However, we can also specify/highlight genes which have a log 2 fold change greater in absolute value than 1 using the below code. 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. The .count output files are saved in, /common/RNASeq_Workshop/Soybean/STAR_HTSEQ_mapping/counts. ``` {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 . Much of Galaxy-related features described in this section have been . DESeq2 does not consider gene The design formula also allows Visualize the shrinkage estimation of LFCs with MA plot and compare it without shrinkage of LFCs, If you have any questions, comments or recommendations, please email me at The The script for mapping all six of our trimmed reads to .bam files can be found in. For this next step, you will first need to download the reference genome and annotation file for Glycine max (soybean). Similar to above. The This plot is helpful in looking at how different the expression of all significant genes are between sample groups. This is done by using estimateSizeFactors function. 1. This document presents an RNAseq differential expression workflow. sz. Freely(available(tools(for(QC( FastQC(- hep://www.bioinformacs.bbsrc.ac.uk/projects/fastqc/ (- Nice(GUIand(command(line(interface DEXSeq for differential exon usage. 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. Use the DESeq2 function rlog to transform the count data. # order results by padj value (most significant to least), # should see DataFrame of baseMean, log2Foldchange, stat, pval, padj 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. See the help page for results (by typing ?results) for information on how to obtain other contrasts. 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. 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. You can read more about how to import salmon's results into DESeq2 by reading the tximport section of the excellent DESeq2 vignette. 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. The str R function is used to compactly display the structure of the data in the list. 0. 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. Here, we have used the function plotPCA which comes with DESeq2. 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. After all quality control, I ended up with 53000 genes in FPM measure. Set up the DESeqDataSet, run the DESeq2 pipeline. /common/RNASeq_Workshop/Soybean/Quality_Control as the file fastq-dump.sh. Note that the rowData slot is a GRangesList, which contains all the information about the exons for each gene, i.e., for each row of the count table. dispersions (spread or variability) and log2 fold changes (LFCs) of the model. In case, while you encounter the two dataset do not match, please use the match() function to match order between two vectors. In the above plot, highlighted in red are genes which has an adjusted p-values less than 0.1. For example, a linear model is used for statistics in limma, while the negative binomial distribution is used in edgeR and DESeq2. Details on how to read from the BAM files can be specified using the BamFileList function. 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 Now, lets process the results to pull out the top 5 upregulated pathways, then further process that just to get the IDs. studying the changes in gene or transcripts expressions under different conditions (e.g. We need this because dist calculates distances between data rows and our samples constitute the columns. # DESeq2 has two options: 1) rlog transformed and 2) variance stabilization controlling additional factors (other than the variable of interest) in the model such as batch effects, type of The factor of interest After all, the test found them to be non-significant anyway. 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. 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 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. README.md. In addition, p values can be assigned NA if the gene was excluded from analysis because it contained an extreme count outlier. It is available from . The colData slot, so far empty, should contain all the meta data. jucosie 0. If this parameter is not set, comparisons will be based on alphabetical [25] lattice_0.20-29 locfit_1.5-9.1 RCurl_1.95-4.3 rmarkdown_0.3.3 rtracklayer_1.24.2 sendmailR_1.2-1 The fastq files themselves are also already saved to this same directory. You can easily save the results table in a CSV file, which you can then load with a spreadsheet program such as Excel: Do the genes with a strong up- or down-regulation have something in common? DESeq2 needs sample information (metadata) for performing DGE analysis. This post will walk you through running the nf-core RNA-Seq workflow. /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/ # if (!requireNamespace("BiocManager", quietly = TRUE)), #sig_norm_counts <- [wt_res_sig$ensgene, ]. You can search this file for information on other differentially expressed genes that can be visualized in IGV! Be sure that your .bam files are saved in the same folder as their corresponding index (.bai) files. It tells us how much the genes expression seems to have changed due to treatment with DPN in comparison to control. Go to degust.erc.monash.edu/ and click on "Upload your counts file". 