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RNA-seq of tomato chlorotic spot virus from peanut
Total RNA was extracted from symptomatic leaf samples using an RNeasy kit (Qiagen, Hilden, GmbH) following the manufacturers protocol. Total RNA was quantified using a Nanodrop spectrophotometer (ThermoFisher Scientific, Waltham, MA). The quality of total RNA was evaluated using an Agilent 2100 bioanalyzer (Agilent Technologies). RNA preparations with RNA Integrity Numbers (RIN) above 7.0 were used for downstream applications. The ribo-depletion of total RNA, library preparation and NGS procedures were carried out by the Huntsman Cancer Institute, University of Utah, Salt Lake City, UT using Illumina HiSeq 2500 platform.After trimming adapters, the 250-bp paired end sequences generated by NGS were quality filtered to remove reads with a read length below 125-bp using the CLC Genomics Workbench 8.0 software (Qiagen, Hilden, GmbH). These reads were mapped to the reference genomes of A. durensis (NC_029772.1) and A. ipaensis (NC_029785.1), and subsequently, de novo assembly of the quality filtered 125-bp paired end reads were assembled into contigs using default parameters and annotated against the reference virus and viroid genome sequence database by BLASTn program (http://www.ncbi.nlm.nih.gov/blast). Depth of coverage was calculated by mapping the number of bases at each site as a proportion of total reads matching to the reference genome sequence
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SUB5390886
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