TITLE: Automated high throughput Qiagen MagAttract High Molecular Weight DNA extraction from mosquitoes
AUTHORS: Fiona Teltscher, Mara Lawniczak

[DESCRIPTION]
This is an automated protocol for the 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). Due to small specimen size, we need to maximize DNA yield. so we also do a double 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 Anopheles mosquito preserved using best practices (snap frozen from living) weighing 2-3 mg yields about 600-800 ng of DNA using this protocol. Following shearing using g-tubes (Covaris) 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 100% ethanol and DESS (DMSO salt solution) preserved specimens held at room temperature for as long as a week, as long as the specimen was gently squished to ensure rapid penetration of the preservative.

This protocol has been used successfully on a wide range of arthropod species as part of the Tree of Life Programme at the Wellcome Sanger Institute. Please use the commenting function to share whether this protocol worked or not for your species. If you make modifications to improve this protocol so it works better for your species, please use the forking function in protocols.io to share these adjustments. 

[BEFORE_START]
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.

[STEPS]
SECTION: Sample preparation
1. Prepare an open insulated box of dry ice to store sample tubes on whilst working through steps 2-4
SECTION: Sample preparation
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: 200 µL   ; 20 µL    (Mix by inverting the tube 5 times);  4 µL   and 150 µL    (mix by inversion)

SECTION: Sample preparation
3. For each sample, add 374 µL of the mastermix from step 2 into a new 1.5 ml DNA LoBind tube.

SECTION: Sample preparation
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 sample into the mastermix in tubes (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 70-100% ethanol.
 
 
 
 
SECTION: Sample preparation
5. Repeat step 4 for the remaining samples
SECTION: Sample preparation
6. Briefly spin all samples in a minicentrifuge or similar to collect solution at bottom before next step
SECTION: Sample preparation
7. Incubate the sample at 25 °C (or room temperature if no heat block is available) for 120 min

SECTION: Preparation of plates for the Kingfisher Apex
8. Unpack 9 Kingfisher Deepwell 96 well plates
 

 

SECTION: Preparation of plates for the Kingfisher Apex
10. Prepare and fill the plates with  ,  ,  ,  ,   and  as suggested below:

 

 

 

Only fill the wells that you intend to use, but the same well will have to be used for each plate. E.g. you start with a new plate and have 4 samples which you intend to run in wells A1, A2, A3 and A4, so you fill those wells in every plate.

 

 

SECTION: Preparation of plates for the Kingfisher Apex
9. Prepare wash buffers included in the   by adding Ethanol to them.

SECTION: Preparation of plates for the Kingfisher Apex
11. Download the programme onto a computer and transfer it to your KingFisher Apex:   
 



SECTION: Preparation of plates for the Kingfisher Apex
12. Start your KingFisher Apex and select the correct protocol to run.
 


 

SECTION: Preparation of plates for the Kingfisher Apex
13. The software allows you to input batch/lot information for each reagent. This is optional but highly recommended.
 
 
SECTION: Preparation of plates for the Kingfisher Apex
14. Load all but the sample plate onto the instrument.
SECTION: Preparation of plates for the Kingfisher Apex
15. Once the 120 min incubation time for the sample has elapsed add the sample to the sample plate using wide-orifice pipette tips. Make sure to keep a record which sample was added in which well.

SECTION: Preparation of plates for the Kingfisher Apex
16. Load the sample plate including the sample onto the instrument and start the programme. It takes about 50 min.
 
 


SECTION: Post-extraction
17. Once the KingFisher Apex is finished the plates can be removed as prompted. Elution 1 and 2 for each sample can be combined into the same tube (making sure to use wide-orifice tips).
 
Used plates that still have unused wells can be reused in future runs. Either carefully tip out the waste without contaminating unused wells or keep buffers in. 
 
Mark which wells were used and cover plates with a film, such as  . When reusing plates that still have used buffers from previous runs in some wells make sure you only run the same programme with the same volume to avoid spillage in the machine.
SECTION: Post-extraction
18. 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