5th September 2016 - Microbiology Society
Enrichment of DNA by hybridisation is an important tool which enables users to gather target-focused next-generation sequence data in an economical fashion. Current in-solution methods capture short fragments of around 200 - 300 nt, potentially missing key structural information such as recombination or translocations often found in viral or bacterial pathogens. The increasing use of long-read third-generation sequencers requires methods and protocols to be adapted for their specific requirements. Here, we present a variation of the traditional bait-capture approach which can selectively enrich large fragments of DNA or cDNA from specific bacterial and viral pathogens, for sequencing on long-read sequencers. We enriched cDNA from cultured influenzavirus A, human cytomegalovirus (HCMV) and genomic DNA from two strains of Mycobacterium tuberculosis (M.tb) from a background of cell line or spiked human DNA. We sequenced the enriched samples on the Oxford Nanopore MinION and the Illumina MiSeq platform and present an evaluation of the method, together with analysis of the sequence data. We found that unenriched influenza A and HCMV samples had no reads matching the target organism due to the high background of DNA from the cell line used to culture the pathogen. In contrast, enriched samples sequenced on the MinION had 57% and 99% best-quality on-target reads respectively.