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Sequencing of pathogen DNA directly from clinical samples offers the possibilities of rapid diagnosis, faster antimicrobial resistance prediction and enhanced outbreak investigation. The approach is especially advantageous for infections caused by species which grow very slowly in culture, such as Mycobacteria tuberculosis. Since the pathogen of interest may represent as little as 0.01% of the total DNA, enrichment of the input material for target sequences by specific amplification and, or depletion of non-target DNA (human, other bacteria) is essential for success. Here, we investigated the potential of isothermal multiple displacement amplification by Phi29 polymerase. We directed the amplification reaction towards Mycobacteria DNA in sputum samples by exploiting in our oligonucleotide primer design, their high GC content (approximately 65%) relative to human DNA. Amplified DNA was then sequenced using the Oxford Nanopore Technology MinION. In addition, a model system comprising standardised ‘mock clinical samples’ was designed. Pooled infection negative human sputum samples were spiked with enumerated Mycobacterium bovis (BCG) Pasteur strain at concentrations spanning the typical range at which Mycobacterium tuberculosis is found in human sputum samples (106 - 101 BCG cells/ml). To assess the amount of BCG sequence enrichment achieved, sample DNA was sequenced both before, and after amplification. Reads from amplified samples, which mapped to a BCG reference genome, comprised short repeated sequences - apparently transcribed multiple times from the same fragment of BCG DNA. Therefore post-amplification, the samples were enriched for BCG sequences relative to unamplified sequences (8,101 BCG reference mapped reads, increasing to 28,617 at 106 BCG cells/ml sample), but BCG genome coverage declined markedly (for example 89.4% to 4.1%). In summary, the use of standardised mock clinical samples allowed direct comparison of data from different Mycobacteria enrichment experiments and sequencing runs. However, optimal conditions for multiple displacement amplification of minority Mycobacteria DNAs, remain to be identified. |
