TITLE: High-Throughput gDNA Extraction of Mosquito Tissues using QIAcube HT
AUTHORS: Jack Dorman, Ilinca Ciubotariu, Maria Levy, Abebe Fola, Giovanna Carpi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Genomic DNA (gDNA) is crucial to the study of many important aspects of vector-parasite interactions of vector-borne diseases. Many studies operate at the population level of those diseases and as such, it is of immense value to have a high-throughput method of gDNA extraction for field collected mosquitoes that maintains sample integrity. Here we present such a protocol that has presently been optimized for the study of </span><span style = "font-style:italic;">Anopheles gambiae</span><span>, the primary vector of the malaria parasite </span><span style = "font-style:italic;">Plasmodium falciparum. </span><span>All mosquitoes used in optimization were of the KEELE strain, insectary-raised, unfed, and stored in desiccant to mimic the preservation method generally used for entomological samples in malaria endemic areas. </span></div><div class = "text-block">This protocol provides an automated DNA extraction method from dried mosquito thorax and/or abdomen using the QIAcube HT instrument. The QIAcube HT is intended to perform automated, medium- to high-throughput purification of nucleic acids for molecular biology applications such as gDNA sequencing, qPCR of malaria parasite, target capture, etc. The QIAcube HT delivers high performance and reliability, enabling purification of high-quality nucleic acids from 8–96 samples per run. Yield is further maximized and efficiency increased through homogenization via TissueLyser II (QIAGEN) as compared to manual homogenization with pestles. </div><div class = "text-block">Genomic DNA quantity and quality was assessed via Qubit 1x dsDNA High Sensitivity quantification and Genomic DNA ScreenTape for the Agilent TapeStation 4150. For thoraxes (n=12), the average gDNA concentration was ~2.4 ng/uL (range: 1.13-3.89 ng/uL) while for abdomens (n=20) it was ~4.0 ng/uL (range: 1.28-8.14) in an elution volume of 100 uL. While concentrations were outside the recommended ranges for the Genomic DNA ScreenTape (or sometimes outside the functional range) for the TapeStation DIN calculation, values ranged from 4-6 with fragment peaks around 7000-9000 bp for TissueLyser-homogenized samples. Attached are TapeStation, and Rmarkdown reports, and an Excel sheet containing the IDs and conditions of the samples in the run.</div></div>

[STEPS]
?. [Sample Homogenization]
Using clean tweezers, separate the head and abdomen of a mosquito from the thorax on a sterile petri dish. Move either the thorax or abdomen (depending on which will be used for extraction) to the 2 mL RB tube with a 5mm stainless steel bead in it. Store the other 2 segments in the other 2 RB tubes and place in -20 or -80 C.
?. [Sample Homogenization]
Set a water bath to  and check Buffer ATL for precipitates. If there are precipitates visible, incubate buffer for  minutes.
56 °C
?. [Sample Homogenization]
Create a working solution (in a 15-50 mL Falcon tube depending on number of samples) of Lysis Buffer which is composed of  Buffer ATL and  Reagent DX per mosquito. Vortex well (2000 rpm for at least  seconds). Consider including an extra 10% volume to account for buffer loss.
180 µl
1.3 µl
?. [Sample Homogenization]
Using an Eppendorf Repeater, dispense  of well-vortexed Lysis Buffer into each tube containing a steel bead and the body segment to be extracted.
180 µl
?. [Sample Homogenization]
Visually balance samples in a TissueLyser adapter set and load into the TissueLyser II. Lyse Abdomens at 30 Hz for  seconds. Lyse Thoraxes at 30 Hz for  minute. Spin down tubes for  seconds at 15,000 rpm.
?. [Sample Homogenization]
Add  Proteinase K to each sample tube.  Vortex for  seconds at 2000 rpm and spin down for  seconds at 15,000 rpm.
20 µl
?. [Sample Homogenization]
Load samples into floating racks and place in   water bath. Incubate for  hour.
56 °C
?. [QIAcube Extraction]
After incubation of mosquito solution is complete, spin down the tubes for  seconds at 15,000 rpm.
?. [QIAcube Extraction]
While the samples are incubating, start the QIAcube instrument and select the DNeasy InsectLysates200ul AL-EtoH_tv file. Use default parameters provided by instrument. Follow software (version 1.6.61) and manual instructions for setting up the run in addition to the following notes (as relevant):Check Buffer AL for precipitate and incubate at  for around  minutes if precipitate is presentDispense buffers (volumes provided by QIAcube software based on the number of samples) in reagent troughs under a biosafety hoodConsider including an extra 10% of AL/Ethanol to account for loss in transferThoroughly mix AL/Ethanol solution in 50 mL Falcon tube by inverting the solution at least 5 times (or until solution is homogenous)If running less than 96 samples, make sure to hermetically seal unused columns/wells with a Tape PadMake sure all components (Elution plate, QIAamp plate, and when ready the S-Bock) are pushed firmly to the upper left portion of their carriages
56 °C
?. [QIAcube Extraction]
Move tubes to a magnetic rack to immobilize 5mm beads. Using a 200 uL pipette, immediately transfer as much of the   supernatant as possible to the S-Block. [Some loss may be observed on occasion (5-10 uL) but it should not have a significant impact on DNA yield].
200 µl
?. [QIAcube Extraction]
Place the properly labelled S-Block on to the QIAcube carriage. Labels should include information such as date of extraction, sample type, project name, etc. After placing the S-Block in its carriage in the instrument, follow the QIAcube software and manual instructions for starting the DNA extraction run. It is advisable to save the Pre-Run report in the event that troubleshooting is needed.
?. [QIAcube Extraction]
Once the run is complete, save the Post-Run report and transfer the properly labelled Elution Plate (now containing the extracted DNA) to storage (at  for short-term storage, and  for long-term storage) unless doing immediate quality assessment.
-20 °C
-80 °C
?. [QIAcube Extraction]
Follow software and manual for cleanup instructions.