Sep 29, 2023
Sanger Tree of Life HMW DNA Extraction: Automated MagAttract v.2
- 1Tree of Life, Wellcome Sanger Institute, Hinxton, Cambridgeshire, CB10 1SA
- Tree of Life at the Wellcome Sanger Institute
- Earth BioGenome Project
Protocol Citation: Graeme Oatley, Amy Denton, Caroline Howard 2023. Sanger Tree of Life HMW DNA Extraction: Automated MagAttract v.2. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3y4dg8j/v1
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: September 05, 2023
Last Modified: September 29, 2023
Protocol Integer ID: 87380
Keywords: HMW DNA extraction, magnetic bead extraction, MagAttract, automated DNA extraction, KingFisher, solid phase reversible immobilisation, reference genome, long read sequencing, life hmw dna extraction, sanger tree of life hmw dna extraction, spri of hmw dna, qiagen magattract hmw dna extraction kit, hmw dna fragmentation, hmw dna pooling, hmw dna, dna extraction, acronyms hmw, automated extraction, taxonomic group, extraction, read sequencing, high molecular weight spri, genome size of the species, dna, tree of life programme, automated magattract
Funders Acknowledgements:
Wellcome Trust
Grant ID: 218328
Wellcome Trust
Grant ID: 206194
Gordon and Betty Moore Foundation
Grant ID: GBMF8897
Abstract
This protocol describes the automated extraction and SPRI of HMW DNA from multiple different tissue samples from a variety of species intended for long-read sequencing using the Qiagen MagAttract HMW DNA extraction kit and the Thermo Fisher KingFisher™ Apex. This process is effective for a wide variety of taxonomic groups covered by the Tree of Life Programme, excluding plants and fungi. The output of this protocol is HMW DNA, which depending upon yield and genome size of the species, can be directed towards either HMW DNA Pooling, HMW DNA Fragmentation: Diagenode Megaruptor®3 for LI PacBio or HMW DNA Fragmentation: g-Tube for ULI PacBio. This protocol was adapted from Sanger Tree of Life HMW DNA Extraction: Automated MagAttract v.1 to include a pre-shear SPRI of the HMW DNA extracted.
Acronyms
HMW: high molecular weight
SPRI: solid-phase reversible immobilisation
LI: low input
ULI: ultra-low input
Guidelines
- For the lysis buffer master mix, prepare enough for n+1 samples to allow for pipetting errors.
- Keep samples on dry ice to maintain temperature and prevent nucleic acid degradation until the lysis buffer is ready to be added to them.
- For the 0.45X SPRI, the DNA and AMPure beads should not sit together in the sample plate for more than 5 minutes before starting the KingFisher™ Apex.
- An experienced operator can expect to comfortably process up to 32 samples, with approximately 2–3 hours handling time over a start to finish period of 4–5 hours. This estimation includes the utilisation of the KingFisher™ Apex for both the extraction and SPRI protocols, and excludes subsequent QC checks.
Additional Notes:
- FluidX tubes are used throughout the Tree of Life programme in order to track samples, therefore rather than the microcentrifuge tubes which have been mentioned in this protocol for DNA storage, all routine DNA extracts are stored in FluidX tubes.
- Both the KingFisher™ Apex protocol scripts and the KFX.files have been made available for this protocol – the KFX.files require ‘BindIx software for KingFisher Apex’ to allow the KingFisher™ Apex protocols to be viewed on a PC or laptop. Alternatively, the files can be transferred directly onto a KingFisher™ Apex instrument using a USB flash drive.
