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

Summary

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.

Abstract

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.

Introduction

Recent development of an improved DNA extraction protocol¹ has allowed many high-impact downstream studies involving whole genome sequencing^2,3,4,5,6. 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 >10 ng of high-quality DNA.

Protocol

1. General sample storage and preparations prior to DNA extraction

1. Hydrate the sample in 100 µL PCR-grade water for 1 h (or overnight) at 4 °C if the sample has been stored in >70% alcohol to soften the tissue.

2. Sample disruption

1. Set an incubator or shaking heat block at 56 °C.
2. Make proteinase K (PK) buffer/enzyme mix. 2 µL of Proteinase K (100 mg/mL) and 98 µL of Proteinase K Buffer (total 100 µL) 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.
3. If samples were hydrated prior to extraction, discard water from each sample tube.
4. In a 1.5 mL microcentrifuge tube containing mosquito tissue, add 100 µL of PK buffer/enzyme mix.
5. Homogenize the tissue using microcentrifuge tube pestle.
6. Centrifuge the samples for 1 min at 9,600 x g at room temperature.
7. Incubate the sample for 2-3 h at 56 °C.
8. During incubation, prepare the other reagents using the DNA extraction step (step 3).

3. DNA extraction

1. Make a magnetic bead master mix. For each sample, mix 100 µL of lysis buffer, 100 µL of Isopropanol, and 15 µL of magnetic beads (total 215 µL). 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.
2. After the incubation time (step 2.7), transfer each lysate to a clean microcentrifuge tube or a microplate well using pipette.
3. Add 100 µL lysate and 215 µL of the magnetic bead master mix to each sample.
4. 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.
5. Place the tube/plate on the magnetic bead separator and wait until the solution is clear.
6. 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.
7. Move the tube/plate away from magnetic bead separator and add 325 µL of washing buffer 1 to each well.
8. Mix thoroughly by pipetting and incubate for 1 min at room temperature.
9. Repeat steps 3.5-3.6.
10. Move the tube/plate away from magnetic bead separator and add 250 µL of washing buffer 1 to each well.
11. Mix thoroughly by pipetting and incubate for 1 min at room temperature.
12. Repeat steps 3.5-3.6.
13. Move the tube/plate away from magnetic bead separator and add 250 µL of washing buffer 2 to each sample.
14. Mix thoroughly and incubate for 1 min at room temperature.
15. Repeat steps 3.5-3.6.
16. Move the tube/plate away from magnetic bead separator and add 250 µL of washing buffer 2 to each well.
17. Mix thoroughly and incubate for 1 min at room temperature.
18. Repeat steps 3.5-3.6.
19. Move the tube/plate away from the magnetic bead separator and add 100 µL of elution buffer to each well.
20. Mix thoroughly and incubate for 2 min at room temperature.
21. Move the tube/plate over on the magnetic bead separator and wait until the solution is clear.
22. Pipette the supernatant into a new clean 0.5 microcentrifuge tube or microplate well.
23. Store DNA samples at 4 °C (up to 1 week) or -80 °C. If a microplate is being used, cover it with parafilm.
24. Determine the DNA yields using any appropriate method.
    NOTE: In this study, DNA fluorometer reading and absorbance at 260/280 nm were utilized.

Results

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 construction^1,7. 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 variabilit...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
AE Buffer	Qiagen	19077	Elution buffer
AL Buffer	Qiagen	19075	Lysis buffer
AW1 Buffer	Qiagen	19081	Washing buffer 1
AW2 Buffer	Qiagen	19072	Washing buffer 2
MagAttract Suspension G	Qiagen	1026901	magnetic bead
Magnetic bead separator	Epigentek	Q10002-1
Nanodrop	ThermoFisher	ND-2000	microvolume spectrophotometer
PK Buffer	ThermoFisher	4489111	Proteinase K buffer
Proteinase K	ThermoFisher	A25561
Qubit	Invitrogen	Q33238	fluorometer