TITLE: Extraction of high molecular weight insect DNA for long-read sequencing
AUTHORS: Bérénice Lafon, Marie-Ka Tilak, Fabien L. Condamine

[DESCRIPTION]
The objective of this protocol is to extract insect DNA with minimal fragmentation to optimize long fragment sequencing with Oxford Nanopore Technology (ONT). The DNA manipulation and centrifugation steps have been minimized all along the protocol, which have also been simplified for implementing its application in the field. We have applied this protocol to a wide range of insects such as butterflies (Papilionidae [adults and larvae] and Nymphalidae), moths (Uraniidae), ants (Formicidae), beetles (Carabidae), and cicadas (Cicadidae). We have generally obtained on average 7 μg (ranging from 2 to 13 μg) of high-quality DNA for long-read sequencing. As the obtained DNA pellet is not purified during this extraction, it will require purification on beads before constructing the library. Such DNA extractions have further been successfully used for ONT sequencing on non-model insect species.

[STEPS]
SECTION: Step 1: Preparing and selecting the tissues
2. Depending on how the specimen has been sampled and preserved, one may adapt the DNA extractions as follows:
SECTION: Step 1: Preparing and selecting the tissues
2.3. If the tissue sample was preserved in RNAlater, pour the contents of the tube into a Petri dish. Blot the RNAlater onto a sterile pad, then place the tissue pieces into a 1.5 mL tube with 1 mL of PBS to wash out the RNAlater. Blot again on a sterile pad before placing the tissue in the mortar.
SECTION: Step 1: Preparing and selecting the tissues
2.2. If the specimen was preserved in ethanol, pour the contents of the tube into a Petri dish. Before placing the tissue in the mortar, lightly blot it with a sterile pad.
SECTION: Step 1: Preparing and selecting the tissues
2.1. If the specimen is alive or has been killed and stored at -80°C, dissect it in absolute ethanol in a Petri dish. Lightly blot the tissue and cut it into small pieces on a sterile compress before placing it in the mortar. 
SECTION: Step 1: Preparing and selecting the tissues
1. Select tissues for extraction (avoid cuticle, bristles, gut, defensive glands, eggs...). Give priority to muscles and respiratory system (tracheae).

 

SECTION: Step 2: Grinding the tissues
4. Place 500 μL lysis buffer (ATL from the Qiagen DNeasy Blood & Tissue Kit containing EDTA and SDS) in a mortar.
SECTION: Step 2: Grinding the tissues
5. Grind with a pestle for a few minutes until the tissue is well crushed.
SECTION: Step 2: Grinding the tissues
6. Recover all the crushed tissue with a P1000 pipette. If the grind is not sufficiently fluid, cut off the tip of the P1000 cone to facilitate collection. Transfer to a 2 mL tube.
SECTION: Step 3: Digesting the tissues
8. Add Proteinase K (included in the Qiagen DNeasy Blood & Tissue Kit) at 10% of the buffer volume (e.g. 50 µL Proteinase K to 500 µL buffer).
SECTION: Step 2: Grinding the tissues
7. Depending on the amount of tissue, adjust the volume by adding lysis buffer to completely submerge the tissue.
SECTION: Step 3: Digesting the tissues
9. Gently mix by swirling the tube (do not vortex).
SECTION: Step 3: Digesting the tissues
10. Add RNase A to digest the RNA. Qiagen recommends 4 μL RNase A for 200 μL (buffer + Proteinase K), so adjust the volume of RNase A accordingly (e.g. add 11 μL RNase A for 550 μL volume).
SECTION: Step 3: Digesting the tissues
11. Mix by gentle swirling of the tube (do not vortex). Centrifuge for a maximum of 3 seconds.
SECTION: Step 4: Precipitating DNA
13. Centrifuge at 11,000 g for 10 minutes at room temperature to pellet the remaining tissue.
SECTION: Step 4: Precipitating DNA
14. Collect the supernatant, avoiding any floating fat layer, and transfer to a fresh 2 mL tube. Tip: Pipet in 100 µL steps to determine the final volume of supernatant (e.g. you hope to pipet between 450 and 500 µL). 
SECTION: Step 3: Digesting the tissues
12. Incubate at 56°C (in a Eppendorf ThermoMixer F1.5 at 300 rpm; agitation is not mandatory) for 1 to 2 hours, depending on the progress of the digestion.
SECTION: Step 4: Precipitating DNA
15. Important for subsequent steps: If the volume of supernatant obtained exceeds 600 μL, separate this volume into two tubes.
SECTION: Step 4: Precipitating DNA
16. Add sodium acetate (3 M) to 10% of the supernatant volume (e.g. 50 μL for 500 μL supernatant).
SECTION: Step 4: Precipitating DNA
17. Mix gently by swirling the tube (do not vortex).
SECTION: Step 4: Precipitating DNA
18. Add absolute ethanol equal to twice the volume of the supernatant (e.g. 1,000 μL ethanol for 500 μL supernatant).
SECTION: Step 4: Precipitating DNA
19. Mix gently by inversion of the tube until the DNA pellet forms (approximately 20 inversions).
SECTION: Step 5: Washing DNA
21. If the DNA forms a pellet:
SECTION: Step 5: Washing DNA
21.1. Collect it and transfer it to a 1.5 mL tube containing 1 mL of 70% ethanol. Tip: Collect the DNA pellet by aspiration with a P1000 cone, taking care not to aspirate the pellet completely so as not to break it. Otherwise, remove the supernatant by gentle pipetting without aspirating the pellet.
 

