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Page 4 of 20 Linehan et al. Microbiome Res Rep. 2025;4:24 https://dx.doi.org/10.20517/mrr.2024.92

Table 1. Clinical characteristics of the 18 mother-infant dyads in this study
Variables
Infant characteristics Cohort (n = 18)
Gender
Male 10/18 (55.6%)
Female 8/18 (44.6%)
Delivery mode
Caesarean 8/18 (44.4%)
Vaginal 10/18 (55.6%)
Birth weight (g), mean (SD) 3667.2 (386.6)
Gestational age (in weeks), mean (SD) 39 (0.97)
AGPAR, mean (SD) 9 (1.15)
PROM
Yes 7/18 (38.9%)
No 11/18 (61.1%)
Feed type
Breast fed 10/18 (55.6%)
Formula 6/18 (33.3%)
Not noted 2/18 (11.1%)
Maternal characteristics Cohort (n = 18)
Intrapartum antibiotic
Yes 10/18 (55.6%)
No 8/18 (44.4%)
Amoxicillin (Augmentin) 1/18 (5.6%)
Flucloxacillin 1/18 (5.6%)
16/18 (88.8%)
Type of antibiotic not noted
Gravida, mean (SD) 3 (1.5)
Parity, mean (SD) 2 (1.3)
Continuous variables are presented as mean (+SD). Categorical variables are presented as number of participants (percentages). SD: Standard
deviation; APGAR: appearance, pulse, grimace, activity, and respiration; PROM: premature rupture of membranes.

16S rRNA gene amplification and MiSeq sequencing
The V3-V4 hypervariable region of the 16S rRNA gene was amplified from 90 DNA samples following the
Illumina 16S Metagenomic Sequencing Library Protocol. Negative extraction blanks and no-template
controls were included throughout. PCR amplification utilized V3-V4 specific primers [Supplementary
Table 1]. Reactions included 5 ng/μL template DNA, primers (5 μM), 12.5 μL KAPA2G Robust HotStart
ReadyMix and PCR-grade water. PCR conditions were as follows: 95 °C initial denaturation (3 min), 30
cycles (95 °C, 30 s; 55 °C, 30 s; 72 °C, 30 s), followed by 72 °C (5 min), and a 4 °C hold. Amplicons were
verified by gel electrophoresis and cleaned with AMPure XP magnetic beads. Illumina sequencing adapters
and dual-index barcodes were added in a second PCR. Samples were pooled equimolarly and verified with a
Bioanalyzer before sequencing on an Illumina MiSeq (2 × 300 cycle V3 kit).

Bioinformatics analysis
Raw sequences were assessed using MultiQC and trimmed with Cutadapt (v2.10). Quality filtering, error
[23]
correction, denoising, merging of paired reads, and chimera removal were conducted using DADA2
[24]
(v1.14) . Taxonomic classification of amplicon sequence variants (ASVs) was conducted using the SILVA
database (v138) , with genus- and species-level matches made via assignTaxonomy and assignSpecies
[25]
functions. Phyloseq (v1.24) facilitated the integration of metadata, ASV tables, phylogenetic trees, and
taxonomic assignments . The decontam package (v1.16.0) was used to identify and remove contaminants
[26]