Step 4: calculate the normalized count values using the normalization factor. The first step in the DE analysis workflow is count normalization, which is necessary to make accurate comparisons of gene expression between samples. Count normalization with DESeq2 | Introduction to DGE - ARCHIVED Therefore, we cannot directly compare the counts for XCR1 (or any other gene) between sampleA and sampleB because the total number of normalized counts are different between samples. For each gene, a pseudo-reference sample is created that is equal to the geometric mean across all samples. Using RPKM/FPKM normalization, the total number of RPKM/FPKM normalized counts for each sample will be different. Lets start by creating the DESeqDataSet object and then we can talk a bit more about what is stored inside it. This is performed for each sample in the dataset. ### Check that sample names match in both files. Our count matrix input is stored inside the txi list object, and so we pass that in using the DESeqDataSetFromTximport() function which will extract the counts component and round the values to the nearest whole number. We can take a look at the normalization factor applied to each sample using: Now, to retrieve the normalized counts matrix from dds, we use the counts() function and add the argument normalized=TRUE. Normalization is the process of scaling raw count values to account for the "uninteresting" factors. If your data did not match, you could use the match() function to rearrange them to be matching. The median value (column-wise for the above table) of all ratios for a given sample is taken as the normalization factor (size factor) for that sample, as calculated below. These custom data structures are similar to lists in that they can contain multiple different data types/structures within them. To perform the median of ratios method of normalization, DESeq2 has a single estimateSizeFactors() function that will generate size factors for us. In the example, Gene X and Gene Y have similar levels of expression, but the number of reads mapped to Gene X would be many more than the number mapped to Gene Y because Gene X is longer. NOTE: Since we had created a data variable in the last lesson which contains the counts, we could have also used that as input. Step 1: creates a pseudo-reference sample (row-wise geometric mean). Usually these size factors are around 1, if you see large variations between samples it is important to take note since it might indicate the presence of extreme outliers. gene count comparisons between replicates of the same samplegroup; counts per length of transcript (kb) per million reads mapped. Notice that the differentially expressed genes should not affect the median value: normalization_factor_sampleA <- median(c(1.28, 1.3, 1.39, 1.35, 0.59)), normalization_factor_sampleB <- median(c(0.78, 0.77, 0.72, 0.74, 1.35)). We will also need to specify a design formula. For our dataset we only have one column we are interested in, that is ~sampletype. The median of ratios method makes the assumption that not ALL genes are differentially expressed; therefore, the normalization factors should account for sequencing depth and RNA composition of the sample (large outlier genes will not represent the median ratio values). Since tools for differential expression analysis are comparing the counts between sample groups for the same gene, gene length does not need to be accounted for by the tool. gene count comparisons within a sample or between samples of the same sample group; gene count comparisons between genes within a sample; counts divided by sample-specific size factors determined by median ratio of gene counts relative to geometric mean per gene, gene count comparisons between samples and for, uses a weighted trimmed mean of the log expression ratios between samples, Explore different types of normalization methods, Understand how to normalize counts using DESeq2. These are open access materials distributed under the terms of the Creative Commons Attribution license (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This column has three factor levels, which tells DESeq2 that for each gene we want to evaluate gene expression change with respect to these different levels. However, in that case we would want to use the DESeqDataSetFromMatrix() function. The counts in Sample B would be greatly skewed by the DE gene, which takes up most of the counts. Step 4: calculate the normalized count values using the normalization factor This is performed by dividing each raw count value in a given sample by that sample's normalization factor to generate normalized count values. For example, suppose we wanted the original count matrix we would use counts() (Note: we nested it within the View() function so that rather than getting printed in the console we can see it in the script editor) : As we go through the workflow we will use the relevant functions to check what information gets stored inside our object. These normalized counts will be useful for downstream visualization of results, but cannot be used as input to DESeq2 or any other tools that peform differential expression analysis which use the negative binomial model. For every gene in a sample, the ratios (sample/ref) are calculated (as shown below). DESeq2 will output an error if this is not the case. Basically, for a typical RNA-seq analysis, you would not run these steps individually. The figure below illustrates the median value for the distribution of all gene ratios for a single sample (frequency is on the y-axis). NOTE: The steps below describe in detail some of the steps performed by DESeq2 when you run a single function to get DE genes. This is performed for all count values (every gene in every sample). This is performed by dividing each raw count value in a given sample by that samples normalization factor to generate normalized count values. Count normalization with DESeq2 | Introduction to DGE - ARCHIVED We will use the function in the example below, but in a typical RNA-seq analysis this step is automatically performed by the DESeq() function, which we will see