Laboratory contamination in airway microbiome studies

Christine Drengenes,Per Bakke,Harald Wiker,Ingvild Haaland,Sverre Lehmann,Øistein Svanes,Tomas Eagan,Gunnar Husebø,Rune Grønseth,Eli Nordeide

doi:10.1183/13993003.congress-2016.pa3984

Christine Drengenes, Per Bakke + Show 8 more

Open Access

https://doi.org/10.1183/13993003.congress-2016.pa3984

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Publication Date: Sep 1, 2016
Citations: 1	License type: other-oa

Affiliation: Haukeland University Hospital, University of Bergen

Abstract

Background: Several ongoing studies on the low-biomass airway microbiome may be hampered with an hithereto unestimated contamination by laboratory processing. Methods: We collected negative control samples (NCS), oral wash (OW), small volume lavage, protected specimen brushes and bronchoalveolar lavage (PSB, PBAL) in 16 subjects with COPD and 8 controls. We sequenced the V3-V4 region of the 16S rRNA on an Illumina MiSeq. Bioinformatic analyses were performed with QIIME. We estimated operational taxonomic units (OTUs) seen in NCS and compared the effect of keeping (1) or removing (2) NCS OTUs from the airway samples. Results: We were left with 10.5 and 2.5 million sequences and 803 and 777 OTUs using approach (1) and (2), respectively. Ralstonia dominated airway samples in approach (1). whereas the taxonomic distribution was more heterogeneous in approach (2) (Figure1). The impact of keeping NCS contaminants was also evident in principle coordinate analyses of β-diversity (Figure 2). Conclusion: Contamination from sample handling and sequencing steps is likely to influence the composition of airway microbiota, and needs to be addressed in future studies.

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

Introduction

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])

Methods

Results

Discussion

Conclusion