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Tuesday, July 28 • 13:30 - 13:35
Comparison of extraction methods and sequence platforms for complex water sample

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Viruses are one of the most important threats to agriculture. The diversity and impact of viruses present in various sources of irrigation water, such as rivers or processed wastewater, remains understudied. High-throughput sequencing is the most well rounded approach for exploring the virome of irrigation water.. However, water as a sample for metagenomics analysis is incredibly complex. It can contain large amounts of very diverse genetic material that is not only limited to viruses. This makes it challenging to successfully detect individual viral species with certainty. On the other hand, the amount of plant virus genomic material present in water samples is very low and we must employ multiple concentration and purification steps to maximize the sequence yield and quality, without introducing too much bias. In preparation for large-scale irrigation water sampling, we have been optimizing individual steps of our analysis workflow. Here, we describe the comparative analysis done on a single sample of a Serbian river water used for irrigation. We compared two nucleic acid extraction protocols on the same original sample. The modified Trizol protocol for RNA extraction has the advantage of producing high quantities of good-quality RNA with relatively high length of nucleic acids fragments, however, it can be time consuming. On the other hand, Qiagen’s QIAmp MinElute Virus Spin Kit is a faster, more user-friendly alternative that consists of a well-standardized extraction method that targets both DNA and RNA. Assessment of the extraction methods was done using the Illumina MiSeq platform. Samples were normalized based on read count and compared in terms of viral families’ richness, with special consideration for RNA viruses, as the majority of plant viruses have RNA as their genetic material. We have identified several viral species that were detected in these samples and compared the genome coverage obtained with the two extraction methods. The bioinformatics analysis was done using Qiagen’s CLC Genomic Workbench software for pre-processing of reads and majority of individual species mappings, Diamond blastx for similarity search of obtained sequencing reads against GenBank nonredundant database, and MEGAN6 for visualization and comparison of Diamond results. Results indicated the advantage of using the modified Trizol extraction protocol for detection of plant viruses. Having this in mind, we have also assessed and compared the performance of Oxford Nanopore Technologies MinION platform using the Flongle flow cell paired with Ligation Sequencing Kit for library preparation of a Trizol-extracted RNA from the same sample in order to evaluate ability of a long read sequencing platform to increase reliability of detection.

avatar for Olivera Maksimović Carvalho Ferreira

Olivera Maksimović Carvalho Ferreira

Young researcher, National Institute of Biology Slovenia, IPS "Jožef Stefan" Slovenia
Hello everyone,I am a young researcher at the National Institute of Biology in Slovenia working under INEXTVIR project. For the most part I am working with plant viruses and water and their mutual relationship. At the moment we are predominantly testing irrigation water (via HTS sequencing... Read More →

Tuesday July 28, 2020 13:30 - 13:35 MSK
Zoom Conference https://zoom.us/j/94321101353?pwd=QlJBb09uM0NVVnVyK0FkbTJ3Nkcrdz09