COVID-19 Genomics

SARS-CoV2, the virus that causes Covid-19 disease, can mutate and evolve over time, leading to the emergence of variants such as the Alpha (B.1.1.7, UK), Beta (B.1.351, S. Africa), Gamma (P1, Brazil), Delta (B.1.617.2, India) and Omicron (B.1.1.529, S. Africa) variants. In many cases, mutations are inconsequential for this virus, butsome could have the potential to increase transmissibility and disease severITy, as well as negatively impact on the performance of diagnostics, therapeutics and vaccines.

Genomic surveillance consists to analyze similarities and differences among the whole genome sequences obtained for each specimen in a population over a defined timeframe. It allows visibility on the variants that circulate and if necessary to adapt the health measures taken according to these epidemiological data. This can help indicate routes of transmission enable identification and investigation of clusters and help inform strategies to control the spread of the virus. This real-time surveillance is possible by rapidly sequencing and sharing SARS-CoV2 genomic data.

Our LUX-GEMM unit plays a key role in the fight against Covid19 and this since the beginning of the epidemic. From early 2020, the laboratory rapidly implemented an in-house next generation sequencing protocol with Illumina technology adapted for whole-genome sequencing of SARS-CoV2. This could be done quickly thanks to our long-term experience in microbial sequencing and with an adapted technical platform and bioinformatics expertise. In April 2020, the first sequencing results were already delivered.

Thanks to this expertise and the implementation of our sequencing process and strategy, we are able to perform whole genome sequencing of the virus on a large selection of positive samples and to report these results regularly allowing visibility of SARS-CoV-2 mutations and variants through time and space in Luxembourg.

Currently, for sequencing the whole genome of SARS-CoV2, we use an amplicon-based sequencing approach with two technologies of sequencing (Illumina and Oxford Nanopore technologies). Both methods employ a PCR tiling approach in which the viral genome is amplified in overlapping sections, maximising coverage across the full genome.

The Illumina technology corresponding to the sequencing by synthesis of small fragments of DNA is the first technique that has been implemented in our lab from the beginning of the crisis. The extracted RNA is converted to complimentary DNA. After amplification, purified and quantified DNA is cut up into fragments, end-repaired and A-tailed. Then, short segments of genetic material called adapters are ligated to the DNA fragments. This step is called “Tagmentation”. After clean-up, sequences from each sample are identified with a specific couple of indexes and after clean-up of the final libraries, the samples are pooled then quantitated and normalized before loading onto a flow cell adapted to the sequencer used. It is possible with this technology to pool several samples at the same time (for example max 384 on Next-Seq-1000). The preparation of the DNA and the library for sequencing take around two working days. The preparation of the library is a step that can be realized manually or by automation using the Hamilton NGS star device. Once the sequencer is started, the sequencing reaction on its own takes 20 to 24 hours.

In 2022, in parallel to Illumina technology, we have implemented a new technology using a third generation sequencer, the Gridion from Nanopore (ONT for Oxford Nanopore Technologies). The sequencing is done by passing the entire DNA molecule through an electrical pore in the sequencer. The preparation for sequencing is performed using the Rapid/Midnight SARS-CoV2 kit from Nanopore. After conversion of the RNA into DNA and amplification of different overlapping segments of the genome, each amplicon is barcoded with a unique barcode per sample. This allows the pool of different samples on one run. After clean-up and quantification, each fragment is linked to an adapter allowing the passage through the pore. Sample preparation for sequencing can be done within one day and the sequencing reaction on Gridion takes 20 hours.

After launching each sequencer, a large amount of data (up to millions of sequences of letters/nucleotides) which are then assembled together and aligned with a reference sequence. Then, bioinformatics programs compare the new sequence data to the reference sequence and identify variations in the genome sequenced. To assign a lineage to a genome according to these variants, our bioinformatic team use their pipeline updated regularly with the last version of Pangolin available. Then, these data are shared on public scientific database such as GISAID, so internationally available.

As the National Reference Laboratory for Acute Respiratory Infections, LNS receives SARS-CoV2 positive samples from the national network of laboratories with the aim of sequencing a representative sample of Luxembourg cases. Priority for sequencing is given to samples from hospital laboratories, the sentinel network to estimate community circulation and samples that are part of clusters and outbreaks. The national laboratory works closely together with Division de l’inspection sanitaire to optimize sample selection. Sequencing coverage and variant distribution in Luxembourg as well as all relevant epidemiological data are reported in our Revilux report (see some examples of data published).

We are continuously trying to increase our sequencing capacity in order to be able to provide the maximum amount of information for genomic surveillance in Luxembourg and to be ready to respond to eventually sequencing needs of European countries and more widely to international partners. We benefits support from HERA "Health Emergency Preparedness and Response Authority" incubator grant. Moreover, we have been recently nominated as WHO reference laboratory for COVID19 and ECDC recognized us as training centre for Microbial sequencing.

We have gone from a capacity of 380 at the end of 2020 to 1200 at the end of 2021 and in the course of 2022, our maximum capacity can reach at least 2500 sequencing per week. This can be achieved by the automatisation part of the Illumina sequencing samples preparation and also the implementation of the new Nanopore technology in parallel to the already installed Illumina technology.

We are also evolving by working on new sequencing approaches by the implementation of metagenomics and target enrichment approaches. These broadly approaches will enable us to have more information and to be more specific in the diagnosis of certain infections, for example in the case of emergence of a new virus or in the diagnosis of co-infections.