Droplet-based whole genome amplification: a novel approach for sequencing minute amounts of Mycobacterium tuberculosis DNA

Abstract

Abstract Implementation of whole genome sequencing (WGS) for patient care is hindered by limited Mycobacterium tuberculosis (Mtb) in clinical specimens and slow Mtb growth. We evaluated droplet multiple displacement amplification (dMDA) for amplification of minute amounts of Mtb DNA to enable WGS as an alternative to other Mtb enrichment methods. Purified genomic Mtb-DNA (0.1, 0.5, 1, and 5pg) was encapsulated and amplified using the Samplix Xdrop-instrument and sequenced alongside a control sample using standard Illumina protocols followed by MAGMA-analysis. The control and 5pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400ng DNA from the 0.1-5pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7× for 0.1pg input DNA to ≥ 60× for 5pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5pg input DNA. Nanopore sequencing of the 5pg dMDA sample presented excellent coverage depth, breadth, and accurate strain characterization, albeit elevated false positive and false negative variants compared to Illumina-sequenced dMDA sample with identical Mtb DNA input. dMDA coupled with Illumina WGS for samples with ≥ 5pg DNA offers precision for drug resistance, phylogeny, and transmission insights.