A Method for Targeted 16S Sequencing of Human Milk Samples

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• High-throughput sequencing enables widespread microbial community studies.
• Reproducible results require robust laboratory workflows, particularly for low-biomass samples prone to contamination.

Purpose of the Study:

• To detail a semi-automated workflow for microbial identification in human breast milk.
• To address challenges in low-biomass sample analysis, ensuring data accuracy and reproducibility.

Main Methods:

• Targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region.
• Sample preparation including lysis, nucleic acid extraction, amplification, and library preparation.
• Incorporation of quality control measures, positive/negative controls, and contamination mitigation strategies.

Main Results:

• A detailed protocol for processing human breast milk samples for microbial identification.
• Consideration of critical factors for low-biomass samples, such as PCR inhibitor removal and contamination control.
• Demonstration of a workflow adaptable to various sample types.

Conclusions:

• The described workflow enhances the reliability of microbial identification in human breast milk.
• The protocol provides essential guidance for reproducible low-biomass sample analysis.
• This method is adaptable for diverse sample types, broadening its applicability in microbial research.