Optimising whole genome amplification for genome sequencing of minute amounts of Mycobacterium tuberculosis DNA

Whole genome sequencing (WGS) can be used for tuberculosis surveillance and outbreak identification. Use of WGS for patient care is hindered by low numbers of Mycobacterium tuberculosis (Mtb) present in clinical specimens and slow growth of Mtb. Current methods to enrich Mtb or Mtb-DNA are complex and costly. We hypothesised that whole genome amplification (WGA) can accurately and efficiently amplify Mtb-DNA to enable WGS from paucibacillary samples. Purified genomic Mtb-DNA of a pan-susceptible lineage 2 clinical isolate, at four amounts (0.1, 0.5, 1.0, and 5.0pg), was encapsulated in micro-droplets using the SAMPLIX instrument for multiple displacement amplification (MDA). The amplified DNA and one control sample (DNA extracted from sub-culture of the clinical isolate) were sequenced using standard Illumina protocols, followed by analysis with the XBS pipeline. WGA generated 105 to 2400ng DNA from the 0.1 to 5pg input DNA, respectively. The mean sequencing depth increased from 9x for 0.1pg unput DNA to 70x for 5pg input DNA and the control sample. The average breadth of coverage at =5x was 48% for 0.1pg input DNA and =93% for =0.5pg input DNA and the control. Phylogenetic analysis suggested that MDA resulted in an unacceptably high number (179 and 47) of false positive and false negative (429 and 25) variants when 0.1 and 0.5pg input DNA was amplified, respectively. Starting from =5pg of input DNA, the SAMPLIX method can sufficiently and accurately amplify Mtb-DNA for drug resistance inference, phylogeny and assessment of transmission.