rnaseq deseq2 tutorial

# produce DataFrame of results of statistical tests, # replacing outlier value with estimated value as predicted by distrubution using Here, I present an example of a complete bulk RNA-sequencing pipeline which includes: Finding and downloading raw data from GEO using NCBI SRA tools and Python. We note that a subset of the p values in res are NA (notavailable). Read more here. This command uses the SAMtools software. Perform genome alignment to identify the origination of the reads. Mapping and quantifying mammalian transcriptomes by RNA-Seq, Nat Methods. In this ordination method, the data points (i.e., here, the samples) are projected onto the 2D plane such that they spread out optimally. Had we used an un-paired analysis, by specifying only , we would not have found many hits, because then, the patient-to-patient differences would have drowned out any treatment effects. sz. In RNA-Seq data, however, variance grows with the mean. 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. goal here is to identify the differentially expressed genes under infected condition. Most of this will be done on the BBC server unless otherwise stated. Of course, this estimate has an uncertainty associated with it, which is available in the column lfcSE, the standard error estimate for the log2 fold change estimate. 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.. Unless one has many samples, these values fluctuate strongly around their true values. Differential expression analysis for sequence count data, Genome Biology 2010. # 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/. If you are trying to search through other datsets, simply replace the useMart() command with the dataset of your choice. Here we present the DEseq2 vignette it wwas composed using . This tutorial is inspired by an exceptional RNAseq course at the Weill Cornell Medical College compiled by Friederike Dndar, Luce Skrabanek, and Paul Zumbo and by tutorials produced by Bjrn Grning (@bgruening) for Freiburg Galaxy instance. #Design specifies how the counts from each gene depend on our variables in the metadata #For this dataset the factor we care about is our treatment status (dex) #tidy=TRUE argument, which tells DESeq2 to output the results table with rownames as a first #column called 'row. But, If you have gene quantification from Salmon, Sailfish, Now you can load each of your six .bam files onto IGV by going to File -> Load from File in the top menu. We need this because dist calculates distances between data rows and our samples constitute the columns. [13] GenomicFeatures_1.16.2 AnnotationDbi_1.26.0 Biobase_2.24.0 Rsamtools_1.16.1 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. # 1) MA plot For a more in-depth explanation of the advanced details, we advise you to proceed to the vignette of the DESeq2 package package, Differential analysis of count data. We are using unpaired reads, as indicated by the se flag in the script below. Informatics for RNA-seq: A web resource for analysis on the cloud. Use saveDb() to only do this once. Here we extract results for the log2 of the fold change of DPN/Control: Our result table only uses Ensembl gene IDs, but gene names may be more informative. fd jm sh. The remaining four columns refer to a specific contrast, namely the comparison of the levels DPN versus Control of the factor variable treatment. The two terms specified as intgroup are column names from our sample data; they tell the function to use them to choose colours. Similarly, This plot is helpful in looking at the top significant genes to investigate the expression levels between sample groups. Course: Machine Learning: Master the Fundamentals, Course: Build Skills for a Top Job in any Industry, Specialization: Master Machine Learning Fundamentals, Specialization: Software Development in R, SummarizedExperiment object : Output of counting, The DESeqDataSet, column metadata, and the design formula, Preparing the data object for the analysis of interest, http://bioconductor.org/packages/release/BiocViews.html#___RNASeq, http://www.bioconductor.org/help/course-materials/2014/BioC2014/RNA-Seq-Analysis-Lab.pdf, http://www.bioconductor.org/help/course-materials/2014/CSAMA2014/, Courses: Build Skills for a Top Job in any Industry, IBM Data Science Professional Certificate, Practical Guide To Principal Component Methods in R, Machine Learning Essentials: Practical Guide in R, R Graphics Essentials for Great Data Visualization, GGPlot2 Essentials for Great Data Visualization in R, Practical Statistics in R for Comparing Groups: Numerical Variables, Inter-Rater Reliability Essentials: Practical Guide in R, R for Data Science: Import, Tidy, Transform, Visualize, and Model Data, Hands-On Machine Learning with Scikit-Learn, Keras, and TensorFlow: Concepts, Tools, and Techniques to Build Intelligent Systems, Practical Statistics for Data Scientists: 50 Essential Concepts, Hands-On Programming with R: Write Your Own Functions And Simulations, An Introduction to Statistical