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J Microbiol Methods. 2012 Nov;91(2):231-9. doi: 10.1016/j.mimet.2012.08.016. Epub 2012 Sep 4.

Development of a standardized approach for environmental microbiota investigations related to asthma development in children.

Author information

1
Division of Gastroenterology, University of California, San Francisco, CA 94143, United States.

Abstract

Standardized studies examining environmental microbial exposure in populations at risk for asthma are necessary to improve our understanding of the role this factor plays in disease development. Here we describe studies aimed at developing guidelines for high-resolution culture-independent microbiome profiling, using a phylogenetic microarray (PhyloChip), of house dust samples in a cohort collected as part of the NIH-funded Inner City Asthma Consortium (ICAC). We demonstrate that though extracted DNA concentrations varied across dust samples, the majority produced sufficient 16S rRNA to be profiled by the array. Comparison of array and 454-pyrosequencing performed in parallel on a subset of samples, illustrated that increasingly deeper sequencing efforts validated greater numbers of array-detected taxa. Community composition agreement across samples exhibited a hierarchy in concordance, with the highest level of agreement in replicate array profiles followed by samples collected from adjacent 1×1 m(2) sites in the same room, adjacent sites with different sized sampling quadrants (1×1 and 2×2 m(2)), different sites within homes (living and bedroom) to lowest in living room samples collected from different homes. The guidelines for sample collection and processing in this pilot study extend beyond PhyloChip based studies of house-associated microbiota, and bear relevance for other microbiome profiling approaches such as next-generation sequencing.

PMID:
22975469
PMCID:
PMC3615718
DOI:
10.1016/j.mimet.2012.08.016
[Indexed for MEDLINE]
Free PMC Article

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