Introduction
This protocol is a low-cost alternative to the Illumina DNA Prep protocol (previously Nextera DNA Flex). The paper describing Hackflex can be found here. Hack Flex is similar to the official protocol, but uses diluted Bead-Linked Transposomes and lab-made buffers.
The Illumina DNA Prep protocol can be found here.
Reagents
Reagent | Vendor | Catalog | Storage |
Illumina DNA Prep Kit | Illumina | 20060059 | Various |
IDT for Illumina DNA/RNA UD Indexes Set A | Illumina | 20027213 | -20°C |
IDT for Illumina DNA/RNA UD Indexes Set B | Illumina | 20027214 | -20°C |
IDT for Illumina DNA/RNA UD Indexes Set C | Illumina | 20042666 | -20°C |
IDT for Illumina DNA/RNA UD Indexes Set D | Illumina | 20042667 | -20°C |
80% Ethanol, freshly prepared | NA | NA | RT |
Molecular-Grade (Nuclease-Free) Water | Sigma | W4502-1L | RT |
Tris HCI Buffer, pH 7.6, 1 M | Crystalgen | 221-230 | 4°C |
MgCl2, 1 M | VWR | E525-100ML | RT |
N,N-DIMETHYLFORMAMIDE >99%, liquid | Sigma | D4551-250ML | RT, in flammable cabinet |
UltraPure 10% SDS | ThermoFisher | 15553027 | RT |
PrimeSTAR GXL DNA Polymerase Kit | Takara | R050A | -20°C |
Tris-EDTA (TE) Buffer | FisherScientific | BP2473-1 | RT |
SPRI (Homemade) or AmpureXP Beads | 4°C |
Plasticware | Vendor | Catalog |
96-well Hard Shell Plates (HSP) for Zephyr | REVVITY | 6008870 |
150 ul filter tips for Zephyr | REVVITY | 111426 |
StorPlate 450 ul V bottom plates for Zephyr | REVVITY | 6008290 |
2 ml Deep Well Plate (DWP) for Zephyr | REVVITY | 6008880 |
Lids for Zephyr | REVVITY | 6000030 |
12-Column Reservoir Seahorse DWP for Zephyr | REVVITY | 6008700 |
287 ml Reservoir for Zephyr | REVVITY | 6008730 |
Protocol
Prepare Reagents
Bead-Linked Transposomes (BLT) (Stored at 4°C)
Bring to room temperature. Vortex to mix. Do not centrifuge before pipetting.
NEVER put BLT on ice. Do not use BLT stored below 2°C.
Lab-Made Tagmentation Buffer 1 (LTB) (Stored -20°C)
(Split between 2 50 mL conicals)
Bring to room temperature. Vortex to mix.
Reagent | [Stock] | [Final] | Volume (ml) |
Tris HCI Buffer, pH 7.6, 1 M | 1 M | 20 mM | 1 |
MgCl, 1 M | 1 M | 20 mM | 1 |
N,N-DIMETHYLFORMAMIDE >99%, liquid | >99% | 50% | 25 |
Molecular Grade Water | NA | NA | 23 |
Total | NA | NA | 50 |
Lab-Made Tagment Stop Buffer (LTSB) (Stored Room Temperature)
If precipitates are observed, heat at 37°C for 10 minutes and then vortex until dissolved.
Reagent | [Stock] | [Final] | Volume (ml) |
Ultrapure SDS | 10% | 0.2% | 1 |
Molecular Grade Water | NA | NA | 49 |
Total | NA | NA | 50 |
Lab-Made Tagment Wash Buffer (LTWB) (Stored Room Temperature)
Filter through 0.22 um filter before use. Use at room temperature
Reagent | [Stock] | [Final] | Amount |
PEG 8000 | Solid | 10% | 25 g |
NaCl | 5 M | 0.25 M | 12.5 ml |
Tris-EDTA (TE) buffer (10 mM Tris and 1 mM EDTA) | 1X | NA | to 250 ml |
Total | NA | NA | 250 ml |
PrimeSTAR GXL DNA Polymerase kit (Stored -20°C)
Index Adapters (Stored -20°C)
Thaw at room temperature. Spin briefly before use.
Resuspension Buffer (RSB) (Stored 4°C)
Thaw and bring to room temperature. Vortex to mix.
SPRI beads or AmpureXP (Stored 4°C)
Equilibrate to RT for at least 30 min. Vortex and invert to mix.
Dilute DNA
This should be done immediately before library prep, and no more than 24 hours in advance.
Create an Excel document to dilute your extracted DNA to 1 ng/ul. The total volume (DNA + EB/Water) needs to be between 12 and 170 ul. This is a snippet of the Excel document that needs to be uploaded to the Zephyr computer in C:\Pooling:
Normalization and Pooling | |||
Sample Well ID | Sample Volume | Diluent Volume | Destination Well ID |
A01 | 5 | 148 | A01 |
B01 | 5 | 76 | B01 |
C01 | 9 | 4.23 | C01 |
D01 | 5 | 19 | D01 |
E01 | 5 | 149.5 | E01 |
F01 | 5 | 156.5 | F01 |
… | … | … | … |
Open Maestro Workstation and open the “Pooling and Normalization” protocol:
(File → Open Application → Pooling and Normalization).
Push the green Start arrow.
Load deck as follows:
Item | Position | Plasticware | Contents |
150 ul Filter Tips | A2 | Box Filter Tips | Full Box |
DNA Source Plate | A3 | 96-well HSP | Extracted DNA (thawed, vortexed and spun down) |
DNA Destination Plate | B3 | 96-well HSP | Empty |
EB/Water | B4 | 287 ml Reservoir | EB or Water or C6 |
Grease the 96-well plate head (only 1x per day).
