AMPure XP Solid Phase Reversible Immobilization (SPRI) Bead PCR Clean-Up (optional)

This protocol is modified from Beckman Coulter Life Sciences’ protocol for typical PCR cleanup of DNA barcoding PCR products using solid phase reversible immobilization (SPRI) beads (i.e., AMPure XP beads). This protocol will suitably remove the remnants of PCR, selecting for PCR products in the range of 400-800 base pairs, which is consistent with most standard DNA barcode marker regions (e.g., rbcL, COI, etc.). The intent of this protocol is to prepare DNA amplicons for rapid sequencing with ONT’s Rapid Barcoding Kit. Note that it is “optional,” because once all samples are pooled, bead clean-up will still need to be conducted on the pooled amplicon library.

1. Re-suspend AMPure XP beads by using the pipette to mix.
2. Add the entire PCR reaction (~20 μL) directly to a labeled 1.5-mL microfuge tube containing 36 μL AMPure XP beads. Use the pipette to carefully mix the beads and DNA.
3. Incubate for 5 minutes at room temperature on a microfuge tube rack.
4. Spin down the tube for 15 seconds and place the tube on a magnetic rack. Wait for about 1 minute for the beads to completely pellet near the magnet. Note that the liquid
5. Keeping the tube on the magnet, pipette off the supernatant and discard without disturbing the pellet.
6. Keeping the tube on the magnet, add 200 μL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
7. Repeat Step 5.
8. Remove the tube from the magnetic rack and spin down the tube for 15 seconds. Replace the tube on the magnetic rack and allow the pellet to reform.
9. Keeping the tube on the magnet, remove any residual liquid (~10-30 μL). With the tube open, allow the pellet to dry for 30 seconds. Caution: Do not allow the pellet to dry to the point of cracking. Be prepared to immediately move on to Step 10.
10. Remove the tube from the magnetic rack and resuspend the pellet in 10 μL of EB. Use the pipette to continuously wash the EB over the pellet until it completely dissociates.
11. Incubate the tube for 5 minutes away from the magnetic rack.
12. Spin down the tube for 15 seconds and replace the tube on the magnetic rack. Wait for about 1 minute for the beads to completely pellet near the magnet. Note that the liquid supernatant should be completely clear.
13. Remove and retain 10 μL of supernatant. Pipette the supernatant into a thin-walled PCR tube. Discard the tube containing the pellet. The PCR product is now ready for the ONT Rapid Barcode Kit.

Rapid Barcode Index Attachment

After the optional SPRI bead clean-up step, DNA amplicons are ready for rapid barcode index attachment. Note that up to 24 DNA amplicons can be uniquely tagged during a single library prep and sequencing run using the Rapid Barcode Kit 24 V14. ONT manufactures another kit with up to 96 unique barcode indexes.

1. Obtain one unique Rapid Barcode (RB) mix for each PCR amplicon. Record each sample number and its associated RB.
2. Spin down RB tubes. Pipette 1.5 μL of each unique RB directly into a PCR tube containing 10 μL of its associated DNA amplicon. Vortex briefly or pipette to mix.
3. Ensure that PCR tubes are sealed shut, and then incubate tubes in a thermal cycler using the following conditions:

Temperature	Time
30° C	2:00 minutes
80° C	2:00 minutes
4° C	hold

4. Spin down PCR tubes. DNA amplicons are now uniquely tagged using the ONT RBs.
5. Pipette each tagged DNA amplicon (~11.5 μL) into a single class-wide 1.5-mL microfuge tube. This represents the pooled amplicon library.

Library Clean-Up with Solid Phase Reversible Immobilization (SPRI) Beads

Library clean-up will remove remnants of PCR (e.g., primers, polymerase, etc.), residual transposome and unused rapid barcodes, as well as any dyes or indicators used during previous steps (e.g., Cresol red). Note that this step must be carried out even if the optional PCR clean-up step was performed with a class.

