TITLE: DNA extraction and Nanopore library prep from single wild-caught drosophilids
AUTHORS: Bernard Y Kim

[DESCRIPTION]
We have been assembling the genomes of many Drosophila species. This protocol fork allows for the sequencing of single wild-caught flies using Nanopore MinION sequencers, in contrast to our previous protocol that originally used gDNA extracted from tens or hundreds of flies from an inbred line.

While read lengths are not nearly close to what we were seeing before, libraries range from 2-10kb read N50 and typically produce genome assemblies with contig N50 >1 Mbp.

The protocol for ultra-long read sequencing from large pools of flies can be found here: dx.doi.org/10.17504/protocols.io.bdfqi3mw

For now, please cite the original genomes paper if you use this protocol: https://doi.org/10.7554/eLife.66405

An update that includes many single wild-caught fly genomes is in progress.

[GUIDELINES]
Flies were collected from the wild and stored in 95% ethanol at -20C. We have successfully assembled genomes from samples ranging from 4 months to 15 years old. Younger samples perform better, of course. Ideally the samples are no older than 5 years and stored properly (e.g. alcohol has not evaporated).

Apart from the gDNA extraction steps, this protocol mostly follows the standard LSK110 protocol. Slight modifications were made to improve library recovery and pore occupancy.

[STEPS]
SECTION: (Optional) Hydration of ethanol-fixed tissue
1. Place flies on a sheet of filter paper and briefly dab with a Kimwipe to remove excess ethanol, then transfer the fly to a 1.5 mL LoBind tube.

  
SECTION: (Optional) Hydration of ethanol-fixed tissue
2. Add 500 µL Buffer STE to the tube with the fly. 
 
SECTION: (Optional) Hydration of ethanol-fixed tissue
3. Incubate on a platform rocker or rotator for 15 min  at low to medium speed.
SECTION: (Optional) Hydration of ethanol-fixed tissue
4. Replace the solution with 500 µL of fresh Buffer STE and incubate for another 15 min  on the rocker at low to medium speed.
 

SECTION: Tissue homogenization
6. Add 283 µL lysis buffer to the tube with the fly.

 

SECTION: Tissue homogenization
7. Using a pellet pestle, manually homogenize the fly tissue. While it's important to squish the fly enough so that soft tissue can be digested, it is okay for some bigger pieces of tissue, particularly those held together by tough cuticle like the legs, to remain.
 

 





SECTION: Phenol chloroform extraction
10. Spin down 1 2mL phase lock gel tube per sample at 15000 x g for 30 s .
 

 

 
  

SECTION: Phenol chloroform extraction
11. Briefly spin down the homogenate/lysis solution, then transfer to the phase lock gel tube by decanting or pipetting with a wide-bore tip.
SECTION: Phenol chloroform extraction
12. Add an equal volume (about 350 µL) of Tris-saturated phenol chloroform isoamyl alcohol (PCI) to the phase lock tube.
 

 

SECTION: Phenol chloroform extraction
13. Mix by placing tubes on a rocker at medium speed for 8 min .
 

SECTION: Phenol chloroform extraction
14. Centrifuge the phase lock tube at 16000 x g for 8 min. Phase lock layer should now separate aqueous and organic layers.

SECTION: Phenol chloroform extraction
15. Perform one more PCI extraction:   .
 

SECTION: Phenol chloroform extraction
16. Add an equal volume (usually 350 µL) of chloroform to the tube.
 

 

SECTION: Phenol chloroform extraction
17. Mix by placing tubes on a rocker at medium speed for 8 min .
SECTION: Phenol chloroform extraction
18. Centrifuge the phase lock tube at 16000 x g for 8 min. Phase lock layer should now separate aqueous and organic layers.

SECTION: Phenol chloroform extraction
19. Quickly decant the aqueous (top) layer into a fresh 1.5 mL LoBind tube.
 

 

  


SECTION: DNA precipitation, wash, and resuspension
20. Chill 100% ethanol on ice and make 500 µL of fresh 70% ethanol using nuclease-free water.

SECTION: DNA precipitation, wash, and resuspension
21. Add 0.1 volume (typically 30 µL) of 3M sodium acetate to the sample. Gently swirl to mix.
 


SECTION: DNA precipitation, wash, and resuspension
22. Add 2-2.5 volumes (typically 675 µL) of cold 100% ethanol to the tube, and mix with careful swirling and gentle rocking. It will be difficult to see precipitated DNA, but extractions from larger flies will usually have a few visible strands.
 


SECTION: DNA precipitation, wash, and resuspension
23. Centrifuge the tube at 5000 x g for 5 min to pellet the DNA.

