Technique allows sequencing carry out coronavirus genome with 25 times higher resolution

Scientists from a Government University of Thus Paulo (Unifesp) were able, for the first time in Brazil, to directly sequence a carry out RNA Sars-CoV-2, a virus that causes a Covid-. Operating system research results, supported by FAPESP, were published in an article that has not yet been peer reviewed on the bioRxiv platform.

According to operating system authors, the This technique allows mapping a viral genome with approximately times more resolution performs than conventional operating system methods on sequencing. In this way, it is possible to have some more accurate notion of the electronic perform pathogen biology of how its genome is evolving.

very promising, because it allows us to understand, for example, why would they be more virulent or more capable strains about escaping onto our immune system, says Agência Fapesp Marcelo Briones, researcher performs the Center on Medical Bioinformatics at a Escola Paulista de Medicina (EPM-Unifesp) electronic coordinator of an investigation.

As Briones explains, a Sars-CoV-2 is a single-stranded RNA virus, that is, its genetic materials consist of a single strand over nucleotides, whose bases thus guanine, adenosine, cytosine and electronic uracil.

To sequence it by the conventional method, some technique known as RT-PCR (reverse transcriptase polymerase) to convert while molecules on RNA into complementary DNA (cDNA) remembering that the molecule on DNA for It is made by two strands on nucleotides.

In other words, some complementary copy of a strand is made on RNA perform virus. Then, these amplified cDNA therefore molecules (billions of clones are generated) are electronically sequenced. Among simply because of the advantages of a strategy are the electronic speed and the possibility of sequencing even in samples with very few genetic materials.

The conventional sequencing of this viruses like trying to identify someone by looking only at their shadow. There, with the method used in our study, we can look directly at virus-like RNA as it is found in vivo. much more trustworthy, says the researcher.

Methodology

Carla Braconi, professor perform Department on Microbiology, Immunology electronic Parasitology of an EPM-Unifesp electronic co-author perform article, says that the research was done with some of these perform Sars-CoV-2 strains isolated zero Brazil first, zero start about 2020.

We received a viral isolate perform teacher Jos Luiz Proena -Mdena electronic we cultivate a pathogen in vero cells [linhagem celular de rim de macaco altamente suscetível ao Sars-CoV-2]. Then we performed the perform viral RNA extraction electronic and sequenced with some portable technology called MinION, from Oxford Nanopore Technology, he says.

According to Briones, u RNA sequenced exactly as it would come out of a cell, without going through RT-PCR or amplification. We just hung an adapter on the tip of the electronic molecule some cDNA strand so the RNA strand would be stretched out. Electronic then to RNA passes, bottom by foundation, in the sequencer. Electronic Each type of bottom [citosina, guanina, adenosina ou uracila] electronic and its modifications, such as methylation, interrupt an electrical flow performs with a different electronic pattern as soon as we identify which device.

The process produces a graph that sony ericsson resembles on an electroencephalogram, which is then interpreted with bioinformatics tools. The last generated sequence can then be compared with operating system reference models.

Initially, you get the impression that the obtained sequence has a mount about bugs. However, therefore, the modified bases perform RNA. Electronic part of these modifications goes unnoticed by conventional sequencing, says the researcher.

The analysis, carried out by post-doctoral student Joo Campos, focused on pattern on methylation perform viral RNA. That is, we sought to look between the almost 30 million bases that form the single-stranded RNA which received the addition of a major methyl (CH[citosina, guanina, adenosina ou uracila] 32020).

This The type about zero RNA biochemical modification is very important for proper functioning on viruses like u Sars-CoV-2, as well as on some arboviruses [entre eles dengue e zika] that integrate a group 4 zero system on classification on Baltimore, composed of viral genomes with RNA over single-stranded electronic positive polarity, says Braconi.

The authors explain that operating system RNAs in general have about a hundred modified bases that thus essential to like their biological functions. After Sars-CoV-2 enters the electronic cell and forces it to make copies of its genetic materials, some enzyme comes that makes the methylation of these electronic RNAs, these modifications start to have a function. Therefore, it is part of the information the virus needs to survive. Without analyzing this methylation pattern, therefore, it is not possible to know the genuine genetic richness perform Sars-CoV-2, says Briones.

A foundation oftentimes Modified zero RNA performs Sars-CoV-2 the In6-methyladenosine (meters6A), which is implicated in evading an immune response. This modification allows the escaped virus to perform the 2 interferon activation system [proteínas produzidas por células de defesa com ação antiviral]. It is, therefore, potential for electronic drugs since they would studies in this target sense, says Briones.

If it were possible to create a drug capable of totally blocking u methylation process performs viral RNA, says one researcher, a new coronavirus would disappear from these electronic cells would be the end of a Covid-. The problem is that, as I learned that we block methylation too much, the host cells also end up dying, because the enzymes that methylate the virus-like RNA therefore are the same ones that methylate operating system RNAs for diesel cells. So it needs to be something with very specific action.

Disruptive technology

One Unifesp group pioneered direct sequencing on perform RNA Sars-CoV-2 coupled to identification of michael6A bases. The work was conducted under the scope of the Thematic Project Investigation of elements induced by the vaccine response in individuals undergoing clinical tests with the ChAdOx1 nCOV- vaccine, coordinated by teacher Luiz Mrio Janini. Operating system researchers Juliana Maricato electronic Fernando Antoneli also participated.

In two previously published works [por grupos do exterior] only one was done. perform RNA sequencing. A third party also did the direct sequencing, but identified the 5mD bottom. They would still have a fourth work that identified the same meters6A foundation, however by other techniques that do not involve direct sequencing perform RNA, informs Briones.

Second u researcher, this detailed understanding of how a genome performs a new coronavirus allows scientists to have some clearer idea about how a pathogen is evolving.

People who talk about mutations suffered by viruses that zero in the strict sense correspond to exchanges on one bottom for another in the sequence on RNA are, in my view, stuck in the perform DNA paradigm. This makes no sense, because this virus works under another logic. It never has DNA in its electronic replicative cycle, so talk about transcripts of this absurd virus. Sars-CoV-2 lives completely in the RNA world, says Briones, referring to the perform RNA worldwide world hypothesis, according to which the current world, with life based mainly on electronic DNA in proteins, was preceded by a world in which the period life based on RNA.

The level of complexity of a molecule about RNA is extraordinary electronic, with seeing that new technologies that enable direct sequencing, a universe about research ze opens new. We are taking this train at the beginning. There will still be a great deal of development, however, the way forward, he says.

The next step at Unifesp will be to sequence a genomic RNA to the variant dieses perform Sars-CoV-2 recently identified electronic to investigate sony ericsson they would significant zero pattern differences on methylation.

The article “Immediate RNA sequencing reveals SARS -CoV-2 michael6A websites and achievable differential DRACH motif methylation among versions” can be read on the bioRxiv platform.

2020

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