585 Specificities of Microbiota From Sensitive Skin
Twenty microliter reactions were performed in triplicate with the following reactants: 10 µL
PrimeTime™ Gene Expression Master Mix, 0.8 µL of forward primer, 0.8 µL of reverse
primer, 0.4 µL of probe, 6 µL of nuclease-free water, and 2 µL of DNA template. A reaction
was performed with a blank extraction as a DNA template, and another was performed
with nuclease-free water as a DNA template. The reactions were performed in white, low
profile 96-well plates, sealed with thermoresistant optical films. The qPCR was performed
in Agilent Aria Mx machine with the following cycling conditions: initial denaturation
95°C—3 minutes—1 cycle denaturation 95°C—5 seconds annealing/extension 60°C—
40 cycles final extension 60°C for 5 minutes and 1 cycle (Agilent, Les Ulis, France).
PACBIO LIBRARY PREPARATION
The primers used to amplify the full-length 16S ribosomal ribonucleic acid (rRNA) gene
were composed of the specific regions 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and
1492R (5′-CGGTTACCTTGTTACGACTT-3′) combined to asymmetrical barcodes. PCRs
were performed with the following solution mix: 10 µL of Q5 reaction buffer, 1 µL of
10 mM deoxinucleotides triphosphates (dNTPs), 2 µL of forward primer at 10 µM, 2 µL
of reverse primer at 10 µM, 2 µL of DNA template at 10-2 ng/µL, 0.5 µL of Q5 high-
fidelity DNA polymerase, 32.5 µL of nuclease-free water. The cycling conditions were as
follows: initial denaturation 95°C—during 30 seconds for 1 cycle denaturation at 95°C for
5 seconds during 30 cycles annealing at 59°C during 30 cycles extension at 72°C for 45
seconds and final extension at 72°C for 2 minutes during 1 cycle.
Amplifications were checked by electrophoresis on 1% v/v agarose gel for a 30 minute
migration, using 100 mV to assess the success of PCR reactions. Nucleic acid fragments
were separated by their length while moving through the agarose matrix then visualized
under ultraviolet light after adding intercalating agent like ethidium bromide (a band
was expected around 1,500 base pairs). Samples were then purified using AMPure™ PB
beads (PacBio, SanDiego, CA, USA) following manufacturer recommendations. The entire
PCR volume was mixed in equal ratio with AMPure™ XP beads, and two washes were
performed with 80% ethanol. Finally, DNA was eluted in 25 µL of nuclease-free water.
DNA concentration was measured using Qubit™ dsDNA HS assay kit (Thermofisher
Scientific) following manufacturer recommendations. Three µL of eluted DNA was used
for quantification. Samples were pooled in equal amounts to reach a final DNA mass of
1,000 ng. In total, 3 DNA pools were generated to ensure sufficient sample coverage. Pools
were then kept at −20°C until completion of library preparation.
Pools were sent to Maryland Genomics (Baltimore, United States) to perform a PacBio short-
insert library preparation, which included DNA damage repair, end repair/A-tail, ligation,
and AMPure™ PB bead purifications. Each pool was sequenced in a PacBio Sequel II 8M
run, 30-hour movie, to generate circular consensus sequences. Samples were demultiplexed
and exported in FASTQ format. FASTQ format is a text-based format for storing both a
biological sequence (usually nucleotide sequence) and its corresponding quality scores.
MOCK COMMUNITY PREPARATION
To assure the quality of the sequencing and bioinformatic workflows, as a positive control
a mock community composed of 14 wild species previously isolated from the skin was

586 JOURNAL OF COSMETIC SCIENCE
built. The community includes the following species: Cutibacterium: C. acnes, C. avidum,
C. namnetense, C. Granulosum, Staphylococcus: S. epidermidis, S. capitis, S. aureus, S. Hominis,
Corynebacterium: C. tuberculostearicum, C. kroppenstedtii Micrococcus luteus Kocuria palustris
and rhizophila and Bacillus thuringiensis. Species were plated in BHI agar supplemented with
0.1% of Tween80 at 37°C in aerobic (Staphylococcus, Micrococcus, Corynebacterium, and Kocuria
species), or anaerobic conditions (Cutibacterium species). A single colony was picked from
each plate and grown in BHI, Thermo Fischer Scientific) liquid medium for all bacteria
except Corynebacterium species that were cultivated in a specific liquid medium (confidential
composition). Fifty µL of each culture was pooled in a single tube. The bacterial density of
each culture was assessed as colony forming units per volume unit (CFU/mL) after growth
in agar medium under the appropriate conditions (as described above). The pool of cultures
was subjected to two DNA extractions using Zymo BIOMICS™ DNA miniprep (Zymo
Research). These two DNA solutions from the same mock community were prepared for
sequencing using the same protocol as for the skin samples. Two amplification replicates
were performed for each DNA solution.
BIOINFORMATIC ANALYSIS
Samples were analyzed with the bioinformatics pipeline DADA2,18 which consists of a series
of steps to filter the raw sequences obtained through Illumina sequencing and a final step
to obtain the taxonomy of the sequences that have been filtered for microbial community
study. A metadata sheet was used to gather all information specific to samples, such as
participant characteristics (age, sex, skin phenotype), skin sites, and the corresponding
sequencing run. Sequences were filtered based on size, quality, and presence of primers.
The error rate was determined by gathering samples sequenced in the same run, and
the subsequent denoising was performed with the pseudo-pooling method. Taxonomic
assignment was achieved with the database Silva 138.1 (online resource for quality checked
and aligned ribosomal RNA sequence data) at the species level. If the algorithm did not
manage to provide a classification at the species level, the species annotation was built with
the corresponding genus combined to the mention spp (species plural, e.g. Staphylococcus
spp.). Amplicon sequence variants (ASVs) that were not assigned to any genera were excluded
from the analysis. ASVs classified as mitochondria and chloroplast at the family and class
level, respectively, were also discarded from the analysis. To make the data manipulation
easier and to exclude potential sequencing artifacts, ASVs having less than 10 reads in
the overall study or present in a single sample were also discarded. A cutoff of 5,000
reads was selected to avoid the loss of rare species. Rarefaction curves were generated to
ensure sufficient sequencing depth with the vegan package from the R program. The vegan
package provides tools for descriptive community ecology. It has the most basic functions
of diversity analysis, community ordination, and dissimilarity analysis.
NORMALIZATION OF THE SEQUENCING DATA
Sequencing data were treated according to four different workflows to check the impact of
normalization methods. Total Sum Scaling (TSS) was achieved with the function transform_
sample_counts and rarefied counts, with and without replacement, were achieved with the
rarefy_even_depth function. Both functions are from the phyloseq package. Phyloseq is