 

SECTION: DNA precipitation, wash, and resuspension
24. While being careful not to disturb the pellet, pipette off the ethanol.
SECTION: DNA precipitation, wash, and resuspension
25. Add 200 µL of 70% ethanol to wash the DNA. Gently swirl to mix.

SECTION: DNA precipitation, wash, and resuspension
26. Centrifuge the tube at 5000 x g for 2 min. 

SECTION: DNA precipitation, wash, and resuspension
27. While being careful not to disturb the pellet, pipette off the ethanol.
 

SECTION: DNA precipitation, wash, and resuspension
28. Wash the pellet once more:   .

SECTION: DNA precipitation, wash, and resuspension
29. Allow the tube to air dry no longer than 1 min. Do not over-dry.

SECTION: DNA precipitation, wash, and resuspension
30. Resuspend in 42 µL 10mM Tris and incubate at 50 °C for 30 min.
 

  

SECTION: DNA precipitation, wash, and resuspension
31. Briefly spin down tube to gather any condensation and store at 4 °C.

SECTION: DNA resuspension
32. Pipette mix slowly 5X with a P200 tip to ensure proper resuspension.

SECTION: DNA resuspension
33. Check sample concentration and quality of 1 µL aliquots using Qubit and Nanodrop.
 


SECTION: Reserve DNA for short-read sequencing
34. Reserve at least 20 ng gDNA in 6 µL Tris for Illumina library prep. Store at -20 °C until ready for library prep.

 

 

SECTION: DNA repair and end-prep
35. Thaw NEBNext repair and dA-tailing mixes and buffers from the Nanopore Companion Module. Mix buffers by vortexing and enzyme mixes by flicking. Spin down tubes and keep chilled on ice.

 

SECTION: DNA repair and end-prep
36. Add all non-reserved DNA (up to 40 µL) to a PCR tube. Dilute the HMW DNA with water to a final volume of 48.5 µL. Add 3.5 µL of FFPE DNA repair buffer, 3.5 µL of end-prep reaction buffer, 2 µL of FFPE DNA repair mix, and 3 µL of end-prep reaction mix to the tube. Mix tube with gentle flicking (or very gentle pipetting with a cut-off P200 tip).
 

 

SECTION: DNA repair and end-prep
37. In a thermal cycler, incubate at 20 °C for 60 min then 65 °C for 30 min. After this, sample can be held at 4 °C temporarily until ready to proceed.

SECTION: DNA repair and end-prep
38. Using a cut-off P200 tip (a wide bore will be too small to fit in the PCR tube), gently transfer sample to a 1.5 mL DNA LoBind tube. Add 60 µL AMPure beads. Using a wide-bore P200 tip, quickly but gently mix the tube.

 
 
  



SECTION: DNA repair and end-prep
39. Allow the sample to sit at least 5 min at Room temperature to allow the beads to bind the DNA. Meanwhile, prepare 500 µL fresh 70% ethanol with nuclease-free water.

SECTION: DNA repair and end-prep
40. Pellet the beads by placing the tube onto a magnetic tube rack for 5 min or until sample has completely cleared.
 



SECTION: DNA repair and end-prep
41. Pipette off the supernatant, taking care not to disturb the bead pellet.

SECTION: DNA repair and end-prep
42. Add 175 µL of 70% ethanol. Pipette slowly, with the tip touching the front wall of the tube, so that the pellet is not disturbed.
SECTION: DNA repair and end-prep
43. Pipette off the supernatant, taking care not to disturb the DNA pellet.
SECTION: DNA repair and end-prep
44. Wash the pellet once more with 70% ethanol . 

SECTION: DNA repair and end-prep
45. Briefly spin the sample tube down and place back onto the magnet. Remove any remaining drops on the bottom of the tube with a P10.
 

SECTION: DNA repair and end-prep
46. Immediately resuspend bead pellet in 31 µL nuclease-free water.

SECTION: DNA repair and end-prep
47. Incubate the tube on the heat block at 50 °C for 30 min. Every 5 minutes, gently flick the tube to encourage any settled beads to resuspend.

SECTION: DNA repair and end-prep
48. Briefly spin down the tube to collect condensation then place tube back on the magnetic rack. Allow tube to sit for
 5 min or until sample has completely cleared.

SECTION: DNA repair and end-prep
49. With a cut-off P200 tip, transfer 31 µL eluate to a fresh 1.5 mL LoBind tube.
  


SECTION: DNA repair and end-prep
50. Check for recovery with Qubit using 1 µL of sample. Recovery should be at least 150 ng DNA at this point.

SECTION: Adapter ligation
51. Thaw AMXII, T4 ligase, LNB, and LFB from the NEBNext Nanopore Companion Module and the Nanopore LSK110 kit. Mix AMXII, T4 ligase, and LFB by flicking. Mix LNB by pipetting. Briefly spin the tubes down and keep chilled on ice.
 

