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

This method can help answer key questions in the field of vector-borne disease ecology such as, what role does the vector microbiome play in pathogen dynamics, and how do vector microbiomes form and vary? The main advantages of this technique are that it allows for high replication, and high resolution and identification, allowing the user to accurately characterize vector microbiome under varying ecological and environmental conditions. Visual demonstration of this method is important, as the microbiome sequencing setup steps are difficult to learn, and there are many different products available, and techniques, but few available resources for learning these steps.

Begin this procedure with tick collection, as described in the text protocol. To remove surface contaminants from the ticks, add 500 microliters of hydrogen peroxide, and vortex for 15 seconds. Repeat this wash with 70%ethanol, and then double-distilled water.

Place the ticks into a new PCR tube, and allow them to air dry. In this tube, mechanically disrupt tick tissues by crushing the ticks with a mortal and pestle. Purify DNA from individual ticks, following the instructions provided in a commercially available DNA extraction kit.

Elute to a final volume of 100 microliters. Creating a negative control at the extraction step is critical for later differentiating suspected contaminants from true vector microbes. Using sterile technique during the extraction step and later PCR steps is also necessary to minimize the impact of contamination common in 16S sequencing.

Set up the amplicon PCR in a 27.5 microliter reaction containing five microliters of each primer at one micromolar and 12.5 microliters of commercially available PCR mix. Also include five microliters of DNA extracted from individual ticks at a five nanograms per microliter concentration. Move the tubes to a thermocycler and run the program listed in the text protocol.

Once the PCR is done, visualize the PCR product by loading four to six microliters per sample on a 1.5%agarose gel. Look for a band at 460 base pairs to confirm amplification. Using a new PCR tube for each sample, combine the PCR product with double-distilled water to obtain a total of 60 microliters.

For samples with low DNA concentrations, perform amplicon PCR in triplicate to reduce amplification bias and pool the samples to concentrate. Bring paramagnetic beads to room temperature and vortex them well before use. Add 48 microliters of paramagnetic beads to 60 microliters of the sample and incubate for five minutes.

Place the tubes on a magnetic rack for five minutes until the solution becomes clear. Then, remove the supernatant. With tubes on the magnetic rack, add 500 microliters of freshly prepared 80%ethanol.

Immediately after adding ethanol to all tubes, pipette out the liquid, do not remove the beads. Repeat the ethanol addition and removal one more time. Air dry the samples to remove excess ethanol by leaving the tubes open on the magnetic rack for five minutes or until small cracks are visible in the beads.

Now, add 20 microliters of TE buffer and incubate the samples off the magnet at room temperature. After five to 10 minutes place the tubes back on the magnet. Once the beads and liquid are separated, transfer the supernatants to a fresh tube to obtain the cleaned PCR product.

Assign unique primer combinations to each sample, by selecting either forward or reverse primers or both from a commercially available library index kit. To attach the dual primers or indices to the samples, prepare a PCR reaction containing 2.5 microliters of each primer, 12.5 microliters of commercially available PCR Master Mix, five microliters of double-distilled water, and 2.5 microliters of cleaned amplicon product. Move the tubes to a thermocycler programmed as detailed in the text protocol.

Once the PCR is done, visualize the PCR product by loading four to six microliters per sample on a 1.5%agarose gel. Look for a band at 550 base pairs to confirm amplification before performing the clean-up procedure as before. To perform accurate quantification of each purified barcoded product run qPCR on each sample and standard in triplicate and run each sample at three or more dilution levels.

Prepare 10 microliter qPCR reactions containing six microliters of qPCR Master Mix, two microliters of double-distilled water, and two microliters of the sample or standard. Move the qPCR plate to a real-time PCR instrument programmed as described in the text protocol and perform the run. Following quantification, create the combined library by adding equal volumes of all individual libraries into a single tube.

After diluting and denaturing the combined library and the sequencing control, load the combined mixture onto the sequencing flow cell. Thoroughly clean the flow cell prior to loading for optimum optics. Then, load the flow cell onto the sequencer and perform amplicon sequence analysis.

Rarefaction curves for sequence count normalization are shown here. These curves indicate that rarefying to 2000 sequences per sample should be sufficient to capture the full diversity of observed operational taxonomic units. After rarefying and quality filtering the sequence reads, alpha and beta diversity output is performed.

Shown here is an automatically generated principle coordinates analysis plot depicting how tick microbiome composition differs based on treatment. Further, visualization of the microbes present within each sample, shown here at they phylum level, are automatically created for all samples. While attempting this procedure, it's important to remember to maintain sterile technique to minimize the effect of microbial contamination which can critically impact sequence data interpretation.

Following this procedure, other methods like histological staining can be performed to answer additional questions such as, where are the observed microbes located within the vector? After its development, this method paved the way for researchers in the field of molecular ecology to explore the role of microbial interactions within the vector and investigate how this may effect pathogen transmission.