Impact of sample processing on human airways microbial metagenomes

- Sufficient DNA is collected by throat swabs for airway metagenome analysis.
- One day storage in transport medium distorts the metagenome profile.
- Specimens for metagenome analysis should be immediately processed or frozen.
- Process metagenome samples with less than 10 ng DNA by sonication.
- Check the microbial metagenome of chemicals and devices used during sample processing.

Whole metagenome shotgun sequencing provides information about the gene content and the composition of microbial communities provided that the processing of the samples does not introduce a methodology-driven bias. We tested the impact of DNA isolation and storage period on the metagenome profile. Deep throat swabs were collected from healthy adults and an infected infant. DNA was isolated by sonification or enzymatic lysis either immediately or after 24 h storage in agar gel Amies transport medium at room temperature. Disruption of cells and subsequent fragmentation of DNA by sonification was as suitable as the common enzymatic lysis to generate high-quality metagenomes particularly for low total DNA input of less than ten nanograms. Conversely, storage of samples for 24 h produced severely distorted metagenomes. The majority of species became less abundant or even extinct, whereas a few Streptococcus, Neisseria and Haemophilus spp. proliferated so that the total number of bacterial reads increased at the expense of human reads. We recommend that samples for metagenome analysis should be immediately processed or frozen at −80 °C.