DNA extraction and Nanopore library prep from single wild-caught drosophilids

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 tube.

Add 500µL Buffer STE to the tube with the flies.

Incubate on a platform rocker or rotator for 0h 15m 0s at low to medium speed.

Replace the solution with 500µL of fresh Buffer STE and incubate for another 0h 15m 0s on the rocker at low to medium speed.

Transfer the fly to a 1.5 mL LoBind tube.

Add 283µL lysis buffer to the tube with the fly.

Using a pellet pestle, manually homogenize the fly tissue.

Equipment

Value	Label
Pellet Pestle	NAME
Kimble Kontes	BRAND
K749520-1590	SKU
1.5 mL Polypropylene w/o Microtubes RNAse DNase free	SPECIFICATIONS

Spin down 1 phase lock gel tube per sample at 15000x g,0h 0m 0s for 0h 0m 30s .

Equipment

Value	Label
5PRIME Phase Lock Gel tube, light, 2mL	NAME
Quantabio	BRAND
2302820	SKU
Light	SPECIFICATIONS

Briefly spin down the homogenate/lysis solution, then transfer to the phase lock gel tube by decanting or pipetting with a wide-bore tip.

Add an equal volume (about 350µL) of Tris-saturated phenol chloroform isoamyl alcohol (PCI) to the phase lock tube.

Mix by placing tubes on a rocker at medium speed for 0h 8m 0s .

Centrifuge the phase lock tube at 16000x g,0h 0m 0s for 0h 8m 0s. Phase lock layer should now separate aqueous and organic layers.

Perform one more PCI extraction: .

Decant aqueous (top) layer into the phase lock tube. Add an equal volume (usually 350µL) of chloroform to the tube.

Mix by placing tubes on a rocker at medium speed for 0h 8m 0s .

Centrifuge the phase lock tube at 16000x g,0h 0m 0s for 0h 8m 0s. Phase lock layer should now separate aqueous and organic layers.

Quickly decant the aqueous (top) layer into a fresh 1.5 mL LoBind tube.

Equipment

Value	Label
DNA LoBind tubes, 1.5 mL	NAME
Tubes	TYPE
Eppendorf	BRAND
022431021	SKU
1.5 mL	SPECIFICATIONS

Chill 100% ethanol on ice and make 500µL of fresh 70% ethanol using nuclease-free water.

Add 0.1 volume (typically 30µL) of 3M sodium acetate to the extract from Step 24. Gently swirl to mix.

Add 2-2.5 volumes (typically 675µL) 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.

Centrifuge the tube at 5000x g,0h 0m 0s for 0h 5m 0s to pellet the DNA.

While being careful not to disturb the pellet, pipette off the ethanol.

Add 200µL of 70% ethanol to wash the DNA. Gently swirl to mix.

Centrifuge the tube at 5000x g,0h 0m 0s for 0h 2m 0s.

While being careful not to disturb the pellet, pipette off the ethanol.

Wash the pellet once more: .

Allow the tube to air dry no longer than 0h 1m 0s. Do not over-dry .

Resuspend in 42µL 10mM Tris and incubate at 50°C for 1h 0m 0s.

Briefly spin down tube to gather any condensation and store at 4°C.

Pipette mix slowly 5X with a P200 tip to ensure proper resuspension.

Check sample concentration and quality of 1µL aliquots using Qubit and Nanodrop.

Reserve at least 20ng gDNA in 6µL Tris for Illumina library prep. Store at -20°C until ready for library prep.

Thaw NEBNext repair and dA-tailing mixes and buffers from the Nanopore Companion Module. Mix buffers by vortexing and mixes by flicking. Spin down tubes and keep chilled on ice.

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

In a thermal cycler, incubate at 20°C for 1h 0m 0s then 65°C for 0h 30m 0s. After this, sample can be held at 4°C temporarily until ready to proceed.

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 60µL AMPure beads. Using a wide-bore P200 tip, quickly but gently mix the tube.

