NGS Analysis of Pathogens in Broncho-Alveolar-Lavage

The partial removal of host DNA from a broncho-alveolar lavage (BAL) sample using Molzym’s Ultra-Deep Microbiome Prep isolation protocol prior to whole-metagenome shotgun sequencing (WMGS) increases detection sensitivity of bacterial and fungal species and the sequence coverage of the bacteria reported by clinical microbiology tests while decreasing that for viruses.

These findings are shown by Leo et al.  [1] in a study evaluating the effect of microbial enrichment during DNA preparation. DNA was isolated from BAL samples with either mechanical lysis (NucleoSpin Soil Kit, Macherey-Nagel, Germany) or selective enrichment of bacterial and fungal DNA (Ultra-Deep Microbiome Prep kit, Molzym, Germany). Paired-end sequencing was performed on Illumina HiSeq platforms. Compared to total DNA, extraction sequential lysis resulted in a significant increased (about 230-fold and 310-fold) proportion of bacterial DNA over human DNA. Consistently, the percentage of reads mapped to bacterial genomes from enriched samples reached 23.7% and 26.44%, while being <0.1% in unenriched sample dataset.

WMGS allows the identification of microorganisms that were not searched for in routine tests, such as anaerobic bacteria. Moreover, it is complementary with routine diagnostic tests based on culture and molecular approaches for bacterial and fungal identification, providing deeper insights in the composition of the microbiota in clinical samples.

Reduced sensitivity in identifying microorganisms due to the high proportion of human DNA fragments relative to microbial DNA fragments in clinical samples is one of the main reasons why WMGS is not used in clinical routine analyses yet. Leo et al. [1] showed that a solution could be Molzym’s Ultra-Deep Microbiome Prep kit.