Press the Start button.
Open the input Excel file (located in C:\Pooling).
Press the Continue button. The program takes about 90 minutes.
Once the program finishes, cover the DNA dilution plate with foil, vortex, and spin down.
Cover the DNA source plate in foil and return to the -20C freezer.
Run the “Nextera Flex Tagmentation and PCR Setup” Protocol on Zephyr
Open Maestro Workstation and open the “HackFlex - DNA Prep No Wash” protocol:
(File → Open Application → Illumina Flex - DNA Prep).
Push the green Start arrow.
Select the appropriate number of columns and then click the “Nextera Flex Tagmentation and PCR Setup” button on the popup.
Load the deck as follows using the prompts:
All volumes are for a full 96-well plate. Adjust as necessary using the HackFlex Zephyr Workbook.xls document (C:\ProgramData\CaliperLS\Maestro\Workbooks).
Item | Position | Plasticware | Contents |
150 ul Filter Tips | A1, A2, B1, B2 | Box Filter Tips | Full box |
BLT/LTB Broadcasting Plate | A3 | 96-well HSP | Empty |
LTSB Broadcasting Plate | A3 | 96-well HSP | Empty |
Waste Reservoir | B4 | 96-well DWP | Empty |
Reagent Plate | C2 on CPAC adapter | StorPlate 450 ul V-bottom with Lid | Column 1: 1:50 BLT/LTB Mix (477 ul per well)
* BLT: 22.4 ul
* H2O: 1098 ul (2x 549 ul)
* LTB: 2800 ul
Column 3: LTSB (137 ul per well)
Column 5: PrimeStar PCR Mix (244 ul per well)
* 5x GXL Buffer: 1244 ul (2x 622 ul)
* dNTPs: 498 ul
* Polymerase: 249 ul |
Diluted DNA Plate | C3 on magnet | 96-well HSP | 10 ul per well of sample DNA at 1 ng/ul |
i5/i7 Index Primer Plate | B3 (after prompt) | 96-well HSP | 10 ul per well of index from IDT for Illumina DNA/RNA UD Index plate |
Water Plate | A3 (after prompt) | 96-well HSP | 75 ul per well |
Press the Finish button to start the run. The program takes about 80 minutes.
When prompted (after ~55 minutes) add the i5/i7 Index Primer and Water Plates.
When prompted (after ~70 minutes) add more 150 ul filter tips.
When the protocol finishes, seal the plate with Microseal ‘B’, and centrifuge at 280 × g for 30 seconds. Place on the thermal cycler and run the BLT PCR program with a 45 uL volume and 100°C lid:
- 68°C for 3 min
- 98°C for 3 min
- 12 cycles of:
- 98°C for 45 sec
- 62°C for 30 sec
- 68°C for 2 min
- 68°C for 1 min
- 10°C hold
This is a safe stopping point. If you stop here, seal the plate and store at -20°C for up to 3 days. Do not leave in the thermal cycler overnight or else the samples will evaporate.
Run the “Standard Post-PCR Size Selection” Protocol on Zephyr
This protocol will automatically start after the previous protocol as long as the application isn’t closed. If it was closed, restart the application, select the appropriate number of columns, and then press the “Standard Post-PCR Size Selection” button.
Load the deck as follows using the prompts:
All volumes are for a full 96-well plate. Adjust as necessary using the HackFlex Zephyr Workbook.xls document (C:\ProgramData\CaliperLS\Maestro\Workbooks).
Item | Position | Plasticware | Contents |
150 ul Filter Tips | A1, A2, B1, B2 | Box Filter Tips | Full box |
Diluted SPB Plate | A3 | 96-well HSP | Empty (stacked) |
Undiluted SPB Plate | A3 | 96-well HSP | Empty (stacked) |
SPB Plate | B3 (on shaker) | 96-well HSP | 57 ul per well of IBP/AmpureXP/SPRI beads |
Waste Reservoir | B4 | 2 ml DWP | Empty |
RSB Plate | C2 on CPAC adapter | 96-well HSP with Lid | 32 ul per well of RSB or Water THIS WILL BE THE FINAL ELUTION PLATE |
Water Plate | C3 on magnet | 96-well HSP | Saved from previous protocol. |
Ethanol Plate | C4 | 12-column Reservoir Seahorse Deepwell | 4 ml per well of 80% Ethanol (freshly made) |
Sample Plate | (after prompt) | 96-well HSP | From PCR Machine. Spin down before loading. |
Press the Finish button to start the run. The program takes about 60 minutes.
When prompted (~5 minutes), add the Sample Plate.
When prompted (~50 minutes), add more 150 ul filter tips.
This is a safe stopping point. If you stop here, seal the plate with foil and store at -20°C for up to 30 days.
Next Steps
- Libraries with DNA Inputs of 100-500 ng generated in the same experiments do not require quantification individually. These can be pooled in a microcentrifuge tube. Add 5 ul of each library (up to 384), vortex to mix, and centrifuge briefly before quantifying the pool.
- Libraries with DNA Inputs of <100 ng (i.e. HackFlex libraries!) require individual quantification.
- Use the HS DNA 5000 tape and the appropriate reagent buffer and ladder. A successful library preparation will have a peak of around 500 bp. See below for example traces. Calculate the average fragment size of the individual libraries.
- Quantify the libraries with the Picogreen.
- Use the concentration and average fragment size to calculate the molarity of individual samples.
- Pool equal molar concentrations of each library.
- Calculate pool molarity by measuring concentration via Qubit and average fragment size via TapeStation.
- Dilute to 2 nM (NextSeq) or 1 nM (MiniSeq) before preparing the run
HackFlex Tapestation Trace