Laboratory contamination in airway microbiome studies

Abstract Background Studies on the airway microbiome have been performed using a wide range of laboratory protocols for high-throughput sequencing of the bacterial 16S ribosomal RNA (16S rRNA) gene. We sought to determine the impact of number of polymerase chain reaction (PCR) steps (1- or 2- steps) and choice of target marker gene region (V3 V4 and V4) on the presentation of the upper and lower airway microbiome. Our analyses included lllumina MiSeq sequencing following three setups: Setup 1 (2-step PCR; V3 V4 region), Setup 2 (2-step PCR; V4 region), Setup 3 (1-step PCR; V4 region). Samples included oral wash, protected specimen brushes and protected bronchoalveolar lavage (healthy and obstructive lung disease), and negative controls. Results The number of sequences and amplicon sequence variants (ASV) decreased in order setup1 > setup2 > setup3. This trend appeared to be associated with an increased taxonomic resolution when sequencing the V3 V4 region (setup 1) and an increased number of small ASVs in setups 1 and 2. The latter was considered a result of contamination in the two-step PCR protocols as well as sequencing across multiple runs (setup 1). Although genera Streptococcus, Prevotella, Veillonella and Rothia dominated, differences in relative abundance were observed across all setups. Analyses of beta-diversity revealed that while oral wash samples (high biomass) clustered together regardless of number of PCR steps, samples from the lungs (low biomass) separated. The removal of contaminants identified using the Decontam package in R, did not resolve differences in results between sequencing setups. Conclusions Differences in number of PCR steps will have an impact of final bacterial community descriptions, and more so for samples of low bacterial load. Our findings could not be explained by differences in contamination levels alone, and more research is needed to understand how variations in PCR-setups and reagents may be contributing to the observed protocol bias.

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DNA extraction from concrete v2

10.17504/protocols.io.b2i5qcg6 ◽

2021 ◽

Author(s):

Anders Kiledal ◽

Julia A Maresca

Keyword(s):

Dna Extraction ◽

Dental Plaque ◽

Illumina Miseq ◽

Amplicon Sequencing ◽

Metagenomic Sequencing ◽

Sample Analysis ◽

Larger Sample ◽

Laboratory Contamination ◽

Biomass Sample ◽

Over Time

This is a protocol for extracting DNA from concrete, based on the protocol developed by L. S. Weyrich, et al. for extraction of DNA from ancient calcified dental plaque. We have scaled it up for larger sample sizes and made some additional modifications for the chemistry of concrete. DNA extracted using this method is suitable for metagenomic sequencing by Illumina MiSeq and NextSeq, as well as amplicon sequencing. This protocol should yield 10 ng to 5 μg DNA per 10 g of concrete, depending on the age and integrity of the sample. Reference: L. S. Weyrich et al., Laboratory contamination over time during low-biomass sample analysis. Mol. Ecol. Resour. 19, 982–996 (2019).

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Airway microbiome is associated with respiratory functions and responses to ambient particulate matter exposure

Ecotoxicology and Environmental Safety ◽

10.1016/j.ecoenv.2018.09.079 ◽

2019 ◽

Vol 167 ◽

pp. 269-277 ◽

Cited By ~ 9

Author(s):

Liping Wang ◽

Haoxiang Cheng ◽

Dongbin Wang ◽

Bo Zhao ◽

Jushan Zhang ◽

...

Keyword(s):

Particulate Matter ◽

Ambient Particulate Matter ◽

Airway Microbiome ◽

Particulate Matter Exposure

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Environmental shaping of the bacterial and fungal community in infant bed dust and correlations with the airway microbiota

10.21203/rs.3.rs-21642/v1 ◽

2020 ◽

Author(s):

Shashank Gupta ◽

Mathis H. Hjelmsø ◽

Jenni Lehtimäki ◽

Xuanji Li ◽

Martin Steen Mortensen ◽

...

Keyword(s):

Environmental Factors ◽

Fungal Community ◽

Fungal Diversity ◽

Environmental Exposures ◽

Amplicon Sequencing ◽

Living Environment ◽

Crucial Importance ◽

Healthy Development ◽

Airway Microbiome ◽

Sampling Season

Abstract Background From early life children are exposed to a multitude of environmental exposures, which may be of crucial importance for a healthy development. Here, the environmental microbiota may be of particular interest as it represents the interface between environmental factors and the child. As infants in modern societies spend a considerable amount of time indoors, we hypothesize that the indoor bed dust microbiota might be an important factor for the child and for the colonization of the early airway microbiome. To explore this hypothesis, we analysed the influence of environmental exposures on 577 dust samples from children’s beds (age 6 months) together with 542 airway samples (age 3 months) from the Copenhagen Prospective Studies on Asthma in Childhood 2010 cohort. Results The bed dust and airway microbiota were both profiled with bacterial amplicon sequencing while also fungal community was profiled from bed dust. Bacterial and fungal diversity in the dust was positively correlated. We observed that bacterial bed dust microbiota was influenced by multiple environmental factors, such as type of home (house or apartment), rural or urban living environment and pets (cat and/or dog), whereas fungal bed dust microbiota was majorly influenced by the sampling season. We further observed minor associations between bed dust and airway microbiota compositions among infants, but no evidence of transfer of individual taxa between the two departments. Conclusions Our finding demonstrate that bed dust microbiota is influenced by environmental exposures and could represent an interface between environment and child.

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Airway microbiome clusters and heterogeneous interactions of microbial communities in adult asthma

World Allergy Organization Journal ◽

10.1016/j.waojou.2020.100197 ◽

2020 ◽

Vol 13 (8) ◽

pp. 100197

Author(s):

Hye-Ryun Kang ◽

Young-Chan Kim ◽

Kyoung-Hee Sohn ◽

Suh-Young Lee ◽

Min-Suk Yang ◽

...

Keyword(s):

Microbial Communities ◽

Adult Asthma ◽

Airway Microbiome ◽

Heterogeneous Interactions

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Nutritional immunity: the impact of metals on lung immune cells and the airway microbiome during chronic respiratory disease

Respiratory Research ◽

10.1186/s12931-021-01722-y ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Claire Healy ◽

Natalia Munoz-Wolf ◽

Janné Strydom ◽

Lynne Faherty ◽

Niamh C. Williams ◽

...

Keyword(s):

Immune System ◽

Trace Metals ◽

Lung Disease ◽

Chronic Lung Disease ◽

Immune Cells ◽

Immune Cell ◽

Redox Activity ◽

Nutritional Immunity ◽

Airway Microbiome ◽

The Impact

AbstractNutritional immunity is the sequestration of bioavailable trace metals such as iron, zinc and copper by the host to limit pathogenicity by invading microorganisms. As one of the most conserved activities of the innate immune system, limiting the availability of free trace metals by cells of the immune system serves not only to conceal these vital nutrients from invading bacteria but also operates to tightly regulate host immune cell responses and function. In the setting of chronic lung disease, the regulation of trace metals by the host is often disrupted, leading to the altered availability of these nutrients to commensal and invading opportunistic pathogenic microbes. Similarly, alterations in the uptake, secretion, turnover and redox activity of these vitally important metals has significant repercussions for immune cell function including the response to and resolution of infection. This review will discuss the intricate role of nutritional immunity in host immune cells of the lung and how changes in this fundamental process as a result of chronic lung disease may alter the airway microbiome, disease progression and the response to infection.

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