Assessment of two-pool multiplex long-amplicon nanopore sequencing of SARS-CoV-2.
J Med Virol
; 94(1): 327-334, 2022 01.
Article
in English
| MEDLINE | ID: covidwho-1410052
ABSTRACT
Genomic surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays an important role in COVID-19 pandemic control and elimination efforts, especially by elucidating its global transmission network and illustrating its viral evolution. The deployment of multiplex PCR assays that target SARS-CoV-2 followed by either massively parallel or nanopore sequencing is a widely-used strategy to obtain genome sequences from primary samples. However, multiplex PCR-based sequencing carries an inherent bias of sequencing depth among different amplicons, which may cause uneven coverage. Here we developed a two-pool, long-amplicon 36-plex PCR primer panel with ~1000-bp amplicon lengths for full-genome sequencing of SARS-CoV-2. We validated the panel by assessing nasopharyngeal swab samples with a <30 quantitative reverse transcription PCR cycle threshold value and found that ≥90% of viral genomes could be covered with high sequencing depths (≥20% mean depth). In comparison, the widely-used ARTIC panel yielded 79%-88% high-depth genome regions. We estimated that ~5 Mbp nanopore sequencing data may ensure a >95% viral genome coverage with a ≥10-fold depth and may generate reliable genomes at consensus sequence levels. Nanopore sequencing yielded false-positive variations with frequencies of supporting reads <0.8, and the sequencing errors mostly occurred on the 5' or 3' ends of reads. Thus, nanopore sequencing could not elucidate intra-host viral diversity.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Genome, Viral
/
Multiplex Polymerase Chain Reaction
/
Whole Genome Sequencing
/
Nanopore Sequencing
/
SARS-CoV-2
Type of study:
Prognostic study
Limits:
Humans
Language:
English
Journal:
J Med Virol
Year:
2022
Document Type:
Article
Affiliation country:
Jmv.27336
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