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ncPRO-seq is a tool for annotation and profiling of ncRNAs from smallRNA sequencing data. It aims to interrogate and perform detailed analysis on small RNAs derived from annotated non-coding regions in miRBase, Rfam and repeatMasker, and regions defined by users. The ncPRO pipeline also has a module to identify regions significantly enriched with short reads that can not be classified as known ncRNA families.