Aug 25, 2020
Optimized HT gDNA extraction from Dried Blood Spot using QIAcube HT for Malaria Genomic Epidemiology Studies
- Abebe A. Fola 1,
- Jack Dorman1,
- Maria Levy1,
- Ilinca Ciubotariu1,
- Giovanna Carpi1
- 1The Department of Biological Sciences, Purdue University
Protocol Citation: Abebe A. Fola , Jack Dorman, Maria Levy, Ilinca Ciubotariu, Giovanna Carpi 2020. Optimized HT gDNA extraction from Dried Blood Spot using QIAcube HT for Malaria Genomic Epidemiology Studies. protocols.io https://dx.doi.org/10.17504/protocols.io.bh69j9h6
Manuscript citation:
1. QIAamp® DNA Mini Kit and QIAamp DNA Blood Mini Kit Handbook. 2. QIAcube HT User Manual 07/2013. 3. Stangegaard M, et al. Evaluation of Four Automated Protocols for Extraction of DNA from FTA Cards. Journal of Laboratory Automation 2013 Oct;18(5):404-10. doi: 10.1177/2211068213484472. 4. Kumar A et al. Optimization of extraction of gDNA from DBS: Potential application in epidemiological research & biobanking. Gates Open Research . 2019 Nov 14;2:57. doi: 10.12688/gatesopenres.12855.3.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 02, 2020
Last Modified: August 25, 2020
Protocol Integer ID: 38849
Keywords: Dried blood spot (DBS), genomic DNA, laboratory automation, malaria genomics,
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Abstract
Genomic DNA (gDNA) extracts from human dried blood spots (DBS) are commonly used for infectious diseases surveillance, genomic epidemiology studies of targeted pathogens, and Biobanking. In particular, DBS samples are routinely collected during cross-sectional and large-scale epidemiological studies (i.e. Malaria Indicator Survey) in malaria-endemic countries for malaria surveillance and genomic studies. DBS samples have multiple advantages over venous blood, including less invasive sample blood collection (finger prick) and the recommended short- and long-term storage at room temperature, which facilitates sample transportation (domestically or internationally). Here we present an optimized high throughput gDNA extraction protocol to recover Plasmodium gDNA from human DBS (collected on Whatman filter paper) using field samples from Zambia and stored at room temperature with desiccant pellet (up to 2 years).
Using the QIAcube HT instrument and QIAmp DNA 96-well kit, this protocol allows for gDNA extraction from human DBS samples in a manner that is automated, reproducible, and fast when compared to other methods. After gDNA extraction is completed, genomic DNA quantity and integrity is assessed using Qubit 1x dsDNA High Sensitivity Assay and Genomic DNA ScreenTape on the Agilent TapeStation 4150, respectively. In this case, gDNA samples that were obtained from field collected human DBS samples and extracted using this optimized protocol were then used for Plasmodium falciparum whole-genome sequencing by hybrid capture.
Using this protocol, we first compared gDNA concentration between 2X6mm and 3X3mm Uni Core full punch (100% blood punch) and found a statistically significant difference in DNA concentration obtained from 2x6mm (ng/µL) vs 3x3mm DBS punches (range =0.44-2.01ng/µL) (P-value= 0.0003) with a median increased of ~4-fold in the gDNA yield when using 2x6mm hole punches. See the attached Comparison of DNA concentration 2X6mm vs 3X3mm Uni Core Punch documents. Furthermore, we tested and demostrated that using this protocol with 2X6mm punches, it is feasible to successfully extract sufficient gDNA for P. falciparum whole genome capture and deep sequencing from DBS samples (n=93) that had been stored at RT for up to 2 years with variable level of parasitemia. While generally concentrations were outside the recommended range for the Genomic DNA ScreenTape for the TapeStation DIN calculation, from those samples DIN was estimated, values ranged from 6.2 - 8.7 with average fragment peaks of 35,325 bp, indicating that DNA integrity is suitable for downstream analysis. Becuase these were field DBS samples, we reported high variation in gDNA concentration (ranges= 0.50-4.88ng/µL) which reflected the variation in blood volume and adsorbance across DBS samples. See the attached gDNA concentration for 93 DBS field samples documents.
Guidelines
Follow standard molecular biology techniques including:
- Change gloves frequently and especially when there is suspicion of any contamination
- Have solutions of freshly prepared 10% bleach and 70% ethanol readily available for decontamination of surfaces/equipment
- Use only filter tips
Materials
MATERIALS
Ethanol 200 ProofDecon LabsCatalog #2716
Qubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
QIAamp 96 DNA QIAcube HT Kit QiagenCatalog #51331
QIAcube HTQiagenCatalog #9001793
QIAcube HT PlasticwareQiagenCatalog #950067
Reagent Trough with lid Vf=70mLQiagenCatalog #990554
Reagent Trough with lid Vf=170mLQiagenCatalog #990556
Uni-Core 6.00mmGe Life SciencesCatalog #WB100040
Genomic DNA ScreenTape®Agilent TechnologiesCatalog #5067-5366
- BioSafety Cabinet Class II
- Centrifuge (Eppendorf 5424R)
- Plate centrifuge (Sigma 4-16)
- Thermomixer C with Smartblock (Eppendorf 5382000023, 5363000039)
- Water bath (only for dissolving Qiagen ATL reagents ifp ecipitates are visible)
- Vortex mixer
- Pipette (P20, P200, P1000)
- 8-channel pipette (20-300 µL)
- Pipette filter tips (20 µL , 200 µL, 1000 µL)
- Serological Pipettes (25mL, 50 mL)
- Reagent reservoir (50 mL)
- Conical centrifuge tubes (50 mL)
Safety warnings
- Refer to the QIAcube HT manual for further safety warnings concerning instrument operation and cleaning.
