Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

Summary

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

Abstract

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Introduction

The resurgence and spread of vector-borne diseases in recent decades pose a serious threat to global human and wildlife health. Effective vaccines are lacking for a majority of these diseases, and control efforts are hindered by the complex biological nature of vectors and vector-host interactions. Understanding the role of microbial interactions within a vector in pathogen transmission can allow for the development of novel strategies which circumvent these challenges. In particular, interactions between vector-associated microbial commensals, symbionts, and pathogens, referred to as the microbiome, may have important consequences for pathogen transmission. Overwhelm....

Protocol

1. Tick Collection and Surface Sterilization

1. Collect ticks by dragging a 1 m² white cloth over a tick-associated habitat, removing ticks attached to host species, or rearing ticks in the lab^15,16. Use fine forceps to manipulate ticks and store them at -80 °C.
2. Place ticks in the individual PCR tubes and remove surface contaminants by vortexing for 15 s successively with 500 μL of hydrogen peroxide (H₂O₂), 70% ethanol, and ddH₂O.
3. Place the ticks in a new PCR tube and allow them to air-dry.
4. In this tube, mechanically d....

Results

A total of 42 ticks from three separate egg clutches and two environmental exposure periods, 0 and 2 weeks in soil, were processed for microbiome sequencing. Each treatment group, considered to be a single clutch and exposure time, contained 6-8 replicate tick samples. These processed tick extracts were loaded onto a next-generation sequencer and yielded 12,885,713 paired-end reads passing filter. Included in this run were 3 negative controls from the extraction step, yielding a total of .......

Discussion

Next-generation sequencing of 16S rRNA has become a standard approach for microbial identification and enabled the study of how vector microbiomes affect pathogen transmission. The protocol outlined here details the use of this method to investigate microbial community assembly in I. pacificus, a vector species for Lyme disease; however, it can easily be applied to study other tick species or arthropod vector species.

Indeed, 16S rRNA sequencing for microbiome analysis has been used b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Item	Name of Material/Equipment	Company	Catalog #
1	DNeasy Blood & Tissue Kit	Qiagen	69504
2	Qubit 4 Fluorometer	ThermoFisher Scientific	Q3326
3	NanoDrop 8000 Spectrophotometer	ThermoFisher Scientific	ND-8000-GL
4	2x KAPA HiFi HotStart ReadyMix	Kapa Biosystems	KK2501
5	AMPure XP beads	Agen Court	A63880
6	Magnetic Rack	ThermoFisher Scientific	MR02
6	TE buffer	Teknova	T0223
7	Nextera Index Kit	Illumina	FC-121-1011
8	KAPA Library Quantification Kit	Roche	KK4824
9	MiSeq System	Illumina	SY-410-1003
10	MiSeq Reagent Kit v3	Illumina	MS-102-3001
11	10 mM Tris-HCl with 0.1% Tween 20	Teknova	T7724