Norovirus whole genome sequencing by SureSelect target enrichment: a robust and sensitive method

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

Use of Whole-genome Sequencing of Adenovirus in Immunocompromised Paediatric Patients to Identify Nosocomial Transmission and Mixed-genotype Infection

Background: Adenoviruses are significant pathogens for the immunocompromised, arising from primary infection or reinfection. Serotyping is insufficient to support nosocomial transmission investigations. We investigate whether whole-genome sequencing (WGS) provides clinically relevant information on transmission among patients in a paediatric tertiary hospital. Methods: We developed a target-enriched adenovirus WGS technique for clinical samples and retrospectively sequenced 107 adenovirus-positive residual diagnostic samples, including viraemias (>5x104 copies/ml), from 37 patients collected January 2011 - March 2016. WGS was used to determine genotype and for phylogenetic analysis. Results: Adenovirus sequences were recovered from 105/107 samples. Full genome sequences were recovered from all 20 non-species C samples and from 36/85 species C viruses, with partial genome sequences recovered from the rest. Whole genome phylogenetic analysis suggested linkage of three genotype A31 cases and uncovered an unsuspected epidemiological link to an A31 infection first detected on the same ward four years earlier. In nine samples from one patient who died we identified a mixed genotype adenovirus infection. Conclusions: Adenovirus WGS from clinical samples is possible and useful for genotyping and molecular epidemiology. WGS identified likely nosocomial transmission with greater resolution than conventional genotyping, and distinguished between adenovirus disease due to single or multiple genotypes