Impact of DNA extraction methods and culture-independent approaches oncanine lung mycobiota analysis

peer reviewed / There is growing evidence, at least in humans, that fungi play a role in lung health preservation and in disease development and progression. Our understanding of fungal implication in such conditions relies on accurate and reproducible data acquisition. One of the critical steps in mycobiota analysis concerns DNA extraction as fungi are protected by complex cell wall that resists to classical lysis protocol. There is also a need to limit biases introduced by contaminant DNA, susceptible to result in a wrong mycobiota representation. This concern is of particu- lar importance in healthy lungs where fungi are rare.In this study, we compared 2 protocols of DNA extraction and 2 sequencing approaches to analyze the lung mycobiota (LMy) of 8 healthy dogs. DNA from bronchoalveolar lavage fluid samples were extracted using either the DNeasy Blood and Tissue kit with the pre- treatment for Gram-positive bacteria preceded by a mechanical lysis on FastPrep-24 (Protocol A), or the QIAsymphony DSP DNA Midi kit preceded by a mechanical lysis on TissueLyser and an enzymatic lysis (Protocol B). DNA were then analyzed by amplicon sequencing target- ing the internal transcribed spacer (ITS) 2. DNA extracted with proto- col B were also analyzed by shotgun metagenomics analysis (MetaMIC). Except for the step of DNA extraction, sequencing and data analysis were performed for all samples at the same time and in the same laboratory. Comparison between extraction protocols using ITS amplicon profiling revealed that β -diversity was significantly different (P = 0.013) with a greater inverse Simpson index in protocol A compared to B (median [interquartile range]: 6.2 [5.4 – 7.6] versus 1.8 [1.6 – 2.3]; P = 0.008, respectively). Only 2 phyla, Ascomycota and Basidiomycota, were found with protocol B versus 6 with protocol A. In only 2 samples, a similar predominant genus (Malassezia) was identified with the 2 proto- cols. Shotgun analysis resulted in a small number of fungal DNA frag- ment identification. It might partly be due to bioinformatics techniques used to process sequences that were designed for human samples. No real correlation was found between ITS amplicon profil- ing and shotgun results. In conclusion, the DNA extraction protocol and the techniques used to sequence DNA and process sequences have a great impact on LMy determination. Accordingly, LMy comparison between studies using different extraction and sequencing techniques is not recommended. The use of bioinformatic tools design for dogs is warranted. The rarity of the LMy of healthy dogs may explain the difficulty in obtaining accurate and reproducible data.