TITLE: Preparing multiplexed WGS/MetaG SMRTbell libraries with the Express TPK2.0 for the PacBio Sequel2
AUTHORS: André M Comeau, Gina V Filloramo

[DESCRIPTION]
The preparation of (meta)genomic libraries using the PacBio Express Template Kit 2.0 (TPK2.0) with the Barcoded Adapter Plate 3.0 at the IMR.

Based upon PacBio protocol 101-696-100 (Dec.2019).

[STEPS]
SECTION: gDNA Shearing with Covaris g-TUBEs
1. Dilute 1 µg gDNA with water into 100 µL to get a final concentration of 10 ng/µL.
SECTION: gDNA Shearing with Covaris g-TUBEs
2. Transfer gDNA to the g-TUBE and centrifuge (cap up) at 860 x g, 5 min (MBI centrifuge D3024 fixed-angle rotor) to achieve a target mode size of 9-10 kb. Repeat the spin until the entire gDNA sample has passed through the orifice (this may take 2-3 spins).

 

 

SECTION: gDNA Shearing with Covaris g-TUBEs
3. Invert the g-TUBE and repeat the above spin/procedure.
SECTION: gDNA Shearing with Covaris g-TUBEs
4. Collect the sheared gDNA from the 48 individual g-TUBEs and transfer them to the first 6 columns of a new 96-well PCR plate.
SECTION: AMPure PB Clean-up
5. Add 0.4X volume of AMPure PB beads to each sheared gDNA sample in the 96-well plate, then seal the plate and pulse vortex it to mix. Finally, quickly spin down the plate (for 2-3 seconds) to collect the bead mixture at the bottom of the plate.
SECTION: AMPure PB Clean-up
6. Incubate the beads on the benchtop for 10 min at Room temperature.

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

 

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

With the plate on the magnetic rack, slowly fill the plate 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
9. Repeat Step 8 above for the 2nd ethanol wash.
SECTION: AMPure PB Clean-up
10. Remove any residual 80% ethanol:

Remove the plate 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
11. Remove the plate from the magnetic rack and allow the beads to air-dry for 2 min.

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

Seal the plate and pulse vortex it, followed by a quick spin down.

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

Place the plate back on the magnetic rack and wait for the supernatant to clear completely, then, without disturbing the bead pellet, transfer the supernatant to a new 96-well plate and label as "Clean sheared gDNA".
SECTION: Sheared gDNA Quantification and Sizing
13. Quantify the Clean sheared gDNA using the Invitrogen Quant-iT dsDNA HS assay kit on the fluorescent 96-well plate reader. Use 1 µL of 1:10 diluted gDNA of each sample (1 µL of gDNA + 9 µL of water) to quantify. Use the remaining 9 µL of the 1:10 dilution to evaluate the size distribution of the sheared gDNA on an analytical platform: the IMR uses 0.5% precast gels with 3+20 kb DNA markers on the Coastal Genomics Nimbus Select robot.

SECTION: SMRTbell Library Construction
14. Remove Single-Strand Overhangs

Aliquot at least 480 ng of each sheared gDNA and add Elution Buffer to make a total volume of  14.6 µL for each into a new 96-well plate. PacBio low input for the Sequel II System (1 sample) requires a minimum of > 400 ng. PacBio low input for multiplexing with the Sequel II System (2 samples up to 600 Mbp per genome) requires a minimum of > 300 ng per sample.

Dilute the DNA Prep Additive 1:5 with Enzyme Dilution Buffer as follows:

 

Prepare DNA Prep Master Mix as follows:

 

For each sample, add 3.3 µL of the DNA Prep Master Mix to the above 14.6 µL of sheared gDNA.

Seal the plate and pulse vortex it, followed by a quick spin down.

Incubate at 37 °C for 15 min, then return the reactions to 4 °C. Proceed to the next step.
SECTION: SMRTbell Library Construction
15. DNA Damage Repair

Prepare DNA Damage Master Mix as follows:

 

For each sample, add 1 µL of the DNA Damage Master Mix to the above 17.9 µL of digested gDNA.

Seal the plate and pulse vortex it, 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
16. End-Repair/A-Tailing

For each sample, add 1 µL of End Prep Mix to the above 18.9 µL of damage-repaired gDNA.

Seal the plate and pulse vortex it, followed by a quick spin down.

Incubate at 20 °C for 10 min, followed by 65 °C for 30 min, then return the reaction to 4 °C. Proceed to the next step.
SECTION: SMRTbell Library Construction
17. Ligate Barcoded Overhang Adapters

Add 2 µL of Barcoded Overhang Adapters from the SMRTbell Barcoded Adapter Plate 3.0 to the above 19.9 µL of A-tailed gDNA.

Seal the plate and pulse vortex it, followed by a quick spin down.

Prepare Ligation Master Mix as follows:

 

For each sample, add 10.7 µL of Ligation Master Mix to the above 21.9 µL of barcoded gDNA.

Seal the plate and pulse vortex it, 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
18. Follow Steps 5-12 above with the addition of 14.7 µL (0.45X) of AMPure PB beads to the above 32.6 µL of final SMRTbell Templates and using 20 µL of Elution Buffer to elute the DNA from the beads.

Quantify the eluted Purified SMRTbell Templates as above in Step 13.

Purified SMRTbell Templates are stored at -20 °C.
SECTION: Anneal, Bind and Clean Final SMRTbell Templates (ABC)
20. For primer annealing and polymerase binding, follow the instructions in the SMRT Link Sample Setup print-out. Use the Sequel II Binding Kit 2.0 and Sequencing Primer v4 for all libraries.
SECTION: Pooling Calculation and Final Quantification
19. Pool purified SMRTbell Template libraries in equimolar amounts, if so desired, or use the PB Multiplex Calculator for more complex cases.

Quantify the final library pool in preparation for ABC below.

(Optional): Proceed with a gel size-selection (via 0.5% precast gel on the Coastal Genomics Nimbus Select robot, Pippin-Prep, or similar) of the final library pool to ensure absolute removal of any potential inhibitors (see note below).