Genome sequencing Q & A

ESR has been leading the way in genome sequencing, but what is it exactly? 

What is genome sequencing?

If a person tests positive for COVID-19 their sample or an extract may be referred to ESR for genome sequencing. Genome sequencing for COVID-19 is about developing a complete picture of a virus’s RNA. It involves obtaining positive COVID-19 samples and generating a complete RNA sequence of that virus from that sample. The complete viral genome (~30000 nucleotides) is extracted from the sample and sequenced, allowing us to read the genome of the virus.

ESR is now sequencing and analysing up to 100 genomes a week and are the only ones providing this service in New Zealand.

Are all positive samples genome tested?
ESR is sequencing a subset of positive samples, which can provide vital clues for the current investigation and future investigations. 

Sequencing typically runs once a week, but can be processed more urgently at  the request of the Ministry of Health or a Public Health Unit.

Urgent samples come from cases where it is unclear how they might have been infected, or when additional evidence is needed to confirm which cluster they belong to.

How long does it take to complete a genomic sequence?
For rapid/urgent samples we typically have a result within 24 hours. For the other samples we have now moved to a weekly schedule to reduce cost and workload.

What do we do with the information?
The last, and most important part of the process, is the interpretation, which is how we wrap intelligence around the results to be used to inform the response, especially with difficult case investigations. This can vary widely between scenario’s and involves many experts inside and outside of ESR, from epidemiologists to phylogenetic experts and modellers. We’re very fortunate to have strong partnerships with the public health units, the Ministries and collaborations with our academic colleagues to answer these sometimes-tricky questions.

When we analyse the genomes, we look for links between genomes both locally and globally to better understand the situation. The virus can mutate as it is passed on from person to person. Based on those mutations the virus is leaving us a breadcrumb trail that we can follow back through the cases and together with epidemiological data (on movement, symptoms, etc.) helps us determine who may have been infected by whom. For previous investigations these results have at times provided crucial information to avoid measures like lockdowns, for example connecting the Auckland student In November with the Defence Force worker, having that link established provided decision makers with the confidence that the situation was under control and that there was no undetected community spread.

Also, ESR uploads the RNA genome of the virus to international databases for viral sequences, (NCBI & GISAID) to support the global response to COVID-19 and allow data integration and visualisation through platforms like link). We can compare our own sequencing with others around the world through platforms like this to see how the virus is mutating over time.

Why are we doing it?
ESR is uniquely placed to do this work and the information obtained assists us to understand how specific versions of the virus are spreading in the community and helps us to understand how outbreaks are occurring and how the virus is spreading in general.

Determining the RNA genome provides very rich information for both surveillance and research purposes. Sharing of this data is very important; it enables scientists and doctors to study how the virus can enter human cells and informs vaccine development. Health officials and government need accurate data on the virus to allow them to respond appropriately to the pandemic. It is crucial while we have cases to have a clear picture of where infections might have happened to inform appropriate community testing and contact tracing. One example is linking a case of unknown origin to a cluster.  Genomics can help to link a sample with a cluster by showing the virus of someone within the cluster is similar or identical to another case with known links.

How does the virus differ depending on where it came from (Iran, Europe or China, for example)?
The genomes of viruses change slightly over time and between people. You might have heard about different strains of the virus. This refers to People are talking about one or a few small changes in a specific region of the genome of the virus. These small localised changes help us to identify links between the viruses we find here and the travel links that were established when our borders where still open. Now we can use them to link cases to know outbreaks emerge and to follow up with additional questions about how a particular case might have come in contact with that outbreak.

Is that something ESR has been doing with every case or just specific requests?
The Ministry of Health and ESR have committed to whole genome sequencing a number of pandemic viruses, including in specific cases which may be the key to understanding community transmission and large outbreaks. ESR is collecting as many positive samples as possible to ensure the relevant samples can be sequenced when a question is asked. We are also working together with a wider group of researchers across New Zealand with the aim of sequencing as many as possible of all the positive cases in New Zealand. We are in a unique position to be able to do this comprehensive study and it will provide us with a detailed overview of which strains were introduced into New Zealand and how they have spread.

Can you please explain what high CT values are and how they affect samples?
The cycle threshold value (CT value), is essentially a measure of the amount of the virus in a sample. More virus in the sample means more virus for us to extract a complete genome from. When we have a full genome, it allows us to suggest what lineage/type that particular virus is.

The 'CT Value' comes from the technique used in the test to detect the virus. Basically, scientists copy parts of the virus thousands of times, which gives off a fluorescent light that is measured by a machine in cycles. These cycles continue until a detection threshold is met. Samples that take fewer cycles to meet that threshold (a low cycle threshold value) are more suitable for genome sequencing, there is more virus in the sample for us to read and extract a genome from.

On the flipside, if it takes more cycles to detect the virus (a high CT value) we know there is not much virus in the sample, and it is going to be much harder to extract a full genome. We might fail to extract a genome completely, but we also might be able to extract only a partial genome. This can still provide clues for us on lineage/type of that virus but not the full picture. For these partial genomes, the usefulness of the data depends on which parts we get, kind of like only getting a few pieces of the jigsaw puzzle and trying to figure out what the complete picture is. You might get lucky with the right parts, like an eye or an ear in a photo, and you might not, like a piece of the white border.

Now that we know what mutations we are looking for (based on the genomes from the community cases) we have designed a more sensitive assay that can work on lower concentrations and shorter pieces of RNA to be used for these situations.

How are the variants of COVID-19 determined?

The variants of COVID-19 are determined by Whole Genome Sequencing, this process reads the RNA of the virus and identifies the variants present which determine the lineage of that virus. This lineage indicates if a genome is a non-Delta, Delta or Delta plus variant as per WHO guidelines(external link). To assign a lineage to a genome according to these variants we use the Pangolin software(external link) which for Delta is the B.1.617.2 lineage(external link). The Delta plus variants are slight variations on the original Delta these are named AY.1 / AY.2 / AY.3 variants and can be compared to the original Delta variant on outbreak. For more information click here(external link).

These lineages and definitions are being continuously reviewed to adapt to new and emerging variants and ensure we have a good understanding of which variants are present in Aotearoa and across the world. We are in the privileged position where every sample from a case is subjected to genome sequencing which means we have a very good understanding of the different lineages that are present.

Who at ESR is working on this?
ESR are able to accept samples for urgent and routine sequencing in Auckland, Wellington, and our Christchurch facility is also able to accept samples for urgent sequencing.

Various teams from ESR work on sequencing from the sequencing itself to analysis and reporting.

Also, the team are working with scientists across New Zealand and the globe to interpret this data and provide input into the COVID-19 response team.