Sequence!Sequence!Sequence!

Apr 03, 2020

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3 reasons why India needs to sequence more samples

Studies have shown that infectious diseases spread differently in different populations. This is based on genetic predisposition and results in different clinical presentations. Sequencing of the viral genome present in Indian samples and also those who have tested positive will help us better understand SARS Cov-2 spread in India. This is also important for the following three reasons –

1. Making sure that the RT PCR test is valid as mutations take place across the viral genome.
2. Finding out the viral sample origin and mutation as they infect people across India to see if we can predict infection rates/severity.
3. GWAS study of Indian human samples tested positive to see if there is a trend/marker that helps in overcoming the disease and also look for any drug or treatment regimen that works well in the Indian population.

Also Read: 5 things India can do to scale up Covid Testing

RT PCR Test Validity.

As multiple studies have shown, viral mutations can influence the results of RT PCR tests. RT PCR tests are the most common confirmatory tests for the Sars Cov-2 virus. In our experience with the H1N1 test kit that we created in 2009, one has to verify the primers and probes designed by other countries before using them in India. The reason, viral mutations in Indian samples could be a challenge and can interfere with the testing and can potentially give false results. CDC has released primers and probes to detect the novel coronavirus, SARS Cov-2. They are designed to amplify/detect three distinct regions within the ‘N’ gene. A sample is called ‘positive’ for SARS Cov-2 if the primers produce products of approximately 70 bases in the RT-PCR and ‘negative’ if they fail to produce a product. This is currently the standard followed by most commercial companies offering test kits or reagents for labs to use worldwide. Two Indian sequences were submitted by Dr Varsha Potdar of National Institute of Virology (NIV) Pune, to GISAID.org The two Indian samples came from cases detected in Kerala in January. There were seven mutations detected in these two samples submitted.

We ran a bioinformatics search to see if these mutations interfere with the RT-PCR reaction when using CDC primer and probe sets. We observed that there is no effect of these mutations on the regions which are amplified in the PCR reaction. These mutations are quite upstream to the actual regions which are being amplified and they do not hamper the amplification/detection of SARS Cov-2.

We need to keep checking if any mutations interfere with the primers and probes regions and hinder the PCR process. If they do, we need to redesign the primers and probes so that the reaction does not give a false negative, which could be disastrous in controlling the spread of COVID 19. Every kit or reagent approved must publish their primers and probes on ICMR site or any agency site that is giving approval. And we need to keep sequencing more samples that test positive in India so that we know that these tests are effective.

Also Read: The Immune system warriors: GAMED antibodies

Assess Viral Mutation

The two Indian samples sequenced and submitted to the GISAID site came from one of the first two samples that were positively identified from Kerala. They had a travel history to Wuhan and we cannot draw any conclusions about the mutations on those samples as there were other Indian samples which have not been sequenced (or at least published) that came in from across the world.

Dr Dinesh Gupta et al from IGIB published a paper analyzing one of the Indian samples with four other samples from across the world (Italy, USA, Wuhan, Nepal). They were able to glean important information regarding the Indian sample, including that a mutation was seen on the Spike protein-expressing part of the viral genome. Though nothing can be said conclusively without a lot more sample sequencing, this is a good starting point in understanding the various strains of the virus in the Indian population.

Subsequent positive samples came from all over the world including Italy, Iran, Dubai and the US. This means that we have different strains of the virus from different sources currently in India. Hence it is very important to sequence those samples to understand any mutations in the viral genome and not draw any conclusions based on one sample. Once we sequence and understand the viral genome we can also trace the infection rates of various mutations and understand different clusters across the country better. This could help in setting up customized treatment protocols.

GWAS of Indian Human samples

Genome-Wide Association Studies (GWAS) of Indian human samples tested positive for SARS Cov-2 can give us an understanding of genetic predisposition to the disease. A better understanding of genetic predisposition can provide prevention and treatment strategies, an important consideration for personalized treatment especially in times of a pandemic against an unknown virus.

At Mapmygenome, we analyzed our entire customer database and generated reports that help our customers understand their risk of infection, the severity of infection, their drug response if they get infected, and also their immunity profile. Pharmacogenomics, the ability to assess drug toxicity and response from genomic markers has been well studied and can help Indian patients especially in a scenario where various drugs are being tried in patients from Hydroxychloroquine to antivirals on a trial and error basis as this is a novel disease. Patients with a pharmacogenomics profile will be easier to treat as their drug response and toxicity profile is already known.

Genetic factors are a basis of our defensive immunity, the functional states of the nervous and endocrine systems, nutritional status, psychological factors, age, gender, etc. Human MHC or the HLA system can be studied to understand specific ’immunity genes’ if any that might be specific to Indian populations against SARS Cov-2.

Institutes such as NIBMG, IGIB, CCamp, and CCMB are well equipped to do large scale genome sequencing studies. Mapmygenome and other private sector companies can help as well. This will also give us the ability to understand differences between Indian and other country samples to see if any differences in markers can help Indians or hurt Indians.

Also Read: Covid: To test or not to test, is that a question?

Conclusion

Given the above reasons, we should quickly evolve a system to sequence both the viral genomes in the Indian positive samples and also the human samples. Any information that we can glean out of here can help personalize treatment, isolate clusters based on strains and also advise doctors on drugs that might work in Indian population better. The biotech industry and academic labs are well equipped to take on this challenge similar to the exceptional work done by ICMR on the testing and treatment challenges posed by this pandemic.

Since this is a national emergency, it is imperative that all the sequences be published publicly every week so that everyone can contribute. We are all in it together. Together we can fight COVID.

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