Harnessing Next Generation Sequencing to Detect SARS-CoV-2 …and Prepare for the Next Pandemic

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96-Well Plates

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96-well plates. Credit: Peter Duchek

Researchers at the Vienna BioCenter developed a screening procedure for SARS-CoV-2 that can process 10s of countless samples in less than 48 hours. The approach, called SARSeq, is released in the journal Nature Communications and might be adjusted to a lot more pathogens.

The COVID-19 pandemic has actually lasted more than a year and continues to affect our lives enormously. Although some nations have actually released fast vaccination projects, numerous still wait for massive immunization plans and reliable antiviral treatments — prior to that occurs, the world urgently requires to restore a form of normalcy.

One method to bring us closer to that point is huge parallel screening. Molecular tests that discover the existence of SARS-CoV-2 have actually ended up being the very best method to separate favorable cases and include the spread of the infection. Several techniques have actually stepped forward, some that discover viral proteins from nasopharyngeal swabs (such as antigen tests), and some that discover the existence of viral RNA from swabs, swish samples, or saliva samples (such as reverse transcription and polymerase domino effect checks, or RT-PCR).

Although antigen tests help with some logistical elements of mass screening, their detection power is reasonably weak — contaminated people bring low quantities of infection stay undiscovered and can continue to contaminate other individuals. PCR tests, on the other hand, are more delicate since they increase pieces of the viral genome prior to scanning samples for the infection. However, they depend on the detection of fluorescent labels that tag viral series, which implies that pooling samples originating from various individuals makes the procedure rather ineffective: if a swimming pool tests favorable, all the samples within the swimming pool need to be evaluated once again separately to recognize the source of the fluorescent signal. Too numerous devices required, too pricey, too sluggish.

During the extremely first lockdown, researchers at the Vienna BioCenter were mulling over the scenario: there needed to be a method to scale up screening. Ulrich Elling, group leader at the Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), and Luisa Cochella, group leader at the Research Institute of Molecular Pathology (IMP), chose to direct their aggravation into an ingenious service. IMP group leader Alexander Stark and IMBA postdoc Ramesh Yelangandula joined their efforts, and the job removed.

Combining their competence in genomics, RNA biochemistry and information analysis, they established an approach that might make it possible for big groups to be evaluated for SARS-CoV-2 with the very same level of sensitivity as routine PCR tests. SARSeq, or ‘Saliva Analysis by RNA sequencing’, attains high level of sensitivity, uniqueness, and the power to process approximately 36,000 samples in less than 48 hours. The approach is now released in the journal Nature Communications.

The screening concept is conceptually basic: specific client samples are gathered into the wells of a screening plate — one well for each sample. Then, a piece of viral RNA distinct to SARS-CoV-2 — the nucleocapsid gene — is selectively transformed to DNA and PCR-amplified in any well which contains it.

“Amplifying the viral material from individual samples to a maximum homogenizes its quantity across positive samples, making SARSeq highly sensitive,” describes Luisa Cochella. “Within the thousands of samples that we could test simultaneously, some may contain up to 10 million times more coronavirus particles than others — if we pooled such samples before amplification, those with high amounts of viral material could mask other positive cases.”

What differentiates this primary step to the typical PCR test is that each sample gets a unique set of brief DNA series — or barcodes — that connect to the enhancing viral DNA. In a 2nd amplification action, all the samples from one plate are pooled into one well, which gets a 2nd set of distinct DNA barcodes. The contents of several plates can be pooled again, as the DNA particles from each sample bring a unique mix of 2 sets of barcodes. This pooling and barcoding technique makes SARSeq extremely particular and scalable.

“We combine the sensitivity of PCR with the high throughput of Next Generation Sequencing technology, or NGS, the same used to sequence the human genome. The NGS machine processes the pooled samples and tells us which samples contained any SARS-CoV-2 material. The barcodes allow us to distinguish each positive sample from the others, and trace it back to a patient,” states Ramesh Yelagandula, very first author of the research study. Moreover, the NGS-based approach enables to evaluate a number of Registered NurseAs in parallel, consisting of Registered NurseAs that manage the sample quality or Registered NurseAs from other pathogens for differential diagnostics.

“The Next Generation Sequencing facility and other colleagues at the Vienna BioCenter were of tremendous help to develop and optimize the method,” states Alexander Stark. “With our machines, home-made enzymes, and analysis pipeline, we expect each test to cost less than five Euro.”

The screening treatment can run in parallel to existing diagnostics, while being independent of the traffic jams in supply chains. Therefore, it does not take on other screening techniques for reagents or devices.

“We developed SARSeq to try and circumvent the limitations of other tests, and to process thousands of samples in parallel. Not only is it an excellent method to detect SARS-CoV-2, but it can also be applied to other respiratory pathogens like the flu virus, the common cold rhinoviruses, and potentially many others,” states Ulrich Elling.

The concepts behind SARSeq are basic and versatile to any breathing pathogen. As the world’s population escalates together with our distance to animals, advanced diagnostic techniques like SARSeq will be essential to avoid future illness from spreading out like wildfire.

Reference: “Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq” by Ramesh Yelagandula, Aleksandr Bykov, Alexander Vogt, Robert Heinen, Ezgi Özkan, Marcus Martin Strobl, Juliane Christina Baar, Kristina Uzunova, Bence Hajdusits, Darja Kordic, Erna Suljic, Amina Kurtovic-Kozaric, Sebija Izetbegovic, Justine Schaeffer, Peter Hufnagl, Alexander Zoufaly, Tamara Seitz, VCDI, Manuela Födinger, Franz Allerberger, Alexander Stark, Luisa Cochella and Ulrich Elling, 25 May 2021, Nature Communications.
DOI: 10.1038/s41467-021-22664-5