miR-PREFeR: microRNA PREdiction From small RNAseq data
Rationale and background:
Plant microRNA prediction tools that utilize small RNA sequencing data are emerging quickly. These existing tools have at least one of the following problems:
miR-PREFeR workflow uses expression patterns of miRNA and follows the criteria for plant microRNA annotation to accurately predict plant miRNAs from one or more small RNA-Seq data samples of the same species. miR-PREFeR was tested on several plant species and the results show that miR-PREFeR is sensitive, accurate, fast, and has low memory footprint. The miR-PREFeR paper is published on Bioinformatics. http://bioinformatics.oxfordjournals.org/content/30/19/2837.abstract. Please cite the paper if you use the tool in your work.
In this example we will identify microRNA
compare gene transcript abundance drought sensitive sorghum line under drought stress(DS) and well-watered (WW) condition. The expression of drought-related genes was more abundant in the drought sensitive genotype under DS condition compared to WW.
We will use RNAseq to compare expression levels for genes between DS and WW- samples for drought sensitive genotype IS20351 and to identify new transcripts or isoforms. from a Genome-wide profiling of small RNAs in Arabidopsis seedlings under salt and cold stresses experiment. For AtCold library, plants were treated under 5 ?C for 24 hours; For AtPdep and AtPind libraries, plants were treated with 300mM NaCl for 5 hours. Total RNA was isolated from shoots and fractionated on 15% denaturing polyacrylamide gel. RNA molecules ranging from 18 to 30 nt were excised. For AtCold and AtPdep libraries, excised RNAs were ligated to a preadenylated 3' adaptor and a 5’- adaptor using T4 RNA ligase. For AtPind library, excised RNAs were first de-phosphorylated at the 5’ end. After 3’ end adaptor was ligated, the 5’ end was phosphorylated before 5’ adaptor was ligated. Ligation products were purified in polyacrylamide gels, followed by RT-PCR. The small RNA libraries were sequenced using Illumina Genome Analyzer
In this tutorial, we will use data stored at the NCBI Sequence Read Archive.