What a targeted sequencing protocol

Different targeted methods have been developed to library preparation and sequence full-length SARS-CoV-2 genomes from specimens containing both viral and human nucleic acids. Due to the size difference between the SARS-CoV-2 (~30kb) and human (~6.4 Gb) genomes, sequencing the sample without enriching or amplifying the viral genome could result in >99% of the sequencing reads being human.

SARS-CoV-2 WGS – amplicon sequencing methods

In amplicon sequencing, the SARS-CoV-2 genome is amplified by RT-PCR (reverse transcription-polymerase chain reaction) on overlapping fragments, which can differ in length. The most commonly used primers are the ARTIC primers designed by Josh Quick (University of Birmingham) which produce 400bp fragments. Other primer designs include those used in the Oxford Nanopore Midnight protocol. Amplifying longer amplicons (as achieved with Entebbe primers) has the advantage of requiring less primer pairs to cover the full-length genome (which may reduce the number of times the primer design needs to be updated to incorporate new variants), although they are unlikely to amplify SARS-CoV-2 in degraded samples as well as the ARTIC primers. However, more amplicons mean more opportunities for amplicon dropout leading to technical artefacts in the consensus sequence. Amplicons are prepared for sequencing using commercially available library preparation kits or open-source protocols (see the Resources of this course).

The tailed PCR method is a variation of the standard amplicon sequencing approach in which a second PCR step replaces the need for library preparation. Non-homologous sequences containing the information required for NGS are added to the end of the ARTIC primers (‘tail’) and are therefore incorporated into the amplified fragments. A second round of PCR using indexing primers generates fragments ready to be sequenced. This approach is cost-effective as there is no library preparation although the likelihood of contamination occurring may increase compared to some methods due to the second round of PCR.

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Figure 1 – Overview of the ARTIC protocol (top) and tailed amplicon method (bottom). Source: BMC Genomics

The Wellcome Sanger Institute provided large-scale and high-throughput sequencing of SARS-CoV-2 for COG-UK using a different tailed amplicon approach, whereby the first PCR step was with the ARTIC primers followed by a second PCR with a mix of tailed versions of the ARTIC primers plus the indexing primers.

The tailed PCR method has been further developed using a reverse complement-PCR strategy (RC-PCR) in which indexed libraries ready for Illumina sequencing are generated in a single PCR step. This approach has been used in the UK for monitoring SARS-CoV-2 in wastewater with an open-source protocol available .

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Figure 2 – Schematic representation of the RC-PCR technology. The protocol consists of one single PCR-like reaction consisting of 2 steps. A. Two types of oligos are present, 1) the universal tail which includes a Unique Dual Index (UDI), sequence adapter, and universal sequence. 2) The RC primer contains an extension blocker, universal sequence, and the reverse complement of the SARS-CoV-2 genomic target sequence. B. The universal sequences anneal and form a SARS-CoV-2 specific PCR primer. C – E. A regular PCR in which the SARS-CoV-2 specific amplicons are created. F. The final amplicons are ready to sequence on an Illumina sequencer. Source Journal of Clinical Virology

SARS-CoV-2 WGS – bait/probe capture

In bait/probe capture the library is enriched for the viral genome using small fragments (baits/probes) complementary to the viral sequence which are then captured on a solid support. Various probe panels (both RNA and DNA) have been designed against SARS-CoV-2. An advantage of using this approach is that one bait set can be designed to target multiple viruses, for example, multiple respiratory viruses, meaning coinfections can be detected and sequenced from the same sample. Combined with deep sequencing, this approach can identify minority populations within a sample, allowing for analysis of the evolution of resistance mutations and informing treatment.

Examples of commercially available SARS-CoV-2 panels are included in the Resources.

References:

CoronaHiT: high-throughput sequencing of SARS-CoV-2 genomes

Rapid turnaround multiplex sequencing of SARS-CoV-2: comparing tiling amplicon protocol performance

SARS-CoV-2 whole-genome sequencing using reverse complement PCR: For easy, fast and accurate outbreak and variant analysis

Mini-XT, a miniaturized tagmentation-based protocol for efficient sequencing of SARS-CoV-2

A rapid, cost-effective tailed amplicon method for sequencing SARS-CoV-2

Clinical and biological insights from viral genome sequencing

Reverse Complement PCR: A novel one-step PCR system for typing highly degraded DNA for human identification

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore

SARS-CoV-2 genomes recovered by long amplicon tiling multiplex approach using nanopore sequencing and applicable to other sequencing platforms