title: Impact of sample processing on human airways microbial metagenomes.
abstract: 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 24h 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 24h 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.
authors.affiliation: Clinical Research Group, OE 6711, Medizinische Hochschule Hannover, D-30625 Hannover, Germany; Core Unit 'Next Generation Sequencing', Medizinische Hochschule Hannover, D-30625 Hannover, Germany; Institute for Human Genetics, Medizinische Hochschule Hannover, D-30625 Hannover, Germany. Electronic address: Wiehlmann.Lutz@mh-hannover.de.
authors.forename: Lutz
authors.initials: L
authors.lastname: Wiehlmann
authors.affiliation: Clinical Research Group, OE 6711, Medizinische Hochschule Hannover, D-30625 Hannover, Germany. Electronic address: Pienkowska.Katarzyna@mh-hannover.de.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Pienkowska
authors.affiliation: Clinical Research Group, OE 6711, Medizinische Hochschule Hannover, D-30625 Hannover, Germany. Electronic address: Jansen.Silke@mh-hannover.de.
authors.forename: Silke
authors.initials: S
authors.lastname: Hedtfeld
authors.affiliation: Clinical Research Group, OE 6711, Medizinische Hochschule Hannover, D-30625 Hannover, Germany; Core Unit 'Next Generation Sequencing', Medizinische Hochschule Hannover, D-30625 Hannover, Germany. Electronic address: Boehm.Marie@mh-hannover.de.
authors.forename: Marie
authors.initials: M
authors.lastname: Dorda
authors.affiliation: Clinical Research Group, OE 6711, Medizinische Hochschule Hannover, D-30625 Hannover, Germany; Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research, D-30625 Hannover, Germany. Electronic address: Tuemmler.Burkhard@mh-hannover.de.
authors.forename: Burkhard
authors.initials: B
authors.lastname: Tümmler
mesh_terms: D016477:Artifacts; D015373:Bacterial Typing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D008168:Lung / Q000382:microbiology*; D054892:Metagenome / Q000235:genetics*; D059013:Microbial Consortia / Q000235:genetics*; D015203:Reproducibility of Results; D018401:Sample Size; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*
chemical_list: D004247:DNA
keywords: Airways; Metagenome; Microbiome; Next generation sequencing