Computational protocol: Differential Characteristics of Viral siRNAs between Leaves and Roots of Wheat Plants Naturally Infected with Wheat Yellow Mosaic Virus, a Soil-Borne Virus

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Protocol publication

[…] About 5 μg of total RNA was extracted from each of the leaves and roots of virus-free and WYMV-infected plants for the preparation of small RNA (sRNA) libraries using the Illumina TruSeq Small RNA Sample Preparation Kit (Illumina, United States), while the remaining RNA was used for reverse transcription-quantitative PCR (RT-qPCR) analysis. sRNA high-throughput sequencing was carried out on an Illumina HiSeq 2500 at LC-BIO (Hangzhou, China). Preliminary treatment of raw data was performed as described previously (). Briefly, after removal of the 3′ adaptor, low quality and junk sequences using the FASTX-Toolkit, sRNAs with length of 18- to 30-nt were extracted and collapsed for further bioinformatics analysis. To identify WYMV-derived siRNAs, processed reads derived from both leaves and roots of infected and virus-free wheat libraries were mapped to the WYMV genome (NCBI accession No.: PRJNA15358) using Bowtie software allowing for one mismatch. To facilitate the comparisons between different sized libraries, identified vsiRNA raw reads counts were scaled to “Reads Per Million” (RPM) based on the total sRNA read numbers of the corresponding library. Downstream analyses for the vsiRNAs were carried out using custom perl scripts and Linux bash scripts. […]

Pipeline specifications

Software tools FASTX-Toolkit, Bowtie
Application sRNA-seq analysis
Organisms Triticum aestivum, Wheat yellow mosaic virus
Chemicals Nucleotides