Jun 03, 2026

5. Loading SMRTbell 3.0 Libraries onto the Sequel IIe

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx3py4g8j/v1
  • 1CFSAN/FDA;
  • 2USDA;
  • 3US FDA
Jae Hee Jang
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygx3py4g8j/v1
Protocol Citation: Jae Hee Jang, Kathryn Judy, Ellie Meeks, Maria Hoffmann 2026. 5. Loading SMRTbell 3.0 Libraries onto the Sequel IIe. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3py4g8j/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 11, 2023
Last Modified: June 03, 2026
Protocol  Integer ID: 84856
Keywords: PacBio, Pacific Biosciences, Sequel IIe, SMRTbell, 3.0, Loading, Library, Libraries, library samples onto the sequel iy, loading smrtbell, smrtbell library prep, pacbio smrtbell library prep, prepared library sample, sequel iy, library prep on the sciclone, sequel iie this protocol, library, library prep
Abstract
This protocol outlines the workflow for loading prepared library samples onto the Sequel IIe. It is recommended to use this protocol after "PacBio SMRTbell Library Prep 3.0 (Manual Version)" or after library prep on the Sciclone. Samples should be around 12-15 kb in size and between 20 and 60 ng/µL.
Materials
Reagents
  • Sequel II Binding Kit 3.2 (PacBio Cat# 102-194-100)
  • SMRTbell Cleanup Beads (PacBio Cat# 102-158-300)
  • Sequel II Sequencing Kit 2.0 (1 rxn) or (4 rxn) (PacBio Cat# 102-194-400 or 101-820-200)
  • SMRT Cell 8M Tray (PacBio Cat# 101-389-001)
  • SMRT Cell Oil (PacBio Cat# 100-621-300)
  • optional Qubit 1x dsDNA High Sensitivity Kit, (Thermo Fisher Scientific Cat# Q33231 or Q33230)

Supplies
  • Pipette Tips, Sterile, Filtered (P2, P20, P200), any manufacturer
  • Wide-bore Pipette Tips, Sterile, Filtered (P200) (Mettler Toledo Cat# 30389241 or equivalent)
  • DNA Low-binding microcentrifuge tubes, 1.5mL, sterile, (Eppendorf Cat# 022431005 or equivalent)
  • optional Qubit Assay Tubes (Thermo Fisher Scientific Cat# Q32856)
  • Sequencer Pipette Tips (PacBio Cat# 100-667-601)
  • Tube Septa (PacBio Cat# 100-667-700)
  • Sequel Sample Plate Foil (PacBio Cat# 100-667-400)
  • Sample Plate (PacBio Cat# 000-448-888)
  • Sequel Mixing Plate (PacBio Cat# 100-667-500)
  • Nitrogen supply, >500psi remaining, installed

Equipment
  • SMRT Link software
  • Micropippettes (P2, P20, P200), any manufacturer
  • Microcentrifuge (Benchmark Scientific Cat# C1012 or equivalent)
  • Vortex (Scientific Industries Cat# SI-A236 or equivalent)
  • Vortex Deepwell Microplate Adapter and Platform Attachment (Scientific Industries Cat# 504-0232-00and 146-6005-00, or equivalent)
  • Magnetic Separation Rack (Thermo Fisher Scientific Cat# 12321D or equivalent)
  • Ice bucket, any manufacturer
  • optional Qubit Fluorometer (4.0, 3.0, or 2.0) (Thermo Fisher Scientific Cat# Q33238)
  • Microplate centrifuge (Eppendorf Cat# 022620596 or equivalent)
  • Microplate Heat Sealer (Thermo Fisher Scientific Cat# AB1443-A or equivalent)
  • Sequel IIe Sequencer (PacBio)
Safety warnings

Safety information
Compressed Gas Warning: The Sequel IIe instrument requires gaseous nitrogen, supplied by a nitrogen cylinder. Compressed nitrogen gas may cause rapid suffocation (by displacement of oxygen) or frostbite (from contact with rapidly expanding gas) if released. Cylinders may explode if heated, causing injury or death and releasing the compressed gas. Keep nitrogen cylinders tightly closed until properly connected to the equipment (properly rated for cylinder pressure). Always keep cylinders upright and strapped to the wall.

Chemical Safety Warning: Take proper precautions and wear appropriate PPE when handling potentially hazardous chemicals. Ensure that chemicals, spent containers, and unused contents are disposed of in accordance with governmental safety standards.

PacBio Sequel II Binding Kit 3.2: No components of the PacBio Sequel II Binding Kit 3.2 are classified as hazardous substances or mixtures. See PacBio SDSs for further information.

