Name: tick microbiome characterization by next-generation 16s rrna amplicon sequencing
Uploaded: 2018-08-25T14:45:00
Description: Watch this Scientific Journal Video about Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing at JoVE.com

This work was supported by National Science Foundation grants to A.S. (DEB #1427772, 1745411, 1750037).

1. Tick Collection and Surface Sterilization
<ol>
	<li>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 lab15,16. Use fine forceps to manipulate ticks and store them at -80 &#176;C.</li>
	<li>Place ticks in the individual PCR tubes and remove surface contaminants by vortexing for 15 s successively with 500 &#956;L of hydrogen peroxide (H2O2</s...

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...

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. Overwhelming evidence now supports this assertion, with examples demonstrating a link between the vector microbiome and competence for diseases such as malaria, Zika virus, and Lyme disease1,2,3. However, translating these findings into strategies for disease control requires a far more detailed understanding of the structure, function, and origin of vector microbiomes. Identification and characterization of the vector microbial community under varying ecological and experimental conditions constitute an important path forward in this field.
A procedure for identifying the microbial residents of a pathogen vector is provided here by utilizing the Western black-legged tick, Ixodes pacificus, a vector species of the Lyme disease pathogen Borrelia burgdorferi. While ticks harbor more types of human pathogens than any other arthropod, relatively little is known about the biology and community ecology of tick microbiomes4. It is evident that ticks harbor a diverse array of viruses, bacteria, fungi, and protozoans which include commensals, endosymbionts, and transient microbial residents5,4. Prior work has demonstrated strong variations in Ixodes microbiomes associated with geography, species, sex, life stage, and blood meal source6,7,8. However, the mechanisms underlying this variation remain unknown and warrant more detailed investigations of the origin and assembly of these microbial communities. Ticks can acquire microbes through vertical transmission, contact with hosts, and uptake from the environment through the spiracles, mouth, and anal pore9. Understanding the factors shaping the initial formation and development of the tick microbiome, specifically the relative contribution of vertical and environmental transmission, is important for understanding the natural patterns and variations in tick microbiome diversity and how these communities interact during pathogen transmission, with possible applications to disease or vector control.
Powerful molecular techniques, such as next-generation sequencing, now exist for identifying microbial communities and can be employed to characterize vector microbiomes under diverse environmental or experimental conditions. Prior to the advent of these high-throughput sequencing approaches, the identification of microbes relied predominantly on microscopy and culture. While microscopy is a rapid and easy technique, morphological methods for identifying microbes are inherently subjective and coarse and limited by low sensitivity and detection10. Culture-based methods are broadly used for microbial identification and can be used to determine susceptibility of microbes to drug treatments11. However, this method also suffers from low sensitivity, as it has been estimated that fewer than 2% of environmental microbes can be cultured in a laboratory setting12. Histological staining approaches have also been employed to detect and localize specific microbes within vectors, enable investigations of various taxa distributions within the tick, and study hypotheses about microbial interactions. However, prior knowledge of microbial identity is required for selecting the appropriate stains, making this approach ill-suited for microbial characterization and identification. Furthermore, histological staining is a highly time-intensive, laborious process and does not scale well for large sample sizes. Traditional molecular approaches such as Sanger sequencing are similarly limited in their sensitivity and detection of diverse microbial communities.
Next-generation sequencing allows for the rapid identification of microbes from a large number of samples. The presence of standard marker genes and reference databases further enables enhanced taxonomic resolution, often to the genus or species level. Small subunit ribosomal RNAs are frequently used to achieve this goal, with 16S rRNA being the most common due to the presence of conserved and variable regions within the gene, allowing for the creation of universal primers with unique amplicons for each bacterial species13,14. This report details a procedure for identifying taxa in the tick microbiome through 16S rRNA next-generation sequencing. In particular, this protocol emphasizes the steps involved in preparing samples for sequencing. More generalized details on the sequencing and bioinformatics steps are provided, as there are a variety of sequencing platforms and analysis programs currently available, each with extensive existing documentation. The overall feasibility of this next-generation sequencing approach is demonstrated by applying it to an investigation of microbial community assembly within a key disease vector.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Item</td>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog #</td>
		</tr>
		<tr>
			<td>1</td>
			<td>DNeasy Blood &#38; Tissue Kit</td>
			<td>Qiagen</td>
			<td>69504</td>
		</tr>
		<tr>
			<td>2</td>
			<td>Qubit 4 Fluorometer</td>
			<td>ThermoFisher Scientific</td>
			<td>Q3326</td>
		</tr>
		<tr>
			<td>3</td>
			<td>NanoDrop 8000 Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-8000-GL</td>
		</tr>
		<tr>
			<td>4</td>
			<td>2x KAPA HiFi HotStart ReadyMix</td>
			<td>Kapa Biosystems</td>
			<td>KK2501</td>
		</tr>
		<tr>
			<td>5</td>
			<td>AMPure XP beads</td>
			<td>Agen Court</td>
			<td>A63880&#160;</td>
		</tr>
		<tr>
			<td>6</td>
			<td>Magnetic Rack</td>
			<td>ThermoFisher Scientific</td>
			<td>MR02</td>
		</tr>
		<tr>
			<td>6</td>
			<td>TE buffer</td>
			<td>Teknova</td>
			<td>T0223</td>
		</tr>
		<tr>
			<td>7</td>
			<td>Nextera Index Kit</td>
			<td>Illumina</td>
			<td>FC-121-1011</td>
		</tr>
		<tr>
			<td>8</td>
			<td>KAPA Library Quantification Kit</td>
			<td>Roche</td>
			<td>KK4824</td>
		</tr>
		<tr>
			<td>9</td>
			<td>MiSeq System</td>
			<td>Illumina</td>
			<td>SY-410-1003</td>
		</tr>
		<tr>
			<td>10</td>
			<td>MiSeq Reagent Kit v3&#160;</td>
			<td>Illumina</td>
			<td>MS-102-3001</td>
		</tr>
		<tr>
			<td>11</td>
			<td>10 mM Tris-HCl with 0.1% Tween 20</td>
			<td>Teknova</td>
			<td>T7724</td>
		</tr>
	</tbody>
</table>

tick microbiome characterization by next-generation 16s rrna amplicon sequencing

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.

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 ...

Watch this Scientific Journal Video about Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing at JoVE.com

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

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.

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

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