TITLE: DNA extraction and Nanopore library prep from 15-30 whole flies- V.3.2
AUTHORS: Bernard Y Kim, Hannah Gellert

[DESCRIPTION]
This protocol is optimized for rapid and cost-effective (about $150) genome assembly of Drosophila species from laboratory lines using ONT PromethION sequencers. Following this protocol, a typical Drosophila Nanopore sequencing run should have read N50 of 20-40kbp. 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, 10-40 whole male flies should be starved for 1 day then quickly frozen in a -80 freezer. If flies must be transported but shipping on dry ice is an issue, preserve flies in 95% ethanol. We have sequenced flies shipped through the postal service (7 days in transit) without any major issues. While yield of the very long fragments (>100kb) will be smaller, it is not critical to have pristine samples for good Nanopore runs. Flies should ideally be preserved less than 6 months ago.

Initially, we utilized nuclear extractions thinking that it was critical to maximize the quality of extracted gDNA for Nanopore sequencing. However we realized that this required too many flies, making sample prep difficult and increasing haplotype diversity in the library. Additionally and somewhat counterintuitively, library prep will be less effective when DNA is too long. Therefore, some shearing of very high quality gDNA is essential. Given these issues, we now extract gDNA from a smaller number of whole flies and this is more than sufficient for the preparation of multiple Nanopore libraries and a short read prep.

The amount of starting material required can vary depending on the size of the fly and sample quality. For fresh, flash-frozen flies, we usually start with 30 D. melanogaster-sized flies for a yield of about 5ug of HMW gDNA. Half of that is used for library prep, so an effective library prep could certainly be done with 15 and possibly even fewer flies. For larger species like Hawaiian Drosophila, we have used as few as 6 flies. For these specimens, DNA yield is improved by clipping off the wings before running this protocol.



[GUIDELINES]
This protocol is used to prepare ~1-2 ug 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 1.0 mL 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 25-50 flies to a LoBind tube

 
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 
SECTION: Lysis
7. Incubate lysis tube at 50 °C for 180 min to 240 min. Mix the tube with gentle rocking and inversion, until solution appears relatively homogeneous, at 45 min intervals.

 


 

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

 

 

SECTION: Phenol chloroform extraction
9. Transfer the homogenate/lysis solution to the phase lock gel tube by pipetting with a wide-bore tip.
SECTION: Phenol chloroform extraction
10. Add an equal volume (about 400 µL) of Tris-saturated phenol chloroform isoamyl alcohol (PCI) to the phase lock tube.
 

 

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

SECTION: Phenol chloroform extraction
12. 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
13. Add an equal volume (usually 400 µL) of chloroform to the tube.
 

 

SECTION: Phenol chloroform extraction
15. 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
14. Mix by placing tubes on a rocker at medium speed for 8 min .
SECTION: Phenol chloroform extraction
16. Quickly decant the aqueous (top) layer into a fresh 2.0 mL LoBind tube.
 

 

 

SECTION: DNA precipitation, wash, and resuspension
17. Chill 100% ethanol on ice and make 1 mL per sample of fresh 70% ethanol using nuclease-free water.

SECTION: DNA precipitation, wash, and resuspension
18. Add 0.1x volume (typically 50 µL) of 3M sodium acetate to the extract from Step 4.18. Gently swirl to mix.

  

SECTION: DNA precipitation, wash, and resuspension
19. Add 2-2.5x volumes (typically 1100 µL) of cold 100% ethanol to the tube, and mix with careful swirling and gentle rocking. 
 

 


SECTION: DNA precipitation, wash, and resuspension
20. Using a P200 pipette and a wide-bore tip, transfer the stringy clump to a fresh 1.5 mL LoBind tube.
 

 

SECTION: DNA precipitation, wash, and resuspension
23. Add 150 µL of 70% ethanol to wash the DNA. Gently swirl to mix.

SECTION: DNA precipitation, wash, and resuspension
24. Centrifuge the tube at 2000 x g for 5 min. 

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

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

SECTION: DNA precipitation, wash, and resuspension
32. Allow the DNA to air dry right until the moment it becomes translucent (usually 5 min). Do not over-dry the pellet.
 


SECTION: DNA precipitation, wash, and resuspension
33. Depending on fly size, resuspend in 31-90uL of   and incubate at 50 °C for at least 60 min.

 

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

SECTION: DNA resuspension
35. Keep the DNA at 4C for at least 1 week to obtain proper resuspension. Every ~48 hours, mix the DNA gently with a P1000 or P200 pipette with a normal tip. This will encourage DNA to resuspend and make sure it is adequately sheared for library prep. We usually mix three times:

1. 10X with a P1000
2. 5X with a P1000
3. 5X with a P200
 

If there is a lot of DNA that doesn't seem to be resuspending, shearing with a blunt-end needle may be necessary. When using a blunt-end needle, be sure to 'unstick' the plunger before using it on DNA. Aspirate DNA into the needle (not the plunger) 3x times maximum. 
SECTION: DNA resuspension
36. Check sample concentration and quality of 1 µL aliquots using Qubit and Nanodrop.
 


