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How is SARS-CoV-2 sequencing done?

An example workflow for SARS-CoV-2 sequencing sequencing
© COG-Train

There are many sequencing technologies used for SARS-CoV-2 (Figure 1). In this step, we will approach a broadly used protocol based on viral whole-genome sequencing (WGS). The method consists of viral RNA isolation, Library preparation, Sequencing run and Analysis.

Viral RNA Isolation

1. Viral inactivation
Sequencing of SARS-CoV-2 first requires inactivation of live virus either by heat or inactivating buffer so that the laboratory worker can handle the sample without putting themselves at risk. This procedure is carried out at various Biosafety Levels (BSL) worldwide. In the UK, prior to the current pandemic, this viral isolation would have been carried out at BSL3 facilities, but in early 2020, Public Health England issued a directive allowing this work to be completed at BSL2+. At which BSL is SARS-CoV-2 inactivation carried out in your country? Comment below.

2. Extraction of nucleic acid
The sample requires extraction of the nucleic acid so there is nothing else in the sample which could inhibit the sequencing. Extraction is normally performed by lysing (breaking down) the virus. Extraction can be performed using commercial kits or using magnetic beads to separate the nucleic acids from other materials in the sample. A range of buffers and alcohol suspensions are used to elute the nucleic acid into a final solution which is usually molecular-grade water.

This is a figure about methods used for SARS-CoV-2 genome sequencing. DNA strands are represented by thick black lines. The figure is made up of three separate diagrams labelled A, B, and C. Diagram A is a workflow detailing Illumina’s Nextera DNA Flex Enrichment protocol. The first step of this workflow shows a strand of DNA, the First-strand, which forms a map from which cDNA, (complementary DNA) strands will be synthesised. The second step of this workflow shows that the complementary strands are now the maps for which another set of complementary DNA strands are synthesised. This is called Second strand DNA synthesis. The third step in the workflow is called Bead-linked tagmentation. Strands of DNA are shown with small grey extensions representing molecular tags. The fourth step is called Indexing PCR, and the small grey tags are now represented in red. These are labelled: Sequencing library. The fifth step shows Eppendorf tubes containing a small volume of liquid being added together - these are Pooled samples. The sixth step shows a magnet with a probe being used to match the red-tagged DNA fragments. The seventh step shows an Eppendorf tube which is labelled: Enriched Library. The eighth step is called QC and sequence and shows that the Enriched Library can be used in an Illumina sequencing machine. Diagram B is a workflow for the ARTIC protocol. The first step of this workflow shows a strand of DNA, the First-strand, which forms a map from which cDNA strands will be synthesised. The second step of this workflow is called Multiplex PCR (2 pools), Untailed Primers. Two Eppendorf tubes are shown being added together, and are labelled: Combine pools and QC. The third step is labelled Barcode addition / NGS library preparation. Strands of DNA are shown with small red extensions and are labelled: sequencing library. The fourth step is labelled: Normalise, QC and sequence, and shows that the sequencing library can be used in Oxford Nanopore and Illumina machines. Diagram C is a workflow for the Tailed Amplicon Method. The first step of this workflow shows a strand of DNA, the First-strand, which forms a map from which cDNA strands will be synthesised. The second step of this workflow is called Multiplex PCR (2 or 4 pools), Tailed primers. Four Eppendorf tubes with small volumes of liquid are shown being combined. The third step is called indexing PCR, and strands of DNA with small red extensions are shown. These are labelled: Sequencing Library. The fourth step is called Normalise, QC and sequence, and shows that the sequencing library can be used in Illumina machines

Figure 1 – Methods for SARS-CoV-2 genome sequencing. a.) In Illumina’s Nextera DNA Flex Enrichment protocol cDNA is tagmented and made into barcoded sequencing libraries, which are then enriched using sequence capture with a respiratory virus panel containing probes against SARS-CoV-2. b.) In the ARTIC protocol, first-strand cDNA is enriched by amplifying with two pools of primers to generate amplicons tiling the SARS-CoV-2 genome. These amplicons are then subjected to either Illumina or Oxford Nanopore library preparation, using methods that either directly add adapters to the ends of the amplicons or to fragment them to enable sequencing on a wider variety of Illumina instruments. c.) The tailed amplicon approach, developed here, enriches first-strand cDNA using ARTIC v3 primers containing adapter tails. This allows functional sequencing libraries to be created through a second indexing PCR reaction that adds sample-specific barcodes and flow cell adapters. Source: BMC Genomics

Library preparation

1. Complementary DNA synthesis (not used for all technologies)
RNA extracts must be converted to cDNA because amplification methods, such as PCR, require DNA molecules as a template. A reverse transcriptase enzyme is used to synthesise the cDNA.

2. PCR Set-up and Amplification (not used for all technologies)
The cDNA is then added to a reaction mix including PCR reagents and SARS-CoV-2 specific primers, each targeting small regions of the viral genome. A specific primer targeting strategy is used to ensure that the entire genome is covered by these small regions. Samples are placed in a thermocycler, where amplification takes place. At the end of the process, millions of viral DNA fragments are generated.

3. Index PCR Set-up
NGS technologies allow multiplexing ie. pooling several different samples in a single sequencing run. In this process oligonucleotide indexes are added to each individual sample, ‘barcoding’ them, using a PCR procedure. This allows the individual samples to be separated computationally after sequencing.

4. Sample Pooling
The concentration of the DNA of the barcoded samples is measured and equal amounts of DNA of each sample are pooled in a single tube.

5. Clean-Up
NGS methodologies are highly sensitive. Inhibitors and reagent residues must be removed through DNA purification. It is strongly recommended to use magnetic beads purification protocols for the clean-up step.

Sequencing run

Depending on the technology used, the samples are loaded onto a flow cell and run on a sequencer that will generate fluorescent reads which can be captured by a camera or one which passes the entire molecule through a Nanopore and captures the sequence in real-time.

Analysis

Sequencing data is demultiplexed and the viral genome is assembled (similar to an alignment) using bioinformatics tools. There are many analysis pipelines, most commonly using command line, but there are also graphic interface tools.

SARS-CoV-2 ARTIC Protocols

There are specific ARTIC Network-derived protocols available for both Illumina and Oxford Nanopore Technologies platforms.

We will address the details of Bioinformatics and Sequencing in upcoming courses of this series.

If you are interested in seeing a sequencing workflow in a laboratory, watch this video (note, the video is silent) :

This is an additional video, hosted on YouTube.

Please note: This video is for educational purposes only, filmed at UCL Genomics, for MSc training. Dr Paola Niola performed denaturation and dilution of libraries, and Charlotte Williams performed MiSeq set up, filmed and edited by Sandra Cantilena.

© COG-Train
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Making sense of genomic data: COVID-19 web-based bioinformatics

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