  


SECTION: Adapter ligation
52. Add 30 µL prepared DNA sample, 2.5 µL AMX, and 5 µL T4 ligase to a fresh 1.5 mL DNA LoBind tube. Gently flick the tube to mix.
 

 

 

 


SECTION: Adapter ligation
53. Add 12.5 µL LNB to the sample. Working quickly, mix by gentle pipetting with a wide-bore tip. DNA precipitation is normal, but if the DNA precipitates before you finish mixing it will stick to your pipette tip and you will lose a significant amount of library.

  

 

SECTION: Adapter ligation
54. Incubate the reaction mixture at Room temperature for 30 min. 

SECTION: Adapter ligation
55. Add 20 µL AMPure beads and mix gently by flicking until uniform in color. Let sample sit for at least 5 min, with occasional gentle mixing if necessary, so the DNA can bind to the beads.

  

SECTION: Adapter ligation
56. Pellet the beads by placing the tube onto a magnetic tube rack for 5 min or until sample has completely cleared.

 

SECTION: Adapter ligation
57. Pipette off the supernatant, being careful not to disturb the bead pellet.
SECTION: Adapter ligation
58. Remove tube from magnetic rack and add 100 µL of LFB to the tube, flicking to mix.

  
 



SECTION: Adapter ligation
59. Pellet the beads by replacing the tube onto the magnetic tube rack for 5 min or until sample has completely cleared.
 


SECTION: Adapter ligation
60. Being careful not to disturb the pellet, pipette off all the supernatant.
SECTION: Adapter ligation
61. Remove the tube from the magnet and briefly spin down. Replace tube onto magnet and remove any remaining drops of supernatant with a P10.
SECTION: Adapter ligation
62. Wash the pellet with LFB one more time.   

SECTION: Adapter ligation
63. Resuspend beads in 26 µL EB.
 


SECTION: Adapter ligation
64. Incubate beads on the heat block at 37 °C for 15 min. Every 5 minutes, gently flick the tube to resuspend any settled beads. 

SECTION: Adapter ligation
66. Place sample tube onto magnetic rack for at least 5 min or until sample has cleared.

 

SECTION: Adapter ligation
67. Using a cut-off P200 tip, transfer 26 µL eluate into a fresh 1.5 mL LoBind tube.

  


SECTION: Adapter ligation
68. Quantify library concentration with Qubit using 1 µL of the prepared library.

SECTION: Tips for sequencing the library
69. Thaw 1 tube sequencing buffer SBII (SQK-LSK110), 1 tube loading solution LS (SQK-LSK110), 1 tube flush buffer FB (EXP-FLP002), and 1 tube flush tether FLT (EXP-FLP002). Mix SBII, LS, and FB by flicking. Mix FLT with a pipette. Spin down and keep reagents on ice until ready to sequence.
 

  

SECTION: Tips for sequencing the library
70. With a cut off P200 tip, transfer at least 100 ng of prepared library to a fresh 1.5mL LoBind tube. This should not exceed 25 µL in volume (i.e. the volume of the eluate from Step 64).
 

  


SECTION: Tips for sequencing the library
71. Add the volume of LS that will result in a total volume of 25 µL in the LoBind tube. For example, if 15 µL of prepared library was initially added to the tube, add 10 µL of LS for a total 25 µL library + LS.

  


SECTION: Tips for sequencing the library
72. Add 25 µL SBII to the tube for a total volume of 50 µL.

 

  

SECTION: Tips for sequencing the library
73. Follow the official instructions to prime the flow cell, then add the prepared library to the flow cell. When loading the library, be sure to use a wide-bore pipette tip. Gently pipette mix the library before loading to ensure even distribution of the library across the flow cell membrane.
 

SECTION: Tips for sequencing the library
74. Over the course of a sequencing run, pores will get clogged and become inactive. If you have enough library, we recommend to flush the flow cell at 10-14 hour intervals to make these pores available again. 
 

SECTION: Tissue lysis
8. Add 7.5 µL 20 mg/mL Proteinase K, 7.5 µL 10% SDS, and 2 µL 20 mg/mL RNAse A to the tube. Gently flick and swirl to mix.

  
  
  

SECTION: Tissue lysis
9. Incubate tube for 120 min to 240 min at 50 °C. Gently mix tube every 45 min.
 


SECTION: (Optional) Hydration of ethanol-fixed tissue
5. Use a pipette to remove as much of the liquid as possible, but do not dry the fly. 
SECTION: Adapter ligation
65. Briefly spin down the tube to collect condensation.