Methods and Strategies to Examine the Human Breastmilk Microbiome

: It has recently been discovered that breastmilk is not sterile, but contains a vast array of microbes, known collectively as the breastmilk microbiome. The breastmilk microbiome field is in its infancy, but over the last decade, our understanding of the microbial communities that inhabit the human body has increased exponentially, due in large part to novel next-generation sequencing technologies. These culture-independent, high-throughput molecular technologies have allowed biologists to investigate the entirety of microbiota present in breastmilk, which was previously poorly known. These approaches are novel and the methodologies surrounding the exploration of the breastmilk microbiota remain in flux. The objectives of this chapter are to outline what is known thus far and detail the optimal methods and strategies to conducting a breastmilk microbiome study from subject recruitment and milk collection to DNA extraction, high-throughput sequencing and bioinformatics analyses.