Pooling Strategy for Massive Viral Sequencing

Skums, Pavel; Artyomenko, Alexander; Glebova, Olga; Ramachandran, Sumathi; Campo, David S.; Dimitrova, Zoya; Măndoiu, Ion; Zelikovsky, Alexander; Khudyakov, Yury

doi:10.1002/9781119272182.ch3

Cited by 1 publication

(1 citation statement)

References 38 publications

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“…According to the literature, the pool composition of different metagenomic studies for plant viromes varied from four to 50 individual samples per pool [ 9 , 12 , 35 , 36 , 37 , 38 , 39 ]. Skums et al [ 40 ] provided a mathematical approach for a pooling strategy for the massive sequencing of human viruses, since the complex nature of these samples imposed restrictions on the pool design [ 40 ]. Here, we tried to address the problem of minimum reads needed to detect the less abundant viruses in a pool by following an in silico approach.…”

Section: Discussionmentioning

confidence: 99%

Use of High-Throughput Sequencing and Two RNA Input Methods to Identify Viruses Infecting Tomato Crops

Maachi¹,

Torre²,

Sempere³

et al. 2021

Microorganisms

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We used high-throughput sequencing to identify viruses on tomato samples showing virus-like symptoms. Samples were collected from crops in the Iberian Peninsula. Either total RNA or double-stranded RNA (dsRNA) were used as starting material to build the cDNA libraries. In total, seven virus species were identified, with pepino mosaic virus being the most abundant one. The dsRNA input provided better coverage and read depth but missed one virus species compared with the total RNA input. By performing in silico analyses, we determined a minimum sequencing depth per sample of 0.2 and 1.5 million reads for dsRNA and rRNA-depleted total RNA inputs, respectively, to detect even the less abundant viruses. Primers and TaqMan probes targeting conserved regions in the viral genomes were designed and/or used for virus detection; all viruses were detected by qRT-PCR/RT-PCR in individual samples, with all except one sample showing mixed infections. Three virus species (Olive latent virus 1, Lettuce ring necrosis virus and Tomato fruit blotch virus) are herein reported for the first time in tomato crops in Spain.

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