Protocol to study the inter-relationship between phageome and lipidome in low-volume preterm milk

Bacteriophages and lipids in human milk may benefit preterm infant health by modulating gut microbiomes. Here, we present a protocol for analyzing the phageome and lipidome in preterm milk using shotgun metagenomics and untargeted lipidomics approaches, respectively. We describe steps for extracting phages and lipids in low-volume milk, characterizing phageome using an in-house bioinformatic pipeline, and statistical analysis to correlate the phageome and lipidome. Finally, we detail an in vitro assay to examine the associations between fatty acid chain length and phage morphotype. For complete details on the use and execution of this protocol, please refer to Yew et al.¹.