Materials
- 1.5 mL DNA Lo-Bind microcentrifuge tubes (Eppendorf Cat. no. 0030108418)
- 2 mL DNA Lo-Bind microcentrifuge tubes (Eppendorf Cat. no. 0030108078)
- 1.5 mL BioMasher II tubes and pestles (sterile) (Cat. no. 9791A)
- Thermo Fisher KingFisher™ 1 mL 96-well Deep-well plates (Thermo Fisher Cat. no. 95040450)
- Thermo Fisher KingFisher™ 200 µL standard 96-well plate (Thermo Fisher Cat. no. 97002084)
- Thermo Fisher KingFisher™ 96 Tip Comb (Thermo Fisher Cat. no. 97002570)
- Qiagen MagAttract HMW DNA extraction kit (Qiagen Cat. no. 67563)
- Dry ice
- 1x phosphate-buffered saline (PBS)
- 100% absolute ethanol
- 15 mL or 50 mL centrifuge tubes
- AMPure PB beads (Pacific Biosciences Cat. no. 100-265-900)
- Buffer EB (Qiagen Cat. no. 19086)
Equipment:
- Pipettes for 0.5 to 1000 μL and filtered tips
- Wide-bore tips (200 μL and 1000 μL, filtered if available)
- Diagnocine PowerMasher II tissue disruptor (Cat. no. FNK-891300)
- Thermo Fisher KingFisher™ Apex instrument (Cat. no. 5400930)
- Eppendorf ThermoMixer C (Cat. no. 5382000031)
- Eppendorf SmartBlock 2.0 ml (Cat. no. 5362000035)
- Vortexer (Vortex Genie™ 2 SI-0266)
KingFisher™ Apex DNA Extraction Protocol Script:
KFX file: 
Qiagen MagAttract Standard.kfx2.7KB
Qiagen MagAttract Standard.kfx2.7KB - Pick Up Tip - Tip Plate
- DNA Binding - Sample Plate Pre-collect beads: Off Release beads: Off Heating & Cooling: Off Mixing 1# 00:05:00 Fast Postmix: Off Collect beads: On 5 Count 2 Seconds
- Collect Beads 1 - Sample Plate Collect beads: Count 5 Collect time: 1 Second
- Wash 1 - MW1 Wash 1 Plate Pre-collect beads: Off Release beads: On 00:00:10 Bottom mix Heating & Cooling: Off Mixing 1# 00:01:00 Fast Postmix: Off Collect beads: On 5 Count 1 Second
- Collect Beads 2 - MW1 Wash 1 Plate Collect beads: Count 5 Collect time: 1 Second
- Wash 2 - MW1 Wash 2 Plate Pre-collect beads: Off Release beads: On 00:00:10 Bottom mix Heating & Cooling: Off Mixing 1# 00:01:00 Fast Postmix: Off Collect beads: On 5 Count 1 Second
- Collect Beads 3 - MW1 Wash 2 Plate Collect beads: Count 5 Collect time: 1 Second
- Wash 3 - PE Wash 1 Plate Pre-collect beads: Off Release beads: On 00:00:10 Bottom mix Heating & Cooling: Off Mixing 1# 00:01:00 Fast Postmix: Off Collect beads: On 5 Count 1 Second
- Collect Bead 4 - PE Wash 1 Plate Collect beads: Count 5 Collect time: 1 Second
- Wash 4 - PE Wash 2 Plate Pre-collect beads: Off Release beads: On 00:00:10 Bottom mix Heating & Cooling: Off Mixing 1# 00:01:00 Fast Postmix: Off Collect beads: On 5 Count 1 Second
- Collect Bead 5 - PE Wash 2 Plate Collect beads: Count 5 Collect time: 1 Second
- Water Rinse - NFW Plate Pre-collect beads: Off Release beads: Off Heating & Cooling: Off Mixing 1# 00:00:00 Postmix: Off Collect beads: On 5 Count 1 Second
- Dry - NFW Plate Duration: 00:01:00 Dry Type: Above Well
- Elute 1 - Elution Plate 1 Plate Pre-collect beads: Off Release beads: On 00:00:00 Heating & Cooling: On 25°C Pre-heat: Off Mixing 1# 00:01:00 Paused Looping: 1 2# 00:05:00 Slow Tip Position: Above Well Postmix: Off Collect beads: On 3 Count 1 Seconds
- Elute 2 - Elution Plate 2 Plate Pre-collect beads: Off Release beads: On 00:00:00 Heating & Cooling: On 25°C Pre-heat: Off Mixing 1# 00:01:00 Paused Looping: 1 2# 00:05:00 Slow Tip Position: Above Well Postmix: Off Collect beads: On 3 Count 1 Seconds
