TITLE: DNA extraction and Nanopore library prep from single flies
AUTHORS: Bernard Y Kim, Hannah Gellert

[DESCRIPTION]
This protocol is optimized for rapid and cost-effective (about $150) genome assembly of Drosophila species from single flies using ONT PromethION sequencers. Following this protocol, a typical Drosophila Nanopore sequencing run should have read N50 of 5-20kbp. Sequencing is halted at about 40-60X depth of coverage (10-14 Gbp on MinKNOW for most species, assuming ~20% of data is removed by a quality filter).

[BEFORE_START]
This protocol is for DNA extraction from whole Drosophila. Before starting the protocol, individual flies are collected into 95% ethanol or other nucleic acid preservation liquid. We have sequenced flies shipped through the postal service (7 days in transit) without any major issues. Flies should ideally be preserved less than 6 months ago but the protocol has worked for 20 year old samples.



[GUIDELINES]
This protocol is used to prepare 10-1000 ng of Nanopore library from a single reaction. The amount loaded onto the flow cell depends on the quality of the library. Larger amounts of longer libraries should be loaded to keep the molar concentration of adapted ends consistent. However, longer libraries tend to clog the flow cell more quickly, necessitating frequent DNase flushing and reloading and reducing throughput. Two libraries with the same N50 but where one has a larger number of >100kb fragments will sequence differently.

Ballpark estimates of R10.4.1 library loads maintaining good pore occupancy are:
Read N50 1kb: 10-15 ng library
Read N50 5kb: 25 ng library
Read N50 10kb: 50 ng library
Read N50 20kb: 100 ng library
Read N50 30kb: 200 ng library
Read N50 40kb+: 300 ng library

To maximize read lengths, one should not wait until all active pores have been depleted to flush and reload. A DNase flush should take place as soon as sequencing throughput starts to decrease, or about every 8 hours. A flow cell with loaded library can be stored at 4C overnight with no ill effects. 

[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 flies to a 1.5 mL tube.
SECTION: (Optional) Hydration of ethanol-fixed tissue
2. Add 300 µL Buffer STE to the tube with the flies. 
 

SECTION: (Optional) Hydration of ethanol-fixed tissue
3. Incubate at room temperature for at least 15 min.
SECTION: Tissue homogenization
5. Add 1 fly to a LoBind tube

 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
SECTION: Lysis
8. Incubate lysis tube at 50 °C for 180 min. Mix the tube with gentle rocking and inversion, until solution appears relatively homogeneous, at 30 min intervals.


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

 

 

SECTION: Phenol chloroform extraction
11. Transfer the homogenate/lysis solution to the phase lock gel tube by pipetting with a wide-bore tip.
SECTION: Phenol chloroform extraction
12. Add an equal volume (about 200 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 10000 x g for 8 min. Phase lock layer should now separate aqueous and organic layers.

SECTION: Phenol chloroform extraction
16. Add an equal volume (usually 200 µL) of chloroform to the tube.
 

 

SECTION: Phenol chloroform extraction
18. Centrifuge the phase lock tube at 15000 x g for 8 min. Phase lock layer should now separate aqueous and organic layers.

SECTION: Phenol chloroform extraction
17. Mix by placing tubes on a rocker at medium speed for 8 min .
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 per sample of fresh 70% ethanol using nuclease-free water.

SECTION: DNA precipitation, wash, and resuspension
21. Add 0.1x volume (typically 20 µL) of 3M sodium acetate to the extract from Step 19. Gently tap to mix.

  

SECTION: DNA precipitation, wash, and resuspension
22. Add 2-2.5x volumes (typically 440 µL) of cold 100% ethanol to the tube, and mix with careful swirling and gentle rocking. 
 

 


SECTION: DNA precipitation, wash, and resuspension
23. Centrifuge the tube at 10000 x g for 10 min. 

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
28. Wash the pellet once more:  .

SECTION: DNA precipitation, wash, and resuspension
31. Allow the DNA to air dry right until the moment it becomes translucent (usually 2 min). Do not over-dry the pellet.
 


SECTION: DNA precipitation, wash, and resuspension
32. Resuspend in 30uL of   and incubate at 50 °C for at least 60 min.

 

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

SECTION: DNA resuspension
34. Keep the DNA at 4C for at least, one night depending on previous pellet size. It could be left to resuspend for even 1 week to obtain proper resuspension if need be.
 

