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Rare Event Detection Using Error-corrected DNA and RNA Sequencing
Published on: August 3, 2018
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SARS-CoV-2 sequencing artifacts associated with targeted PCR enrichment and read mapping
Kirsten Maren Ellegaard1, Vithiagaran Gunalan2, Raphael Sieber1
1Department of Sequencing and Bioinformatics, Statens Serum Institut, Copenhagen, Denmark.
Plos One
|October 16, 2025
Summary
Primer choice and reference genome selection significantly impact SARS-CoV-2 genome sequencing accuracy. Continuous monitoring and updating of primer schemes and bioinformatics pipelines are crucial for reliable variant surveillance.
Area of Science:
- Genomics
- Virology
- Bioinformatics
Background:
- SARS-CoV-2 genome sequencing protocols were rapidly developed during the pandemic.
- Targeted enrichment by PCR followed by shotgun sequencing and reference-based assembly is a common approach.
- Global SARS-CoV-2 surveillance is shifting to a lower intensity, prompting a review of established methods.
Purpose of the Study:
- To investigate the impact of different primer schemes and reference genomes on SARS-CoV-2 sequencing.
- To analyze the temporal changes in ambiguous base calls during the emergence of the BA.2.86.x variant.
Main Methods:
- Sequencing of samples using multiple primer schemes (Artic V3, V4.1, V5.3.2).
- Re-processing of sequencing reads using multiple reference genomes.
- Analysis of ambiguous base calls and their temporal development.
Main Results:
- Primer schemes can lead to recurrent ambiguous base calls, especially with new variants.
- PCR artifacts and amplicon drop-out cause consistent base-calling errors.
- Misalignments and partially mapped reads result in ambiguous calls and omitted mutations.
Conclusions:
- Targeted enrichment by PCR and reference-based assembly have limitations for SARS-CoV-2 sequencing.
- Regular monitoring and updating of primer schemes and bioinformatics pipelines are essential for accurate genomic surveillance.
