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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

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

Published: April 15th, 2021

DOI:

10.3791/62354

1Florida Medical Entomology Laboratory, Department of Entomology and Nematology, Institute of Food and Agricultural Sciences, University of Florida, 2U. S. Fish and Wildlife Service, Pacific Islands Fish and Wildlife Office, 3Department of Entomology and Nematology, University of California Davis

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 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.

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 ....

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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 eac.......

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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 variabilit.......

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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.......

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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.

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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

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

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