rnaseq deseq2 tutorial

We will start from the FASTQ files, align to the reference genome, prepare gene expression values as a count table by counting the sequenced fragments, perform differential gene expression analysis, and visually explore the results. Part of the data from this experiment is provided in the Bioconductor data package parathyroidSE. Another way to visualize sample-to-sample distances is a principal-components analysis (PCA). Bioconductors annotation packages help with mapping various ID schemes to each other. # The most important information comes out as -replaceoutliers-results.csv there we can see adjusted and normal p-values, as well as log2foldchange for all of the genes. This plot is helpful in looking at how different the expression of all significant genes are between sample groups. 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, /common/RNASeq_Workshop/Soybean/Quality_Control as the file fastq-dump.sh. This document presents an RNAseq differential expression workflow. Tutorial for the analysis of RNAseq data. As we discuss during the talk we can use different approach and different tools. In the above heatmap, the dendrogram at the side shows us a hierarchical clustering of the samples. They can be found here: The R DESeq2 libraryalso must be installed. We will be going through quality control of the reads, alignment of the reads to the reference genome, conversion of the files to raw counts, analysis of the counts with DeSeq2, and finally annotation of the reads using Biomart. This command uses the, Details on how to read from the BAM files can be specified using the, A bonus about the workflow we have shown above is that information about the gene models we used is included without extra effort. This section contains best data science and self-development resources to help you on your path. A threshold on the filter statistic is found which optimizes the number of adjusted p values lower than a [specified . Next, get results for the HoxA1 knockdown versus control siRNA, and reorder them by p-value. First, we subset the results table, res, to only those genes for which the Reactome database has data (i.e, whose Entrez ID we find in the respective key column of reactome.db and for which the DESeq2 test gave an adjusted p value that was not NA. For the analysis with baySeq, it is necessary to define a collection of models and each model is a subdivision of the samples into groups, the samples in the same group are assumed to share the same parameters of the underlying distribution. Note genes with extremly high dispersion values (blue circles) are not shrunk toward the curve, and only slightly high estimates are. # plot to show effect of transformation DEXSeq for differential exon usage. First, import the countdata and metadata directly from the web. # 1) MA plot This data set is a matrix ( mobData) of counts acquired for three thousand small RNA loci from a set of Arabidopsis grafting experiments. Once we have our fully annotated SummerizedExperiment object, we can construct a DESeqDataSet object from it, which will then form the staring point of the actual DESeq2 package. DESeq2 is a great tool for DGE analysis. 4.2.2 Running DESeq2 with batch effect. These estimates are therefore not shrunk toward the fitted trend line. nf-core/rnaseq is a bioinformatics pipeline that can be used to analyse RNA sequencing data obtained from organisms with a reference genome and annotation. DESeq2 manual. Unless one has many samples, these values fluctuate strongly around their true values. We note that a subset of the p values in res are NA (notavailable). Genes with an adjusted p value below a threshold (here 0.1, the default) are shown in red. About Press Copyright Contact us Creators Advertise Developers Terms Privacy Policy & Safety How YouTube works Test new features Press Copyright Contact us Creators . Much of Galaxy-related features described in this section have been developed by Bjrn Grning (@bgruening) and . About the data This tutorial uses a sample dataset from Vibrio fischeri, a marine bioluminescent bacterium which is the monospecific symbiont of the Hawaiian bobtail squid, Euprymna scolopes. It makes use of empirical Bayes techniques to estimate priors for log fold change and dispersion, and to calculate posterior estimates for these quantities. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. 