Laboratory contamination in airway microbiome studies

Area of Science:

• Microbiology
• Bioinformatics
• Genomics

Background:

• Studies on the airway microbiome are susceptible to contamination from bacterial DNA introduced during sample collection and laboratory procedures.
• Low bacterial loads in lung samples exacerbate the risk of contamination impacting study results.

Purpose of the Study:

• To investigate the impact of laboratory contamination on lower airway microbiome samples.
• To explore in silico methods for identifying and managing contamination post-sequencing.

Main Methods:

• Protected bronchoscopy was used to collect samples from the lower airways.
• Quantitative PCR and targeted amplicon sequencing of the bacterial 16S rRNA gene were performed.
• The Decontam R package was utilized for in silico contaminant identification.

Main Results:

• Bacterial load varied significantly by sample type (oral wash > bronchoalveolar lavage fractions > protected specimen brush).
• An estimated 10-50% of bacterial community profiles in lower airway samples were attributed to laboratory contamination.
• The DNA extraction kit (FastDNA Spin Kit) was identified as a primary source of contamination.
• The Decontam R package effectively balanced the retention and removal of taxa present in both negative controls and study samples.

Conclusions:

• Laboratory contamination levels can vary substantially between airway microbiome studies.
• Utilizing contaminant identification tools like Decontam R, based on statistical models, can reduce researcher subjectivity.
• Reporting contaminant estimates and employing bioinformatics tools can improve the accuracy of inter-study comparisons in airway microbiome research.