Introduction
The Illumina DNA Prep (previously Nextera DNA Flex) protocol is a versatile and efficient approach for producing high-quality sequencing libraries from DNA samples. This protocol utilizes bead-linked tagmentation technology, which integrates DNA fragmentation and adapter tagging into a single enzymatic reaction. The kit offers flexibility in accommodating a broad range of DNA inputs. If the total input falls below 100 ng, it is necessary to quantify and normalize the input DNA. However, if the input DNA quantity is between 100-500 ng, normalization is not required.
This protocol has been adapted from the Illumina DNA Prep protocol, which can be found here (full protocol) and here (checklist protocol). We also have a protocol for a low-cost variation called HackFlex, 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 Water | Sigma | W4502-1L | RT |
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.
Tagmentation Buffer 1 (TB1) (Stored -20°C)
Bring to room temperature. Vortex to mix.
Tagment Stop Buffer (TSB) (Stored Room Temperature)
If precipitates are observed, heat at 37°C for 10 minutes and then vortex until dissolved.
Tagment Wash Buffer (TWB) (Stored Room Temperature)
Use at room temperature
Enhanced PCR Mix (EPM) (Stored -20°C)
Thaw on ice. Invert to mix, then briefly centrifuge.
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.
Illumina Purification Beads (IPB) (Stored Room Temperature)
Vortex and invert to mix.
Tagment DNA
Add 2–30 µl DNA to each well of a 96-well PCR plate so that the total input amount is 1–500 ng. If input is <100 ng, quantify and normalize.
If DNA volume < 30 µl, add nuclease-free water to the DNA samples to bring the total volume to 30 µl.
Vortex BLT vigorously for 10 sec to resuspend. Repeat as necessary as beads settle.
Prepare Tagmentation Master Mix (10% overage is already included):
Reagent | Volume | 96 Samples | x Samples |
BLT | 11 ul | 1,056 | |
TB1 | 11 ul | 1,056 |
Vortex the Tagmentation Master Mix thoroughly to resuspend.
Transfer 20 μl Tagmentation Master Mix to each well of the plate containing a sample. If prepping a large number of samples, you may divide the mix volume equally into an 8-tube strip and use a multichannel pippette to transfer.
Pipette each sample 10 times to resuspend.
Seal the plate with Microseal ‘B’, place on the preprogrammed thermal cycler, and run the TAG-FLEX program (55°C for 15 min, hold at 10°C, 50 uL volume and 100°C lid). All programs are under CHMI→NEXTERAFLEX on the thermocycler.
Post-Tagmentation Cleanup
Add 10 µl TSB to the tagmentation reaction.
Slowly pipette each well 10 times to resuspend the beads.
Seal the plate with Microseal ‘B’, place on the preprogrammed thermal cycler, and run the PTC-FLEX program (37°C for 15 min, hold at 10°C, 60 uL volume, 100°C lid).
After removing from the thermocycler, place the plate on the magnetic stand and wait until liquid is clear (~3 minutes).
Using a multichannel pipette, remove and discard supernatant.
Wash with TWB twice, as follows:
Remove the sample plate from the magnetic stand and very slowly add 100 µl TWB directly onto the beads, avoiding foaming.
Pipette slowly until beads are fully resuspended.
Place the plate on the magnetic stand and wait until the liquid is clear (~3 minutes).
Using a multichannel pipette, remove and discard supernatant.
Remove the plate from the magnetic stand and use a slowly pipette to add 100 µl TWB directly onto the beads.
Pipette each well slowly to resuspend the beads.
Seal the plate and place on the magnetic stand until the liquid is clear (~3 minutes). Keep on the magnetic stand until the appropriate step in the next section.
Amplify Tagmented DNA
Prepare PCR Master Mix, per sample (overage is already included):
Reagent | Volume (ul) | x # Samples |
EPM | 22 | |
Molecular-grade Water | 22 |
Vortex, and then centrifuge the PCR master mix at 280 × g for 10 seconds.
With the plate on the magnetic stand, remove the seal and use a 200 µl multichannel pipette to remove and discard supernatant (containing TWB). Foam that remains on the well walls does not adversely affect the library.
Remove the plate from the magnet.
Immediately add 40 µl PCR Master Mix directly onto the beads in each sample well.
Immediately pipette to mix until the beads are fully resuspended. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
Seal the sample plate with foil and centrifuge at 280 × g for 3 seconds.
Add 10 ul of the appropriate index adapter to each sample.
Using a pipette set to 40 µl, pipette 10 times to mix. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
Seal the plate with Microseal ‘B’, and then centrifuge at 280 × g for 30 seconds. Place on the thermal cycler and run the BLT PCR program with a 50 uL volume and 100°C lid:
- 68°C for 3 min
- 98°C for 3 min
- X cycles of (see table below):
- 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 2°C to 8°C for up to 3 days. Do not leave in the thermal cycler overnight or else samples will evaporate.
Clean Up Libraries
Centrifuge PCR plate at 280 × g for 1 minute to collect contents at the bottom of the well.
Place the plate on the magnetic stand and wait until the liquid is clear (~5 minutes).
Transfer 45 µl supernatant from each well of the PCR plate to the corresponding well of a new deep-well plate.
Vortex IPB and invert to mix.
Add 40 µl nuclease-free water to each well containing supernatant.
Add 45 µl beads to each well containing supernatant.
Pipette each well 10 times to mix. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
Seal the plate and incubate at room temperature for 5 minutes.
Place on the magnetic stand and wait until the liquid is clear (~5 minutes).
During incubation, thoroughly vortex IPB, and then add 15 µl to each well of a new deep-well plate.
Transfer 125 µl supernatant from each well of the first plate into the corresponding well of the second plate (containing 15 µl IPB).
Pipette each well in the second plate 10 times to mix. Alternatively, seal the plate and use a plate shaker at 1600 rpm for 1 minute.
Discard the first plate.
If your starting material is anything other than gDNA, consult the manual. Smaller input DNA lengths may require a different quantity of beads.
Incubate at room temperature for 5 minutes.
Place on a magnetic stand and wait until the liquid is clear (~5 minutes).
Remove and discard all supernatant from each well without disturbing the beads.
Wash 2 times as follows:
Add 190 μl fresh 80% EtOH to each well.
Incubate on the magnetic stand for 30 seconds.
Remove and discard all supernatant from each well without disturbing the beads.
Using a 20 μl pipette, remove residual 80% EtOH from each well.
Air-dry on the magnetic stand for 5 minutes or until beads just start to dry.
Remove from the magnetic stand.
Add 32 μl RSB to each well.
Pipette to resuspend.
Incubate at room temperature for 2 minutes.
Place on a magnetic stand and wait until the liquid is clear (~2 minutes).
Transfer 30 μl supernatant to a new plate.
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.
Quality Check
- 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 require individual quantification.
- Use the HS DNA 5000 or HS DNA 1000 tape and the appropriate reagent buffer. A successful library preparation will have a peak around 500 bp. See below for example traces.
- Quantify the sample with the HS DNA Qubit kit or PicoGreen.
Standard TapeStation Trace