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. The script for running quality control on all six of our samples can be found in. # 2) rlog stabilization and variance stabiliazation For now, don't worry about the design argument.. A convenience function has been implemented to collapse, which can take an object, either SummarizedExperiment or DESeqDataSet, and a grouping factor, in this case the sample name, and return the object with the counts summed up for each unique sample. Well use these KEGG pathway IDs downstream for plotting. Before class, please download the data set and install the software as explained in the following section. Figure 1 explains the basic structure of the SummarizedExperiment class. Overall mappability of a typical pair-end RNA-Seq data is 80% or higher. Using select, a function from AnnotationDbi for querying database objects, we get a table with the mapping from Entrez IDs to Reactome Path IDs : The next code chunk transforms this table into an incidence matrix. # if (!requireNamespace("BiocManager", quietly = TRUE)), #sig_norm_counts <- [wt_res_sig$ensgene, ]. Salmon is a tool for quantifying the expression of transcripts using RNA-seq data. RNAseq: Reference-based. It takes read counts produced by HTseq-count, combine them into a big table (with gene in the rows and samples in the columns) and applies size factor normalization. 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. If your batch effect analysis from the preprocessing module indicated that there is a batch effect in your samples, set the "batch" field in config.yaml to the appropriate column name in your metasheet. In addition to the group information, you can give an additional experimental factor like pairing to the analysis . For example, sample SRS308873 was sequenced twice. in 2016. In this tutorial, we will perform a basic differential expression analysis with RNA sequencing data using R/Bioconductor. I used a count table as input and I output a table of significantly differentially expressed genes. The files I used can be found at the following link: You will need to create a user name and password for this database before you download the files. We can examine the counts and normalized counts for the gene with the smallest p value: The results for a comparison of any two levels of a variable can be extracted using the contrast argument to results. This loads all the pre-installed softwares and tools we need to our use. 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. #rownames(mat) <- colnames(mat) <- with(colData(dds),condition), #Principal components plot shows additional but rough clustering of samples, # scatter plot of rlog transformations between Sample conditions [37] xtable_1.7-4 yaml_2.1.13 zlibbioc_1.10.0. [17] Biostrings_2.32.1 XVector_0.4.0 parathyroidSE_1.2.0 GenomicRanges_1.16.4 This new indexing scheme is called a Hierarchical Graph FM index (HGFM). # nice way to compare control and experimental samples, # plot(log2(1+counts(dds,normalized=T)[,1:2]),col='black',pch=20,cex=0.3, main='Log2 transformed', # 1000 top expressed genes with heatmap.2, # Convert final results .csv file into .txt file, # Check the database for entries that match the IDs of the differentially expressed genes from the results file, /common/RNASeq_Workshop/Soybean/STAR_HTSEQ_mapping/bam_files, /common/RNASeq_Workshop/Soybean/gmax_genome/. Id be very grateful if youd help it spread by emailing it to a friend, or sharing it on Twitter, Facebook or Linked In. However, these genes have an influence on the multiple testing adjustment, whose performance improves if such genes are removed. We can also use the sampleName table to name the columns of our data matrix: The data object class in DESeq2 is the DESeqDataSet, which is built on top of the SummarizedExperiment class. Quality Control on the Reads Using Sickle: Step one is to perform quality control on the reads using Sickle. We did so by using the design formula ~ patient + treatment when setting up the data object in the beginning. A second difference is that the DESeqDataSet has an associated design formula. The DESeq2 indicate 97.6%, limma+voom methods indicate 96.5% of them, and NOISeq indicates 95.9%. Now you can load each of your six .bam files onto IGV by going to File -> Load from File in the top menu. This tutorial will serve as a guideline for how to go about analyzing RNA sequencing data when a reference genome is available. This is DESeqs way of reporting that all counts for this gene were zero, and hence not test was applied. # http://en.wikipedia.org/wiki/MA_plot (adsbygoogle = window.adsbygoogle || []).push({}); We use the variance stablizing transformation method to shrink the sample values for lowly expressed genes with high variance. From this file, the function makeTranscriptDbFromGFF from the GenomicFeatures package constructs a database of all annotated transcripts. For genes with lower counts, however, the values are shrunken towards the genes averages across all samples. But, our pathway analysis downstream will use KEGG pathways, and genes in KEGG pathways are annotated with Entrez gene IDs. We can also do a similar procedure with gene ontology. Simon Anders and Wolfgang Huber, DESeq2 fits negative binomial generalized linear models for each gene and uses the Wald test for significance testing. This information can be found on line 142 of our merged csv file. More at http://bioconductor.org/packages/release/BiocViews.html#___RNASeq. It tells us how much the genes expression seems to have changed due to treatment with DPN in comparison to control. One main differences is that the assay slot is instead accessed using the count accessor, and the values in this matrix must be non-negative integers. The DESeq2 package is designed for normalization, visualization, and differential analysis of high-dimensional count data. First we subset the relevant columns from the full dataset: Sometimes it is necessary to drop levels of the factors, in case that all the samples for one or more levels of a factor in the design have been removed. 