TY - JOUR
T1 - Analysis of microbial composition and sharing in low-biomass human milk samples
T2 - a comparison of DNA isolation and sequencing techniques
AU - Spreckels, Johanne E
AU - Fernández-Pato, Asier
AU - Kruk, Marloes
AU - Kurilshikov, Alexander
AU - Garmaeva, Sanzhima
AU - Sinha, Trishla
AU - Ghosh, Hiren
AU - Harmsen, Hermie
AU - Fu, Jingyuan
AU - Gacesa, Ranko
AU - Zhernakova, Alexandra
N1 - © 2023. The Author(s).
PY - 2023/12
Y1 - 2023/12
N2 - Human milk microbiome studies are currently hindered by low milk bacterial/human cell ratios and often rely on 16S rRNA gene sequencing, which limits downstream analyses. Here, we aimed to find a method to study milk bacteria and assess bacterial sharing between maternal and infant microbiota. We tested four DNA isolation methods, two bacterial enrichment methods and three sequencing methods on mock communities, milk samples and negative controls. Of the four DNA isolation kits, the DNeasy PowerSoil Pro (PS) and MagMAX Total Nucleic Acid Isolation (MX) kits provided consistent 16S rRNA gene sequencing results with low contamination. Neither enrichment method substantially decreased the human metagenomic sequencing read-depth. Long-read 16S-ITS-23S rRNA gene sequencing biased the mock community composition but provided consistent results for milk samples, with little contamination. In contrast to 16S rRNA gene sequencing, 16S-ITS-23S rRNA gene sequencing of milk, infant oral, infant faecal and maternal faecal DNA from 14 mother-infant pairs provided sufficient resolution to detect significantly more frequent sharing of bacteria between related pairs compared to unrelated pairs. In conclusion, PS or MX kit-DNA isolation followed by 16S rRNA gene sequencing reliably characterises human milk microbiota, and 16S-ITS-23S rRNA gene sequencing enables studies of bacterial transmission in low-biomass samples.
AB - Human milk microbiome studies are currently hindered by low milk bacterial/human cell ratios and often rely on 16S rRNA gene sequencing, which limits downstream analyses. Here, we aimed to find a method to study milk bacteria and assess bacterial sharing between maternal and infant microbiota. We tested four DNA isolation methods, two bacterial enrichment methods and three sequencing methods on mock communities, milk samples and negative controls. Of the four DNA isolation kits, the DNeasy PowerSoil Pro (PS) and MagMAX Total Nucleic Acid Isolation (MX) kits provided consistent 16S rRNA gene sequencing results with low contamination. Neither enrichment method substantially decreased the human metagenomic sequencing read-depth. Long-read 16S-ITS-23S rRNA gene sequencing biased the mock community composition but provided consistent results for milk samples, with little contamination. In contrast to 16S rRNA gene sequencing, 16S-ITS-23S rRNA gene sequencing of milk, infant oral, infant faecal and maternal faecal DNA from 14 mother-infant pairs provided sufficient resolution to detect significantly more frequent sharing of bacteria between related pairs compared to unrelated pairs. In conclusion, PS or MX kit-DNA isolation followed by 16S rRNA gene sequencing reliably characterises human milk microbiota, and 16S-ITS-23S rRNA gene sequencing enables studies of bacterial transmission in low-biomass samples.
U2 - 10.1038/s43705-023-00325-6
DO - 10.1038/s43705-023-00325-6
M3 - Article
C2 - 37945978
SN - 2730-6151
VL - 3
JO - ISME Communications
JF - ISME Communications
IS - 1
M1 - 116
ER -