Sammendrag
Objectives: Direct genotyping of adenovirus or enterovirus from clinical material
using polymerase chain reaction (PCR) followed by Sanger sequencing is often
difficult due to the presence of multiple virus types in a sample, or due to varying
efficacy of PCR amplifying the capsid gene on the background of foreign nucleic acids.
Here we present a simple protocol for virus genotyping using massive parallel
amplicon sequencing.
Methods: The protocol utilized a set of 16 tailed degenerate primers flanking the
seventh hypervariable region of the adenovirus hexon gene and 9 tailed degenerate
primers targeted to the proximal portion of the enterovirus VP1 gene. Subsequent
addition of dual indices enabled simultaneous sequencing of 384 different samples on
an Illumina MiSeq instrument. Downstream bioinformatic analysis was based on
remapping to a set of references representative of the presently known repertoire of
virus types.
Results: After validation with known virus types, the sequencing method was applied
on 301 adenovirus‐positive samples and 350 enterovirus‐positive samples from a
longitudinally collected series of stools from 83 children aged 3 to 36 months. We
detected 7 different adenovirus types and 27 different enterovirus types. There were
37 (6.2%) samples containing more than one genotype of the same viral genus. At
least one dual infection was experienced by 23 of 83 (28%) of the children observed
over the 3 years' observation period.
Conclusions: Amplicon sequencing with a multiplex set of degenerate primers seems
to be a rapid and reliable technical solution for genotyping of large collections of
samples where simultaneous infections with multiple strains can be expected.
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