Molzym Blog

Metagenomic Shotgun Sequencing of Respiratory Pathogens

29 November 2019

blog 13715924 s 142

Clinically diagnosed ventilator-associated pneumonia (VAP) cannot be confirmed microbiologically in up to 60% of cases. Therefore Qi et al. [1] evaluated 67 samples by metagenomic shotgun sequencing (MSS) in comparison to reference culture.

For MSS, host DNA was depleted and microbial DNA isolated from 2ml bronchoalveolar lavage samples (BAL) using the Ultra-Deep Microbiome Prep kit (Molzym, Germany). After library preparation with the Nextera XT DNA library prep kit (Illumina®, USA), the samples were loaded onto the NextSeq500 sequencer.

The paired-end 2x150 base sequencing reads resulted in a highly variable number of reads with an average of 10.9 million reads per sample, whereof approximately 22% of reads mapped to microorganisms in the reference database.

Of the 67 samples, 22 grew positive in culture and the species determined by culture were also identified with MSS. Moreover, MSS revealed additional species in 8 of 16 samples. For the 45 culture-negative samples, potential pathogens were determined in 40 samples (89%) by MSS including six unique bacterial species and one human herpesvirus 1. In three samples, Tropheryma whipplei accounted for many reads which was not considered a typical pneumonia pathogen.

The proof-of-concept study revealed that the benefit of MSS accounts for culture-negative cases. MSS of human DNA-depleted samples can be considered a powerful means of non-selective and highly sensitive detection of disease-relevant bacteria, fungi and viruses in BAL.


[1] Qi C, Pickens CO, Walter JM, Kruser JM, Singer, BD, Seed P. Green SJ, Wunderink RG (2019) Detection of respiratory pathogens in clinical samples using metagenomic shot-gun sequencing. J Med Microbiol 68, 996-1002. (link)

Brochure                       Product Information