- Leave Tip - NFW Plate
KingFisher™ Apex 0.45X SPRI Protocol Script:
KFX file: Pre-shear 0.45X SPRI.kfx2KB
Pre-shear 0.45X SPRI.kfx2KB - Pick Up Tip - Tip Plate
- Mix - Sample Plate Pre-collect beads: Off Release beads: On 00:00:00 Heating & Cooling: Off Mixing: 1# 00:01:00 Slow 2# 00:01:00 Medium 3# 00:08:00 Paused Looping: 1 Tip position: Tip edge in liquid Postmix: Off Collect beads: On 10 Count 30 Seconds
- Wash 1 - Ethanol Wash Plate Pre-collect beads: On Release beads: Off Heating & Cooling: Off Mixing 1# 00:00:30 Slow Postmix: Off Collect beads: Off
- Wash 2 - Ethanol Wash Plate
Pre-collect beads: Off
Release beads: Off
Heating & Cooling: Off
Mixing 1# 00:00:30 Slow
Postmix: Off
Collect beads: Off
5. Dry - Ethanol Wash Plate
Duration: 00:01:00 Above well
6. Elute - Elution Plate
Pre-collect beads: Off
Release beads: On 00:01:00 Slow
Heating & Cooling: On 37℃ Preheat: On
Mixing: 1# 00:07:00 Slow
2# 00:08:00 Paused
Looping: 1 Tip position: Tip edge in liquid
Postmix: Off
Collect beads: On 4 Count 30 Seconds
7. Leave Tip - Ethanol Wash Plate
Protocol PDF: Sanger Tree of Life HMW DNA Extraction_ Automated MagAttract v.2.pdf94.4KB
Sanger Tree of Life HMW DNA Extraction_ Automated MagAttract v.2.pdf94.4KB Safety warnings
- The operator must wear a lab coat, powder-free nitrile gloves and safety specs to perform the laboratory procedures in this protocol. Cotton glove liners are strongly recommended when handling the samples on dry ice.
- Waste needs to be collected in a suitable container (e.g. plastic screw-top jar or Biobin) and disposed of in accordance with local regulations.
- Liquid waste needs to be collected in a suitable container (e.g. glass screw-top jar) and disposed of in accordance with local regulations.
- Do not open the door of the KingFisher™ Apex instrument whilst it is in operation.
Before start
- Add 100% ethanol to the MW1 and PE wash buffers as per manufacturer’s instructions.
- Remove the AMPure PB beads from the fridge 30 minutes before starting the 0.45X SPRI KingFisher™ Apex protocol to bring them to room temperature.
Sample lysis
Prepare a lysis buffer master mix:
| Reagent | Volume per sample | |
| Phosphate-buffered saline (PBS) | 200 µL | |
| Proteinase K | 20 µL | |
| RNase A | 4 µL | |
| Buffer AL | 150 µL |
Set a heat block to 25 °C.
For samples which have been cryogenically homogenised:
- Transfer 25 mg of the sample into a 2 mL microcentrifuge tube, then hold on dry ice to keep the sample frozen.
- Add 374 µL of the lysis buffer master mix to sample, then homogenise the sample and master mix by gently pipetting 10 times with a wide-bore pipette tip.
For PowerMashed samples (weight less than 25 mg):
- Transfer sample into a 1.5 mL BioMasher II tube and add 374 µL lysis buffer.
- Disrupt sample in lysis buffer using the Diagnocine PowerMasher II tissue disruptor and BioMasher pestle, until no large pieces remain or sample cannot be disrupted further. (For more detailed instructions regarding PowerMashing, please refer to the Sanger Tree of Life Sample Homogenisation: PowerMash protocol.)
