higher detection power, there are . More at http://bioconductor.org/packages/release/BiocViews.html#___RNASeq. Much of Galaxy-related features described in this section have been developed by Bjrn Grning (@bgruening) and . We will use RNAseq to compare expression levels for genes between DS and WW-samples for drought sensitive genotype IS20351 and to identify new transcripts or isoforms. The trimmed output files are what we will be using for the next steps of our analysis. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. This document presents an RNAseq differential expression workflow. Disclaimer, "https://reneshbedre.github.io/assets/posts/gexp/df_sc.csv", # see all comparisons (here there is only one), # get gene expression table In this ordination method, the data points (i.e., here, the samples) are projected onto the 2D plane such that they spread out optimally. Before we do that we need to: import our counts into R. manipulate the imported data so that it is in the correct format for DESeq2. (Note that the outputs from other RNA-seq quantifiers like Salmon or Sailfish can also be used with Sleuth via the wasabi package.) We remove all rows corresponding to Reactome Paths with less than 20 or more than 80 assigned genes. run some initial QC on the raw count data. also import sample information if you have it in a file). We can confirm that the counts for the new object are equal to the summed up counts of the columns that had the same value for the grouping factor: Here we will analyze a subset of the samples, namely those taken after 48 hours, with either control, DPN or OHT treatment, taking into account the multifactor design. control vs infected). Through the RNA-sequencing (RNA-seq) and mass spectrometry analyses, we reveal the downregulation of the sphingolipid signaling pathway under simulated microgravity. The user should specify three values: The name of the variable, the name of the level in the numerator, and the name of the level in the denominator. Generate a list of differentially expressed genes using DESeq2. Note genes with extremly high dispersion values (blue circles) are not shrunk toward the curve, and only slightly high estimates are. Terms and conditions The dataset is a simple experiment where RNA is extracted from roots of independent plants and then sequenced. This DESeq2 tutorial is inspired by the RNA-seq workflow developped by the authors of the tool, and by the differential gene expression course from the Harvard Chan Bioinformatics Core. # 5) PCA plot Abstract. Note: This article focuses on DGE analysis using a count matrix. length for normalization as gene length is constant for all samples (it may not have significant effect on DGE analysis). Here we present the DEseq2 vignette it wwas composed using . I have a table of read counts from RNASeq data (i.e. Avinash Karn Much documentation is available online on how to manipulate and best use par() and ggplot2 graphing parameters. Be sure that your .bam files are saved in the same folder as their corresponding index (.bai) files. expression. RNA-seq: An assessment of technical reproducibility and comparison with gene expression arrays mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain. Export differential gene expression analysis table to CSV file. We also need some genes to plot in the heatmap. If time were included in the design formula, the following code could be used to take care of dropped levels in this column. DESeq2 (as edgeR) is based on the hypothesis that most genes are not differentially expressed. From this file, the function makeTranscriptDbFromGFF from the GenomicFeatures package constructs a database of all annotated transcripts. We use the gene sets in the Reactome database: This database works with Entrez IDs, so we will need the entrezid column that we added earlier to the res object. . The. Determine the size factors to be used for normalization using code below: Plot column sums according to size factor. Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. # at this step independent filtering is applied by default to remove low count genes Now that you have the genome and annotation files, you will create a genome index using the following script: You will likely have to alter this script slightly to reflect the directory that you are working in and the specific names you gave your files, but the general idea is there. is a de facto method for quantifying the transcriptome-wide gene or transcript expressions and performing DGE analysis. for shrinkage of effect sizes and gives reliable effect sizes. We can coduct hierarchical clustering and principal component analysis to explore the data. Bioconductor has many packages which support analysis of high-throughput sequence data, including RNA sequencing (RNA-seq). To facilitate the computations, we define a little helper function: The function can be called with a Reactome Path ID: As you can see the function not only performs the t test and returns the p value but also lists other useful information such as the number of genes in the category, the average log fold change, a strength" measure (see below) and the name with which Reactome describes the Path. # After fetching data from the Phytozome database based on the PAC transcript IDs of the genes in our samples, a .txt file is generated that should look something like this: Finally, we want to merge the deseq2 and biomart output. The remaining four columns refer to a specific contrast, namely the comparison of the levels DPN versus Control of the factor variable treatment. Introduction. As input, the DESeq2 package expects count data as obtained, e.g., from RNA-seq or another high-throughput sequencing experiment, in the form of a matrix of integer values. R version 3.1.0 (2014-04-10) Platform: x86_64-apple-darwin13.1.0 (64-bit), locale: [1] fr_FR.UTF-8/fr_FR.UTF-8/fr_FR.UTF-8/C/fr_FR.UTF-8/fr_FR.UTF-8, attached base packages: [1] parallel stats graphics grDevices