Brown, JR;
Roy, S;
Ruis, C;
Yara Romero, E;
Shah, D;
Williams, R;
Breuer, J;
(2016)
Norovirus whole genome sequencing by SureSelect target enrichment: a robust and sensitive method.
Journal of Clinical Microbiology
, 54
(10)
pp. 2530-2537.
10.1128/JCM.01052-16.
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Abstract
Norovirus full genome sequencing is challenging due to sequence heterogeneity between genomes. Previous methods have relied on PCR amplification, which is problematic due to primer design, and RNASeq which non-specifically sequences all RNA in a stool specimen, including host and bacterial RNA.Target enrichment uses a panel of custom-designed 120-mer RNA baits which are complementary to all publicly available norovirus sequences, with multiple baits targeting each position of the genome, thus overcoming the challenge of primer design. Norovirus genomes are enriched from stool RNA extracts to minimise sequencing non-target RNA.SureSelect target enrichment and Illumina sequencing was used to sequence full genomes from 507 norovirus positive stool samples with RT-qPCR Ct values 10-43. Sequencing on an Illumina MiSeq in batches of 48 generated on average 81% on-target-reads per sample and 100% genome coverage with >12,000-fold read depth. Samples included genotypes GI.1, GI.2, GI.3, GI.6, GI.7, GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.13, GII.14 and GII.17. Once outliers are accounted for, we generate over 80% genome coverage for all positive samples, regardless of Ct value.164 samples were tested in parallel with conventional PCR genotyping of the capsid shell domain. 164/164 samples were successfully sequenced, compared to 158/164 that were amplified by PCR. Four of the samples that failed capsid PCR had low titres, suggesting target enrichment is more sensitive than gel-based PCR. Two samples failed PCR due to primer mismatches; target enrichment uses multiple baits targeting each position, thus accommodating sequence heterogeneity between norovirus genomes.
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