SECTION: Step 5: Washing DNA
21.2. Gently mix by inverting the tube.
SECTION: Step 5: Washing DNA
21.3. Repeat the previous step a second time to make two washes with 70% ethanol.
SECTION: Step 5: Washing DNA
21.4. Remove the 70% ethanol by pipetting without aspirating the DNA pellet.
SECTION: Step 5: Washing DNA
21.5. Centrifuge for a maximum of 3 seconds.

SECTION: Step 5: Washing DNA
21.6. Remove the remaining ethanol with a P10 pipette.
SECTION: Step 5: Washing DNA
21.7. Allow to dry for 5 minutes with the tube open to allow the 70% ethanol to evaporate.

SECTION: Step 5: Washing DNA
22. If a DNA pellet is not present:
SECTION: Step 5: Washing DNA
22.1. Centrifuge the tube at 11,000 g for 10 minutes to pellet.
SECTION: Step 5: Washing DNA
22.2. Pipette off the supernatant. 
SECTION: Step 5: Washing DNA
22.3. Add 1 mL of 70% ethanol to wash the DNA pellet. Tap the tube to dislodge and resuspend.
SECTION: Step 5: Washing DNA
22.4. Remove the 70% ethanol and repeat the previous step to perform two washes.
SECTION: Step 5: Washing DNA
22.5. Remove the ethanol by pipetting without aspirating the DNA pellet.
SECTION: Step 5: Washing DNA
22.6. Centrifuge for a maximum of 3 seconds.

SECTION: Step 5: Washing DNA
22.7. Remove the remaining ethanol with a P10 pipette.
SECTION: Step 6: Eluting DNA
23. Resuspend the DNA pellet in water (between 100 μL and 200 μL, depending on the size of the pellet) to allow it to settle properly.

 

SECTION: Step 6: Eluting DNA
24. If the DNA pellet does not dissolve completely (presence of fat or protein), incubate at 37°C for 30 minutes with minimal agitation (Thermomixer).
SECTION: Step 6: Eluting DNA
25. Leave it overnight at room temperature.
SECTION: Step 6: Eluting DNA
26. The next day, if impurities remain, centrifuge for 5 minutes at 11,000 g and collect the supernatant. If any fat remains, pipette the supernatant, taking care not to collect the fat, and transfer to a new 1.5 mL tube.
SECTION: Step 7: DNA quality controls
27. Perform a Qubit BR DNA assay.
SECTION: Step 7: DNA quality controls
28. Load on a 1% agarose gel. A large band above 10 kb should normally be observed. Degraded RNAs (bands below 200 bp) are often left over for purification during library construction.
SECTION: Step 7: DNA quality controls
29. Purification with AMPure 1X beads. Make a Qubit BR and Nanodrop assays.

 

SECTION: Step 1: Preparing and selecting the tissues
3. Video showing the selection of tissues as in the case 2.1. This is a specimen of Carabus hispanus that was freshly collected and dissected in absolute ethanol. Here we see the selection of internal tissues (mostly muscles) after the specimen has been cut into two parts. The tissues are lightly blotted before being placed in the mortar.
 

SECTION: Step 4: Precipitating DNA
20. Video showing the formation of the DNA pellet as expected from a high molecular weight insect DNA.