Droplet-based Whole Genome Amplification: Successfully Sequencing Minute Amounts of Mycobacterium Tuberculosis DNA, Upcoming Webinar Hosted by Xtalks
In this free webinar, delve into the significance of whole genome sequencing (WGS) in advancing tuberculosis (TB) surveillance. WGS is crucial for 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. Attendees will learn how droplet-based multiple displacement amplification (dMDA) coupled with WGS helped successfully sequence minute amounts of Mtb DNA.
TORONTO, Feb. 28, 2024 /PRNewswire-PRWeb/ -- 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.
Join Dr. Anzaan Dippenaar, Senior Researcher, University of Antwerp, for the live webinar on Wednesday, March 20, 2024, at 10:30am EDT (3:30pm CET/EU-Central).
For more information, or to register for this event, visit Droplet-based Whole Genome Amplification: Successfully Sequencing Minute Amounts of Mycobacterium Tuberculosis DNA.
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