RNA-Seq DE (Differential Expression)

“open” button – opens files with extensions: .txt, .fasta (.fas, .fa, .fsa, .ffn, .fna), .fastq.

  • Genes differential expression
  • miRNAs differential expression

Genes differential expression
The differential gene expression analysis performs for two conditions designated as the control and the induced conditions. 
First, open the read files (.fasta or .fastq) in the corresponding table (windows): “Induced RNA sample”, “Control RNA sample”. Use the “delete” button to remove files from the tables. Each replicate of the induced sample should be provided together with a control sample replicate.
Then open a genbank file of the genome to generate a list of genes for analysis. Use the “clear” button to empty the list of genes. Press the “gene types” button to choose the types of genes. Select the corresponding rows in the “Genome” table and press the “add” button to select a gene for analysis. The selected genes will appear in the table “Selected genes”. Use the “delete” button to remove the selected genes. Housekeeping genes can be selected using the corresponding rows in the “Genome” table and pressing the “add” button above the table “Housekeeping genes”. Use the “delete” button to remove the selected housekeeping genes. The selected section of the “Housekeeping genes” should be On. If the selected section is Off, the further analysis will be performed without a reference to housekeeping genes.
Set the number of seeds and the seed size (in bp). The seed size should be less than the read size and the number of seeds is limited by the ratio of the gene length to seed size. See the explaining scheme below. For instance, for two seeds, the number of exact matches of seed 1 in the reads will be added up to the number of exact matches of seed 2. The resulting numbers will be used to calculate the relative expression of genes between the induced and control samples. The calculation for higher number of seeds is more reliable but it takes more time. 
In the case of several replicates the average and standard deviation values will be calculated. 

Pressing the “find” button starts the calculation. The process can be cancelled using the “cancel” button. When the process is completed, press the button “up/down genes Graph/Table” to see the the graphic representation of up and downregulated genes. To see the list of regulated genes press the button “table of up/down genes”. To set the minimum regulation fold use the popup menu “fold greater than”.

miRNAs differential expression
The differential miRNAs expression analysis performs for two conditions designated as the control and the induced conditions. First, open the reads files (.fasta or .fastq) in corresponding tables: “Induced miRNA sample” and “Control miRNA sample”. Use the “delete” button to remove the files from the tables. Each induced sample replicate should be provided with a control sample replicate.
Set the corresponding organism from the list of the organisms provided (third level, right side). The search field can help to find the organism of interest. Double click on the organism row to open a window with settings for default (from miRBase.org) and custom miRNA databases. Alternatively, double click on table header when the desired organism is not in the table. For generation of custom database set “miRNA name”, “Organism”, “miRNA sequence” and then press the “add” button. To export, import or to delete the custom miRNA database file (fasta file with .fa extension) use corresponding buttons.

Press the “find” button to find exact matches of miRNAs in the reads. Use the “cancel” button to stop the process. To set the minimum of regulation fold use the popup menu “fold greater than”. To see the found miRNAs in the sample, select the corresponding row in the table of samples. The found miRNAs will be displayed in the window “Found miRNAs”. To search for a particular miRNA use the search field. Press the button “up/down miRNAs Graph/Table” to see the graphic representation of up and downregulated miRNAs. To see the lists of regulated miRNAs press the button “table of up/down miRNA”.  

To print or save the graphic representation as a pdf file, use the “print” or “save” buttons. To save the tables of up and downregulated miRNAs use the “save” button. 
Note. The presented values of fold-change of up/downregulated genes/miRNAs will be normalised according to the read library sizes of induced and control samples, i.e. the non-normalised folds will be multiplied by the normalisation factor calculated as the ratio of total numbers of reads of control sample to total numbers of reads of induced sample. This procedure is equivalent to the Reads Per Million Reads (RPM) normalisation.

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