SARS-CoV-2 sequencing workflow

Here we outline one potential ARTIC workflow example. It must be noted that many different approaches, kits and methods have been used throughout the pandemic to good effect. Commercial kits provide an off-the-shelf option that can allow labs to start sequencing quickly. However, the lack of visibility around the primer design process increases the risk that all VOCs are not covered, and how quickly new VOCs are included is unclear. COG-UK in particular was able to be so adaptive due to the ongoing work of the ARTIC network and constant primer design activities led by the University of Birmingham.

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Figure 1 – SARS-CoV-2 next-generation sequencing workflow, from the sample to sequence analysis.

For each sample, the LunaScript RT Supermix kit (New England Biolabs, NEB, United States) is used to obtain single-strand cDNA from RNA extracts using random hexamers and poly-dT primers, allowing even coverage across the length of the RNA targets. RT was performed at 25°C for 2 min, 55°C for 10min and 95°C for 1 min. Pools of specific primer sets were used to generate amplicons from the cDNA using the Q5 Hot Start High-Fidelity DNA Polymerase (NEB, United States). These multiplex primer sets (divided into two separate pools) were designed using the Primal scheme software on the SARS-CoV-2 reference sequence (Genbank accession number NC_045512) to sequentially amplify either 400bp (ARTIC protocol) or 1200bp (Midnight protocol) using the following PCR conditions: 98°C for 30 s, 40 cycles of 98°C for 15 s, 65°C for 5 min, and ended by 65°C for 5 min. PCR products from both pools are mixed into one tube after successful PCR amplification.

Clean-up of PCR products was performed with AMPure XP magnetic beads (Beckman Coulter, United States). The quantity of amplicons was measured with the Qubit 2.0 fluorometer using the dsDNA HS Assay Kit (Thermo Fisher Scientific, United States), and the quality was assessed by electrophoresis. The amplicons obtained from the two clinical samples and their corresponding isolates were normalised to equal concentrations, multiplexed (X2), and sequenced using the MinION and Illumina System

SARS-Cov-2 viral genomes from patient samples:

Step 1: Extraction of viral RNA genomes

The viral RNA genome is typically extracted from various VTM solutions of clinical COVID-19 samples. Samples should be stored at −80°C, thawed on ice, and kept cold to maintain viral genome integrity

Step 2: Reverse transcription of viral RNA to cDNA

Following extraction, RNA is converted to cDNA as soon as possible, preferably without a freeze-thaw cycle, to prevent potential RNA degradation. Lunascript RT SuperMix kit (New England Biolabs) is commonly used because of the short reaction time and single master mix containing dye, which accelerates and simplifies the workflow and reduces contamination potential.

Step 3: qPCR to approximate viral load

To quantify approximate viral load in each sample, qPCR is performed using cDNA as template using any commercially available or in-house kit

Step 4: Multiplex ARTIC PCR to amplify viral genome sequences

Once qPCR is performed, viral genome cDNA is amplified to increase the copy number of viral genomes. Two pools of primers are designed by the ARTIC Network, which are commercially available from IDT as the ARTIC nCoV-2019 V3 primer set. These require two pools of PCR reactions for every sample, with pool 1 containing 55 primer sets and pool 2 containing 54 primer sets. Two primer pools are used to create overlapping amplicons that reduce interference during PCR and prevent short overlapping products from being preferentially produced. Through this tiling amplicon scheme, complete coverage of the genome is achieved. Q5 High-Fidelity DNA Polymerase from New England Biolabs (NEB) is used commonly for PCR reactions

Step 5: Pooling and purification of ARTIC PCR products

Before library preparation, pool 1 and pool 2 of ARTIC PCR reactions are combined for every sample and the ~400 base-pair (bp) amplicons are purified from contaminating PCR reactions using SPRI AMPure XP beads (Beckman-Coulter).

Step 6: Fluorescence-based qubit quantification of pooled and purified ARTIC PCR products

The purified PCR pools are quantified using a Qubit 2.0 Fluorometer and Qubit dsDNA High Sensitivity (HS) Assay Kit following the manufacturer’s recommended protocol

Step 7: Preparation of DNA ends for barcoding

Following the approximation of pooled PCR product concentration by Qubit, 60 ng of pooled and SPRI purified PCR amplicons from SPRI clean-up are added to the end preparation reactions designed to create compatible ends of the DNA amplicons for sample barcoding. These are referred to as end-prep reactions. The DNA is first end-repaired followed by dA-tailing and inactivation of end-repair enzymes. Ultra II End-Prep kit from NEB most widely used following the manufacturer’s recommended protocol. After this step, DNA can be frozen at −20°C but is most often kept at 4°C for all downstream library preparation steps.

Step 8: DNA library sample barcoding and adapter ligation

To sequence a full 96-well plate of samples, each sample must be uniquely barcoded and later demultiplexed by allocating reads to samples with the matching barcode. This reduces the overall cost per sample and allows sequencing of up to 96 samples in a single run. To achieve this, Rapid Barcoding Expansion 96 kit (EXP-NBD110) from ONT is widely used for nanopore sequencing and Illumina based CovidSeq kit is used for Illumina platform. After individual barcoding reactions are performed in a 96-well plate, all samples are pooled and cleaned with SPRI beads. After barcoding and clean-up, ONT adapters are then ligated to each amplicon end for the pooled libraries in a single reaction.

Step 9: Loading and running the MinION/Illumina system:

For optimal performance on a MinION instrument, 20 ng of adapter-ligated library should be loaded onto the flow cell. Overloading the flow cell results in lower throughput. Therefore, a final Qubit quantification is performed. The DNA sequencing library is then prepared using ONT reagents/Illumina reagents following recommended protocols.

Step 10: Sequencing, real-time visualisation, and data analysis

Step 11: Phylogenetic analysis, variant calling, and database deposition of SARS-CoV-2 genomes

Do you know a different sequencing workflow? Use the comments section to share your experiences.