Posted in Literature

Characterizing New Viruses in Rice

Novel mastreviruses identified in Australian wild rice

Simona Kraberger, Andrew D.W. Geering, Matthew Walters, Darren P. Martin, Arvind Varsani

Virus Research, vol 238, pg 193-197, 15 June 2017. Link to article.

The deep sequencing techniques made possible by next-generation sequencing technologies enable research into, among other things, viral diversity. Considering the vast majority of viruses in the world do not affect human existence at all, this might appear to be a rather esoteric pursuit. However, increased understanding of viral populations leads to increased understanding of the characteristics that make certain viruses more or less destructive, or that restrict their targeted host species, or that create vulnerabilities and weaknesses in the virus’s defense.

In this case, the researchers sampled wild rice plants around Australia for viruses, specifically searching for a type of virus called mastrevirus that infects grasses and economically important crops such as chickpeas and maize. Because the genome of this particular type of virus is circular single-stranded DNA. a specific extraction kit could be used that selected for viral DNA to the exclusion of any plant or bacterial contaminant. The resulting DNA was sequenced on an Illumina machine to determine whether any mastrevirus-specific sequence was present, and primers were designed based off of those identified sequence to enable the circular viral genome from each rice sample to be sequenced individually using Sanger sequencing. Based on this, two complete and previously unknown mastrevirus genomes were discovered – the first two mastreviruses shown to infect any variety of rice.

As the authors conclude, the “discovery of novel mastreviruses within both cultivated grasses and their uncultivated relatives [… is] an essential first step in identifying instances where these viruses are in the process of emerging as pathogens of economic significance.”

Posted in Sequencing Technology and Methods

Nucleic Acid Fragmentation: Three Methods

An essential step in preparing total RNA or genomic DNA samples for sequencing is cutting them down into usable fragments. For Illumina instruments, the bridge amplification aspect of the sequencing process works best when these fragments are between 100 and 1000 nucleotides long, and quality is improved by using fragments within a narrow size range (ideally no more than 200bp spread). There are several different techniques for accomplishing this: nebulization, sonication, and enzymatic fragmentation.

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Posted in Sequencing Technology and Methods

Adapter Ligation

For a DNA fragment to be sequenced on an Illumina instrument, it first has to attach to the Illumina flow cell. The interior of the fluidics lane on each flow cell is printed, top and bottom, with a lawn of single-stranded oligonucleotides. All our DNA of interest needs to have is a complementary region on each end for clustering and sequencing to take place.

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