QSeq supports loading miRNA sequence data for miRNA discovery and quantification. To use QSeq for miRNA discovery, choose File > Import Experiments > miRNA, and then select your miRNA files to import during the Add Experiments to Import step of the Project Setup Wizard. (For a list of supported file formats, see Supported File Types.) The wizard will then prompt you to load or download your reference sequences, also called templates, during the Set Up Preprocessing step , as well as give you access for defining processing parameters.
By default, each miRNA sequence file that is imported is treated as its own experiment and is processed individually. Alternatively, files can be merged, allowing the sequence data in multiple files to be treated as a single experiment. During processing, reads are mapped to a target, such as a genomic template. QSeq then looks for peaks, or regions where more reads than expected map to the target.
QSeq uses mers, defined by a sliding window of a specified number of bases (default is 15) to determine where reads are mapped to the template. Several options are available for determining the minimum requirements necessary for a read to be considered as a match to a template. QSeq uses one of three peak detection algorithms to determine where the peaks are located based on the read mapping. You may adjust these parameters, and others, in the QSeq Advanced Options dialog. QSeq will look for peaks along the entire genome for miRNA peak discovery.
Once the reads have been processed, signal values are created based upon the number of reads within each peak. By default, ArrayStar uses the log2 of these values for all visualizations and calculations. QSeq also calculates the start and end positions of each peak along the genome as well as the maximum height (in reads) of each peak. Depending upon what Peak Detection algorithm you use, QSeq may also calculate a P-value for each peak.
If you wish to use QSeq to quantify your miRNA sequence data against known miRNA templates, you may wish to use the RNA-Seq workflow. To do this, choose File > Import Experiments > RNA-Seq, and then select your miRNA files to import during the Add Experiments to Import step of the Project Setup Wizard. You may then load or download your miRNA reference target(s) instead of genomic templates during the Set Up Preprocessing step of the wizard.
Note: QSeq uses temporary files during the process of generating and matching mers from individual reads to the template sequences. These temporary files are stored in the directory specified under Edit > Preferences. Since processing can require a significant amount of disk space, it may be helpful to use an external hard drive for very large projects and edit the temporary directory listed under Edit > Preferences to point to the external hard drive location.