We acknowledge funding support from the Pacific Southwest Regional Center of Excellence for Vector-Borne Diseases funded by the U.S. Centers for Disease Control and Prevention (Cooperative Agreement 1U01CK000516), CDC grant NU50CK000420-04-04, the USDA National Institute of Food and Agriculture (Hatch project 1025565), UF/IFAS Florida Medical Entomology Laboratory fellowship to Tse-Yu Chen, NSF CAMTech IUCRC Phase II grant (AWD05009_MOD0030), and Florida Department of Health (Contract CODQJ). The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the U.S. Fish and Wildlife Service.

1. General sample storage and preparations prior to DNA extraction
<ol>
	<li>Hydrate the sample in 100 &#181;L PCR-grade water for 1 h (or overnight) at 4 &#176;C if the sample has been stored in &#62;70% alcohol to soften the tissue.</li>
</ol>
2. Sample disruption
<ol>
	<li>Set an incubator or shaking heat block at 56 &#176;C.</li>
	<li>Make proteinase K (PK) buffer/enzyme mix. 2 &#181;L of Proteinase K (100 mg/mL) and 98 &#181;L of Proteinase K Buffer (total 100 &#181;L) is required for eac...

<ol>
	<li>Nieman, C. C., Yamasaki, Y., Collier, T. C., Lee, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+extraction+protocol+for+improved+DNA+yield+from+individual+mosquitoes.">A DNA extraction protocol for improved DNA yield from individual mosquitoes.</a> F1000Research. 4, 1314 (2015).</li><li>Lee, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+divergence+among+invasive+populations+of+Aedes+aegypti+in+California.">Genome-wide divergence among invasive populations of Aedes aegypti in California.</a> BMC Genomics. 20 (1), 204 (2019).</li><li>Schmidt, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abundance+of+conserved+CRISPR-Cas9+target+sites+within+the+highly+polymorphic+genomes+of+Anopheles+and+Aedes+mosquitoes.">Abundance of conserved CRISPR-Cas9 target sites within the highly polymorphic genomes of Anopheles and Aedes mosquitoes.</a> Nature Communications. 11 (1), 1425 (2020).</li><li>Schmidt, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcontinental+dispersal+of+Anopheles+gambiae+occurred+from+West+African+origin+via+serial+founder+events.">Transcontinental dispersal of Anopheles gambiae occurred from West African origin via serial founder events.</a> Communications Biology. 2, 473 (2019).</li><li>Norris, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+introgression+in+an+African+malaria+mosquito+coincident+with+the+increased+usage+of+insecticide-treated+bed+nets.">Adaptive introgression in an African malaria mosquito coincident with the increased usage of insecticide-treated bed nets.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (3), 815-820 (2015).</li><li>Main, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+basis+of+host+preference+and+resting+behavior+in+the+major+african+malaria+vector,+Anopheles+arabiensis.">The genetic basis of host preference and resting behavior in the major african malaria vector, Anopheles arabiensis.</a> Plos Genetics. 12 (9), 1006303 (2016).</li><li>Yamasaki, Y. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+tools+for+genomic+DNA+library+construction+of+small+insects.">Improved tools for genomic DNA library construction of small insects.</a> F1000Research. 5, 211 (2016).</li><li>Tabuloc, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequencing+of+Tuta+absoluta+genome+to+develop+SNP+genotyping+assays+for+species+identification.">Sequencing of Tuta absoluta genome to develop SNP genotyping assays for species identification.</a> Journal of Pest Science. 92, 1397-1407 (2019).</li><li>Campos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+mitogenome+sequence+of+Anopheles+coustani+from+S&#227;o+Tom&#233;+island.">Complete mitogenome sequence of Anopheles coustani from S&#227;o Tom&#233; island.</a> Mitochondrial DNA. Part B, Resources. 5 (3), 3376-3378 (2020).</li><li>Cornel, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complete+mitogenome+sequences+of+Aedes+(Howardina)+busckii+and+Aedes+(Ochlerotatus)+taeniorhynchus+from+the+Caribbean+Island+of+Saba.">Complete mitogenome sequences of Aedes (Howardina) busckii and Aedes (Ochlerotatus) taeniorhynchus from the Caribbean Island of Saba.</a> Mitochondrial DNA. Part B, Resources. 5 (2), 1163-1164 (2020).</li><li>Lucena-Aguilar, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+source+selection+for+downstream+applications+based+on+dna+quality+indicators+analysis.">DNA source selection for downstream applications based on dna quality indicators analysis.</a> Biopreservation and Biobanking. 14 (4), 264-270 (2016).</li></ol>

