TITLE: Preparing multiplexed 16S/18S/ITS amplicon SMRTbell libraries with the Express TPK2.0 for the PacBio Sequel2
AUTHORS: André M Comeau, Gina V Filloramo

[DESCRIPTION]
The preparation of amplicon libraries using the PacBio Express Template Kit 2.0 (TPK2.0) with fusion primers at the IMR.

Based upon PacBio protocol 101-599-700 (Jun.2019).

[STEPS]
SECTION: Barcoded PCRs
1. Prepare PCRs and verify by Coastal Genomics gels as in the IMR protocol Preparing multiplexed 16S/18S/ITS amplicons for the Illumina MiSeq, except only using Plates 1+2 (F1R1 + F1R2) of the PacBio working primers from the IMR protocol Preparing Combined Indexed Primer Plates (IDT Standard) for the PacBio Sequel2 - Sequel Dual Indices.
SECTION: Barcoded PCRs
2. To pool the 96 samples from the remaining 30 µL of each well in the Aggregated PCR Plate 1, transfer 10 µL of each column into one column of a new 96-well plate named the Amplicon Pool Plate (remaining columns to be used in subsequent poolings). Once complete, pipette 100 µL of each of the 8 wells into one 1.5 mL Eppendorf tube and label it Plate 1 PCR Pool. Repeat this step for the Aggregated PCR Plate 2 and label that tube as the Plate 2 PCR Pool.

SECTION: AMPure PB Clean-up
3. Mix the below volume of AMPure PB beads with 75 µL of the Plate 1 PCR Pool in a new 1.5 mL Eppendorf tube:

 

 

SECTION: AMPure PB Clean-up
4. Gently finger mix (tap the bottom of the tube with your finger) the DNA/bead mixture and then incubate the on the benchtop for 10 min at Room temperature. For improved recovery, you can additionally finger mix the tube 2-3 times during the incubation. After incubation, quickly spin down the tube to collect all the liquid into the bottom of the tube.

SECTION: AMPure PB Clean-up
5. Place the tube on a magnetic bead rack to collect the beads to the side/bottom of the tube and, once cleared, slowly pipette off and discard the cleared supernatant without disturbing the bead pellet.

 

SECTION: AMPure PB Clean-up
6. Wash the beads with freshly prepared 80% ethanol:

With the tube on the magnetic rack, slowly fill the tube with sufficient volume of 80% ethanol without waste or cross-contamination.

Do not disturb the bead pellet.

After 30 s, slowly pipette off the 80% ethanol and discard.

 

SECTION: AMPure PB Clean-up
7. Remove any residual 80% ethanol:

Remove the tube from the magnetic rack and quickly spin to collect the beads.

Place the plate back on the magnetic rack.

Pipette off and discard any remaining 80% ethanol.
SECTION: AMPure PB Clean-up
8. Remove the tube from the magnetic rack and allow the beads to air-dry for 2 min.

SECTION: AMPure PB Clean-up
9. Add 20 µL of Elution Buffer to the beads to elute the DNA:

Gently finger mix the tube.

Elute the DNA by letting the mixture incubate for 2 min at Room temperature.

Quickly spin the tube to collect the bead mixture, then place it back on the magnetic rack.

Wait for the supernatant to clear completely, then, without disturbing the bead pellet, transfer the supernatant to a new 1.5 mL Eppendorf tube and label as Plate 1 Cleaned PCR Pool.
SECTION: Quantification and Combining of Cleaned Pools
11. Quantify both P1+P2 Cleaned PCR Pools using the Invitrogen Qubit dsDNA HS assay as in the IMR standard MiSeq PCR protocol.
SECTION: SMRTbell Library Construction
13. DNA Damage Repair

Prepare DNA Damage Repair reaction ("Reaction Mix 1") as follows in one 0.2 mL PCR tube:

 

Gently finger mix the contents of the tube, followed by a quick spin down.

Incubate at 37 °C for 30 min, then return the reactions to 4 °C. Proceed to the next step.
SECTION: SMRTbell Library Construction
14. End-Repair/A-Tailing

Add 1.5 µL of End Prep Mix to the above 28.5 µL tube of Reaction Mix 1 to make a total volume of 30 µL of "Reaction Mix 2".

Gently finger mix the contents of the tube, followed by a quick spin down.

Incubate at 20 °C for 30 min, followed by 65 °C for 30 min, then return the reaction to 4 °C. Proceed to the next step.
SECTION: SMRTbell Library Construction
15. Adapter Ligation

Prepare the Adapter Ligation Reaction by adding the following components, in order, to the above Reaction Mix 2 tube:

 

Gently finger mix the contents of the tube, followed by a quick spin down.

Incubate at 20 °C for 60 min, followed by 65 °C for 10 min to heat kill the ligation reaction, then return the reaction to 4 °C. Proceed to the next step.

 

SECTION: Purification of SMRTbell Templates
16. Follow Steps 3-9 above with the addition of 58.2 µL (1.2X) of AMPure PB beads to the above 48.5 µL of final SMRTbell Template and using 20 µL of Elution Buffer to elute the DNA from the beads.

Quantify the eluted Purified SMRTbell Template as above in Step 11.

Purified SMRTbell Template is stored at -20 °C.

 

SECTION: Anneal, Bind and Clean Final SMRTbell Templates
17. For primer annealing and polymerase binding, follow the instructions in the SMRT Link Sample Setup print-out. Use the Sequel II Binding Kit 2.1 and Sequencing Primer v4 for all libraries.
SECTION: AMPure PB Clean-up
10. Repeat Steps 3-9 for the Plate 2 PCR Pool and label the final 1.5 mL Eppendorf tube as Plate 2 Cleaned PCR Pool.
SECTION: Quantification and Combining of Cleaned Pools
12. Combine equal molar quantities of both P1+P2 Cleaned PCR Pools in a total mass of at least 350 ng in a new 1.5 mL Eppendorf tube. Adjust the final Combined Pool volume to 23.5 µL to prepare the SMRTbell library.