engleski

Comparison of NGS-based methods in detection of grapevine viruses

Up to date more than 70 different viruses are found in grapevine, making it good model plant for detection of intracellular pathogens using next generation sequencing (NGS). Four Croatian autochthonous grapevine varieties (Babica, Dobričić, Ljutun and Vlaška), suspected to harbour different viruses, were included in comparison trial of different NGS-based methods used for their detection. Isolation of total RNA was done from 0.1 g of leaf petioles, and after removal of host ribosomal RNA sequencing library was constructed on an Apollo 324™at Functional Genomics Lab at QB3-Berkeley Core Research Facility (UC Berkeley). Index addition and library fragment enrichment were done using thirteen cycles of PCR amplification and afterwards 100 paired-end reads were generated on Illumina HiSeq4000 platform at Vincent J. Coates Genomics Sequencing Laboratory (UC Berkeley). FastQC program was used for quality control of genomic data. After trimming with Sickle reeds were initially aligned to a grapevine genome using Bowtie 2 and unaligned reads were used in further analysis. Four NGS-based methods were compared: 1) de novo assembly using SPAdes followed by BLAST nucleotide search (cut-off e-value e-20), 2) mapping to a custom-made library of grapevine-associated viruses 3) detection by automated e-probe-based software Truffle and 4) detection by online virus discovery tool VirFind with default e-value (0.01) and conserved domains search option. All methods compared in this study were effective in detection of most viruses. De novo assembly showed to be very efficient in detection of different viruses with coverage between 0.9x and 7136x. Although efficient in detection, read-mapping showed difficulties in determination of adequate cut-off thresholds. Truffle proved to be an efficient, user friendly and fast tool for detection of known viruses with concerns in detection of viruses with limited sequence information. VirFind proved as a well-balanced tool for detection of known viruses, generating shorter contigs in regards to de novo assembly using SPAdes.

next generation sequencing ; virus ; grapevine

nije evidentirano