Abstract
Whole genome sequencing of viruses directly from clinical samples is integral for understanding the genetics of host-virus interactions. Here, we report the use of sample sparing target enrichment (by hybridisation) for viral nucleic acid separation and deep-sequencing of herpesvirus genomes directly from a range of clinical samples including saliva, blood, virus vesicles, cerebrospinal fluid, and tumour cell lines. We demonstrate the effectiveness of the method by deep-sequencing 13 highly cell-associated human herpesvirus genomes and generating full length genome alignments at high read depth. Moreover, we show the specificity of the method enables the study of viral population structures and their diversity within a range of clinical samples types.
Highlights
Whole genome sequencing of viral genomes directly from clinical samples is critically important for identifying genetic variants which cause disease, including those that are under positive selection pressure through interaction with the host [1]
Direct sequencing of mixed human and viral nucleic acids yields representative proportions of sequence reads that map to viral genomes [3], This represents a significant issue when dealing with samples that contain low proportions of viral nucleic acid and one that has limited such studies from being carried out previously [4,5,6,7]
The presence of PCR-inhibitory secondary structure and the inability of many viral species to thrive in culture present additional difficulties in generating sufficient quantities of viral nucleic acid for whole genome sequencing
Summary
Whole genome sequencing of viral genomes directly from clinical samples is critically important for identifying genetic variants which cause disease, including those that are under positive selection pressure through interaction with the host [1]. Subsequent hybridisation of the RNA baits with sequence library-prepared nucleic acid enables isolation and enrichment of target material (using a minimal number of rounds of PCR) and generating sufficient quantities for sequencing on second-generation platforms (Illumina, Roche, Abi).
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