SECTION: DNA repair and end-prep
47. 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
48. 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 a PCR tube. Add remaining 47.5 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
49. 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
64. Thaw AMXF, Quick T4 Ligase, LNB, and LFB from the NEBNext Nanopore Companion Module and the Nanopore LSK110 kit or LA, Quick T4 Ligase and LNB from Nanopore LSK114 hit . Mix AMXF or LA, Quick T4 ligase, and LFB by flicking. Mix LNB by pipetting. Briefly spin the tubes down and keep chilled on ice.
 
  
  

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

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

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

 
 
SECTION: Adapter ligation
69. Incubate at room temperature for 5 min.

SECTION: Adapter ligation
73. 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
74. Being careful not to disturb the pellet, pipette off all the supernatant.
SECTION: Adapter ligation
75. 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
77. Resuspend pellet in 21 µL EB for R9.4.1 sequencing or in 30 µL for R10.4.1 sequencing.
 


SECTION: Adapter ligation
78. 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
80. 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
81. 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
82. Centrifuge at 10000 x g, 30 min.

SECTION: (Optional) Library size selection with SRE buffer
83. 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
84. 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
85. Centrifuge tube at 10000 x g for 2 min.

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

SECTION: (Optional) Library size selection with SRE buffer
88. 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
89. 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
91. 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
92. 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
93. 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 ng/μL 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
90. Thaw 1 tube SQB (SQK-LSK110), 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
94. 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
95. 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 mL1 Molarity (M) 
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 vial of flies (~25-50 flies), prepare    :
500 µL 
15 µL     (fina concentration of 561  μg/mL)
5 µL  (final concentration of 186 μg/mL)
15 µL (final concentration of 0.28% SDS)
SECTION: Tissue homogenization
6. Homogenize flies with NEB pestle, working quickly to avoid endogenous nuclease digestion of DNA. Add 500 µL to the LoBind tube and mix thoroughly using the pestle.
 


SECTION: DNA precipitation, wash, and resuspension
21. Add 150 µL (or enough to cover the DNA) of 70% ethanol.
SECTION: DNA precipitation, wash, and resuspension
22. Remove and discard the ethanol. Be careful not to discard any DNA.
SECTION: DNA precipitation, wash, and resuspension
26. Add 175 µL of 70% ethanol.
SECTION: DNA precipitation, wash, and resuspension
27. Spin at 10000 rcf for 1 min.
SECTION: DNA precipitation, wash, and resuspension
28. Being careful not to disturb the DNA pellet, remove the ethanol. 
SECTION: DNA precipitation, wash, and resuspension
30. Spin at 10000 rcf for 1 min.
SECTION: DNA precipitation, wash, and resuspension
31. Using a 10uL pipette, remove any excess ethanol.
SECTION: Bead Clean Up
50. Prepare 500 µL of 70% ethanol per sample.

SECTION: Bead Clean Up
51. Transfer sample from PCR tube to a LoBind tube using a cut-off P200 tip. 
SECTION: Bead Clean Up
52. Add equal volume of AmPure XP beads (normally 59.5 µL ) to the sample. Immediately use a P200 wide bore tip to mix 5x.
 

 
  

SECTION: Bead Clean Up
53. Incubate at room temperature for 5 min.

SECTION: Bead Clean Up
54. Place the LoBind tube on a magnetic rack and wait until solution is clear and the beads are pelleted.
SECTION: Bead Clean Up
55. 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
56. Wash by adding 190 µL of 70% ethanol (enough to cover the beads on the wall of the LoBind tube). 

 

SECTION: Bead Clean Up
57. Remove and discard ethanol. 
SECTION: Bead Clean Up
58. Wash again by adding 200 µL of 70% ethanol. 

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

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

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


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

 

SECTION: Short Read Elimination 1
37. Using a cut-off P200 tip (a wide bore will be too small to fit in the PCR tube), gently transfer 30 µL of sample to a 1.5 mL DNA LoBind tube. Add 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
38. 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
39. Pipette off the supernatant, taking care not to disturb the DNA pellet.

 

SECTION: Short Read Elimination 1
40. 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
41. Centrifuge at 10000 x g for 2 min.

SECTION: Short Read Elimination 1
42. 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
43. Repeat wash:   

 

SECTION: Short Read Elimination 1
45. Resuspend pellet in 48.5 µL EB.
 


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

 

SECTION: Short Read Elimination 1
44. Briefly spin sample and use a P10 to remove any remaining ethanol.
SECTION: Adapter ligation
70. Place tubes on magnetic and wait for solution to clear. 
SECTION: Adapter ligation
71. 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
72. Pipette off supernatant with a normal pipette tip, pipetting from the front of the tube away from the pellet. 
SECTION: Adapter ligation
76. While on magnet remove LFB. Briefly spin and use a P10 pipette to remove any remaining excess of LFB.
SECTION: Adapter ligation
79. 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
96. 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
97. Warm the FCF at 37 °C for 30 min

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

 

SECTION: Tips for sequencing the library-R10.4.1
99. Follow the official instructions to prime the flow cell.
SECTION: Tips for sequencing the library-R10.4.1
100. While the flow cell is priming, prepare the library by adding 70 µL of LIS,   library (LIS and library should total to 100uL), and 100 µL 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
101. 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
102. 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).