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

## TL;DR

This paper introduces a protocol to study how bacteriophages and lipids in preterm milk might influence infant gut health.

## Contribution

A novel protocol combining metagenomics and lipidomics to analyze phageome-lipidome interactions in low-volume preterm milk.

## Key findings

- A method for phage and lipid extraction from small milk volumes is described.
- An in-house pipeline characterizes phageome data and correlates it with lipid profiles.
- An in vitro assay links fatty acid chain length to phage morphotype.

## Abstract

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

•Phage isolation and DNA extraction from low-volume milk fat and skim milk•Lipid extraction protocol for LC/MS-based discovery lipidomics•In-house bioinformatic pipeline to characterize phageome•In vitro assay to study associations of fatty acid chain length and phage morphotype

Phage isolation and DNA extraction from low-volume milk fat and skim milk

Lipid extraction protocol for LC/MS-based discovery lipidomics

In-house bioinformatic pipeline to characterize phageome

In vitro assay to study associations of fatty acid chain length and phage morphotype

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.