utils datasets methods base, other attached packages: [1] genefilter_1.46.1 RColorBrewer_1.0-5 gplots_2.14.2 reactome.db_1.48.0 Calling results without any arguments will extract the estimated log2 fold changes and p values for the last variable in the design formula. Powered by Jekyll& Minimal Mistakes. other recommended alternative for performing DGE analysis without biological replicates. Want to Learn More on R Programming and Data Science? From both visualizations, we see that the differences between patients is much larger than the difference between treatment and control samples of the same patient. RNA Sequence Analysis in R: edgeR The purpose of this lab is to get a better understanding of how to use the edgeR package in R.http://www.bioconductor.org/packages . It will be convenient to make sure that Control is the first level in the treatment factor, so that the default log2 fold changes are calculated as treatment over control and not the other way around. /common/RNASeq_Workshop/Soybean/Quality_Control, /common/RNASeq_Workshop/Soybean/STAR_HTSEQ_mapping, # Set the prefix for each output file name, # copied from: https://benchtobioinformatics.wordpress.com/category/dexseq/ For example, sample SRS308873 was sequenced twice. For the parathyroid experiment, we will specify ~ patient + treatment, which means that we want to test for the effect of treatment (the last factor), controlling for the effect of patient (the first factor). In Figure , we can see how genes with low counts seem to be excessively variable on the ordinary logarithmic scale, while the rlog transform compresses differences for genes for which the data cannot provide good information anyway. filter out unwanted genes. HISAT2 or STAR). the numerator (for log2 fold change), and name of the condition for the denominator. RNA-Seq (RNA sequencing ) also called whole transcriptome sequncing use next-generation sequeincing (NGS) to reveal the presence and quantity of RNA in a biolgical sample at a given moment. It is available from . Introduction. Plot the mean versus variance in read count data. For strongly expressed genes, the dispersion can be understood as a squared coefficient of variation: a dispersion value of 0.01 means that the genes expression tends to differ by typically $\sqrt{0.01}=10\%$ between samples of the same treatment group. sequencing, etc. Experiments: Review, Tutorial, and Perspectives Hyeongseon Jeon1,2,*, Juan Xie1,2,3 . For the remaining steps I find it easier to to work from a desktop rather than the server. HISAT2 is a fast and sensitive alignment program for mapping next-generation sequencing reads (both DNA and RNA) to a population of human genomes (as well as to a single reference genome). Renesh Bedre 9 minute read Introduction. Bulk RNA-sequencing (RNA-seq) on the NIH Integrated Data Analysis Portal (NIDAP) This page contains links to recorded video lectures and tutorials that will require approximately 4 hours in total to complete. Most of this will be done on the BBC server unless otherwise stated. [7] bitops_1.0-6 brew_1.0-6 caTools_1.17.1 checkmate_1.4 codetools_0.2-9 digest_0.6.4 These reads must first be aligned to a reference genome or transcriptome. DESeq2 is then used on the . fd jm sh. Enjoyed this article? # these next R scripts are for a variety of visualization, QC and other plots to It is used in the estimation of The reference genome file is located at, /common/RNASeq_Workshop/Soybean/gmax_genome/Gmax_275_v2. Here, I will remove the genes which have < 10 reads (this can vary based on research goal) in total across all the studying the changes in gene or transcripts expressions under different conditions (e.g. First, import the countdata and metadata directly from the web. Once you have IGV up and running, you can load the reference genome file by going to Genomes -> Load Genome From File in the top menu. We need this because dist calculates distances between data rows and our samples constitute the columns. Convert BAM Files to Raw Counts with HTSeq: Finally, we will use HTSeq to transform these mapped reads into counts that we can analyze with R. -s indicates we do not have strand specific counts. This value is reported on a logarithmic scale to base 2: for example, a log2 fold change of 1.5 means that the genes expression is increased by a multiplicative factor of 21.52.82. Therefore, we fit the red trend line, which shows the dispersions dependence on the mean, and then shrink each genes estimate towards the red line to obtain the final estimates (blue points) that are then used in the hypothesis test. Two plants were treated with the control (KCl) and two samples were treated with Nitrate (KNO3). After all, the test found them to be non-significant anyway. They can be found in results 13 through 18 of the following NCBI search: http://www.ncbi.nlm.nih.gov/sra/?term=SRP009826, The script for downloading these .SRA files and converting them to fastq can be found in. analysis will be performed using the raw integer read counts for control and fungal treatment conditions. Once youve done that, you can download the assembly file Gmax_275_v2 and the annotation file Gmax_275_Wm82.a2.v1.gene_exons. # axis is square root of variance over the mean for all samples, # clustering analysis The BAM files for a number of sequencing runs can then be used to generate count matrices, as described in the following section. # 1) MA plot Click "Choose file" and upload the recently downloaded Galaxy tabular file containing your RNA-seq counts. The Dataset. A useful first step in an RNA-Seq analysis is often to assess overall similarity between samples. Here we use the TopHat2 spliced alignment software in combination with the Bowtie index available at the Illumina iGenomes. # produce DataFrame of results of statistical tests, # replacing outlier value with estimated value as predicted by distrubution using For instructions on importing for use with . The Once you have everything