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.

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 protocol 1 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 protocol 1 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.

Representative 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 variability is due to the characteristic of magnetic beads used in the study. Different brands of magnetic beads may produce more consistent concentrations, as indicated in the previous report 1 . It should also be noted that different mosquito species differ in size and that the DNA yield is dependent on those individual and species size ranges. If the DNA concentration is below the typical ranges, one may adjust the incubation period in the proteinase K reaction (step 2.7), but this extension should be no longer than 16 h. Moreover, switching the proteinase K and/or lysis buffer brands can have significant impact on DNA yield 1 .

The typical microvolume fluorometer reading of the final DNA in elution buffer with 0.5 mM EDTA is shown in Figure 1. The average absorbance ratio for 260/280 nm was 2.3 (standard deviation = 0.071). The data was consistent from the samples used in the previous studies for whole genome sequencing 3 , 4 .

Typical reagent and consumable cost for extraction is around $9.50/sample. These cost estimates include reagents listed in the Table of Materials, and other consumables such as pipette tips, tubes, and gloves needed for extraction. The reagent and consumable cost are equivalent to any typical magnetic-bead-based extraction method (Table 1). The cost may vary somewhat depending on bulk purchase discounts available for any given product. The major cost benefit of this protocol comes from not requiring the automated DNA extraction instrument.

Figure 1


Figure 1: Fluorometer output. Typical DNA fluorometer output of mosquito DNA eluted in elution buffer with 0.5 mM EDTA. Please click here to view a larger version of this figure.

Table 1: Cost analysis for different extraction methods. The table lists the costs and the number of samples processed in one working day for different extraction methods. Please click here to download this Table.

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 lycopersicella 2 , 8 , 9 , 10 . It is expected that this protocol will work for these as well as other arthropods.

Ideally, samples should be stored in 70%-80% ethanol at 4 °C prior to DNA extraction. Typically, 80% ethanol is used for storing mosquito samples in Africa. High temperature conditions could accelerate evaporation of ethanol and make ethanol content lower than what is intended (70%). Samples stored in 100% ethanol, denatured alcohol, 75%-90% isopropanol or rubbing alcohol have also resulted in successful whole genome sequencing using the Nieman et al. 1 protocol, and it is expected that this protocol would perform equally well. The DNA extraction protocol described here has also been tried on samples stored dry in silica and frozen at -20 °C. However, there was a higher (15%-30%) chance of failure when stored for >6-12 months, suggesting that these were less than ideal storage conditions.

As DNA extraction without physical sample disruption has a higher chance of failure (33%) 1 , this protocol incorporates a manual physical disruption technique (step 2.5). Sample disruption can also be achieved using steel beads on a tissue lysis instrument 1 .

The reagents and consumable cost for DNA extraction using this protocol is comparable with other extraction methods available (Table 1). However, the protocol described here does not require an automated DNA extraction instrument. Due to manual labor involved in processing DNA extraction, one person can typically process 12-16 samples and finish with DNA quantification in a day. This is significantly less than what automated DNA extraction setup can process per day. Thus, when choosing a protocol, the sample processing capacity should be taken into account. However, the lower cost threshold this protocol offers should enable many resource limited labs and studies to leverage in high-throughput sequencing technology.

In both the Nieman et al. 1 and this protocol, elution buffer can be replaced with the typical TE buffer, 10 mM Tris-Cl, or water depending on the downstream analysis. An elution buffer containing some EDTA may impact enzyme efficiency. Typical enzymatic shearing-based gDNA library preparation protocols include conditioning solutions or enhancers added to compensate for enzyme inhibition by EDTA. It should also be considered that EDTA may change the absorbance at 230 nm wavelength 11 . For example, higher concentrations of EDTA would create a higher absorbance value between 220-240 nm wavelength than that shown in Figure 1 (data not shown).

This protocol can be easily adapted to laboratories with minimal molecular biology tools. The protocol described here attempts to reduce the cost thresholds associated with genome extraction and thereby increase the application of high throughput sequencing to resource limited labs and studies.