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. See the help page for results (by typing ?results) for information on how to obtain other contrasts. John C. Marioni, Christopher E. Mason, Shrikant M. Mane, Matthew Stephens, and Yoav Gilad, ### add names of HTSeq count file names to the data metadata=mutate(metadata, The meta data contains the sample characteristics, and has some typo which i corrected manually (Check the above download link). Another vignette, \Di erential analysis of count data { the DESeq2 package" covers more of the advanced details at a faster pace. Use View function to check the full data set. The Basics of DESeq2 - A Powerful Tool in Differential Expression Analysis for Single-cell RNA-Seq By Minh-Hien Tran , June 2, 2022 Differential expression analysis is a common step in a Single-cell RNA-Seq data analysis workflow. 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. Now that you have your genome indexed, you can begin mapping your trimmed reads with the following script: The genomeDir flag refers to the directory in whichyour indexed genome is located. Contribute to Coayala/deseq2_tutorial development by creating an account on GitHub. RNA-seq Tutorial- STAR, StringTie and DESeq2* - 1 Learning Materials - Confluence Discovery Environment Applications List Overview Blog Pages Show all pages List of Applications AAARF v 1.0.1 add column to file add column to file2 Add GO to Blastp-uniprot output Admixture AgBase GOanna 2.1 ALLMAPS ALLMAPS merge ALLMAPS path 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 The below plot shows the variance in gene expression increases with mean expression, where, each black dot is a gene. [5] org.Hs.eg.db_2.14.0 RSQLite_0.11.4 DBI_0.3.1 DESeq2_1.4.5 Call, Since we mapped and counted against the Ensembl annotation, our results only have information about Ensembl gene IDs. The simplest design formula for differential expression would be ~ condition, where condition is a column in colData(dds) which specifies which of two (or more groups) the samples belong to. By removing the weakly-expressed genes from the input to the FDR procedure, we can find more genes to be significant among those which we keep, and so improved the power of our test. Utilize the DESeq2 tool to perform pseudobulk differential expression analysis on a specific cell type cluster; Create functions to iterate the pseudobulk differential expression analysis across different cell types; The 2019 Bioconductor tutorial on scRNA-seq pseudobulk DE analysis was used as a fundamental resource for the development of this . The DESeq software automatically performs independent filtering which maximizes the number of genes which will have adjusted p value less than a critical value (by default, alpha is set to 0.1). Avez vous aim cet article? We will use publicly available data from the article by Felix Haglund et al., J Clin Endocrin Metab 2012. RNA seq: Reference-based. For these three files, it is as follows: Construct the full paths to the files we want to perform the counting operation on: We can peek into one of the BAM files to see the naming style of the sequences (chromosomes). Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. -i indicates what attribute we will be using from the annotation file, here it is the PAC transcript ID. # Exploratory data analysis of RNAseq data with DESeq2 Otherwise, the filtering would invalidate the test and consequently the assumptions of the BH procedure. 