Allow the sample to sit at least 0h 5m 0s at Room temperature to allow the beads to bind the DNA. Meanwhile, prepare 500µL fresh 70% ethanol with nuclease-free water.

Pellet the beads by placing the tube onto a magnetic tube rack for 0h 5m 0s or until sample has completely cleared.

Equipment

Value	Label
Magnetic 1.5 mL tube rack	NAME
Any	BRAND
NA	SKU

Pipette off the supernatant, taking care not to disturb the bead pellet.

Add 175µL of 70% ethanol. Pipette slowly, with the tip touching the front wall of the tube, so that the pellet is not disturbed.

Pipette off the supernatant, taking care not to disturb the DNA pellet. Make sure all the supernatant is removed and only the pellet remains.

Wash the pellet once more with 70% ethanol

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.

Equipment

Value	Label
Magnetic 1.5 mL tube rack	NAME
Any	BRAND
NA	SKU

Immediately resuspend bead pellet in 31µL nuclease-free water.

Incubate the tube on the heat block at 50°C for 0h 30m 0s. Every 5 minutes, gently flick the tube to encourage any settled beads to resuspend.

Briefly spin down the tube to collect condensation then place tube back on the magnetic rack. Allow tube to sit for

0h 5m 0s or until sample has completely cleared.

With a cut-off P200 tip, transfer 31µL eluate to a fresh 1.5 mL LoBind tube.

Equipment

Value	Label
DNA LoBind tubes, 1.5 mL	NAME
Tubes	TYPE
Eppendorf	BRAND
022431021	SKU
1.5 mL	SPECIFICATIONS

Check for recovery with Qubit using 1µL of sample. Recovery should be at least 150ng DNA at this point.

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.

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

Add 12.5µL 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.

Incubate the reaction mixture at Room temperature for 0h 30m 0s.

Add 20µL AMPure beads and mix gently by flicking until uniform in color. Let sample sit for at least 0h 5m 0s so the DNA can bind to the beads.

Pellet the beads by placing the tube onto a magnetic tube rack for 0h 5m 0s or until sample has completely cleared.

Equipment

Value	Label
Magnetic 1.5 mL tube rack	NAME
Any	BRAND
NA	SKU

Pipette off the supernatant, being careful not to disturb the bead pellet.

Remove tube from magnetic rack and add 100µL of LFB to the tube, flicking to mix.

Safety information

DO NOT USE ETHANOL TO WASH PREPARED LIBRARY. It will denature the motor protein.

Pellet the beads by replacing the tube onto the magnetic tube rack for 0h 5m 0s or until sample has completely cleared.

Equipment

Value	Label
Magnetic 1.5 mL tube rack	NAME
Any	BRAND
NA	SKU

Being careful not to disturb the pellet, pipette off all the supernatant.

Remove the tube from the magnet and briefly spin down. Replace tube onto magnet and remove any remaining drops of supernatant with a P10.

Wash the pellet with LFB one more time.

Resuspend beads in 26µL EB.

Incubate beads on the heat block at 37°C for 0h 15m 0s. Briefly spin down the tube to collect condensation.

Place sample tube onto magnetic rack for at least 0h 5m 0s or until sample has cleared.

Equipment

Value	Label
Magnetic 1.5 mL tube rack	NAME
Any	BRAND
NA	SKU

Using a cut-off P200 tip, transfer 26µL eluate into a fresh 1.5 mL LoBind tube.

Equipment

Value	Label
DNA LoBind tubes, 1.5 mL	NAME
Tubes	TYPE
Eppendorf	BRAND
022431021	SKU
1.5 mL	SPECIFICATIONS

Quantify library concentration with Qubit using 1µL of the prepared library.

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.

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

Note

These libraries tend to be pretty fragmented, with read N50s ranging from 2-10kbp. More than 100 ng library is needed for longer N50 libraries, but it often helps to have 2 library loads so that a flush & reload can be used to improve throughput. So this part is a bit of a guessing game that depends on library yield and sample quality.

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

Add 25µL SBII to the tube for a total volume of 50µL.

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.

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.