Tuberculosis Omics Research Consortium, Department of Family Medicine and Population Health, Global Health Institute, Faculty of Medicine and Health Sciences, University of Antwerp, Antwerp, Belgium.
South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa.
Sci Rep. 2024 Apr 30;14(1):9931. doi: 10.1038/s41598-024-60545-1.
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 5 pg) 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 5 pg input dMDA samples underwent nanopore sequencing followed by Nanoseq and TB-profiler analysis. dMDA generated 105-2400 ng DNA from the 0.1-5 pg input DNA, respectively. Followed by Illumina WGS, dMDA raised mean sequencing depth from 7 × for 0.1 pg input DNA to ≥ 60 × for 5 pg input and the control sample. Bioinformatic analysis revealed a high number of false positive and false negative variants when amplifying ≤ 0.5 pg input DNA. Nanopore sequencing of the 5 pg 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 ≥ 5 pg purified Mtb DNA, equating to approximately 1000 copies of the Mtb genome, offers precision for drug resistance, phylogeny, and transmission insights.
实施全基因组测序（WGS）用于患者护理受到临床标本中结核分枝杆菌（Mtb）含量有限和 Mtb 生长缓慢的阻碍。我们评估了液滴多重置换扩增（dMDA）用于扩增微量 Mtb DNA，以作为其他 Mtb 富集方法的替代方法，从而实现 WGS。使用 Samplix Xdrop 仪器对纯化的基因组 Mtb-DNA（0.1、0.5、1 和 5 pg）进行封装和扩增，并使用标准 Illumina 方案与对照样品一起进行测序，然后进行 MAGMA 分析。对照和 5 pg 输入的 dMDA 样品进行纳米孔测序，然后进行 Nanoseq 和 TB-profiler 分析。dMDA 分别从 0.1-5 pg 输入 DNA 产生 105-2400 ng DNA。在进行 Illumina WGS 后，dMDA 将 0.1 pg 输入 DNA 的平均测序深度从 7×提高到 5 pg 输入和对照样品的≥60×。生物信息学分析表明，当扩增≤0.5 pg 输入 DNA 时，会出现大量假阳性和假阴性变体。尽管与具有相同 Mtb DNA 输入的 Illumina 测序 dMDA 样本相比，纳米孔测序的 5 pg dMDA 样本呈现出出色的覆盖深度、广度和准确的菌株特征，但存在较高的假阳性和假阴性变体。对于具有≥5 pg 纯化 Mtb DNA 的样本，dMDA 与 Illumina WGS 相结合，相当于 Mtb 基因组的大约 1000 个拷贝，可提供耐药性、系统发育和传播见解的准确性。