SMRT Link Sample Setup
Log into SMRT Link and navigate to the Sample Setup page from the dropdown menu at the top left
Click "Add Calculation" and fill out the Sample Calculation form (the landing page for Sample Setup) as follows:
  • Provide a Name for the library
  • Use the dropdown menu to select the Application. For microbial WGS, select Microbial Assembly. For metagenomic samples, select Shotgun metagenomic profiling or assembly
  • Verify the Library type has auto-populated to Standard
  • Select the correct Polymerase / Binding kit, Sequel II Binding Kit 3.2
  • Input the number of samples to prepare. For a single pooled library, the Number of samples is 1. Increase this number to prepare more than one library for loading
  • Input the SMRT Cells per sample. Microbial WGS usually requires only 1 SMRT Cell per library
  • Input the Available volume per sample (library)
  • Provide Sample concentration and Insert size from library pool QC (Qubit, Femto)
  • If desired, adjust Cleanup anticipated yield. The default 75% is acceptable if cleanup yield has not been experimentally determined on similar libraries in the past
  • Input Concentration on plate. 160 picomolar (pM) - 200 picomolar (pM) is effective, though over the recommended concentration according to PacBio

After filling in the fields, instructions for preparing the final library for loading onto the Sequel IIe will populate. These instructions can be printed using the Print icon at the top right of the Sample Setup page
Follow the printed Sample Setup protocol to prepare the final library. Sections 3 - 8 below (Annealing primer - Final loading dilution) provide an example walkthrough of the Sample Setup protocol, but the generated Sample Setup protocol from SMRT Link must be used to prepare the final loading library
Preparations
Retrieve the Sequel II Sequencing Plate 2.0 from frozen storage and remove the plate (in a foil bag) from the box. Place the plate in a (tap) cold water bath for 01:00:00 to thaw. If the plate is removed from the foil bag before placing in the bath, protect from light. Alternatively, the plate can be kept sealed in the foil bag - the (1 rxn) plate will thaw in 60 minutes while still in foil

If multiple SMRT Cells will be used, thaw plate(s) for a sufficient number of reactions. 1 SMRT Cell requires 1 rxn. Sequel II Sequencing Plate 2.0s are available in 1 rxn and 4 rxn configurations, supporting a maximum of 8 SMRT Cells (sequenced in series) at once. See Step 37 for further details on Sequencing Plate options

Turn on the Microplate Heat Sealer to preheat the sealer. Do not touch the heated portions of the sealer. Assume the sealer is hot at all times
Annealing primer

Note
This is a process walkthrough and should not be used as instructions for laboratory work. Follow the Sample Setup protocol generated in Section 1 (SMRT Link Sample Setup) specific to your library

Take out Annealing Buffer, Sequel II Primer 3.2, Sequel II Polymerase Dilution Buffer, ABC buffer, and Sequel II Loading Buffer 3.2 from Binding Kit 3.2 and place at Room temperature . Once thawed, all except the Sequel II Loading Buffer 3.2 should be placed On ice . Keep the Sewquel II Loading Buffer 3.2 at Room temperature

Fully thaw Annealing Buffer and Sequel II Primer 3.2 first. Once thawed, these reagents can be placed On ice

Remove SMRTbell cleanup beads from refrigeration and place at Room temperature . Protect from light. Moving the beads before beginning step 11 provides sufficient time for the beads to fully equilibrate to room temperature (00:30:00 )

Combine the following components at the volumes specified in the Sample Setup calculation in a new low-binding tube and flick the tube to mix
  • Sample
  • Annealing Buffer
  • Sequel II Primer 3.2
Incubate at Room temperature  for 00:15:00 .
15m
During the incubation period, do the following:
  • Return Annealing Buffer and Sequel II Primer 3.2 to -20 °C storage
  • Make sure that Sequel II Polymerase Dilution Buffer is thawed for the next step. Once thawed, place On ice

OPTIONAL: Dilution step before polymerase binding
In the case of a high sample concentration, a dilution step may needed to reach the target concentration for the sample. Refer to the Sample Setup calculation to see if this step is needed. If not, skip to step 17.
Add ABC buffer to the sample, and flick the tube to mix. Refer to the calculation for the specific volume of ABC buffer.
Discard the volume given in the calculation and retain the rest.
Bind Sequencing Polymerase
After the Polymerase Dilution Buffer is thawed, remove Polymerase Stock from the freezer, quick-spin to collect liquid, and place On ice . Do not vortex.

