Droplet-based Whole Genome Amplification: Successfully Sequencing Minute Amounts of Mycobacterium Tuberculosis DNA

Whole genome sequencing (WGS) is crucial for tuberculosis (TB) surveillance, but its implementation in patient care is hindered by the limited amount of Mycobacterium tuberculosis (Mtb) in clinical specimens and the slow growth of Mtb.

The experts evaluated droplet-based multiple displacement amplification (dMDA) for the efficient amplification of minute amounts of Mtb DNA to enable WGS as an alternative to complex and costly Mtb enrichment methods. Purified genomic Mtb-DNA, spanning four amounts (0.1, 0.5, 1.0 and 5.0 pg), was encapsulated and amplified using Samplix’s Xdrop, a benchtop instrument for preparing DNA for highly targeted sequencing.

Sequencing and analysis involved standard Illumina protocols and the maximum accessible genome for Mtb analysis (MAGMA) pipeline for dMDA-DNA and a control sample (undiluted Mtb DNA). The control and 5 pg input dMDA samples underwent nanopore sequencing and were analysed using nanorate sequencing (Nanoseq) and TB-profiler. dMDA generated 105–2400 ng DNA from the 0.1–5.0 pg input DNA. When followed by Illumina WGS, dMDA increased the mean sequencing depth from 7× for 0.1 pg input DNA to ≥60× for 5 pg input and the control sample. The average ≥5× coverage spanned 44 percent for 0.1 pg input DNA and ≥92 percent for ≥0.5 pg input DNA and the control.

Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤0.5 pg input DNA with dMDA. Oxford Nanopore Technologies (ONT) sequencing of the 5 pg dMDA sample presented excellent depth, coverage breadth, as well as accurate lineage and drug susceptibility profiling. However, it showed elevated false positive and false negative variants compared to Illumina-sequenced dMDA samples with identical Mtb DNA input. In summary, dMDA amplification coupled with Illumina WGS for samples with ≥5 pg DNA offers precision for drug resistance, phylogeny and transmission insights.

Register for this webinar today to gain insights into how dMDA amplification coupled with whole genome sequencing helped successfully sequence minute amounts of Mtb DNA.

Speaker

Dr. Anzaan Dippenaar, Senior Researcher, University of Antwerp

Anzaan Dippenaar is a Molecular Biologist with a PhD from Stellenbosch University, South Africa and over 10 years of experience in tuberculosis (TB) research. She is currently employed as a Senior Researcher in the Tuberculosis Omics Research consortium at the University of Antwerp, Belgium. Her research focuses on deciphering the dynamics of TB outbreaks over time, making use of next-generation whole genome sequence data of Mycobacterium tuberculosis (Mtb) isolates. She also investigates the evolution of drug resistance in Mtb clinical isolates and specimens using whole genome and targeted deep sequencing data and optimises methodologies to extract mycobacterial DNA from clinical specimens for downstream NGS applications. Anzaan has extensive experience in NGS data analysis and is particularly interested in its applications in various fields of precision medicine and public health.

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