Name: A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing (Video) | JoVE Journal
Uploaded: 2021-04-15T14:00:00.000+00:00
Duration: 5 min 34 s
Description: Learn a cost-effective magnetic bead-based DNA extraction protocol for individual Aedes aegypti mosquitoes. This method eliminates expensive automated instruments while yielding high-quality DNA suitable for whole genome sequencing.

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5 minutes

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 each individual mosquito extraction. To prepare a master mix for multiple specimens, increase the total amount (combined for all individual specimens) by ~15% to ensure the availability of adequate volume.</li>
	<li>If samples were hydrated prior to extraction, discard water from each sample tube.</li>
	<li>In a 1.5 mL microcentrifuge tube containing mosquito tissue, add 100 &#181;L of PK buffer/enzyme mix.</li>
	<li>Homogenize the tissue using microcentrifuge tube pestle.</li>
	<li>Centrifuge the samples for 1 min at 9,600 x g at room temperature.</li>
	<li>Incubate the sample for 2-3 h at 56 &#176;C.</li>
	<li>During incubation, prepare the other reagents using the DNA extraction step (step 3).</li>
</ol>
3. DNA extraction
<ol>
	<li>Make a magnetic bead master mix. For each sample, mix 100 &#181;L of lysis buffer, 100 &#181;L of Isopropanol, and 15 &#181;L of magnetic beads (total 215 &#181;L). To prepare a master mix for multiple reactions, increase the total amount (combined for all individual specimens) by ~20% to ensure the availability of adequate volume.</li>
	<li>After the incubation time (step 2.7), transfer each lysate to a clean microcentrifuge tube or a microplate well using pipette.</li>
	<li>Add 100 &#181;L lysate and 215 &#181;L of the magnetic bead master mix to each sample.</li>
	<li>Use a pipette to mix it well for 10-20 s and then let it stand for 10 min at room temperature. Gently shake the tube occasionally to maximize the binding of magnetic beads and DNA.</li>
	<li>Place the tube/plate on the magnetic bead separator and wait until the solution is clear.</li>
	<li>Discard the liquid from the tube/plate using pipette. When removing the supernatant, try not to touch or disturb the magnetic beads holding the DNA.</li>
	<li>Move the tube/plate away from magnetic bead separator and add 325 &#181;L of washing buffer 1 to each well.</li>
	<li>Mix thoroughly by pipetting and incubate for 1 min at room temperature.</li>
	<li>Repeat steps 3.5-3.6.</li>
	<li>Move the tube/plate away from magnetic bead separator and add 250 &#181;L of washing buffer 1 to each well.</li>
	<li>Mix thoroughly by pipetting and incubate for 1 min at room temperature.</li>
	<li>Repeat steps 3.5-3.6.</li>
	<li>Move the tube/plate away from magnetic bead separator and add 250 &#181;L of washing buffer 2 to each sample.</li>
	<li>Mix thoroughly and incubate for 1 min at room temperature.</li>
	<li>Repeat steps 3.5-3.6.</li>
	<li>Move the tube/plate away from magnetic bead separator and add 250 &#181;L of washing buffer 2 to each well.</li>
	<li>Mix thoroughly and incubate for 1 min at room temperature.</li>
	<li>Repeat steps 3.5-3.6.</li>
	<li>Move the tube/plate away from the magnetic bead separator and add 100 &#181;L of elution buffer to each well.</li>
	<li>Mix thoroughly and incubate for 2 min at room temperature.</li>
	<li>Move the tube/plate over on the magnetic bead separator and wait until the solution is clear.</li>
	<li>Pipette the supernatant into a new clean 0.5 microcentrifuge tube or microplate well.</li>
	<li>Store DNA samples at 4 &#176;C (up to 1 week) or -80 &#176;C.&#160;If a microplate is being used, cover it with parafilm.</li>
	<li>Determine the DNA yields using any appropriate method. 
	NOTE: In this study, DNA fluorometer reading and absorbance at 260/280 nm were utilized.</li>
</ol>

<ol><li>Nieman, C. C., Yamasaki, Y., Collier, T. C., Lee, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+DNA+extraction+protocol+for+improved+DNA+yield+from+individual+mosquitoes%5BTitle%5D)+AND+%22F1000Research%22%5BJournal%5D)">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/pubmed?term=((Genome-wide+divergence+among+invasive+populations+of+Aedes+aegypti+in+California%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">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/pubmed?term=((Abundance+of+conserved+CRISPR-Cas9+target+sites+within+the+highly+polymorphic+genomes+of+Anopheles+and+Aedes+mosquitoes%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Transcontinental+dispersal+of+Anopheles+gambiae+occurred+from+West+African+origin+via+serial+founder+events%5BTitle%5D)+AND+%22Communications+Biology%22%5BJournal%5D)">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/pubmed?term=((Adaptive+introgression+in+an+African+malaria+mosquito+coincident+with+the+increased+usage+of+insecticide-treated+bed+nets%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((The+genetic+basis+of+host+preference+and+resting+behavior+in+the+major+african+malaria+vector%2C+Anopheles+arabiensis%5BTitle%5D)+AND+%22Plos+Genetics%22%5BJournal%5D)">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/pubmed?term=((Improved+tools+for+genomic+DNA+library+construction+of+small+insects%5BTitle%5D)+AND+%22F1000Research%22%5BJournal%5D)">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/pubmed?term=((Sequencing+of+Tuta+absoluta+genome+to+develop+SNP+genotyping+assays+for+species+identification%5BTitle%5D)+AND+%22Journal+of+Pest+Science%22%5BJournal%5D)">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/pubmed?term=((Complete+mitogenome+sequence+of+Anopheles+coustani+from+S%C3%A3o+Tom%C3%A9+island%5BTitle%5D)+AND+%22Mitochondrial+DNA.+Part+B%2C+Resources%22%5BJournal%5D)">Complete mitogenome sequence of Anopheles coustani from São Tomé 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/pubmed?term=((Complete+mitogenome+sequences+of+Aedes+%28Howardina%29+busckii+and+Aedes+%28Ochlerotatus%29+taeniorhynchus+from+the+Caribbean+Island+of+Saba%5BTitle%5D)+AND+%22Mitochondrial+DNA.+Part+B%2C+Resources%22%5BJournal%5D)">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/pubmed?term=((DNA+source+selection+for+downstream+applications+based+on+dna+quality+indicators+analysis%5BTitle%5D)+AND+%22Biopreservation+and+Biobanking%22%5BJournal%5D)">DNA source selection for downstream applications based on dna quality indicators analysis.</a> Biopreservation and Biobanking. 14 (4), 264-270 (2016).</li>
</ol>

The authors have nothing to disclose.

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><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 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/µL (N = 92, standard deviation 3.513) measured using a fluorometer when eluted using 100 µL 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/µL depending on the mosquito body size and preservation conditions. Some of the high variability is due to the...

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 ...

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