ena-STUDY-Public Health England-25-11-2016-14:08:34:351-2
Using enhanced extraction and library preparation methods, we are maximising representation of reads of RNA virus origin in FASTQ data sets from clinical samples.
RNA viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. Mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false negatives and incomplete characterisation. New sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. Through selective host RNA depletion and compensatory protocol adjustments for ultra-low RNA inputs, we are able to detect three major blood-borne RNA viruses – HIV, HCV and HEV. We recovered complete genomes and up to 43% of the genome from samples with viral loads of 104 and 103 IU/ml respectively. Additionally, we demonstrated the utility of this method in detecting and characterising members of diverse RNA virus families within a human plasma background, some present at levels below the limit of detection of conventional real-time PCR methods. By applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of HCV genotype 6 , and a co-infecting human pegivirus. This method builds upon earlier RNA metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses.
RNA viruses cause significant human pathology and are responsible for the majority of emerging zoonoses. Mainstream diagnostic assays are challenged by their intrinsic diversity, leading to false negatives and incomplete characterisation. New sequencing techniques are expanding our ability to agnostically interrogate nucleic acids within diverse sample types, but in the clinical setting are limited by overwhelming host material and ultra-low target frequency. Through selective host RNA depletion and compensatory protocol adjustments for ultra-low RNA inputs, we are able to detect three major blood-borne RNA viruses – HIV, HCV and HEV. We recovered complete genomes and up to 43% of the genome from samples with viral loads of 104 and 103 IU/ml respectively. Additionally, we demonstrated the utility of this method in detecting and characterising members of diverse RNA virus families within a human plasma background, some present at levels below the limit of detection of conventional real-time PCR methods. By applying this method to a patient sample series, we have simultaneously determined the full genome of both a novel subtype of HCV genotype 6 , and a co-infecting human pegivirus. This method builds upon earlier RNA metagenomic techniques and can play an important role in the surveillance and diagnostics of blood-borne viruses.