The protocol described here can be adapted for other insect species. The original version of the protocol introduced in Nieman et al.1 has been tested on multiple species, including Aedes aegypti, Ae. busckii, Ae. taeniorhynchus, Anopheles arabiensis, An. coluzzii, An. coustani, An. darlingi, An. funestus, An. gambiae, An. quadriannulatus, An. rufipes, Culex pipiens, Cx. quinquefasciatus, Cx. theileri, Drosophila suzukii, Chrysomela aeneicollis Tuta absoluta, and Keiferia lycopersicel...

Recent development of an improved DNA extraction protocol1 has allowed many high-impact downstream studies involving whole genome sequencing2,3,4,5,6. This magnetic bead-based DNA extraction protocol provides reliable DNA yield from individual mosquito samples, which in turn reduces the cost and time associated with acquiring a sufficient number of samples from field collections.
Recent advances in population and landscape genomics are directly correlated with decreasing costs of whole genome sequencing. Though the previous DNA extraction protocol1 increases efficiencies associated with high throughput sequencing, smaller labs/studies without the funds may opt out of using these new powerful landscape and population genomics tools due to costs of implementing the protocol (e.g., costs of specialized instruments).
Here, a modified DNA extraction protocol is presented that uses a similar magnetic bead extraction step as Neiman et al.1 to obtain high purity DNA but does not rely on high-cost instruments for tissue lysis and DNA extraction. This protocol is suitable for experiments requiring &#62;10 ng of high-quality DNA.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AE Buffer</td>
			<td>Qiagen</td>
			<td>19077</td>
			<td>Elution buffer</td>
		</tr>
		<tr>
			<td>AL Buffer</td>
			<td>Qiagen</td>
			<td>19075</td>
			<td>Lysis buffer</td>
		</tr>
		<tr>
			<td>AW1 Buffer</td>
			<td>Qiagen</td>
			<td>19081</td>
			<td>Washing buffer 1</td>
		</tr>
		<tr>
			<td>AW2 Buffer</td>
			<td>Qiagen</td>
			<td>19072</td>
			<td>Washing buffer 2</td>
		</tr>
		<tr>
			<td>MagAttract Suspension G</td>
			<td>Qiagen</td>
			<td>1026901</td>
			<td>magnetic bead</td>
		</tr>
		<tr>
			<td>Magnetic bead separator</td>
			<td>Epigentek</td>
			<td>Q10002-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop</td>
			<td>ThermoFisher</td>
			<td>ND-2000</td>
			<td>microvolume spectrophotometer</td>
		</tr>
		<tr>
			<td>PK Buffer</td>
			<td>ThermoFisher</td>
			<td>4489111</td>
			<td>Proteinase K buffer</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>ThermoFisher</td>
			<td>A25561</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit</td>
			<td>Invitrogen</td>
			<td>Q33238</td>
			<td>fluorometer</td>
		</tr>
	</tbody>
</table>

a magnetic-bead-based mosquito dna extraction protocol for next-generation sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

The average DNA yield per individual mosquito head/thorax tissue was 4.121 ng/&#181;L (N = 92, standard deviation 3.513) measured using a fluorometer when eluted using 100 &#181;L of elution buffer. This is sufficient for the 10-30 ng genomic DNA input requirements necessary for whole genome library construction1,7. The quantity of DNA can vary between 0.3-29.7 ng/&#181;L depending on the mosquito body size and preservation conditions. Some of the high variabilit...

