@article{3029958,
    title = "Recommendations for accurate genotyping of SARS-CoV-2 using
amplicon-based sequencing of clinical samples",
    author = "Kubik, Slawomir and Marques, Ana Claudia and Xing, Xiaobin and Silvery, and Janine and Bertelli, Claire and De Maio, Flavio and Pournaras, Spyros and and Burr, Tom and Duffourd, Yannis and Siemens, Helena and Alloui, and Chakib and Song, Lin and Wenger, Yvan and Saitta, Alexandra and and Macheret, Morgane and Smith, Ewan W. and Menu, Philippe and Brayer, and Marion and Steinmetz, Lars M. and Si-Mohammed, Ali and Chuisseu, Josiane and and Stevens, Richard and Constantoulakis, Pantelis and Sali, Michela and and Greub, Gilbert and Tiemann, Carsten and Pelechano, Vicent and Willig, and Adrian and Xu, Zhenyu",
    journal = "Clinical Microbiology and Infection",
    year = "2021",
    volume = "27",
    number = "7",
    publisher = "Elsevier Sci Ltd, Exeter, United Kingdom",
    issn = "1198-743X",
    doi = "10.1016/j.cmi.2021.03.029",
    keywords = "Amplicon; Coronavirus; Genome; Genotyping; Guidelines; Next-generation
sequencing; NGS; Recommendations; SARS-CoV-2",
    abstract = "Objectives: Genotyping of severe acute respiratory syndrome coronavirus
2 (SARS-CoV-2) has been instrumental in monitoring viral evolution and
transmission during the pandemic. The quality of the sequence data
obtained from these genotyping efforts depends on several factors,
including the quantity/ integrity of the input material, the technology,
and laboratory-specific implementation. The current lack of guidelines
for SARS-CoV-2 genotyping leads to inclusion of error-containing genome
sequences in genomic epidemiology studies. We aimed to establish clear
and broadly applicable recommendations for reliable virus genotyping.
Methods: We established and used a sequencing data analysis workflow
that reliably identifies and removes technical artefacts; such artefacts
can result in miscalls when using alternative pipelines to process
clinical samples and synthetic viral genomes with an amplicon-based
genotyping approach. We evaluated the impact of experimental factors,
including viral load and sequencing depth, on correct sequence
determination.
Results: We found that at least 1000 viral genomes are necessary to
confidently detect variants in the SARS-CoV-2 genome at frequencies of
>= 10%. The broad applicability of our recommendations was validated in
over 200 clinical samples from six independent laboratories. The
genotypes we determined for clinical isolates with sufficient quality
cluster by sampling location and period. Our analysis also supports the
rise in frequencies of 20A.EU1 and 20A.EU2, two recently reported
European strains whose dissemination was facilitated by travel during
the summer of 2020.
Conclusions: We present much-needed recommendations for the reliable
determination of SARS-CoV-2 genome sequences and demonstrate their broad
applicability in a large cohort of clinical samples. (C) 2021 The
Author(s). Published by Elsevier Ltd on behalf of European Society of
Clinical Microbiology and Infectious Diseases."
}