Computational protocol: Identification and Characteristics of microRNAs from Army Worm, Spodoptera frugiperda Cell Line Sf21

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Protocol publication

[…] The high quality reads with read count more than 10 were used for investigating the small RNA population. The small RNA reads were converted into unique read tags and compared against the Noncode database using BLASTN. Those read tags having 100% query coverage with maximum 2 mismatches with the known data were then distributed into different small RNA families.To identify the known miRs, the mature sequences from the species A. gambiae, Aedes aegypti, Culex sp, D. melanogaster, B. mori, A. mellifera, Acyrthosiphon pisum, Tribolium castaneum and C. elegans were downloaded from miRBase v19. Using miRCat, the small RNA reads were mapped against the known set of mature sequences and those reads satisfying the set criteria were separated and named based on their subject annotation. Further, those annotated reads were mapped with minimum 90% query coverage and 90% identity having no gaps to the draft genome assembly of S. frugiperda [] to extract pre miR sequences. The average length of precursor miR was 135 bp and the structure validation was done using MiPred to classify between real and pseudo precursors using random forest prediction model.The small RNA reads not mapped to Noncode and miRBase were used to identify novel miRs. The small RNA reads were mapped against the draft genome assembly [] of S. frugiperda using Bowtie. Once the mature sequences were mapped, the miR loci were identified using miRCat [] and the small RNA reads with higher read counts were considered as the putative. Flanking sequences surrounding the selected miR were extracted with varying window lengths and folded into secondary structures using RNA fold. The secondary structures were further validated using the criteria for novel miR identification []. […]

Pipeline specifications

Software tools BLASTN, miRCat, MiPred, Bowtie
Databases NONCODE miRBase
Application Genome annotation
Organisms Spodoptera frugiperda, Bombyx mori, Drosophila melanogaster, Spodoptera litura, Caenorhabditis elegans