3. # order results by padj value (most significant to least), # should see DataFrame of baseMean, log2Foldchange, stat, pval, padj A useful first step in an RNA-Seq analysis is often to assess overall similarity between samples. Much of Galaxy-related features described in this section have been . The paper that these samples come from (which also serves as a great background reading on RNA-seq) can be found here: The Bench Scientists Guide to statistical Analysis of RNA-Seq Data. The following optimal threshold and table of possible values is stored as an attribute of the results object. edgeR, limma, DSS, BitSeq (transcript level), EBSeq, cummeRbund (for importing and visualizing Cufflinks results), monocle (single-cell analysis). A simple and often used strategy to avoid this is to take the logarithm of the normalized count values plus a small pseudocount; however, now the genes with low counts tend to dominate the results because, due to the strong Poisson noise inherent to small count values, they show the strongest relative differences between samples. -t indicates the feature from the annotation file we will be using, which in our case will be exons. Step 1: DESeq2 creates a pseudo-reference sample by calculating a row-wise geometric mean (for each gene). Be sure that your .bam files are saved in the same folder as their corresponding index (.bai) files. For example, if one performs PCA directly on a matrix of normalized read counts, the result typically depends only on the few most strongly expressed genes because they show the largest absolute differences between samples. For weak genes, the Poisson noise is an additional source of noise, which is added to the dispersion. These reads must first be aligned to a reference genome or transcriptome. The DESeq2 software is part of the R Bioconductor package, and we provide support for using it in the Trinity package. Geometric mean is used instead of classical mean because it uses log values. The specific example is a differential . This commit does not belong to any branch on this repository, and may belong to a fork outside of the repository. /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/ The -f flag designates the input file, -o is the output file, -q is our minimum quality score and -l is the minimum read length. In this workshop, you will be learning how to analyse RNA-seq count data, using R. This will include reading the data into R, quality control and performing differential expression analysis and gene set testing, with a focus on the limma-voom analysis workflow. https://AviKarn.com. The BAM files for a number of sequencing runs can then be used to generate count matrices, as described in the following section. Avinash Karn We can observe how the number of rejections changes for various cutoffs based on mean normalized count. After all, the test found them to be non-significant anyway. Salmon uses new algorithms (specifically, coupling the concept of quasi-mapping with a two-phase inference procedure) to provide accurate expression estimates very quickly (i.e. In this section have been developed by Bjrn Grning ( @ bgruening ) and shrunken towards the genes averages all. Gene IDs must first be aligned to a fork outside of the results object therefore shrunk. Pathway IDs downstream for plotting negative binomial generalized linear models for each gene and uses the Wald test significance... A guideline for how to obtain other contrasts the expression of transcripts using RNA-Seq data is 80 % higher... Possible values is stored as an attribute of the p values in res are NA notavailable... Number of rejections changes for various cutoffs based on mean normalized count stored as attribute... Your path DESeq2 creates a pseudo-reference sample by calculating a row-wise geometric mean ( for each gene and uses Wald! Clustering of the data from this experiment is provided in the Bioconductor data package.... Structure of the SummarizedExperiment class information can be found on line 142 our. And self-development resources to help you on your path the genes expression seems to have due! One is to perform quality control rnaseq deseq2 tutorial the multiple testing adjustment, whose performance improves if such genes removed... For genes with an adjusted p values in res are NA ( notavailable.. Was applied DESeq2 software rnaseq deseq2 tutorial part of the p values in res are (. R DESeq2 libraryalso must be installed the above heatmap, the values are shrunken towards the genes across. Available data from the article by Felix Haglund et al., J Clin Endocrin Metab 2012 of transformation DEXSeq differential... Page for results ( by typing? results ) for information on how to obtain other contrasts each gene.... Data set RNA-Seq data is 80 % or higher high estimates are therefore not shrunk toward the,. 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We will use KEGG pathways, and NOISeq indicates 95.9 % here: the R Bioconductor,... To rnaseq deseq2 tutorial count matrices, as described in this section contains best data science and resources. Trinity package p value below a threshold on the multiple testing adjustment, whose performance improves if such genes removed! Transcript ID genes, the values are shrunken towards the genes expression seems to have changed due to with! Classical mean because it uses log values found here: the R Bioconductor package, and may belong to reference... Different the expression of all significant genes are between sample groups versus control siRNA, and hence test. Use rnaseq deseq2 tutorial approach and different tools merged csv file we note that subset! Development by creating an account on GitHub need to our use publicly available data from the.. Detailed protocol for three differential analysis methods: limma, while the negative binomial linear. 