Researchers of the Oswaldo Cruz Institute (IOC/Fiocruz), in a partnership with the University College London, in the United Kingdom, have developed a new protocol for the genetic sequencing of the novel coronavirus, the SARS-CoV-2. The methodology offers extensive coverage of the entire viral genoma, reducing errors that may occur in the process. In addition, it allows for the sequencing of the entire genoma of the pathogen directly from patient samples, without the need for viral isolation procedures. The technique offers yet another advantage: it can be used to sequence up to 96 genomes at once, in the same sequencing run, which makes this method both quicker and cheaper.
The protocol has been validated for three genetic sequencing platforms: Nanopore (MinION or GridION), Ilumina, and Sanger. Due to their relevance for the scientific community, these data have been published in an article on the BioRxiv pre-print website. The protocol was also shared on the protocols.io page. The work, led by the Laboratory of Respiratory Viruses and Measles of IOC/Fiocruz, relied on the collaboration of the Flavivirus Laboratory of IOC/Fiocruz, the General Coordination of Laboratories of the Ministry of Health in Brazil, and the Great Ormond Street Hospital, in the United Kingdom.
Responsible for developing the protocol, researcher Paola Cristina Resende, of the Laboratory of Respiratory Viruses and Measles, emphasizes the importance of the quality of genetic sequencing for research on the pathogen. “As this virus has only been circulating among humans for a short time, it has accumulated only a few mutations, and the genomas of the virus around the world are very similar. From among the approximately 30,000 bases that make up the RNA [genetic material] of the new coronavirus, we have seen that only a few bases differentiate one strain from another. This is still very little, and is not enough to identify alterations in virus phenotype. However, it does make it possible to recognize and define viral genetic groups that are circulating around the world”, she says.
Researcher Fernando Motta, who worked in the validation of the protocol in the Illumina platform in the Laboratory of Respiratory Viruses and Measles, designated Reference Center for Covid-19 in the Americas for the World Health Organization (WHO), highlights the advantages of the new method in the search for quicker responses to the pandemic. “The protocol has proven to be simple, efficient, and consistent, and can be applied by national and international laboratories that have any of these pieces of equipment”, says the virologist.
Brazilian genomes
In order to validate the new protocol, the specialists decoded 18 complete genomes of the new coronavirus from samples of pacients in six Brazilian states. This works widens the knowledge on the micro organisms that cause Covid-19 in Brazil, significantly expanding the number of genetic sequences available for Rio de Janeiro and Federal District (Brasília), and including, for the first time, data from the states of Alagoas, Bahia, Espírito Santo and Santa Catarina on the GISAID platform, which promotes the sharing of genomic information on influenza viruses, Respiratory Syncytial Virus, and SARS-CoV-2.
According to Resende, who worked in the training of the team of technicians of the Genomic Platform of the University College London to create the new protocol and is one of the curators of the GISAID platform, the analysis of the recently sequenced Brazilian genomes indicates that the virus was introduced to Brazil multiple times by travellers returning to the country. “The phylogeography clearly shows there have been multiple introductions. Taking the recently sequenced samples into account, we have observed they are similar to virus genomes described in Europe, North America, and Oceania, which indicates possible entry routes”, states the specialist.
For Marilda Siqueira, head of the Laboratory of Respiratory Viruses and Measles, it is crucial to follow the evolution of the virus over time. “In addition to tracing dispersion routes in the world and in the hinterlands of Brazil, genomic surveillance integrated to epidemiological surveillance is required to monitor the occurrance of genetic variations that may be associated to disease severity or resistance to drugs”, the virologist explains.