Preparing multiplexed 16S/18S/ITS amplicon SMRTbell libraries with the Express TPK2.0 for the PacBio Sequel2

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 .

To pool the 96 samples from the remaining 30µL of each well in the Aggregated PCR Plate 1, transfer 10µL 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 100µL 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.

Mix the below volume of AMPure PB beads with 75µL of the Plate 1 PCR Pool in a new 1.5 mL Eppendorf tube:

A	B
16S amplicon (~1500 bp)	0.6X
18S amplicon (~1850 bp)	0.6X
ITS amplicon (~750 bp)	1.2X

Gently finger mix (tap the bottom of the tube with your finger) the DNA/bead mixture and then incubate the on the benchtop for 0h 10m 0s 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.

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.

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 0h 0m 30s, slowly pipette off the 80% ethanol and discard.

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.

Remove the tube from the magnetic rack and allow the beads to air-dry for 0h 2m 0s.

Add 20µL of Elution Buffer to the beads to elute the DNA:

• Gently finger mix the tube.

• Elute the DNA by letting the mixture incubate for 0h 2m 0s 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.

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.

Quantify both P1+P2 Cleaned PCR Pools using the Invitrogen Qubit dsDNA HS assay as in the IMR standard MiSeq PCR protocol.

Combine equal molar quantities of both P1+P2 Cleaned PCR Pools in a total mass of at least 350ng in a new 1.5 mL Eppendorf tube. Adjust the final Combined Pool volume to 23.5µL to prepare the SMRTbell library.

DNA Damage Repair

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

A	B
DNA Prep Buffer	3.5 µL
DNA Damage Repair Mix v2	1.0 µL
NAD	0.5 µL
Combined PCR Pool	23.5 µL
Total Volume	28.5 µL

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

• Incubate at 37°C for 0h 30m 0s, then return the reactions to 4°C. Proceed to the next step.

End-Repair/A-Tailing

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

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

• Incubate at 20°C for 0h 30m 0s, followed by 65°C for 0h 30m 0s, then return the reaction to 4°C. Proceed to the next step.

Adapter Ligation

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

A	B
Reaction Mix 2	30.0 µL
Overhang Adapter v3	2.5 µL
Ligation Mix	15.0 µL
Ligation Additive	0.5 µL
Ligation Enhancer	0.5 µL
Total Volume	48.5 µL

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

• Incubate at 20°C for 1h 0m 0s, followed by 65°C for 0h 10m 0s to heat kill the ligation reaction, then return the reaction to 4°C. Proceed to the next step.

Follow Steps 3-9 above with the addition of 58.2µL (1.2X) of AMPure PB beads to the above 48.5µL of final SMRTbell Template and using 20µL 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.

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.