Watch this Scientific Journal Video about A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing at JoVE.com

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

This budget-friendly magnetic bead-based DNA extraction protocol makes high throughput sequencing accessible to resource limited labs and studies. This protocol does not require expensive equipment for high quality DNA extraction and it can be helpful in certain diagnostic or research situations where obtaining a sufficient quantity of DNA with a quality for high throughput sequencing is challenging. This protocol is easy to replicate with a one-time demonstration.It should be tried with a small number of samples first to get familiar with the workflow. To begin, hydrate the mosquito tissue samples stored in greater than 70%alcohol by adding 100 microliters of PCR grade water and incubating for one hour at four degrees Celsius to soften the tissue. After the incubation, discard the water and add 100 microliters of Proteinase K buffer enzyme mix.Then use a microcentrifuge tube pestle to homogenize the tissue. After homogenization, centrifuge the tissue lysate and incubate for two to three hours at 56 degrees Celsius. To extract DNA, pipette 100 microliters of the tissue lysate into a new clean microcentrifuge tube.Then add 215 microliters of the magnetic bead master mix. Using a pipette, mix the lysate and the magnetic beads mixture for 10 to 20 seconds, then let it stand for 10 minutes at room temperature. During the incubation, gently shake the tube occasionally to maximize the binding of the DNA to the magnetic beads.Next, place the tube on the magnetic bead separator until the solution becomes clear. Then using a pipette, gently aspirate the liquid from the tube without disturbing the magnetic beads holding the DNA. Move the tube away from the magnetic bead separator and wash the beads by adding 325 microliters of washing buffer one.Mix thoroughly by pipetting and incubate for one minute at room temperature. After the incubation, place the tube on the magnetic bead separator and remove the supernatant as previously demonstrated, then move the tube away from the magnetic bead separator and wash the beads once with washing buffer one. After the second wash with buffer one, wash the beads twice with 250 microliters of washing buffer two in the same manner.After the last wash, move the tube away from the magnetic bead separator and add 100 microliters of elution buffer. Mix thoroughly by pipetting and then incubate the tube for two minutes at room temperature before placing it back on the magnetic separator. When the solution becomes clear, transfer the supernatant into a new, clean 0.5 milliliter microcentrifuge tube and store appropriately.Shown here as a typical microvolume fluorometer reading of the mosquito DNA in an elution buffer containing 0.5 millimolar EDTA. The average 260 to 280 nanometer absorbance ratio is 2.3. This table shows the costs and the number of samples processed in one working day for different extraction methods.The typical reagent and consumable cost for an extraction by this protocol is around 9.50 per sample. This cost is equivalent to any typical magnetic bead-based extraction method. The major cost benefit of this protocol comes from not requiring the automated DNA extraction instrument.Make sure to change pipette tips between samples to prevent cross-contamination of DNA when working with multiple samples. We use the high quality DNA extracted using this method for whole genome sequencing. We are currently using the sequencing data to identify novel mutations in insecticide resistance or immune genes of concern in public health and disease control.Having affordable solutions for high throughput sequencing opens exciting doors for population genomics and landscape genomics aimed at understanding mosquito dispersal and gene flow which influences the success of many novel genetic control strategies.

a-magnetic-bead-based-mosquito-dna-extraction-protocol-for-next

Research

JoVE Journal

Biology

6.0K visualizações.  University of Florida. Este protocolo de extração de DNA baseado em contas magnéticas amigável ao orçamento torna o sequenciamento de alto rendimento acessível a laboratórios e estudos limitados de recursos. Este protocolo não requer equipamentos caros para extração de DNA de alta qualidade e pode ser útil em certas situações de diagnóstico ou pesquisa onde a obtenção de uma quantidade suficiente de DNA com uma qualidade para sequenciamento de alto rendimento é um desafio. Este protocolo é fácil ...