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

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.