MinION Nanopore Sequencing of Multiple Displacement Amplified Mycobacteria DNA Direct from Sputum

Abstract

37Sequencing of pathogen DNA directly from clinical samples offers the possibilities of rapid 38 diagnosis, faster antimicrobial resistance prediction and enhanced outbreak investigation. The 39 approach is especially advantageous for infections caused by species which grow very slowly 40 in culture, such as Mycobacteria tuberculosis. Since the pathogen of interest may represent as 41 little as 0.01% of the total DNA, enrichment of the input material for target sequences by 42 specific amplification and, or depletion of non-target DNA (human, other bacteria) is 43 essential for success. Here, we investigated the potential of isothermal multiple displacement 44 amplification by Phi29 polymerase. We directed the amplification reaction towards 45 Mycobacteria DNA in sputum samples by exploiting in our oligonucleotide primer design, 46 their high GC content (approximately 65%) relative to human DNA. Amplified DNA was 47 then sequenced using the Oxford Nanopore Technology MinION. In addition, a model 48 system comprising standardised 'mock clinical samples' was designed. Pooled infection 49 negative human sputum samples were spiked with enumerated Mycobacterium bovis (BCG) 50 Pasteur strain at concentrations spanning the typical range at which Mycobacterium 51 tuberculosis is found in human sputum samples (10 6 -10 1 BCG cells/ml). To assess the 52 amount of BCG sequence enrichment achieved, sample DNA was sequenced both before, and 53 after amplification. Reads from amplified samples, which mapped to a BCG reference 54 genome, comprised short repeated sequences -apparently transcribed multiple times from the 55 same fragment of BCG DNA. Therefore post-amplification, the samples were enriched for 56 BCG sequences relative to unamplified sequences (8,101 BCG reference mapped reads, 57 increasing to 28,617 at 10 6 BCG cells/ml sample), but BCG genome coverage declined 58 markedly (for example 89.4% to 4.1%). In summary, the use of standardised mock clinical 59 samples allowed direct comparison of data from different Mycobacteria enrichment 3 60 experiments and sequencing runs. However, optimal conditions for multiple displacement 61 amplification of minority Mycobacteria DNAs, remain to be identified. 4 62 5 87 concentration of target organisms. Mycobacteria DNA, for example, can represent as little as 88 0.01% of the total DNA extracted from sputum [11]. Small scale studies employing direct 89 from sample sequencing have reported 0.002 -0.7% sequence coverage of the M. 90 tuberculosis genome (using differential lysis and a DNA extraction kit) [12], and up to 90% 91 genome coverage with 20x depth (20/24 samples), (using the SureSelect target enrichment 92 method, Agilent, USA) [13, 14]. The study by Brown et al. [13] predicted both Mycobacteria 93 species and antibiotic susceptibility, but the cost ($350 per sample) and duration of the 94 protocol (2 to 3 days) could prevent its use. The ideal 'direct from sample' methodology 95 would be simple, low cost and portable, to facilitate use in remote, low-income settings 96 ...

LAMBDR: Long-range amplification and Nanopore sequencing of theMycobacterium bovis direct-repeat region. A novel method for in-silico spoligotyping ofM. bovisdirectly from badger faeces

“…Partial sequencing was achieved for the direct repeat region from contaminated drinking trough water. While previous studies have shown it possible to undertake whole genome sequencing of pure isolates as well as partial sequencing and assembly of M. tuberculosis from low complexity human sputum samples (George et al, 2018), this is the first study to present a feasible method for resolving a degree of strain level differentiation directly from environmental reservoirs of infection. It is likely that the incomplete spoligotyping of contaminated drinking tough water is due to the low level of M. bovis within the sample which indicates that this strain typing tool is less sensitive than the qPCR method to identify and quantify M. bovis in environmental samples (King et al, 2015, Courtenay et al, 2006).…”

Metagenomic sequencing as a clinical diagnostic tool for infectious diseases: a systematic review and meta-analysis

“…Four studies exclusively used Oxford Nanopore Technologies (ONT) MinION. 26,29,32,39 Six studies compared the use of Illumina sequencing versus ONT MinION. 3,[21][22][23]31,37 Five studies sequenced on the ION Torrent/Proton.…”

“…5,8,20,25,41,43,44 In other studies (n=13) there was no description of how the presence of an organism was confirmed. 19,[21][22][23]26,[31][32][33][34]36,37,45,46 Some studies (n=9) determined contamination without the use of a reported negative control by identifying known pathogens manually as described above (Figure 4). 3,20,22,[26][27][28][29]34,50 Therefore, by use of negative controls and other approaches, 26/36 (72%) studies report contamination of some degree while 9/36 (25%) studies had no details of contamination available.…”

Long-read sequencing in human genetics

“…Antibiotic resistance [7], Ebola [47], gonorrhea [23], West Nile virus, Zika [26], meningitis [6], tuberculosis [19], sepsis…”