TITLE: Manual extraction of High Molecular Weight DNA from single mosquitoes using the Qiagen MagAttract HMW DNA kit
AUTHORS: Fiona Teltscher, Harriet Johnson, Mara Lawniczak

[DESCRIPTION]
This is a protocol for the manual extraction of high molecular weight (HMW) DNA from insects. It uses the Qiagen MagAttract kit and factors in modifications described in the ChromiumTMGenome Reagent Kits User Guide pages 6-8 (https://support.10xgenomics.com/genome-exome/library-prep/doc/user-guide-chromium-genome-reagent-kit-v2-chemistry). It includes further modifications that benefited two particular needs. First, most reagents are halved per extraction (apart from beads) due to small specimen size (2 mg is a typical weight of an Anopheles mosquito, which is far less than the 25 mg of tissue the MagAttract kit can support). Second, due to this small specimen size, we need to maximize DNA yield, so we also perform a second elution to release more DNA.  

DNA resulting from this protocol can be further sheared and cleaned for successful PacBio HiFi sequencing. In our experience, a single fresh Anopheles gambiae mosquito weighing 2-3 mg yields about 600-800 ng of DNA (quantified using qubit HS DNA kit) using this protocol. Following shearing using G-tubes or the MegaRuptor and SPRI based clean up, we typically retain about 200 ng of sheared DNA, which is just about sufficient to reach 25x coverage PacBio HiFi of a 250 Mb genome. The quality and quantity of DNA is best when starting with a snap frozen from living specimen. However, we have also successfully extracted HMW DNA using this protocol from ethanol and DESS preserved specimens held at room temperature for as long as a week, as long as the specimen was punctured or gently squished to ensure rapid penetration of the preservative. See: Squishing insects for preservation of HMW DNA in the field  https://www.protocols.io/view/squishing-insects-for-preservation-of-hmw-dna-in-t-cyp3xvqn 

We normally perform up to 8 DNA extractions in parallel (this will also depend on which magnetic rack is being used). Please add a comment to let the wider community know if it has worked or not on your species. 

NB -- an automated version of this protocol that works on the Kingfisher Apex is also available here on protocols.io

[BEFORE_START]
Ensure all surfaces have been cleaned with 70-80% Ethanol (and ideally bleach before that). Have cleaning wipes for forceps. All kit components, buffers, and RNase A stock solution can be stored at room temperature (15–25°C) for up to 1 year. The box should be labelled with received date. Mix Buffer AL thoroughly by shaking before use. Buffers MW1 and PE are supplied as a concentrate. Before using for the first time, be sure to add the appropriate amount of ethanol (96–100%) as indicated on the bottle. Many components of the kit are also available from Qiagen separately.

[GUIDELINES]
This protocol is an adaptation of Chromium™Genome Reagent Kits User guide and Qiagen's MagAttract® HMW DNA Handbook. The Chromium protocol suggests the use of PBS at the beginning. Also, both SOPs recommend using a thermomixer for wash steps, but we find doing this by hand works fine. Finally, we use 1.5 mL microcentrifuge tubes because the plastic pestles that we use to grind insect tissue do not support effective grinding in the 2 mL microcentrifuge tubes recommended by both original protocols. There are, however, pestles available to use in 2 mL microcentrifuge tubes.

[STEPS]
SECTION: Procedure
1. Prepare an open insulated box of dry ice to store sample tubes on whilst working through steps 2-4.
SECTION: Procedure
2. Make mastermix of reagents for lysis.
 
Calculate the mastermix volumes for the number of samples plus 1 for spare pipetting volume. Volumes per sample are: 100 µL  ; 10 µL   (Mix by inverting the tube 5 times); 2 µL   ; 75 µL    (mix by inversion)



SECTION: Procedure
3. For each sample, add 187 µL of the mastermix from step 2 into a new 1.5 mL DNA LoBind tube.
 

SECTION: Procedure
4. Carefully remove the   from the sample tube using clean forceps. If the   has been stored in a preservation liquid, lightly make contact on a clean piece of tissue to remove surface liquid from the sample. Submerge the   into the mastermix in a tube (see step 3) with clean forceps. Insert a sterile pestle in the tube and smash, smear, squash, twist, grind the tissue against the wall of the tube for 1 min. There should be no recognisable body parts visible following pestle smashing, only flakes. Place the sample in a tube rack on the bench. Clean forceps with 100% ethanol.
 
      

SECTION: Procedure
5. Repeat step 4 for the remaining samples.
SECTION: Procedure
6. Briefly spin all samples in a minicentrifuge or similar to collect solution at bottom before next step.
SECTION: Procedure
7. Incubate the sample at 25 °C for 120 min.