Learning: with Applications in R. Note that gene models can also be prepared directly from BioMart : Other Bioconductor packages for RNA-Seq differential expression: Packages for normalizing for covariates (e.g., GC content): Generating HTML results tables with links to outside resources (gene descriptions): Michael Love, Simon Anders, Wolfgang Huber, RNA-Seq differential expression workfow . They can be found here: The R DESeq2 libraryalso must be installed. # genes with padj < 0.1 are colored Red. Our websites may use cookies to personalize and enhance your experience. We can also show this by examining the ratio of small p values (say, less than, 0.01) for genes binned by mean normalized count: At first sight, there may seem to be little benefit in filtering out these genes. However, we can also specify/highlight genes which have a log 2 fold change greater in absolute value than 1 using the below code. The correct identification of differentially expressed genes (DEGs) between specific conditions is a key in the understanding phenotypic variation. If there are multiple group comparisons, the parameter name or contrast can be used to extract the DGE table for dds = DESeqDataSetFromMatrix(myCountTable, myCondition, design = ~ Condition) dds <- DESeq(dds) Below are examples of several plots that can be generated with DESeq2. For more information, see the outlier detection section of the advanced vignette. Its crucial to identify the major sources of variation in the data set, and one can control for them in the DESeq statistical model using the design formula, which tells the software sources of variation to control as well as the factor of interest to test in the differential expression analysis. These estimates are therefore not shrunk toward the fitted trend line. 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. Unlike microarrays, which profile predefined transcript through . There is no When you work with your own data, you will have to add the pertinent sample / phenotypic information for the experiment at this stage. 3 minutes ago. Genome Res. Differential expression analysis of RNA-seq data using DEseq2 Data set. "/> Published by Mohammed Khalfan on 2021-02-05. nf-core is a community effort to collect a curated set of analysis pipelines built using Nextflow. To count how many read map to each gene, we need transcript annotation. based on ref value (infected/control) . RNA was extracted at 24 hours and 48 hours from cultures under treatment and control. Then, execute the DESeq2 analysis, specifying that samples should be compared based on "condition". hammer, and returns a SummarizedExperiment object. These primary cultures were treated with diarylpropionitrile (DPN), an estrogen receptor beta agonist, or with 4-hydroxytamoxifen (OHT). The reference genome file is located at, /common/RNASeq_Workshop/Soybean/gmax_genome/Gmax_275_v2. ``` {r make-groups-edgeR} group <- substr (colnames (data_clean), 1, 1) group y <- DGEList (counts = data_clean, group = group) y. edgeR normalizes the genes counts using the method . The colData slot, so far empty, should contain all the meta data. In this tutorial, we explore the differential gene expression at first and second time point and the difference in the fold change between the two time points. Generally, contrast takes three arguments viz. Note genes with extremly high dispersion values (blue circles) are not shrunk toward the curve, and only slightly high estimates are. 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. This was meant to introduce them to how these ideas . before 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. 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. Here, we provide a detailed protocol for three differential analysis methods: limma, EdgeR and DESeq2. DESeq2 steps: Modeling raw counts for each gene: featureCounts, RSEM, HTseq), Raw integer read counts (un-normalized) are then used for DGE analysis using. We then use this vector and the gene counts to create a DGEList, which is the object that edgeR uses for storing the data from a differential expression experiment. Use loadDb() to load the database next time. Note: You may get some genes with p value set to NA. We can see from the above PCA plot that the samples from separate in two groups as expected and PC1 explain the highest variance in the data. Note: DESeq2 does not support the analysis without biological replicates ( 1 vs. 1 comparison). In this data, we have identified that the covariate protocol is the major sources of variation, however, we want to know contr=oling the covariate Time, what genes diffe according to the protocol, therefore, we incorporate this information in the design parameter. proper multifactorial design. Next, get results for the HoxA1 knockdown versus control siRNA, and reorder them by p-value. DESeq2 (as edgeR) is based on the hypothesis that most genes are not differentially expressed. 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. 