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Amplicon-based sequencing

This article summarizes amplicon based sequencing and discusses several protocols applied to SARS-CoV-2 amplicon sequencing.
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Introduction

Amplicon sequencing is a highly targeted approach that enables researchers to analyse genetic variation in specific genomic regions. The ultra-deep sequencing of PCR products (amplicons) allows efficient variant identification and characterisation. This method uses oligonucleotide probes designed to target and capture regions of interest, followed by next-generation sequencing (NGS). SARS-CoV-2 whole-genome sequencing (WGS) requires a viral RNA isolation from the clinical samples for sequencing library construction, and there can be orders-of-magnitude differences in viral load across different subjects. A large proportion of clinical samples contain extremely low viral copy number, which may impact the quality of WGS.

A flow diagram showing the processes following RNA extraction and how many days these take. The first flow is ordered as follows: RNA extraction, RT-PCR, PCR Amplification, Library Preparation, Illumina Sequencing, and Sequence Analysis. These processes take three days. The second flow is RNA extraction, qPCR detection, and Report. These processes take two days

Click here to enlarge the image
Figure 1 – Flowchart of a sequencing protocol from viral RNA isolation to sequencing analyses. Source: MDPI

The RNA viral genome is relatively small (~ 30 Kb) and highly heterogeneous. Due to these factors, the copy number of the viral genome is low and many errors can be introduced while sequencing after PCR enrichment. To overcome these hurdles, amplicon-based sequencing is utilised. In this method, the viral genome is amplified using primers which are complementary to known sequences. The amplicon enriched samples are then subjected to sequencing in platforms such as Nanopore or Illumina.

ARTIC protocol

The ARTIC sequencing protocol. The protocol is as follows: A single strand of RNA is completed with cDNA synthesis. This undergoes multiplex PCR with untailed primers. Pools are combined and quality controlled. Barcodes are added or NGS libraries prepared. These form a sequencing library. This library is normalised, quality controlled and sequenced in either Oxford Nanopore or Illumina machinery.

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Figure 2 – The ARTIC sequencing protocol. Source: BMC Genomics

The ARTIC protocol is based on a method that enriches the cDNA generated through reverse transcription of the SARS-CoV-2 genome, using a tiled PCR amplification using two primer pools. A total of 196 primers (98 pairs) were designed to tile the entire SARS-CoV-2 genome. An amplicon size of ~400 bp per target is obtained that then moves to the specific library preparation either for Illumina or Oxford Nanopore sequencing

Midnight protocol

The Midnight protocol approach uses 1200 base pair (bp) tiled amplicons produced by the multiplex PCR. Briefly, two PCR reactions are performed for each SARS-CoV-2 sample which includes two sets of primers. The first set (Pool 1) has thirty primers that generate the odd-numbered amplicons, while the second PCR reaction has twenty-eight primers that generate the even-numbered amplicons (Pool 2). After PCR, the two amplicon pools are combined which can be further processed for sequencing using either Oxford Nanopore or Illumina platforms.

This is a diagram of the Midnight protocol. It shows the steps and the time taken for each. The process begins with RT-PCR (265 minutes), and then pooling (5 minutes). A 1200bp amplicon is rapidly barcoded (15 minutes), and then these are subjected to sample pooling, SPRI clean, quantification, and rapid adapter addition (all taking 35 minutes). Finally, these are loaded into the sequencer (10 minutes) Click here to enlarge the image

Figure 3 – The Midnight protocol. Source: Oxford Nanopore Technologies

Considerations for amplicon-based sequencing

  • Enables researchers to efficiently discover, validate, and screen genetic variants using a highly targeted approach
  • Supports multiplexing of hundreds to thousands of amplicons per reaction so that it can achieve high coverage
  • Delivers highly targeted resequencing, even in difficult-to-sequence areas, such as GC-rich regions
  • Allows flexibility for a wide range of experimental designs
  • Reduces sequencing costs and turnaround time compared to broader approaches such as whole-genome sequencing
  • Since primers cannot capture the very ends of the viral genome, amplicon approaches have the drawback of slightly less complete genome coverage, and mutations in primer binding sites have the potential to disrupt the amplification of the associated amplicon.

References

Clinical and biological insights from viral genome sequencing

Amplicon-Based, Next-Generation Sequencing Approaches to Characterize Single Nucleotide Polymorphisms of Orthohantavirus Species

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

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