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


SECTION: DNA repair and end-prep
46. Thaw NEBNext repair and DNA-tailing mixes and buffers from the Nanopore Companion Module. Vortex buffers and flick mixes after thawing. Spin down tubes and keep chilled on ice.

 

SECTION: DNA repair and end-prep
47. Add 1.75 µL of FFPE DNA Repair Buffer, 1.75 µL of End-Prep Reaction Buffer, 1 µL of FFPE DNA Repair Mix, and 1.5 µL of End-Prep Reaction Mix to a PCR tube. Add remaining 24 µLof DNA sample to the PCR tube using a cut-off P200 tip. Mix tube with gentle flicking (or very gentle pipetting with the cut-off P200 tip), and then briefly spin down. 
 

 

 


SECTION: DNA repair and end-prep
48. 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: Adapter ligation
63. Thaw AMXF, Quick T4 ligase, LNB, and LFB from the NEBNext Nanopore Companion Module and the Nanopore LSK110 kit. Mix AMXF, Quick T4 ligase, and LFB by flicking. Mix LNB by pipetting. Briefly spin the tubes down and keep chilled on ice.
 


 


SECTION: Adapter ligation
64. Add 30 µL prepared DNA sample (the extra 1 µL can be used to Qubit), 2.5 µL AMXF, and 5 µL Quick T4 ligase to a fresh 1.5 mL DNA LoBind tube. Gently flick the tube to mix.
 

 

 


SECTION: Adapter ligation
65. 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
66. Incubate the reaction mixture at room temperature for 20 min. 

SECTION: Adapter ligation
67. Add 20 µL of AmPure XP beads and mix quickly with wide-bore tip.

 
 
SECTION: Adapter ligation
68. Incubate at room temperature for 20 min.

SECTION: Adapter ligation
72. Add 95 µL of LFB to the tube. SFB or a 1:1 dilution of PB can be used here.
 



 

Lightly tap the tube to encourage adapter on the beads to come off, but not necessarily for beads to resuspend. 


SECTION: Adapter ligation
73. Being careful not to disturb the pellet, pipette off all the supernatant.
SECTION: Adapter ligation
74. Wash again using 105 µL of LFB. Pipette LFB on to the beads more quickly to get the pellet off the side of the tube. Lightly tap the tube to mix but not fully resuspend. 

SECTION: Adapter ligation
76. Resuspend pellet in 21 µL EB for R9.4.1 sequencing or in 30 µL for R10.4.1 sequencing.
 


SECTION: Adapter ligation
77. Incubate library on the heat block at 34 °C for 60 min. Briefly spin down the tube to collect condensation then incubate for at least 2880 min before the next step.

SECTION: (Optional) Library size selection with SRE buffer
79. Quantify library concentration using 1 µL of the prepared library with Qubit. This step should not be performed unless library concentration is greater than 40 ng/uL. If the concentration is greater than 100ng/uL the library should be diluted to improve size selection performance.

SECTION: (Optional) Library size selection with SRE buffer
80. Add an equal volume (20 µL) of SRE XL buffer to the library and gently pipette mix using a wide-bore tip.
 

 


SECTION: (Optional) Library size selection with SRE buffer
81. Centrifuge at 10000 x g, 30 min.

SECTION: (Optional) Library size selection with SRE buffer
82. Pipette off the supernatant, being careful not to disturb the DNA pellet at the bottom of the tube.
 

SECTION: (Optional) Library size selection with SRE buffer
83. Add 100 uL of LFB, SFB, or 1:1 diluted PB (similar to step 46) to wash the pellet. It does not really matter which one is used.

 


 

 

SECTION: (Optional) Library size selection with SRE buffer
84. Centrifuge tube at 10000 x g for 2 min.

SECTION: (Optional) Library size selection with SRE buffer
85. Being careful not to disturb the pellet, pipette off all the supernatant.
SECTION: (Optional) Library size selection with SRE buffer
86. Repeat wash:   

SECTION: (Optional) Library size selection with SRE buffer
87. Resuspend pellet in 21 µL EB for R9.4.1 sequencing or in 30 µL for R10.4.1 sequencing.
 


