Abstract
BackgroundThe low bacterial load in samples acquired from the lungs, have made studies on the airway microbiome vulnerable to contamination from bacterial DNA introduced during sampling and laboratory processing. We have examined the impact of laboratory contamination on samples collected from the lower airways by protected (through a sterile catheter) bronchoscopy and explored various in silico approaches to dealing with the contamination post-sequencing. Our analyses included quantitative PCR and targeted amplicon sequencing of the bacterial 16S rRNA gene.ResultsThe mean bacterial load varied by sample type for the 23 study subjects (oral wash>1st fraction of protected bronchoalveolar lavage>protected specimen brush>2nd fraction of protected bronchoalveolar lavage; p < 0.001). By comparison to a dilution series of know bacterial composition and load, an estimated 10–50% of the bacterial community profiles for lower airway samples could be traced back to contaminating bacterial DNA introduced from the laboratory. We determined the main source of laboratory contaminants to be the DNA extraction kit (FastDNA Spin Kit). The removal of contaminants identified using tools within the Decontam R package appeared to provide a balance between keeping and removing taxa found in both negative controls and study samples.ConclusionsThe influence of laboratory contamination will vary across airway microbiome studies. By reporting estimates of contaminant levels and taking use of contaminant identification tools (e.g. the Decontam R package) based on statistical models that limit the subjectivity of the researcher, the accuracy of inter-study comparisons can be improved.
Highlights
The low bacterial load in samples acquired from the lungs, have made studies on the airway microbiome vulnerable to contamination from bacterial DNA introduced during sampling and laboratory processing
In order to establish the bacterial load in protected airway samples collected using different sampling techniques, we included oral washes (OW), two fractions of protected bronchoalveolar lavage (PBAL1 and PBAL2) and protected specimen brushes (PSB) from 23 participants of the MicroCOPD study [12]
Measured amounts of bacteria will vary in lower airway samples collected with different bronchoscopic sampling techniques (e.g. PBAL1, PBAL2, PSB in the current study)
Summary
The low bacterial load in samples acquired from the lungs, have made studies on the airway microbiome vulnerable to contamination from bacterial DNA introduced during sampling and laboratory processing. The most common method used for studying the bacterial communities of the lower respiratory tract is high throughput amplicon sequencing of the bacterial 16S ribosomal RNA (16S rRNA) marker gene [1]. Accurate analysis of the lower respiratory tract microbiome will require separate consideration of both of the aforementioned contamination sources - that from the upper respiratory tract introduced during sampling and that introduced during laboratory processing steps. The impact of laboratory contamination is typically evaluated through the inclusion of negative control samples (NCS) that are processed through all steps of DNA extraction and library preparation for sequencing alongside the study samples. Bioinformatic tools are being developed that aim to wriggle out the authentic microbiota signal using statistical models [9,10,11], but these have yet to be tested on lower respiratory tract sequencing data (e.g. Decontam [9])
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