- Transfer the entire contents of the BioMasher tube to a 2 mL microcentrifuge tube using a wide-bore tip.
Centrifuge sample tubes briefly and incubate on the heat block at 25 °C for 2 hours.
Loading and Running the KingFisher™ Apex for DNA Extraction
While samples are lysing, label nine 1 mL 96-well deep-well KingFisher™ plates and fill the number of wells required for the number of samples in each plate as follows:
| Plate | Reagent(s) required | |
| Tip plate | 96-well tip comb (no reagent) | |
| Elution 2 | 200 µL Buffer AE | |
| Elution 1 | 200 µL Buffer AE | |
| NFW Wash | 500 µL nuclease-free water | |
| PE Wash 2 | 700 µL Buffer PE | |
| PE Wash 1 | 700 µL Buffer PE | |
| MW1 Wash 2 | 700 µL Buffer MW1 | |
| MW1 Wash 1 | 700 µL Buffer MW1 | |
| Sample plate | 15 µL Suspension G magnetic beads + 280 µL Buffer MB |
Once samples have completed lysing, remove sample tubes from the heat block and briefly centrifuge to spin down.
Using a wide-bore pipette tip, set the volume to 380 µL, transfer lysate from the sample tubes to individual wells in the sample plate, taking care not to transfer large pieces of debris if possible.
Select the required DNA extraction protocol in the protocol list on the KingFisher™ Apex (details in KingFisher™ Apex DNA Extraction Protocol Script/attached KFX file in the Materials section) and select using the play button.
Load the filled plates onto the instrument following the instructions provided on screen.
Prior to loading the “Sample Plate”, the instrument will prompt to remove the “Tip Plate”. Once the final plate is loaded, the protocol will automatically begin; this takes approximately 50 minutes.
Once the protocol has completed, follow the on-screen instructions to remove plates from the instrument.
Inspect the elution plates for any magnetic beads in the wells. In the rare instance of magnetic beads remaining in the eluate (possible in viscous samples), these samples will need to be transferred to a 1.5 mL microcentrifuge tube and placed on a magnetic rack. Allow around 5 minutes for the beads to migrate and take the clear eluate containing the DNA using a wide-bore pipette tip.
Using a 200 µL multi-channel pipette and wide-bore tips, pipette eluates from Elution Plate 2 into Elution Plate 1, and gently pipette mix 5–10 times with wide-bore tips to fully homogenise DNA in the eluate. Elution Plate 1 with the combined eluates is now the ‘Sample Plate’ for the 0.45X SPRI.
Loading and Running the KingFisher™ Apex for the 0.45X SPRI
Set-up the KingFisher™ plates for the 0.45X SPRI as detailed below:
| Plate | Plate type | Reagent(s) required | |
| Tip Plate | 1 mL deep-well | 96-well tip comb (no reagent) | |
| Sample Plate (Elution Plate 1 from DNA Extraction Protocol) | 1 mL deep-well | 380 µL DNA + 171 µL AMPure PB beads | |
| Ethanol Wash Plate | 1 mL deep-well | 1000 µL 80% EtOH (freshly made) | |
| Elution Plate | 200 µL standard | 135 µL Buffer EB |
Select the required 0.45X SPRI protocol in the protocol list on the KingFisher™ Apex (details in KingFisher™ Apex 0.45X SPRI Protocol Script/attached KFX file in the Material section) and select using the play button.
Load the filled plates onto the instrument following the instructions provided on screen.
Once the final plate is loaded, the protocol will automatically begin; this will take approximately 40 minutes.
Once the protocol has completed, follow the on-screen instructions to remove plates from the instrument.
Using a wide-bore pipette tip, transfer the 130 µL of eluate from the elution plate into microcentrifuge tubes.
Incubate the DNA at room temperature overnight and perform the required QC the following morning.
Store the DNA at 4 °C.
Protocol references