1. Re-suspend AMPure XP beads by using the pipette to mix or vortexing. Add an appropriate volume of AMPure XP beads to the pooled amplicon library using the following formula:
   VolumeAMPure XP beads= (11.5 μL)(No. of DNA amplicon samples)
   Use a pipette to gently mix the beads and amplicon library such that the beads are evenly distributed throughout the tube.
2. Incubate the mix at room temperature for 10 minutes, flicking occasionally to ensure that beads remain suspended in solution. Note that DNA amplicons are coordinating with SPRI beads during this time.
3. Spin down the mix for 15 seconds to form an initial bead pellet. Then, place the tube on a magnetic rack to pellet the beads. Allow up to one minute for the beads to pellet.
4. Keeping the tube on the magnetic rack, use an appropriate pipette to remove all of the liquid from the tube and discard. Avoid disturbing the pellet. Remember that the DNA is located in the pellet at this point.
5. Keeping the tube on the magnetic rack, add 1000 μL of 80% ethanol to the pellet. Then, remove the supernatant and discard without disturbing the pellet.
6. Repeat Step 5.
7. Remove the tube from the magnetic rack and spin down the tube for 15 seconds. Replace the tube on the magnetic rack and allow the pellet to reform.
8. Keeping the tube on the magnet, remove any residual liquid (~10-30 μL). With the tube open, allow the pellet to dry for 30 seconds. Caution: Do not allow the pellet to dry to the point of cracking. Be prepared to immediately move on to Step 9.
9. Remove the tube from the magnetic rack and resuspend the pellet in 15 μL of EB. Use the pipette to continuously wash the EB over the pellet until it completely dissociates. Note that this may take several repeated washes with the same 15 μL EB.
10. Incubate the tube for 10 minutes away from the magnetic rack at room temperature.
11. Spin down the tube for 15 seconds and replace the tube on the magnetic rack. Wait for about 1 minute for the beads to completely pellet near the magnet. Note that the liquid supernatant should be completely clear.
12. Remove and retain 11 μL of supernatant. Pipette the supernatant into a 1.5-mL microfuge tube. Discard the tube containing the pellet. The pooled amplicon library is now ready for the attachment of rapid adapters and motor proteins.

Rapid Adapter Attachment

The final step of the library preparation is attaching the motor protein to the ends of the DNA. The Rapid Adapter (RA) reagent contains the motor protein and can attach it to the rapid adaptor chemistry on the ends of the DNA which were added by the transposome complex in a previous step.

1. In a 1.5-mL tube dilute Rapid Adapter (RA) as follows:

Reagent	Volume
Rapid Adapter (RA)	1.5 μL
Adapter Buffer (ADB)	3.5 μL
Total	5 μL

2. Add 1 μL of diluted Rapid Adapter to indexed DNA.
3. Incubate for 10 min at room temperature.

Flow Cell Check

The flow cell contains the nanopores which facilitate the sequencing. The number of active pores needs to be determined for every flow cell before it is to be used to ensure that there are an adequate number for the sequencing experiment. Because we are sequencing amplicons and don’t need very high-throughput, we suggest using the Flongle flow cell which has a maximum of 126 pores rather than the Minion, which has maximum of 2048.

1. Open MinKNOW software.
2. Click the “Start” tab on the upper left of the screen.
3. Click “Flow cell check”

The sequencer will take a few minutes to get to the proper temperature and run the flow cell check. When complete, the number of usable pores on the flow cell will be displayed. The flow cell is considered under warranty if it is checked within 2 weeks of the delivery date, was properly stored at 4 degrees, but has less than 50 pores. If under warranty, contact Oxford Nanopore within 2 days of performing the flow cell check.

Flow Cell Priming

Before loading the flow cell with the DNA library, the flow cell needs to be “primed” for sequencing. The reagents required for priming and loading the Flongle flow cell are contained in the Flongle Sequencing Expansion (EXP-FSE002), a separate box from the library prep reagents, which contains glass vials of Sequencing Buffer (SB), Flow Cell Flush (FCF), and Library Beads (LIB).

1. Mix 117 μL FCF (from the Flongle expansion kit) and 3 μL of FCT (from the Rapid Barcoding kit).
2. Peel back the seal from the Flongle flow cell
3. Slowly expel liquid from the pipette by turning the dial clockwise until a bead of liquid forms at the tip.
4. Place the pipette tip in the sample port, keeping the pipette vertical.
5. Turn the dial clockwise to load the flush buffer and tether into the flow cell, stopping right before all liquid is entirely expelled to avoid pipetting air into the flow cell.

Flow cell Loading

1. Make sequencing mix:

Reagent	Volume
Sequencing Buffer (SB)	15 μL
Loading Beads (LIB)	10 μL
DNA Library	5 μL
Total	30 μL

2. Pipette up sequencing mix, slowly expel liquid from the pipette by turning the dial clockwise until a bead of liquid forms at the tip.
3. Place the pipette tip in the sample port, keeping the pipette vertical.
4. Turn the dial clockwise to load the sequencing mix to the flow cell, stopping right before all liquid is entirely expelled to avoid pipetting air into the flow cell.
5. Place the sticker back on.
6. Proceed to start sequencing on MinKNOW software.