Combine specified volumes of Polymerase Dilution Buffer and Polymerase Stock, pipette-mix, and place On ice

Diluted Polymerase must be used immediately, any remaining should be discarded. It is recommended to begin this step as close to the end of the previous incubation as possible
After the annealing incubation (Step 12) is complete, add the specified volume of diluted polymerase to the annealed library and finger tap or pipette-mix with a wide-bore tip to mix
Incubate at Room temperature  for 00:15:00 . Bound complex can be stored at 4 °C for 4 weeks.(*)

(*) Sequencing performance after storage is dependent on DNA sample quality/type and cannot be guaranteed
15m
During the incubation period, do the following:
  • Ensure SMRTbell Clean-up beads are at Room temperature
  • Make sure that Sequel II Loading Buffer 3.2 is thawed and at Room temperature
  • Make sure ABC Buffer is thawed and place On ice

Purification of Polymerase Bound SMRTbell® Complexes
Add the volume of ABC buffer indicated in the Sample Setup calculation to the sample
Add the specified volume of Clean-up Beads to the sample and gently flick or pipette-mix with a wide-bore pipette tip
Incubate at Room temperature for 00:10:00
10m
Place the tube in a magnetic separation rack until the beads collect to the side of the tube and the solution appears clear. Discard the supernatant. DO NOT wash the collected bead pellet
Remove the sample tube from the magnet and immediately resuspend the beads in the specified volume of Room temperature Sequel II Loading Buffer 3.2. Flick or pipette-mix with a wide-bore pipette tip
Incubate at Room temperature for 00:05:00 to elute the polymerase-bound complexes

5m
Place the tube in a magnetic separation rack until the beads collect to the side of the tube and the solution appears clear
Slowly pipette off the eluate without disturbing the beads. Transfer the supernatant to a new tube, discarding the old tube and beads. Place On ice  and protect from light

Optional: If desired, you may quantify sample recovery efficiency using a Qubit instrument, but this is not required. Otherwise, move on to step 30
Internal Control Dilution
During the elution incubation (Step 26), perform three sequential dilution steps using ABC Buffer. These instructions are also described in the Sample Setup protocol.

Use a new low-binding tube for each dilution step. After each dilution step, mix well by flicking tube by hand, pulse spin to collect contents, and keep On ice :

Retrieve Sequel II DNA Internal Control Complex 3.2 from the freezer, quick-spin to collect liquid, and place On ice

Fill 3 DNA Lo-bind tubes with 19 µL ABC Buffer and label them 1, 2, and 3

Perform a first dilution by adding 1 µL Sequel II DNA Internal Control Complex 3.2 to tube 1

Pipette-mix the dilution, quick-spin to collect contents, and place On ice

Perform the second dilution by adding 1 µL of the first dilution (tube 1) to tube 2

Pipette-mix the dilution, quick-spin to collect contents, and place On ice
Perform the third dilution by adding 1 µL of the second dilution (tube 2) to tube 3
Pipette-mix the dilution, quick-spin to collect contents, and place On ice
Discard tubes 1 and 2. Keep tube 3 On ice

Final Loading Dilution
Combine the specified volumes of prepared sample, Sequel II Loading Buffer 3.2, and diluted Internal Control (tube 3) to yield 120 µL final library. Store On ice and protect from light.

Load 115 µL of sample per well and/or store at 4 °C for up to 24 hours before use.(*)

(*) Sequencing performance after storage is dependent on DNA sample quality/type and cannot be guaranteed.
Setting up on the Sequel IIe
1h 2m
Open the Sequel IIe by unlocking the instrument door using the "lock" button at the bottom right of the touchscreen and swinging the door downward: pull the silver handle forward and then down smoothly. Close the Sequel IIe door when not loading / unloading items from the deck. The door will lock automatically when closed. The deck layout is shown below in Figure 1

Figure 1: Sequel IIe layout. Slots 1 to 3 are designated for pipette tip box placement. The sample plate is inserted in slot 4, and the 96 deep well mixing plate is inserted in slot 5. For Slot 6, one row (or two, if necessary) of SMRT cells can be inserted. In the area labelled 7, the oil tubes can be inserted in the circular slots. Slots 8 and 9 are for the reagent plates. Slot 10 belongs to the trash bin, which should be emptied out after every run.

Invert a tube of SMRT Cell Oil several times, spin it down briefly, and replace the cap with a tube septa

Insert the tube in the circular slot in area 7 (refer to Figure 1) labelled "1", making sure that the red line on the tube lines up with the red line on the machine
  • Each oil tube can support up to 4 samples per run. If two Sequencing Plates (in slots 9 and 8) will be used, prepare a second oil tube and place it in the circular slot "3". Circular slots "2" and "4" are not used on the Sequel IIe
  • Discard used tubes after each run
Insert a fresh mixing plate in slot 5 (refer to Figure 1), labelled "MIXING"
Insert an adequate number of SMRT cells into the SMRT cell compartment (slot 6, Figure 1), starting with the slot labelled "CELL 1"
Load 115 µL of the sample into well A01 of a new PacBio Sample Plate. Seal with foil using the preheated microplate sealer. Ensure the foil is aligned with the correct side up as indicated on the packaging. After sealing the plate, turn off the heat sealer. Insert the prepared sample plate in the sample plate compartment (slot 4, Figure 1)

