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Page 4 of 15 Longhi et al. Microbiome Res Rep 2024;3:4 https://dx.doi.org/10.20517/mrr.2023.02

800 µL). After the final wash, the samples were centrifuged at 6,000 g for 3 min, the supernatant discarded,
and the pellet resuspended in 600 µL of PBS. We included three cycles of bead-beating followed by three
steps on ice. For the untreated counterpart of each biological sample, we avoided the whole depletion
protocol, which means that after centrifugation, the bacterial pellet was incubated only with PBS (without
the addition of saponin) and subsequently treated with sterile water and NaCl. We proceeded with
centrifugations, washing, and incubations with PBS (without the addition of Turbo DNase), and finally
bead-beating.

DNA extraction and quantification
The DNA was extracted from the specimens using commercially available kits following the manufacturer’s
instructions. The best-performing kits based on the biological origin matrix were employed for bacterial
DNA extraction. QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) was used to extract exclusively the
bacterial DNA (avoiding other microbiome components such as fungi, archaea, and bacteriophages) from
sputum, saliva, oral and nasopharyngeal swabs and biopsies, and ZymoBIOMICS DNA Miniprep Kit
(Zymo Research, D4300) for vaginal swabs. Then, the DNA concentration and purity were investigated
employing a Picodrop microtiter Spectrophotometer (Picodrop, Hinxton, UK).

Quantitative real-time PCR for bacteria and human DNA
To evaluate the bacterial and eukaryotic abundance in samples treated or not with saponin and DNase, we
performed a qPCR approach with specific primers for bacteria and humans, i.e., Probio_uni
(5′-CCTACGGGRSGCAGCAG-3′) and Probio_rev (5′-ATTACCGCGGCTGCT-3′) for bacterial 16S
b
rRNA , G L B + ( 5 ′ - A C A C A A C T G T G T T C A C T A G C - 3 ′ ) a n d b G L B -
[24]
[25]
(5′-CAACTTCATCCACGTTCACC-3′) for the human β-globin . DNA from the different biological
samples was extracted and diluted to a concentration of 10 ng/µL. qPCR was performed using PowerUp
SYBR Green Master Mix (ThermoFisher Scientific, US) on a CFX96 system (BioRad, CA, USA) following
[26]
previously described protocols . PCR products were detected with SYBR green fluorescent dye and
amplified according to the following protocol: one cycle of 50 °C for 2 min, followed by one process of 95 °C
for 2 min, followed by 40 cycles of 95 °C for 15 s, 55-60 °C for 15 s and 72 °C for 1 min. The melting curve
was 65 to 95 °C with increments of 0.5 °C/s. In each run, negative controls (no DNA) were included. A
standard curve was generated using the CFX96 software (BioRad), and chromosomal DNA belonging to
untreated biological matrices and Bifidobacterium longum were used as qPCR standards for the eukaryotic
and bacterial detection, respectively.

Shallow shotgun sequencing
According to the manufacturer’s instructions, DNA library preparation was performed using the Nextera
XT DNA sample preparation kit (Illumina, San Diego, CA, USA). First, one ng input DNA from each
sample was used for the library preparation, which underwent fragmentation, adapter ligation, and
amplification. Then, Illumina libraries were pooled equimolarly, denatured, and diluted to a concentration
of 1.5 pM. Next, DNA sequencing was performed on a MiSeq instrument (Illumina) using a 2X 250 bp
Output sequencing Kit and a deliberate spike-in of 1% PhiX control library.

Short read taxonomic classification
Sequenced paired-end reads of each sample were subjected to a filtering step removing low-quality reads
(minimum mean quality score 20, window size 5, quality threshold 25, and minimum length 100) using the
fastq-mcf script (https://github.com/ExpressionAnalysis/ea-utils/blob/wiki/FastqMcf.md) to analyze high-
quality sequenced data only. Then, employing the BWA aligner, an additional filtering step was performed
to remove the reads mapping against the Homo sapiens genome sequence, thus removing possible
contaminating human DNA, which was not removed by the previously described depletion protocol (see