SECTION: (Optional) Library size selection with SRE buffer
88. Incubate the tube on the heat block at 37 °C  for 60 min. Briefly spin down the tube to collect condensation, and incubate at least 2880 min at 4 °C before sequencing.

SECTION: Tips for sequencing the library- R9.4.1
90. Quantify the concentration of 1 µL library with Qubit. We usually end up with 1000 ng - 2000 ng of total library at this stage.

SECTION: Tips for sequencing the library- R9.4.1
91. With a cut off P200 tip, transfer about 350 ng of prepared library to a fresh 1.5mL LoBind tube. This should not exceed 35 µL in volume.
 

 




SECTION: Tips for sequencing the library- R9.4.1
92. Add an equal volume of SQB to the tube. Then, add FB from the marked tube (the one that we are not going to prepare the priming mix with) to a final volume of 70 µL.

For example, if 10 µL of 35 1286 library was transferred in step 77, add 10 µL of SQB and 50 µL FB to the tube.
 

 


SECTION: Tips for sequencing the library- R9.4.1
89. Thaw 1 tube SQB (SQK-LSK109), 2 tubes FB (EXP-FLP002), and 1 tube FLT (EXP-FLP002). Mix SQB and FB by flicking. Mix FLT with a pipette. Keep reagents on ice until ready to sequence.
 

 

SECTION: Tips for sequencing the library- R9.4.1
93. 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- R9.4.1
94. Over the course of a sequencing run, pores will get clogged and become inactive. It is essential to flush the flow cell at 10-14 hour intervals to make these pores available again. We recommend Nanopore's Flow Cell Wash Kit (EXP-WSH003).
 

SECTION: Tissue homogenization
4. Prepare   ,   ,   , and   
SECTION: Tissue homogenization
4.1. For   :
5 mL  
2 mL0.5 Molarity (M) 
0.292 g
43 mL 
SECTION: Tissue homogenization
4.2. For   :
5 g SDS
50 mL Distilled H20
SECTION: Tissue homogenization
4.3. For   :
2.461 g 
10 mL 
SECTION: Tissue homogenization
4.4. Per 1 fly, prepare    :
250 µL 
3.13 µL20 mg/mL   (final concentration of 561  μg/mL)
5 µL2 mg/mLfinal concentration of 0.28% SDS)
SECTION: Tissue homogenization
6. Homogenize flies with NEB pestle, working quickly to avoid endogenous nuclease digestion of DNA. Add 200 µL to the LoBind tube and mix thoroughly using the pestle.
 


SECTION: DNA precipitation, wash, and resuspension
25. Add 175 µL of 70% ethanol.
SECTION: DNA precipitation, wash, and resuspension
26. Spin at 12500 x g for 5 min.
SECTION: DNA precipitation, wash, and resuspension
27. Being careful not to disturb the DNA pellet, remove the ethanol. 
SECTION: DNA precipitation, wash, and resuspension
29. Spin at 12500 rcf for 1 min.
SECTION: DNA precipitation, wash, and resuspension
30. Using a 10uL pipette, remove any excess ethanol.
SECTION: Bead Clean Up
49. Prepare 250 µL of 80% ethanol per sample.

SECTION: Bead Clean Up
50. Transfer sample from PCR tube to a LoBind tube using a cut-off P200 tip. 
SECTION: Bead Clean Up
51. Add equal volume of AmPure XP beads (normally 30 µL ) to the sample. Immediately use a P200 wide bore tip to mix 5x.
 

 
  

SECTION: Bead Clean Up
52. Incubate at room temperature for 20 min.

SECTION: Bead Clean Up
53. Place the LoBind tube on a magnetic rack and wait until solution is clear and the beads are pelleted.
SECTION: Bead Clean Up
54. Remove the supernatant by placing pipette tip on the wall of the LoBind tube opposite of the beads. Pipette very slowly to ensure no DNA is pulled off. 
 

SECTION: Bead Clean Up
55. Wash by adding 100 µL of 80% ethanol (enough to cover the beads on the wall of the LoBind tube). 

 

SECTION: Bead Clean Up
56. Remove and discard ethanol. 
SECTION: Bead Clean Up
57. Wash again by adding 115 µL of 70% ethanol. 

SECTION: Bead Clean Up
58. Remove and discard ethanol.
 

SECTION: Bead Clean Up
59. Resuspend sample in 32 µL of nuclease-free water. 

