A Method for Targeted 16S Sequencing of Human Milk Samples

Summary

A semi-automated workflow is presented for targeted sequencing of 16S rRNA from human milk and other low-biomass sample types.

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, it is adaptable to numerous low- and high-biomass sample types, including samples collected on swabs, frozen neat, or stabilized in a preservation buffer.

Introduction

The microbial communities that colonize humans are believed to be critically important to human health and disease influencing metabolism, immune development, susceptibility to disease, and responses to vaccination and drug therapy^1,2. Efforts to understand the influence of the microbiota on human health currently emphasize the identification of microbes associated with defined anatomic compartments (i.e., skin, gut, oral, etc.), as well as localized sites within these compartments^3,4. Underpinning these investigative efforts is the r....

Protocol

For all protocol steps, proper personal protective equipment (PPE) must be worn, and stringent contamination prevention approaches need to be taken. Observe flow of work from pre-amplification work areas to post-amplification work areas to minimize contamination of samples. All supplies used are sterile, free of RNase, DNase, DNA, and pyrogen. All pipette tips are filtered. A flowchart of the protocol steps is provided (Figure 1).

1. Sample Lysis

NOTE: Sample lysis and nucleic acid extraction are performed using a DNA/RNA extraction kit in a clean-room environment where both engineerin....

Results

The protocol presented here includes important quality control (QC) steps to ensure that the data generated meet benchmarks for protocol sensitivity, specificity, and contamination control. The protocol's first QC step follows PCR amplification of the 16S V4 region (Figure 2). One µL of PCR product from each sample was analyzed by electrophoresis to confirm that it was within the expected size range of 315 - 450 bp (Figure 2, red.......

Discussion

Targeted next-generation sequencing of 16S rRNA is a widely used, rapid technique for microbiome characterization¹⁸. However, many factors, including batch effects, environmental contamination, sample cross-contamination, sensitivity, and reproducibility can adversely affect experimental results and confound their interpretation^7,19,20. To best facilitate robust 16S analyses, microbiome workflows must inc.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AllPrep RNA/DNA Mini Kit	Qiagen	80204	DNA/RNA extraction kit
Eliminase	Fisher Scientific	435532	RNase, DNase, DNA decontaminant
Thermo Mixer	Fisher Scientific		temperature-controlled vortexer
Buffer RLT plus	Qiagen	1053393	guanidinium thiocyanate lysis buffer/ Part of Allprep kit
ß-Mercaptoethanol	Sigma Aldrich	63689-25ML-F	ß-ME is a reducing agent that will irreversibly denature RNases by reducing disulfide bonds
LME Beads	MP Biomedicals	116914050	bead tube
QIAgen TissueLyzer	Qiagen	85300	automated sample disruptor adapter set
QIAshredder column	Qiagen	79654
QIAgen RB tube			manufacturer's microcentrifuge tube in kit
QIAcube and related plasticware	Qiagen	9001292	automated DNA/RNA purification instrument
DNA exitus plus	Applichem	A7089	non-enzymatic decontamination solution
EB Buffer	Qiagen	19086	elution buffer
QIAgility and related plasticware	Qiagen	9001532	robotic liquid handler
PCR water	MO BIO	17000-
5PRIME HotMasterMix	Quantabio	2200400
Barcoded reverse primers	Eurofin	No Catalog #'s	designed and ordered
96 well PCR plate	USA scientific	1402-9708
Tapestation 2200 and related plasticware	Agilent	G2964AA	automated DNA/RNA fragment analyzer
D1000 reagents for Tapestation	Agilent	5067-5585	Sample buffer and ladder are part of this kit
OneStep PCR Inhibitor Removal Kit	Zymo Research	50444470	PCR inhibitor removal is done per the manufacturer's instructions.
QIAquick PCR Purification Kit	Qiagen	28104	DNA clean up kit: silica-membrane-based purification of PCR products
Qubit dsDNA HS Assay Kit	Thermo Fisher	Q32854	dimethylsulfoxide-based dilution buffer and dye are part of this kit.
Qubit Fluorometer	Thermo Fisher	Q33216
NanoDrop	Thermo Fisher		microvolume spectrophotometer
MiSeq 300 V2 kit	Illumina	15033624/15033626
MiSeq	Illumina	No Catalog #'s	next generation sequencer