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Whole genome sequencing analysis of M. tuberculosis from direct specimens: a hybrid capture approach

G SK Morgan(1) F Di Marco(1) K Moghaddasi(1) V Batignani(1) A M Cabibbe(1) D M Cirillo(1)

1:San Raffael Scientific Institute

Targeted Next Generation Sequencing (tNGS) approaches efficiently interrogate M. tuberculosis drug resistance in respiratory samples, matching modern treatment regimens, but are limited by pre-selection of target genes and drugs. In addressing these issues, the Qiagen QIAseq xHYB assay on MTB is developed which employs a hybrid capture panel able to enrich the whole MTB genome sequence from indexed libraries.

Sixty-two DNA samples from sputum sediments, previously analysed with a tNGS-based Deeplex MycTB assay, were resequenced using the QIAseq xHYB library preparation assay on an Illumina Nextseq 500 instrument. Briefly, the genomic DNA was fragmented, amplified, and pooled for hybrid capture, then hybridized to M. tuberculosis probes, further amplified, and pooled for sequencing. Obtained Fastqs were analysed using the Kraken2 + Bracken for contamination check and MTBseq for variant calling adopting H37Rv genome as reference.

The analysis showed consistency between mean depth coverage of the QIAseq genome and Xpert MTB/RIF Ct values: 101.35x (Xpert High, Ct < 16, 35 samples), 32.92x (Medium, Ct 16-22, 21 samples), 7.28x (Low, Ct 22-28, 5 samples) and 21.34x (Very low, Ct > 28, 1 sample). The 70% of study samples achieved full coverage of genome breadth suitable for extended analyses such as strain relatedness. We also obtained 100% concordance in drug-resistance–associated target calls when compared with the Deeplex MycTB assay.

The Qiagen assay permits direct MTB sequencing without culturing delay and at full genome resolution. This salient aspect ensures a comprehensive and accurate detection of genome variants supporting outbreak investigation beyond drug resistance detection.

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