later. But, unlike lists they have pre-specified data slots, which hold specific types/classes of data. Suppose we had sample names matching in the counts matrix and metadata file, but they were out of order. By assigning the results back to the dds object we are filling in the slots of the DESeqDataSet object with the appropriate information. Gene expression units explained: RPM, RPKM, FPKM, TPM, DESeq, TMM Ensure the row names of the metadata dataframe are present and in the same order as the column names of the counts dataframe. The next step is to normalize the count data in order to be able to make fair gene comparisons between samples. Step 3: calculate the normalization factor for each sample (size factor). Write the line(s) of code required to create a new matrix with columns ordered such that they were identical to the row names of the metadata. Other genes for Sample B would therefore appear to be less expressed than those same genes in Sample A. Several common normalization methods exist to account for these differences: While TPM and RPKM/FPKM normalization methods both account for sequencing depth and gene length, RPKM/FPKM are not recommended. Bioconductor software packages often define and use a custom class within R for storing data (input data, intermediate data and also results). This method is robust to imbalance in up-/down-regulation and large numbers of differentially expressed genes. The main factors often considered during normalization are: Sequencing depth: Accounting for sequencing depth is necessary for comparison of gene expression between samples. Gene length: Accounting for gene length is necessary for comparing expression between different genes within the same sample. In this way the expression levels are more comparable between and/or within samples. For example, in the table above, SampleA has a greater proportion of counts associated with XCR1 (5.5/1,000,000) than does sampleB (5.5/1,500,000) even though the RPKM count values are the same. RPM is calculated by dividing the mapped reads count by a per million scaling factor of total mapped reads. This requires a few steps: We should always make sure that we have sample names that match between the two files, and that the samples are in the right order. The data stored in these pre-specified slots can be accessed by using specific package-defined functions. To create the object we will need the count matrix and the metadata table as input. Step 2: calculates ratio of each sample to the reference. Therefore, you cannot compare the normalized counts for each gene equally between samples. For example, if the median ratio for SampleA was 1.3 and the median ratio for SampleB was 0.77, you could calculate normalized counts as follows: Please note that normalized count values are not whole numbers. NOTE: This video by StatQuest shows in more detail why TPM should be used in place of RPKM/FPKM if needing to normalize for sequencing depth and gene length. To normalize for sequencing depth and RNA composition, DESeq2 uses the median of ratios method. We can save this normalized data matrix to file for later use: NOTE: DESeq2 doesnt actually use normalized counts, rather it uses the raw counts and models the normalization inside the Generalized Linear Model (GLM). In the example, imagine the sequencing depths are similar between Sample A and Sample B, and every gene except for gene DE presents similar expression level between samples. In this way the expression levels are more comparable between and/or within samples. On the user-end there is only one step, but on the back-end there are multiple steps involved, as described below. The reason is that the normalized count values output by the RPKM/FPKM method are not comparable between samples. Now that we know the theory of count normalization, we will normalize the counts for the Mov10 dataset using DESeq2. Accounting for RNA composition is recommended for accurate comparison of expression between samples, and is particularly important when performing differential expression analyses [1]. The design formula specifies the column(s) in the metadata table and how they should be used in the analysis. While normalization is essential for differential expression analyses, it is also necessary for exploratory data analysis, visualization of data, and whenever you are exploring or comparing counts between or within samples. Reads connected by dashed lines connect a read spanning an intron. The counts of mapped reads for each gene is proportional to the expression of RNA (interesting) in addition to many other factors (uninteresting). Normalization is the process of scaling raw count values to account for the uninteresting factors. You can use DESeq-specific functions to access the different slots and retrieve information, if you wish. The counts of mapped reads for each gene is proportional to the expression of RNA ("interesting") in addition to many other factors ("uninteresting"). This lesson has been developed by members of the teaching team at the Harvard Chan Bioinformatics Core (HBC). However, sequencing depth and RNA composition do need to be taken into account. In the example below, each gene appears to have doubled in expression in Sample A relative to Sample B, however this is a consequence of Sample A having double the sequencing depth. RNA composition: A few highly differentially expressed genes between samples, differences in the number of genes expressed between samples, or presence of contamination can skew some types of normalization methods. RPM (also known as CPM) is a basic gene expression unit that normalizes only for sequencing depth (depth-normalized The RPM is biased in some applications where the gene length influences gene expression, such as RNA-seq. Since the majority of genes are not differentially expressed, the majority of genes in each sample should have similar ratios within the sample. NOTE: In the figure above, each pink and green rectangle represents a read aligned to a gene. This is performed for all count values (every gene in every sample).
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