Assessing the bias linked to DNA recovery from biofiltration woodchips for microbial community investigation by fingerprinting.
In this study, we explored methodological aspects of nucleic acid recovery from microbial communities involved in a gas biofilter filled with pine bark woodchips. DNA was recovered indirectly in two steps, comparing different methods: cell dispersion (crushing, shaking, and sonication) and DNA extraction (three commercial kits and a laboratory protocol). The objectives were (a) to optimize cell desorption from the packing material and (b) to compare the 12 combinations of desorption and extraction methods, according to three relevant criteria: DNA yield, DNA purity, and community structure representation by denaturing gradient gel electrophoresis (DGGE). Cell dispersion was not influenced by the operational parameters tested for shaking and blending, while it increased with time for sonication. DNA extraction by the laboratory protocol provided the highest DNA yields, whereas the best DNA purity was obtained by a commercial kit designed for DNA extraction from soil. After successful PCR amplification, the 12 methods did not generate the same bias in microbial community representation. Eight combinations led to high diversity estimation, independently of the experimental procedure. Among them, six provided highly similar DGGE profiles. Two protocols generated a significantly dissimilar community profile, with less diversity. This study highlighted the crucial importance of DNA recovery bias evaluation.
Cabrol
Léa
L
Centre de Recherche sur l'Eau-Veolia Environnement, Chemin de la Digue, Maisons Laffitte, France. lea.cabrol@gmail.com
Malhautier
Luc
L
Poly
Franck
F
Lepeuple
Anne-Sophie
AS
Fanlo
Jean-Louis
JL
D044822:Biodiversity; D016000:Cluster Analysis; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification*; D016172:DNA Fingerprinting / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005374:Filtration / Q000379:methods; D056186:Metagenomics / Q000379:methods*; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D014934:Wood / Q000382:microbiology*
D004247:DNA