SECTION: Bead Clean Up
60. Place tube on heat block at 50 °C until the pellet has dissolved. 
 


SECTION: Bead Clean Up
61. Place LoBind tube on magnet rack until solution is clear. 
SECTION: Bead Clean Up
62. Using a cut off P200 tip, remove the supernatant containing the aqueous DNA. 
 

 

SECTION: Short Read Elimination 1
36. If sample concentration is greater than 40ng/uL, add equal volume (usually around 30 µL) SRE XL buffer. Using a wide-bore P200 tip, quickly but gently mix the tube. The precipitation buffer described here can be used in place of the SRE buffer but is not as effective at removing small DNA fragments as SRE.

 

 

  

SECTION: Short Read Elimination 1
37. Centrifuge the sample at 10000 x g for 30 min or until DNA has pelleted and solution is no longer viscous. Meanwhile, prepare 500 µL fresh 70% ethanol with nuclease-free water.

SECTION: Short Read Elimination 1
38. Pipette off the supernatant, taking care not to disturb the DNA pellet.

 

SECTION: Short Read Elimination 1
39. Add 150 µL of 70% ethanol. Pipette slowly, with the tip touching the front wall of the tube, so that the pellet is not disturbed.
SECTION: Short Read Elimination 1
40. Centrifuge at 10000 x g for 2 min.

SECTION: Short Read Elimination 1
41. Pipette off the supernatant, taking care not to disturb the DNA pellet. Make sure all the supernatant is removed and only the pellet remains.
SECTION: Short Read Elimination 1
42. Repeat wash:   

 

SECTION: Short Read Elimination 1
44. Resuspend pellet in 25 µL EB.
 


SECTION: Short Read Elimination 1
45. Incubate the tube on the heat block at 50 °C for at least 60 min. Briefly spin down the tube to collect condensation. Incubate for at least 2880 min at 4 °C.

 

SECTION: Short Read Elimination 1
43. Briefly spin sample and use a P10 to remove any remaining ethanol.
SECTION: Adapter ligation
69. Place tubes on magnetic and wait for solution to clear. 
SECTION: Adapter ligation
70. On the magnet, use a cut-off P200 tip to pull the supernatant off the beads very slowly, then dispense the supernatant back onto the bead pellet slowly. Let the sample sit on the magnet for a few minutes. 
SECTION: Adapter ligation
71. Pipette off supernatant with a normal pipette tip, pipetting from the front of the tube away from the pellet. 
SECTION: Adapter ligation
75. While on magnet remove LFB. Briefly spin and use a P10 pipette to remove any remaining excess of LFB.
SECTION: Adapter ligation
78. Place sample on magnet wait until solution is clear. Use a cut off P200 tip to remove sample from beads and place in a new 1.5mL Lo Bind tube. 
SECTION: Tips for sequencing the library-R10.4.1
95. Thaw 1 tube SB (LSK110), 1 tube LIS, 1 tube of FCF per sample, and 1 tube FCT. Mix SQB and FB by flicking. 

SECTION: Tips for sequencing the library-R10.4.1
96. Warm the FCF at 37 °C for 30 min

SECTION: Tips for sequencing the library-R10.4.1
97. Add 30 µL FCT to FCF and pipette 10x to ensure thorough mixing

 

SECTION: Tips for sequencing the library-R10.4.1
98. Follow the official instructions to prime the flow cell.
SECTION: Tips for sequencing the library-R10.4.1
99. While the flow cell is priming, prepare the library by adding 70uL of LIS, 30uL library (LIS and library should total to 100uL), and 100uL SB. Lightly tap to mix until swirls disappear but wait to pipette mix until just before loading. 
SECTION: Tips for sequencing the library-R10.4.1
100. Pipette mix prepared library 2x times and then following official instructions to load the flow cell. 
SECTION: Tips for sequencing the library-R10.4.1
101. Over the course of a sequencing run, pores will get clogged and become inactive. It is essential to flush the flow cell at 10-14 hour intervals to make these pores available again. We recommend Nanopore's Flow Cell Wash Kit (EXP-WSH00).
 

SECTION: Tissue homogenization
7. 
4 µL20 mg/mL  (final concentration of 186 μg/mL)
SECTION: Lysis
9. 
SECTION: Phenol chloroform extraction
15. Repeat Phenol-Chloroform extraction: