Next-generation sequencing (NGS) is a powerful method for rapid whole-genome sequencing of microbial genomes or overall microbial species content in complex samples. However, most biological samples with microbes of interest are dominated by non-microbial DNA. This necessitates extremely deep sequencing in order to accurately resolve microbial genomes, or to fully characterize the variation within microbial communities.
Targeted sequencing, wherein background non-target DNA is excluded from samples prior to sequencing, solves this problem and drastically reduces the overall costs of sequencing and data analysis per sample. Target sequence enrichment typically involves either PCR ampliﬁcation with site-speciﬁc primer pairs or hybridization-based capture with target speciﬁc biotinylated probes. Given the high degree of sequence variation within and between viral or bacterial strains, proper primer binding for amplicon formation can be inconsistent. Thus hybridization capture is currently the most versatile technique currently for comprehensive, cost-effective sequencing of both viruses and bacteria in complex samples.
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