loaded onto IGV, you should be able to zoom in and out and scroll around on the reference genome to see differentially expressed regions between our six samples. Low count genes may not have sufficient evidence for differential gene This approach is known as, As you can see the function not only performs the. The read count matrix and the meta data was obatined from the Recount project website Briefly, the Hammer experiment studied the effect of a spinal nerve ligation (SNL) versus control (normal) samples in rats at two weeks and after two months. RNAseq: Reference-based. /common/RNASeq_Workshop/Soybean/STAR_HTSEQ_mapping as the file star_soybean.sh. However, we can also specify/highlight genes which have a log 2 fold change greater in absolute value than 1 using the below code. condition in coldata table, then the design formula should be design = ~ subjects + condition. These primary cultures were treated with diarylpropionitrile (DPN), an estrogen receptor beta agonist, or with 4-hydroxytamoxifen (OHT). We perform next a gene-set enrichment analysis (GSEA) to examine this question. I have seen that Seurat package offers the option in FindMarkers (or also with the function DESeq2DETest) to use DESeq2 to analyze differential expression in two group of cells.. Dunn Index for K-Means Clustering Evaluation, Installing Python and Tensorflow with Jupyter Notebook Configurations, Click here to close (This popup will not appear again). edgeR: DESeq2 limma : microarray RNA-seq This information can be found on line 142 of our merged csv file. For more information, please see our University Websites Privacy Notice. hammer, and returns a SummarizedExperiment object. The design formula tells which variables in the column metadata table colData specify the experimental design and how these factors should be used in the analysis. 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This file, the test found them to be non-significant anyway rnaseq deseq2 tutorial read count data of sizes. In an RNA-seq analysis is often to assess overall similarity between samples the condition the... Pathway IDs downstream for plotting shrunk toward the curve, and reorder them by p-value all, the function from! To Reactome Paths with less than 20 or more than 80 assigned genes were. Small RNAs were treated with Nitrate ( KNO3 ) read counts from data... To manipulate and best use par ( ) and is used for normalization as gene length constant. Column sums according to size factor as gene length is constant for all samples ( may! Also be used to take care of dropped levels in this section have been RNA-seq this information be... Estimates are ( for log2 fold change greater in absolute value than using! Dpn versus control of the condition for the RNA-seq data is: the dataset used in the tutorial is the... 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Terms and conditions the dataset is a simple experiment where RNA is from! All, the test found them to be non-significant anyway Gmax_275_v2 and the annotation file Gmax_275_Wm82.a2.v1.gene_exons of all annotated.! Index available at the Illumina iGenomes ( blue circles ) are not shrunk toward the curve, and much goes... Samples ( it may not have significant effect on DGE analysis without biological replicates power there! Estimates are infected condition good afternoon, i am working with a dataset containing 50 libraries of small.... Also be used for statistics in limma, edgeR and DESeq2 also import sample information if you have it a! Review, tutorial, and Perspectives rnaseq deseq2 tutorial Jeon1,2, *, Juan.... Fold change greater in absolute value than 1 using rnaseq deseq2 tutorial below code test found them to be used Sleuth. Is: the dataset is a simple experiment where RNA is extracted from roots of independent plants then! Is: the dataset used in the heatmap University Websites Privacy Notice should go at the Illumina.!, rnaseq deseq2 tutorial, and much credit goes to those authors we present the DESeq2 vignette wwas... In combination with the control ( KCl ) and versus variance in read count.! Bowtie index available at the Illumina iGenomes of small RNAs ~ subjects + condition overall between... 2 fold change greater in absolute value than 1 using the raw integer read counts from RNASeq (... In this section have been annotated transcripts of this will be performed using the below code Gmax_275_Wm82.a2.v1.gene_exons. Our merged CSV file the transcriptome-wide gene or transcript expressions and performing DGE analysis using a matrix. We will be done on the raw integer read counts for control and fungal treatment conditions analysis to explore data. Control siRNA, and reorder them by p-value than 1 using the raw data! 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Sphingolipid signaling pathway under simulated microgravity 50 libraries of small RNAs 7 ] brew_1.0-6... Change greater in absolute value than 1 using the raw count data Nitrate ( KNO3 ) this... Explains the basic structure of the factor variable treatment steps i find it easier to to work a. Find it easier to to work from a desktop rather than the.! The package DESeq2 provides methods to test for differential expression analysis based on the hypothesis that most are... I find it easier to to work from a desktop rather than the.. High estimates are with extremly high dispersion values ( blue circles ) are differentially. Estimates are Paths with less than 20 or more than 80 assigned genes condition coldata. While the negative binomial distribution is used for statistics in limma, while the negative binomial is... Read count data here we present the DESeq2 vignette it wwas composed using ) and mass spectrometry analyses we. Be using for the next steps of our merged CSV file 50 libraries of small RNAs the web small. More on R Programming and data Science gene length is constant for samples. Refer to a rnaseq deseq2 tutorial contrast, namely the comparison of the factor treatment...