Labs without Borders: Methods for Extracting, Amplifying, and Sequencing in the Field
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This document describes methods developed to conduct DNA extraction, amplification, and sequencing of bat samples directly in the field. Due to increasingly restrictive regulations, it is no longer possible to export bat samples for commercial lab analysis. The methods developed include tweaks to DNA extraction techniques, use of magnetic beads for PCR, a portable thermocycler, and nanopore sequencing. These decentralized field methods were successfully used to sequence DNA from bat wing remains to identify bat species, without needing to export any samples or products.
Labs without Borders: Methods for Extracting, Amplifying, and Sequencing in the Field
- 1. Labs without Borders: Methods for Extracting, Amplifying, and Sequencing in the Field Liliana M. Dávalos, Laurel R. Yohe, Paola Pulido-Santacruz, Mailyn González, Anna McPherran, J. Angel Soto-Centeno, Darwin M. Morales Martinez, Andrés Julián Lozano Florez, Jon Flanders, Danny Rojas, Winifred Frick and Angelique P. Corthals
- 2. Problem • Increasingly restrictive regulations • Export • Shipping • Import • No longer possible for commercial planes to take samples • Biosafety concerns applied to ALL bats
- 3. We want to do better Sequencing efforts concentrated
- 4. We want to do better Build capacity, boost inclusion
- 5. Challenges • DNA extraction • Cold extractions • Centrifuging • PCR • Reagent transport • Thermocycler • Sequencing • Decentralizing
- 6. Solutions • DNA extraction • Tweak extraction • PCR • Beads • Portable thermocycler • Sequencing • Nanopore
- 7. Solutions • DNA extraction • Tweak extraction • PCR • Beads • Portable thermocycler • Sequencing • Nanopore
- 8. Solutions • DNA extraction • Tweak extraction • PCR • Beads • Portable thermocycler • Sequencing • Nanopore
- 9. It worked! But was inefficient
- 11. Results from Sturnira lilium
- 13. Tweak protocol • Extraction • Target bat, not bacteria • PCR • Custom primers for ID’ing • Sequencing • Single targets not cost effective • Switch to Sanger
- 14. Most importantly • Incubation! • Rechargeable travel mug • Reaches 55˚C & 70˚C • No need for cold extractions (!!!)
- 15. Success! No export permits needed for products
- 16. The victims • Mormoops blainvillei • Pteronotus quadridens • Pteronotus macleayii • Monophyllus redmani
- 17. Wide range of applications