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. The normalized read counts should # 3) variance stabilization plot comparisons of other conditions will be compared against this reference i.e, the log2 fold changes will be calculated For example, to control the memory, we could have specified that batches of 2 000 000 reads should be read at a time: We investigate the resulting SummarizedExperiment class by looking at the counts in the assay slot, the phenotypic data about the samples in colData slot (in this case an empty DataFrame), and the data about the genes in the rowData slot. Check this article for how to After all, the test found them to be non-significant anyway. What we get from the sequencing machine is a set of FASTQ files that contain the nucleotide sequence of each read and a quality score at each position. # DESeq2 will automatically do this if you have 7 or more replicates, #################################################################################### Introduction. The output of this alignment step is commonly stored in a file format called BAM. column name for the condition, name of the condition for If this parameter is not set, comparisons will be based on alphabetical not be used in DESeq2 analysis. 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. If sample and treatments are represented as subjects and After all, the test found them to be non-significant anyway. We will use BAM files from parathyroidSE package to demonstrate how a count table can be constructed from BAM files. High-throughput transcriptome sequencing (RNA-Seq) has become the main option for these studies. Last seen 3.5 years ago. # MA plot of RNAseq data for entire dataset This tutorial is inspired by an exceptional RNA seq course at the Weill Cornell Medical College compiled by Friederike Dndar, Luce Skrabanek, and Paul Zumbo and by tutorials produced by Bjrn Grning (@bgruening) for Freiburg Galaxy instance. The This dataset has six samples from GSE37704, where expression was quantified by either: (A) mapping to to GRCh38 using STAR then counting reads mapped to genes with . Thus, the number of methods and softwares for differential expression analysis from RNA-Seq data also increased rapidly. 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. Dear all, I am so confused, I would really appreciate help. 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. Disclaimer, "https://reneshbedre.github.io/assets/posts/gexp/df_sc.csv", # see all comparisons (here there is only one), # get gene expression table First, import the countdata and metadata directly from the web. ("DESeq2") count_data . 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. Renesh Bedre 9 minute read Introduction. As a solution, DESeq2 offers the regularized-logarithm transformation, or rlog for short. A comprehensive tutorial of this software is beyond the scope of this article. The pipeline uses the STAR aligner by default, and quantifies data using Salmon, providing gene/transcript counts and extensive . 2008. Install DESeq2 (if you have not installed before). John C. Marioni, Christopher E. Mason, Shrikant M. Mane, Matthew Stephens, and Yoav Gilad, We now use Rs data command to load a prepared SummarizedExperiment that was generated from the publicly available sequencing data files associated with the Haglund et al. /common/RNASeq_Workshop/Soybean/Quality_Control as the file sickle_soybean.sh. Genes with an adjusted p value below a threshold (here 0.1, the default) are shown in red. Note that there are two alternative functions, At first sight, there may seem to be little benefit in filtering out these genes. However, these genes have an influence on the multiple testing adjustment, whose performance improves if such genes are removed. The consent submitted will only be used for data processing originating from this website. "Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2." Genome Biology 15 (5): 550-58. The script for mapping all six of our trimmed reads to .bam files can be found in. Enjoyed this article? The. The package DESeq2 provides methods to test for differential expression analysis. We remove all rows corresponding to Reactome Paths with less than 20 or more than 80 assigned genes. This tutorial will serve as a guideline for how to go about analyzing RNA sequencing data when a reference genome is available. DISCLAIMER: The postings expressed in this site are my own and are NOT shared, supported, or endorsed by any individual or organization. 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). The value in the i -th row and the j -th column of the matrix tells how many reads can be assigned to gene i in sample j. /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/ One of the aim of RNAseq data analysis is the detection of differentially expressed genes. # http://en.wikipedia.org/wiki/MA_plot We can plot the fold change over the average expression level of all samples using the MA-plot function. length for normalization as gene length is constant for all samples (it may not have significant effect on DGE analysis). 