Apr 10, 2018

A Method for Targeted 16S Sequencing of Human Milk Samples

Journal of Visualized Experiments : JoVE
Nicole H TobinGrace M Aldrovandi

Abstract

Studies of microbial communities have become widespread with the development of relatively inexpensive, rapid, and high throughput sequencing. However, as with all these technologies, reproducible results depend on a laboratory workflow that incorporates appropriate precautions and controls. This is particularly important with low-biomass samples where contaminating bacterial DNA can generate misleading results. This article details a semi-automated workflow to identify microbes from human breast milk samples using targeted sequencing of the 16S ribosomal RNA (rRNA) V4 region on a low- to mid-throughput scale. The protocol describes sample preparation from whole milk including: sample lysis, nucleic acid extraction, amplification of the V4 region of the 16S rRNA gene, and library preparation with quality control measures. Importantly, the protocol and discussion consider issues that are salient to the preparation and analysis of low-biomass samples including appropriate positive and negative controls, PCR inhibitor removal, sample contamination by environmental, reagent, or experimental sources, and experimental best practices designed to ensure reproducibility. While the protocol as described is specific to human milk samples,...Continue Reading

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Mentioned in this Paper

Pathologic Cytolysis
Buffers
Study
Genes
Environment
Inhibitors
Nucleic Acid Sequencing
Human breast milk
High Throughput Technology
Laboratory

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