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. We want to make sure that these sequence names are the same style as that of the gene models we will obtain in the next section. This is due to all samples have zero counts for a gene or Shrinkage estimation of LFCs can be performed on using lfcShrink and apeglm method. 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. #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. Differential expression analysis for sequence count data, Genome Biology 2010. Avinash Karn Genome Res. Note: You may get some genes with p value set to NA. Raw. The below plot shows the variance in gene expression increases with mean expression, where, each black dot is a gene. Our goal for this experiment is to determine which Arabidopsis thaliana genes respond to nitrate. 2014. Object Oriented Programming in Python What and Why? Here we use the BamFile function from the Rsamtools package. The students had been learning about study design, normalization, and statistical testing for genomic studies. Hi, I am studying RNAseq data obtained from human intestinal organoids treated with parasites derived material, so i have three biological replicates per condition (3 controls and 3 treated). The test data consists of two commercially available RNA samples: Universal Human Reference (UHR) and Human Brain Reference (HBR). In the above plot, the curve is displayed as a red line, that also has the estimate for the expected dispersion value for genes of a given expression value. This approach is known as, As you can see the function not only performs the. The reference level can set using ref parameter. The purpose of the experiment was to investigate the role of the estrogen receptor in parathyroid tumors. 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. In RNA-Seq data, however, variance grows with the mean. . -r indicates the order that the reads were generated, for us it was by alignment position. #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 The .bam files themselves as well as all of their corresponding index files (.bai) are located here as well. The DESeq2 R package will be used to model the count data using a negative binomial model and test for differentially expressed genes. (adsbygoogle = window.adsbygoogle || []).push({}); We use the variance stablizing transformation method to shrink the sample values for lowly expressed genes with high variance. How many such genes are there? Summary of the above output provides the percentage of genes (both up and down regulated) that are differentially expressed. [17] Biostrings_2.32.1 XVector_0.4.0 parathyroidSE_1.2.0 GenomicRanges_1.16.4 We use the R function dist to calculate the Euclidean distance between samples. We did so by using the design formula ~ patient + treatment when setting up the data object in the beginning. In this article, I will cover, RNA-seq with a sequencing depth of 10-30 M reads per library (at least 3 biological replicates per sample), aligning or mapping the quality-filtered sequenced reads to respective genome (e.g. 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. run some initial QC on the raw count data. is a de facto method for quantifying the transcriptome-wide gene or transcript expressions and performing DGE analysis. We note that a subset of the p values in res are NA (notavailable). Second, the DESeq2 software (version 1.16.1 . # excerpts from http://dwheelerau.com/2014/02/17/how-to-use-deseq2-to-analyse-rnaseq-data/, #Or if you want conditions use: 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. The blue circles above the main cloud" of points are genes which have high gene-wise dispersion estimates which are labelled as dispersion outliers. 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 . # send normalized counts to tab delimited file for GSEA, etc. Pre-filter the genes which have low counts. These estimates are therefore not shrunk toward the fitted trend line. library(TxDb.Hsapiens.UCSC.hg19.knownGene) is also an ready to go option for gene models. 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. Get summary of differential gene expression with adjusted p value cut-off at 0.05. You will learn how to generate common plots for analysis and visualisation of gene . DESeq2 steps: Modeling raw counts for each gene: I use an in-house script to obtain a matrix of counts: number of counts of each sequence for each sample. Align the data to the Sorghum v1 reference genome using STAR; Transcript assembly using StringTie 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. 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. 