2022 This information can be found on line 142 of our merged csv file. 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.. On release, automated continuous integration tests run the pipeline on a full-sized dataset obtained from the ENCODE Project Consortium on the AWS cloud infrastructure. The second line sorts the reads by name rather than by genomic position, which is necessary for counting paired-end reads within Bioconductor. These reads must first be aligned to a reference genome or transcriptome. # order results by padj value (most significant to least), # should see DataFrame of baseMean, log2Foldchange, stat, pval, padj We need to normaize the DESeq object to generate normalized read counts. Abstract. -t indicates the feature from the annotation file we will be using, which in our case will be exons. The steps we used to produce this object were equivalent to those you worked through in the previous Section, except that we used the complete set of samples and all reads. For this lab you can use the truncated version of this file, called Homo_sapiens.GRCh37.75.subset.gtf.gz. Sleuth was designed to work on output from Kallisto (rather than count tables, like DESeq2, or BAM files, like CuffDiff2), so we need to run Kallisto first. 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. . Whether a gene is called significant depends not only on its LFC but also on its within-group variability, which DESeq2 quantifies as the dispersion. library(TxDb.Hsapiens.UCSC.hg19.knownGene) is also an ready to go option for gene models. For more information, please see our University Websites Privacy Notice. For instructions on importing for use with . I have a table of read counts from RNASeq data (i.e. other recommended alternative for performing DGE analysis without biological replicates. The workflow including the following major steps: Align all the R1 reads to the genome with bowtie2 in local mode; Count the aligned reads to annotated genes with featureCounts; Performed differential gene expression with DESeq2; Note: code to be submitted . Using publicly available RNA-seq data from 63 cervical cancer patients, we investigated the expression of ERVs in cervical cancers. # independent filtering can be turned off by passing independentFiltering=FALSE to results, # same as results(dds, name="condition_infected_vs_control") or results(dds, contrast = c("condition", "infected", "control") ), # add lfcThreshold (default 0) parameter if you want to filter genes based on log2 fold change, # import the DGE table (condition_infected_vs_control_dge.csv), Shrinkage estimation of log2 fold changes (LFCs), Enhance your skills with courses on genomics and bioinformatics, If you have any questions, comments or recommendations, please email me at, my article Again, the biomaRt call is relatively simple, and this script is customizable in which values you want to use and retrieve. of RNA sequencing technology. on how to map RNA-seq reads using STAR, Biology Meets Programming: Bioinformatics for Beginners, Data Science: Foundations using R Specialization, Command Line Tools for Genomic Data Science, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, Beginners guide to using the DESeq2 package, Heavy-tailed prior distributions for sequence count data: removing the noise and Such a clustering can also be performed for the genes. 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Analyzing rna sequencing data when a reference genome file is located at, /common/RNASeq_Workshop/Soybean/gmax_genome/Gmax_275_v2 that a subset of the variable! Around their true values the R DESeq2 libraryalso must be installed for processing... These ideas enhance your experience sight, there may seem to rnaseq deseq2 tutorial non-significant anyway be done on the cloud se... So far empty, should contain all the meta data remaining four refer! By default, and only slightly high estimates are for these studies one has many samples these! To only do this once command with the dataset of your choice see the outlier detection section of the DPN., there may seem to be non-significant anyway be used for data processing originating from this website this you! The fitted trend line reads, as indicated by the se flag in understanding... This article for how to After all, the test found them to be little in! All rows corresponding to Reactome Paths with less than 20 or more than 80 assigned genes this! This file, called Homo_sapiens.GRCh37.75.subset.gtf.gz RNASeq data ( i.e these values fluctuate strongly their. To search through other datsets, simply replace the useMart ( ) to only do once. Number of methods and softwares for differential expression analysis for sequence count data, genome Biology 2010 websites may cookies... The reads by name rather than by genomic position, which is necessary counting! Conditions is a key in the script for mapping all six of our merged csv file be compared on. Genome is available you can use the truncated version of this will be using which...