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Producing SARS-CoV-2 amplicons

article explaining how to amplify SARS-CoV-2 genomes before sequencing
© COG-Train

Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) hold the genetic information required to make proteins, and therefore all life on earth.

The sequence in which DNA bases occur provides the genetic code. Some viruses, such as SARS-CoV-2, use RNA rather than DNA as their genetic material. To read the genetic code of SARS-CoV-2, its RNA must first be converted to DNA – allowing it to be ‘read’ by the sequencer. The conversion method used is called reverse transcription-polymerase chain reaction (RT-PCR) and it consists of two steps. The first step synthesises complementary DNA (cDNA) from RNA by reverse transcription (i.e the opposite of transcription). In this process an enzyme (reverse transcriptase) adds complementary bases to the single-stranded RNA, creating cDNA. The second step is PCR, which amplifies the cDNA using an enzyme (DNA polymerase), creating enough double-stranded DNA to go into downstream library preparation for sequencing.

Schematic illustration of reverse transcription PCR (RT-PCR). Detailed description in the main text

Click to enlarge

Figure 1 – The reverse-transcription PCR. Source ACS Central Science

SARS-CoV-2 samples contain contaminating human material, so the ARTIC PCR is designed to amplify SARS-CoV-2 cDNA. This is done using a ‘multiplex tiling PCR’. A multiplex PCR is the simultaneous amplification of multiple targets, and ‘tiling’ means overlapping amplicons that can be aligned bioinformatically to determine the full-length genome. The SARS-CoV-2 ARTIC primer pairs each amplify 400b regions of the SARS-CoV-2 genome. The ARTIC primer pairs are split into two pools, with adjacent primer pairs in different pools to prevent primers from competing for the same part of the genome, hence creating a more efficient amplification. The result of the PCR is two pools of 400bp SARS-CoV-2 amplicons which are pooled and subsequently, the library is prepared for sequencing.

Illustrative schematic of a multiplex tiling PCR. A black line represents the SARS-CoV-2 Wuhan-Hu-1 reference genome. Underneath it there are two rows of boxes representing the regions covered by primers set A (boxes A1-7) and set B (boxes B1-7

Click to enlarge

Figure 2 – Schematic of a multiplex tiling PCR. Source: protocols.io

The ARTIC PCR is also different to a typical 3-step (denaturation, annealing, extension) PCR in that it has a combined annealing and elongation, thereby only having two steps. This annealing/elongation step is longer than typical PCR annealing and elongation times, which is required in multiplex PCRs as there are more primers to anneal, and more targets to amplify.

PCR protocol step Number of cycles Typical 3-step Multiplex 2-step
Initial denaturation 1 95°C 30s not applicable
Heat activation 1 not applicable 98°C 30s
Denaturation 30 for Taq; 35 for ARTIC 95°C 15-30s 98°C 15s
Annealing 30 for Taq; 35 for ARTIC 45-68°C 15-60s 65°C 5m
Extension 30 for Taq; 35 for ARTIC 68°C 1m per kb 65°C 5m
Final Extension 1 72°C 5m not applicable

References

Protocol for a Routine Taq PCR

nCoV-2019 sequencing protocol v3 (LoCost) V.3

© COG-Train
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A Practical Guide for SARS-CoV-2 Whole Genome Sequencing

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