SECTION: Procedure
8. Briefly spin samples to collect solution at the bottom of the tube.
SECTION: Procedure
9. Vortex the   for 1 min and add 15 µL to each sample.
 


SECTION: Procedure
10. Add 140 µL  to each sample. Mix by gentle inversion, fully invert but don't shake. If you see the beads making flakey clumps, a little like gold leaf, this is a good sign. This is difficult to do simultaneously as you want to see the mixing. If you are doing 8 samples, give all samples another gentle inversion after the last one. Leave at least 1 min for the beads to bind. Doing multiple samples will often take more time than this.
  

SECTION: Procedure
11. Centrifuge the tube briefly and place on a DynaMagTM-2 Magnetic Rack for 1 min to allow bead capture.
 
 
 
SECTION: Procedure
12. Remove and discard the supernatant. Take care not to disturb the bead pellet.
  

SECTION: Procedure
13. Remove the   from the magnetic rack. Add 350 µL   directly to the bead pellet. Mix by inversion, ensuring that the beads have come away from the side of the tube. This often requires tapping the tube or swilling the contents. Try to be as gentle as possible. Again, this is difficult to do simultaneously as you need to check each sample.

SECTION: Procedure
14. Centrifuge the tube briefly and place on a DynaMagTM-2 Magnetic Rack for 1 min to allow bead capture.

SECTION: Procedure
15. Remove and discard the supernatant. Take care not to disturb the bead pellet.
SECTION: Procedure
16. Repeat steps 13-15 for a total of 2 washes.
SECTION: Procedure
17. Remove the sample from the magnetic rack. Add 350 µL   directly to the bead pellet. Mix by inversion, ensuring that the beads have come away from the side of the tube. This often requires tapping the tube or swilling the contents. Try to be as gentle as possible. Again, this is difficult to do simultaneously as you need to check each sample.

SECTION: Procedure
18. Centrifuge the tube briefly and place on a DynaMagTM-2 Magnetic Rack for 1 min to allow bead capture.

SECTION: Procedure
19. Remove and discard the supernatant. Take care not to disturb the bead pellet.
SECTION: Procedure
20. Repeat steps 17 and 18 for a total of 2 washes but do not remove supernatant immediately, proceed to step 21.
SECTION: Procedure
21. If you have more than four tubes, split them into groups of four or fewer. Perform steps 22-24 (water wash) on the first group of samples, whilst the remaining samples wait in   on the magnet. [While the first group of samples are eluting in step 25-26 below,  it is possible to perform the water wash on the second group of samples.]

SECTION: Procedure
22. Remove and discard the supernatant. Take care not to disturb the bead pellet. With a P20 pipette remove any remaining supernatant. Leave the sample on the magnetic rack for the next step. Do not pipette water directly onto the beads.
 

  

SECTION: Procedure
23. Carefully add 350 µL  down the side of the tube opposite the magnetic pellet. Start a timer counting up from zero. After 15 s add water to the second sample, after 30 s add to the third sample, after 45 s add to the fourth sample. At 1 min remove and discard the water from the first sample, at 75 s remove and discard water from the second sample, at 90 s the third, and 105 s the fourth. This will enable multiple samples to be incubated for exactly 1 min.

SECTION: Procedure
24. Repeat step 23 for a total of 2 washes.
SECTION: Procedure
25. Remove the samples from the magnetic rack. Add 100 µL   directly to the bead pellet of each sample. Ensure that the pellet is submerged and has come away from the side of the tube. Incubate at 25 °C and   shaking for 3 min.

SECTION: Procedure
26. During this 3 min incubation perform steps 22-25 on the second group of samples if present.

SECTION: Procedure
27. Centrifuge each tube briefly and place them on a magnetic rack for 1 min to allow bead capture.

SECTION: Procedure
28. Using a wide-orifice pipette tip, carefully transfer the supernatant containing purified gDNA to a new labelled 1.5 mL LoBind microcentrifuge tube or barcoded (e.g. FluidX) tube.
SECTION: Procedure
29. Second elution: remove the samples from the magnetic rack. Add 100 µL   directly to the bead pellet of each sample. Ensure that the pellet is submerged and has come away from the side of the tube. Incubate at 25 °C and   shaking for 3 min.

 

SECTION: Procedure
30. Using a wide-orifice pipette tip, carefully transfer the supernatant containing purified gDNA to the same 1.5 ml LoBind microcentrifuge tube or Fluidx tube with a final volume of 200 µL.

SECTION: Procedure
31. Store the extracted gDNA sample at 4 °C. Assess the quantity of DNA extracted using the    or   and assess the quality of the DNA using the Femto Pulse .