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RNA-Seeker is a package of scripts that supplement the Bowtie mapping algorithm. The recommended use for RNA-Seeker is to help glean useful information from sets of gene families (e.g. tRNAs), where multiple genes are identical or have identical portions, resulting in RNA-seq reads mapping to multiple locations. RNA-Seeker requires a "Library" file, which contains names, classifications, and genomic locations for the genes that are of interest to the user. There are two primary procedures in the RNA-Seeker package. The Compile Reference Gene Files procedure extracts from the reference genomes the DNA sequences of genes of interest to the user. Additionally, non-coding DNA including any introns and the flanking sequences of the gene within a defined distance are included in the reference files. This allows Bowtie to map processed and unprocessed reads alike without allowing caps during the mapping, a process that raises the possibility of false-positive matches. The Classify Reads procedure addresses the problem of reads mapping to multiple locations in the genome, as is common when dealing with sets of gene families like tRNAs. Instead of discarding these multimapping reads or using a probabilistic approach to distribute them to genes, this procedure notes the relationship of all of the genes that the read maps to and classifies the read to a category of uniqueness based on this. If all of the genes that a read maps to are identical copies of the same gene, this is much more useful to note than the case where a read maps to two or more completely unrelated genes. This procedure distinguishes between these types of multimap reads and more, depending on the user’s customization of the analysis. Importantly, the results of this procedure give only the factual results of the classification, and leave any inference or interpretation of these results to the user.