If multiple samples (libraries) will be run in series, place the first in well A01, the second in B01, and so forth. Note which libraries are placed where for designing the run in SMRT Link. Up to 8 samples can be sequenced in one run (wells A01 through H01)
Prepare the Sequencing Plate 2.0 for Loading
  • if sequencing 1 sample, use a 1 rxn plate
  • if sequencing 2+ samples, use a 4 rxn plate
  • for 2 samples only, it is possible to use two 1 rxn plates in Slots 9 and 8 (Figure 1)
  • if sequencing 5+ samples, two plates are required
Remove the Sequel II Sequencing Plate 2.0 from its cold water bath. If the plate is in foil, remove the foil bag. Check the bottom of the wells and invert to make sure that the reagents are fully thawed
Vortex the plate for 00:01:00 at 1000 RPM, then spin down for 00:01:00 at 2000 RPM

2m
Insert the plate into the slot labeled "REAGENT 1" (slot 9, Figure 1). If two plates are required, insert the second plate into the slot labeled "REAGENT 2" (slot 8, Figure 1). If only one plate is used, fill slot 8 with a sealed blank plate (96 deep well plate with a foil cover)
Set up Run Design on SMRT Link
Log in to SMRT Link and navigate the the Runs page from the dropdown menu at the top left
Use the top center button to + Create New Run
Under Run Information at the left, select the Instrument Type as Sequel IIe
Run Name can optionally be changed. By default, it will include the sequencing date and setup time
Fill out the Sample Information tab. If multiple samples will be sequenced during the run, use the Add Sample button near the top right to add additional samples. For each sample, do the following:
  • Use the dropdown menu to select the Application. For microbial WGS, select Microbial assembly. For metagenomics samples, select Shotgun metagenomic profiling or assembly. Make sure this matches the Application chosen during Sample Setup
  • If sequencing multiple samples, note which Sample Well is selected for library placement. The default is A01, followed by B01, and so forth. Match the Well Sample Name for each to the sample's location from Step 36
  • Verify the SMRTbell Adapter Design is Overhang - SMRTbell Prep Kit 3.0/Kinnex v1
  • Verify the Binding Kit is Sequel II Binding Kit 3.2
  • Use the dropdown menu to select the correct Sequencing Kit based on which sequencing plate was inserted into the Sequel IIe in step 36.3. For sequencing a single library, this should be Sequel II Sequencing Plate 2.0 (1 rxn)
  • Verify the DNA Control Complex is Sequel II DNA Internal Control Complex 3.2
  • Input the library Insert Size (bp)
  • Input the On-Place Loading Concentration (pM) chosen during Sample Setup
  • Verify the Movie Time per SMRT Cell (hours) is 30
Advanced Options:
  • Do not touch Advanced Options
Barcoded Sample Options (if sample is pooled):
  • Select Yes for Sample is Barcoded
  • Specify SMRTbell adapter indexes for Barcode Set
  • Assign Bio Sample Names to Barcodes From a File. Download the formatted CSV file and record the correct sample IDs next to the corresponding barcodes, deleting rows that contain unused barcodes. Save the filled file and Browse to upload the Barcoded Sample Name File
  • Upload file for Assign Bio Sample Names to Barcodes; download formatted Excel sheet and record biosample names next to corresponding barcodes. Delete rows containing unused barcodes.
  • Demultiplex Barcodes should be set to IN SMRT LINK
Click the "Save" button on the upper right hand of the screen. This transfers the run information to the Sequel IIe instrument
On the Sequel IIe screen, select "Select Existing Run", then choose the correct run imported from SMRT Link. If the Run Name was not specified, it will include the date and time of Run setup
From the run preparation page, press Load to indicate the consumables have been added to the instrument, then Scan to have the instrument read the RFID tags
  • If any supplies have expired, the instrument will display a warning screen. The run can still proceed if reagents have been stored properly
Verify the connected nitrogen tank has >500 psi for a single run then press Check Nitrogen to confirm

Select Clear Trash to indicate the trash has been emptied
Start run
Check the run progress approximately 01:00:00 after start to confirm the run is proceeding successfully after initial checks. If there are no error messages on screen, the run is proceeding successfully

1h
If error messages appear, resolve or acknowledge the error(s) when applicable (e.g., select to proceed after acknowledgement of a low humidity warning)
Resetting the run or restarting the machine may resolve other errors
If the run cannot proceed, store sample plate and reagent plate at 4 °C (protected from light), discard oil tube and mixing plate, and contact PacBio tech support