TY - JOUR
TI - Recommendations for accurate genotyping of SARS-CoV-2 using
amplicon-based sequencing of clinical samples
AU - Kubik, Slawomir
AU - Marques, Ana Claudia
AU - Xing, Xiaobin
AU - Silvery,
AU - Janine
AU - Bertelli, Claire
AU - De Maio, Flavio
AU - Pournaras, Spyros
AU - and Burr, Tom
AU - Duffourd, Yannis
AU - Siemens, Helena
AU - Alloui,
AU - Chakib
AU - Song, Lin
AU - Wenger, Yvan
AU - Saitta, Alexandra and
AU - Macheret, Morgane
AU - Smith, Ewan W.
AU - Menu, Philippe
AU - Brayer,
AU - Marion
AU - Steinmetz, Lars M.
AU - Si-Mohammed, Ali
AU - Chuisseu, Josiane
AU - and Stevens, Richard
AU - Constantoulakis, Pantelis
AU - Sali, Michela and
AU - Greub, Gilbert
AU - Tiemann, Carsten
AU - Pelechano, Vicent
AU - Willig,
AU - Adrian
AU - Xu, Zhenyu
JO - Clinical Microbiology and Infection
PY - 2021
VL - 27
TODO - 7
SP - null
PB - Elsevier Sci Ltd, Exeter, United Kingdom
SN - 1198-743X
TODO - 10.1016/j.cmi.2021.03.029
TODO - Amplicon; Coronavirus; Genome; Genotyping; Guidelines; Next-generation
sequencing; NGS; Recommendations; SARS-CoV-2
TODO - 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.
ER -