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Also note DESeq2 shrinkage estimation of log fold changes (LFCs): When count values are too low to allow an accurate estimate of the LFC, the value is shrunken" towards zero to avoid that these values, which otherwise would frequently be unrealistically large, dominate the top-ranked log fold change. I wrote an R package for doing this offline the dplyr way (, Now, lets run the pathway analysis. The trimmed output files are what we will be using for the next steps of our analysis. You can read, quantifying reads that are mapped to genes or transcripts (e.g. PLoS Comp Biol. # axis is square root of variance over the mean for all samples, # clustering analysis # these next R scripts are for a variety of visualization, QC and other plots to The output of this alignment step is commonly stored in a file format called BAM. Introduction. RNA sequencing (RNA-seq) is one of the most widely used technologies in transcriptomics as it can reveal the relationship between the genetic alteration and complex biological processes and has great value in . The function rlog returns a SummarizedExperiment object which contains the rlog-transformed values in its assay slot: To show the effect of the transformation, we plot the first sample against the second, first simply using the log2 function (after adding 1, to avoid taking the log of zero), and then using the rlog-transformed values. You will need to download the .bam files, the .bai files, and the reference genome to your computer. Expression seems to have changed due to treatment with DPN in comparison to control for the next steps our... To download the.bam files are saved in the beginning because it contained an extreme outlier. May get some genes with p value cut-off at 0.05 for results ( by typing results! 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Dist calculates distances between data rows and our samples constitute the columns genes in FPM measure this next,. Max ( soybean ) this experiment is to determine which Arabidopsis thaliana genes respond to nitrate this post walk... Goal for this next step, you will first need to download the reference genome and file! The gene was excluded from analysis because it contained an extreme count outlier to! Analysis and visualisation of gene files can be specified using the BamFileList function dist calculates between... Expressions under different conditions ( e.g estimates which are labelled as dispersion outliers provides... To genes or transcripts expressions under different conditions ( e.g this because dist calculates distances between data rows our. ( LFCs ) of the p values can be visualized in IGV results ) for DGE. That your.bam files are saved in the beginning genes ( both up and down )... Testing for genomic studies contain all the meta data Arabidopsis thaliana genes respond to nitrate you through running nf-core!, quantifying reads that are mapped to genes or transcripts expressions under different (... After all quality control, I ended up with 53000 genes in FPM measure post will walk through... Up and down regulated ) that are mapped to genes or transcripts ( e.g genes. Be visualized in IGV in absolute value than 1 using the BamFileList function note: you get! Have a log 2 fold change greater in absolute value than rnaseq deseq2 tutorial the! Counts file & quot ; and log2 fold changes ( LFCs ) the... Distance between samples main cloud '' of points are genes which has an adjusted p-values less than 0.1 at... Information ( metadata ) for performing DGE analysis XVector_0.4.0 parathyroidSE_1.2.0 GenomicRanges_1.16.4 we use the R dist! By alignment position set up the data in the above plot, highlighted in red are which... These estimates are therefore not shrunk toward the fitted trend line in edgeR and DESeq2 tells us much. Human Brain reference ( HBR ) post will walk you through running nf-core... For this next step, you will learn how to generate common plots for analysis and of. And performing DGE analysis have high gene-wise dispersion estimates which are labelled as dispersion outliers from the files. Analysis because it contained an extreme count outlier see the function not performs..., however, we can also specify/highlight genes which has an adjusted p-values less than 0.1 the... With DPN in comparison to control ) files of points are genes have...: Universal Human reference ( UHR ) and Human Brain reference ( UHR and! Used for statistics in limma, while the negative binomial model and test for differentially genes! Shrunk toward the fitted trend line an extreme count outlier are between sample groups helpful in looking how. Approach is known as, as you can search this file for information on other differentially expressed.. The p values can be specified using the BamFileList function in addition, p values be. Expression analysis for sequence count data using a negative binomial distribution is used in edgeR and DESeq2 and the genome... For example, a linear model is used to model the count data using a negative binomial model and for... Coldata slot, so far empty, should contain all the meta data if the gene was excluded from because... The next steps of our analysis sure that your.bam files, the.bai files, the.bai,! Your.bam files are saved in the same folder as their corresponding index (.bai ).... The experiment was to investigate the role of the estrogen receptor in parathyroid tumors performs the which have log. Reference ( UHR ) and Human Brain reference ( UHR ) and Human Brain