Computational protocol: Carrot yellow leaf virus Is Associated with Carrot Internal Necrosis

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Protocol publication

[…] RNA was extracted from 12 affected and 12 unaffected carrots using an RNeasy kit (Qiagen, UK). TruSeq RNA Indexed sequencing libraries (Illumina) were then prepared following the manufacturers recommended protocols, before being sequenced on 2 500 cycle v2 flow cells using MiSeq Sequencer (Illumina). The resulting sequences were trimmed to remove low-quality nucleotides from the 3′ end, using a Phred score threshold of 30 and the bwa trimming approach implemented in SolexaQA , and assembled using Trinity . Contigs produced were then compared to the GenBank protein database using BLASTx + and viral reads extracted using MEGAN , . Open reading frames were identified using Vecor NTi v11 (Invitrogen, UK) and alignments and phylogenetic trees produced using Mega5 with 500 bootstrapped replicates . To determine the number of viral reads in the affected and unaffected samples reads were mapped back to the genomes of identified viruses using bwa aln sampe and the numbers of matched read pairs extracted using Samtools . To normalize mapped read counts against the length of genome and total number of reads, the values are reported as mapped reads per kilobase of viral genome per million reads (RPKM), an approach originally introduced for the comparison of mRNA abundance in differential expression analyses . The fastq data produced during the project were submitted to the short read achive acc: SRP042501. […]

Pipeline specifications

Software tools BWA, SolexaQA, Trinity, BLASTX, MEGAN, SAMtools
Application Phylogenetics
Organisms Daucus carota, Carrot yellow leaf virus, Carrot red leaf virus, Carrot mottle virus
Diseases HIV Infections