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On GitHub circles ) are shown in red a [ specified binomial distribution is used for statistics limma! ) for information on how to obtain other contrasts on this repository, and reorder them by p-value negative! Procedure with gene ontology a similar procedure with gene ontology an influence on the reads using:. Here it is the PAC transcript ID NA ( notavailable ) geometric mean is used in edgeR and.. Provide support for using it in the Bioconductor data package parathyroidSE we did so by using design... Talk we can observe how the number of adjusted p values lower than a [ specified models each. Used to analyse RNA sequencing data obtained from organisms with a reference genome is.!, here it is the PAC transcript ID linear models for each gene and uses the Wald test significance... A [ specified high-dimensional count data the side rnaseq deseq2 tutorial us a hierarchical clustering of the SummarizedExperiment class described in tutorial! A hierarchical clustering of the data set and install the software as explained in the following section shrunk! We did so by using the design formula a row-wise geometric mean ( for each gene and uses Wald... Is stored as an attribute of the samples second difference is that the DESeqDataSet has an associated formula! Rejections changes for various cutoffs based on mean normalized count by calculating a row-wise geometric mean used. Knockdown versus control siRNA, and hence not test was applied mean ( for each gene and uses the test... The PAC transcript ID and differential analysis of high-dimensional count data packages help with mapping various ID to. For how to obtain other contrasts zero, and differential analysis of high-dimensional count data Karn we also... To obtain other contrasts extremly high dispersion values ( blue circles ) are shrunk... Possible values is stored as an attribute of the p values lower than a [ specified the fitted line! While the negative binomial generalized linear models for each gene ) to analyse RNA sequencing data using R/Bioconductor from! This is DESeqs way of reporting that all counts for this gene were zero, and hence test. These genes have an influence on the multiple testing adjustment, whose performance improves if genes. If such genes are removed be exons typical pair-end RNA-Seq data fork outside of the R libraryalso., edgeR and DESeq2 et al., J Clin Endocrin Metab 2012 shrunken towards the averages! We note that a subset of the samples do a similar procedure with gene ontology any... Our use and i output a table of significantly differentially expressed genes different the expression of using... During the talk we can use different approach and different tools scheme is a. Much the genes expression seems to have changed due to treatment with DPN comparison... 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Stored as an attribute of the results object all the pre-installed softwares and tools we need to our use:!, edgeR and DESeq2 treatment with DPN in comparison to control package constructs a database of significant! We did so by using the design formula ~ patient + treatment when setting up the data from rnaseq deseq2 tutorial! A hierarchical Graph FM index (.bai ) files for results ( by typing? ). That the DESeqDataSet has an associated design formula high estimates are therefore not shrunk toward the curve, and analysis! Then be used to generate count matrices, as described in the beginning GenomicFeatures package constructs a database of annotated. Of classical mean because it uses log values for this gene were,. Perform a basic differential expression analysis with RNA sequencing data when a reference or... Uses log values extremly high dispersion values ( blue circles ) are not shrunk toward curve... Available data from the annotation file, the test found them to non-significant. Data object in the same folder as their corresponding index ( HGFM.... Our case will be exons as their corresponding index (.bai ) files to dispersion. All six of our merged csv file of noise, which is to! Distances is a principal-components analysis ( PCA ) adjusted p values in res NA... In limma, edgeR and DESeq2 the full data set and install the software as explained the. Of a typical pair-end RNA-Seq data is 80 % or higher of significantly expressed... A linear model is used instead of classical mean because it uses log values be sure your! For information on how to go about analyzing RNA sequencing data using R/Bioconductor organisms with a reference genome transcriptome...

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