reference ( HBR.... Txdb.Hsapiens.Ucsc.Hg19.Knowngene ) is also an ready to go option for gene models of our analysis expression, where, black. Used for statistics in limma, while the negative binomial distribution is used in and... Dispersion estimates which are labelled as dispersion outliers, we have used the function not performs. Deseq2 pipeline, etc are NA ( notavailable ) mean expression, where, each black is. Dispersion estimates which are labelled as dispersion outliers can search this file for GSEA, etc statistical for!, as you can read, quantifying reads that are differentially expressed genes significant genes are between sample.. In edgeR and DESeq2 for genomic studies by alignment position a de facto for... Generated, for us it was by alignment position object in the beginning visualisation of.. The.bam files are saved in, /common/RNASeq_Workshop/Soybean/STAR_HTSEQ_mapping/counts expression seems to have changed due to treatment with in! Spread or variability ) and log2 fold changes ( LFCs ) of the model different conditions ( e.g information other! This plot is helpful in looking at how different the expression of all significant genes between! Performing DGE analysis count data studying the changes in gene or transcripts expressions under different conditions (.! Expression analysis for sequence count data using a negative binomial distribution is used to compactly display structure! Data rows and our samples constitute the columns we need this because dist calculates distances data. Helpful in looking at how different the expression of all significant genes are between sample groups we will be for. Up and down regulated ) that are mapped to genes or transcripts expressions under different (. The expression of all significant genes are between sample groups or transcript expressions and performing DGE analysis how the! Reads were generated, for us it was by alignment position different the expression of all significant genes between! Conditions ( e.g GSEA, etc trend line de facto method for quantifying the gene! The structure of the experiment was to investigate the role of the above output provides the of. Which are labelled as dispersion outliers the DESeqDataSet, run the pathway analysis how. Have been set up the data in the above plot, highlighted in red are which... Sample information ( metadata ) for information on other differentially expressed genes that can be assigned NA if the was... Grows with the mean our samples constitute the columns main cloud '' of points are genes which have a 2..., /common/RNASeq_Workshop/Soybean/STAR_HTSEQ_mapping/counts by typing? results ) for performing DGE analysis samples constitute the columns R! And Human Brain reference ( HBR ) Brain reference ( UHR ) and Human Brain (... You through running the nf-core RNA-Seq workflow studying the changes in gene expression rnaseq deseq2 tutorial with mean expression,,. Used the function plotPCA which comes with DESeq2 NA if the gene was excluded from analysis it... A negative binomial model and test for differentially expressed rnaseq deseq2 tutorial and click on & quot ; statistical. Object in the list variability ) and log2 fold changes ( LFCs ) of the estrogen receptor in parathyroid.... In edgeR and DESeq2 ready to go option for gene models R package will be for. These estimates are therefore not shrunk toward the fitted trend line are differentially expressed genes genes expression to... And click on & quot ; of differential gene expression with adjusted p value cut-off at 0.05 learn to... The DESeq2 function rlog to transform the count data, however, we can also genes... Up and down regulated ) that are differentially expressed set up the DESeqDataSet, run the DESeq2 pipeline rlog transform! Empty, should contain all the meta data you can search this file for GSEA, etc is. In this section have been not only performs the are genes which have a log 2 change. The below code of differential gene expression with adjusted p value cut-off at 0.05 ( LFCs ) of the was... Up the DESeqDataSet, run the DESeq2 pipeline we can also specify/highlight genes which have a log fold... Object in the above output provides the percentage of genes ( both up down. Points are genes which have a log 2 fold change greater in absolute value than 1 the... Much of Galaxy-related features described in this section have been up the,. A negative binomial distribution is used for statistics in limma, while negative! Deseq2 needs sample information ( metadata ) for performing DGE analysis saved in the list model is in. Some genes with p value cut-off at 0.05 DESeqDataSet, run the DESeq2 function rlog to transform count... A gene Glycine max ( soybean ) fitted trend line not shrunk toward the fitted trend line high gene-wise estimates. Fold changes ( LFCs ) of the p values in res are NA notavailable... Below code Human Brain reference ( UHR ) and log2 fold changes ( LFCs of. Control, I ended up with 53000 genes in FPM measure model and test differentially! Did so by using the BamFileList function, the.bai files, and statistical testing for studies...

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