- Refer to the Safety Data Sheets (SDS) for health and environmental hazards for QIAGEN reagents.
- Operate in a BSL-2 lab setting
Before start
Clean all relevant surfaces with 10% bleach followed by 70% ethanol and wipe using Kimwipes. If any boxes of filter tips were used previously, UV them for 30 minutes. Select the samples (Dried Blood Spots) to be extarcted and include 1 negative control every 47 samples. For a full plate that means 94 samples and 2 negative controls. The first negative control should contain punches from clean filter paper and all downstream components, while the second should contain lysis buffer in addition to downstream components.
DBS Pretreatment
DBS Pretreatment
20h
20h
Label a new S-Block with important information such as date, project name, sample type, etc. Use the same information to generate .csv file that contains sample ID and well position which will be imported to QIAcube HT robot.
Using clean tweezers, remove a DBS sample from its storage plastic bag and place face-up (the side with the most visible blood) on a cutting mat. Use a clean 6mm Uni-Core punch to remove a circular section and eject the punched section directly over its respective well in the S-block. Repeat so that each well has two punched 6mm circles total per sample in order to obtain as much blood as possible.
Clean a Uni-Core punch after each use by immersing it in 10% bleach followed by 70% ethanol (for 00:00:10 each) and then letting it air dry. Use a rotation of multiple Uni-Cores (at least 4, to allow Uni-Cores to dry without impeding workflow). After each DBS sample, also spray the cutting mat with 70% ethanol and wipe dry. Repeat Step 2 until all selected samples have been processed.
Check Buffer ATL for precipitates. If precipitates are visible, incubate buffer in water bath at 56 °C for 00:05:00 , or until precipitates are fully dissolved.
Create a working solution of Lysis Buffer in a 50 mL Falcon tube by preparing 360 µL Buffer ATL and 20 µL Proteinase K per sample. Include an extra 10% volume to account for buffer loss resulting from the transfer of buffers. Vortex at 2,000 rpm for at least 00:00:10 and transfer solution to a 50 mL reservoir via serological pipette.
Use a multichannel pipette to dispense 380 µL Lysis Buffer into each well in the S-Block containing the dried blood punches. Seal the S-Block with Tape Pad. Caution: use the Lysis Buffer immediately after preparation, as the mix will precipitate within minutes.
Load the S-Block into the Thermomixer set to 56 °C at 900 rpm and cover with lid. Incubate overnight (defined here as 16:00:00 ).
QIAcube HT Extraction
QIAcube HT Extraction
3h
3h
Open the QIAcube control software (version 1.6.61) and select the Buccals_350ul_QCHT_withALEtOHmix_min_V5_dk protocol. Follow instructions provided by the software and manual. Change the final elution volume to 120 µL , but otherwise use default parameters. In addition, consider the following notes as needed:
- Fill the water bath with deionized water. Turn on to preheat at 56 °C .
- Start with a clean QIAcube HT. If left idle for longer than 1 week, run the UV light for 00:30:00 .
- Check Buffer AL for precipitates. If present, incubate in the water bath at 56 °C for at least 00:05:00 .
- Remember to import sample IDs using pre-prepared .csv file. See the HT manual on how to prepare and import a .csv file.
- Reagents volumes are provided by the QIAcube software based on the number of samples to be included in the run, but it is important to add an extra 10% volume for the AL/Ethanol mix.
- Fill the reservoir reagents with the appropriate volumes under a clean and UVed biological safety cabinet (BSC) using sterile serological pipettes.
- Thoroughly mix AL/Ethanol solution in a 50 mL Falcon tube by vortexing at least 1 minute (or until the solution is homogeneous).
- If running less than 96 samples, make sure to hermetically seal unused columns/wells with a Tape Pad to avoid vacuum errors.
- Make sure all components (Elution plate, QIAamp plate, and when ready - the S-Bock) are pushed firmly to the upper left portion of their carriages within QIAcube HT robot.
After overnight incubation is complete, centrifuge S-Block containing the samples at 2,400 RCF for 00:01:00 using a plate centrifuge, properly balanced.
Gently remove Tape Pad seal on the incubating S-Block and use a multichannel pipette to transfer a total of 350 µL (175 µL X 2 - using 200 µL multichannel pipette) of lysate to a new, properly labelled S-Block. Avoid taking debris from filter paper. Note that the gDNA can be re-extracted from the filter paper at a later time, so seal the S-block containing the remaining filter paper and store it at -20 °C for future re-extraction.
Place the new S-Block that contains only the lysate in its QIAcube carriage and follow software instructions for starting the run. Save the Pre-Run Report in case troubleshooting is needed.
Once the run is complete, save the Post-Run Report, apply Elution Cap Strips to the Elution Plate, and transfer to storage (at -20 °C for short-term storage, and -80 °C for long-term storage) unless doing immediate quality assessment or downstream analysis.
After each run, clean the QIAcube instrument by following software and manual instructions.