Preparing multiplexed
amplicon libraries using®
SMRTbell
prep kit 3.0

Page 1

Procedure & checklist

This procedure describes the workflow for constructing amplicon libraries using the SMRTbell prep kit 3.0 for
sequencing on PacBio® Revio™, Sequel® II, and IIe systems. Amplicons may be indexed during PCR, or during library
preparation with SMRTbell indexed adapters.
Overview
PCR-indexed samples

Adapter-indexed samples

Samples per kit

1–24

1–24

Workflow time

3.5 hours

4 hoursSize

250–25,000 bp

250–25,000 bp

DNA input per Revio SMRT® Cell

300–2000 ng per pool

300–2000 ng per sample

DNA input per Sequel II SMRT Cell 8M

150–1000 ng per pool

150–1000 ng per sample© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-359-000 REV03 DEC2023

Page 2

Workflow
Primer-indexed samples
1

Input DNA quality control

Adapter-indexed samples
1

Input DNA quality control& cleanup& cleanup

Safe stop

Safe stop

2

Repair & A-tailing

3

Adapter ligation & cleanup

2

Repair & A-tailing3

Indexed adapter ligation& cleanup

Safe stop

4 Nuclease treatment & cleanup

Safe stop

4 Nuclease treatment & cleanup
Safe stop

5

Pool & concentrate© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-359-000 REV03 DEC2023

Page 3Required materials and equipment
DNA sizing (one or more of the following)
1% agarose gel, an electrophoresis unit, and imager

Any Major Lab Supplier (MLS)

2100 Bioanalyzer

Agilent technologies G2939BA

4200 TapeStation

Agilent technologies G2991BA

5300 or 5400 Fragment analyzer

Agilent technologies M5311AA or M5312AA

FEMTO Pulse system

Agilent Technologies M5330AA

DNA quantitation
Qubit fluorometer

Thermo Fisher Scientific Q33238

Qubit 1X dsDNA HS assay kit

Thermo Fisher Scientific Q33230

SMRTbell library preparation
SMRTbell prep kit 3.0

PacBio 102-182-700

SMRTbell adapter index plate 96A (optional; for indexing)

PacBio 102-009-200

200 Proof ethanol, molecular biology or ACS grade

Any MLS

Nuclease-free water, molecular biology grade

Any MLS8-channel Pipettes, P20 and P200

Any MLS

0.2 mL 8-tube strips

USA Scientific TempAssure 1402-4708

Single-channel Pipettes (P10, P20, P100, P200, and P1000)

Any MLS

Microcentrifuge

Any MLS

Magnetic separation rack compatible with 0.2 mL 8-tube strips

Any MLS

Thermocycler compatible with 0.2 mL 8-tube strips

Any MLS

1.5 mL DNA LoBind® Tubes

Eppendorf 022431021© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-359-000 REV03 DEC2023

Page 4General best practices
DNA input amountThe total amount of DNA input required for constructing the SMRTbell library is dependent on the mean size of theamplicons, or DNA fragment (e.g., plasmid), being sequenced.

PCR-indexed sample input
If samples were indexed during PCR, then the samples can be pooled prior to library prep and the total DNA input
amount will equal the amount of the multiplexed pool. The per sample input will equal the total DNA input divided by
the number of multiplexed samples.
Use no less than 300 ng of total input per Revio SMRT Cell and no less than 150 ng of total input per Sequel II SMRT
Cell 8M to ensure sufficient library yields for optimal SMRT cell loading. Larger amplicons will require more input
material to achieve desired molarity for SMRT Cell loading. Please refer to the tables below for the recommended
minimum total input amounts per SMRT Cell that will be used for the library. Lower total input amounts are possible
but may result in lower sequencing yield if there is not enough SMRTbell library available for optimal SMRT Cellloading.
Mean size

Minimum pooled amount per
Revio SMRT Cell

<3 kb

300 ng

3–12 kb

500 ng>12 kb

750 ngMean size

Minimum pooled amount per
Sequel II SMRT Cell 8M<5 kb

150 ng

5–7 kb200 ng>7 kb

300 ngAdapter-indexed sample input
Use no less than 300 ng of DNA input per sample per Revio SMRT Cell and no less than 150 ng of total DNA input per
sample per Sequel II SMRT Cell 8M to ensure sufficient library yields for optimal SMRT Cell loading. Larger amplicons
will require more input material to achieve desired molarity for SMRT Cell loading. Please refer to the tables below for
the recommended minimum total input amounts per SMRT Cell that will be used for the library. This is to ensuresufficient recovery of each sample at the end of library prep for equal mass or equal molar pooling. Using lower persample amounts, though possible, may result in low library yields and lead to uneven pooling and sequence coverage.
For applications that require lower input amounts, consider using indexed primers so samples can be pooled prior to
library prep.

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102-359-000 REV03 DEC2023

Page 5

Mean size

Minimum per sample amountper Revio SMRT Cell

<3 kb

300 ng

3–12 kb

500 ng>12 kb

750 ngMean size

Minimum per sample amount per
Sequel II SMRT Cell 8M<5 kb

150 ng

5–7 kb200 ng>7 kb

300 ngIndexing and multiplexing
Use the SMRTbell adapter index plates to index samples with the SMRTbell adapter. Quick-spin the plate to collectliquid at bottom of the well prior to use.
Pool amplicons of similar size for optimal sequence yields across all samples.
Pool amplicons ≥3 kb separately from amplicons £3 kb for optimal sequencing yields across all samples.
When amplicons are similar in size, pool an equal mass for each sample. Some experiments may require equal molar
pooling if the mean size differs between samples and similar coverage levels are required.

Sample quality
Using gel-extracted amplicon products may results in lower sequencing performance due to the damage inherently
caused by intercalating dyes such as ethidium bromide and exposure to UV radiation. Sequencing amplicons stainedwith SYBR dyes from ThermoFisher Scientific is untested, and therefore cannot be recommended. If working with agel-extracted product that has been stained with a dye, it is recommended to bring it through additional rounds ofamplification to remove damage and/or dyes prior to library prep and sequencing.

Reagent handling
Room temperature is defined as any temperature in the range of 18–23°C for this protocol.
Thaw the repair buffer, nuclease buffer, and elution buffer at room temperature.
Mix reagent buffers and SMRTbell adapter with a brief vortex prior to use. Enzyme mixes do not require vortexing.
Quick-spin all reagents in microcentrifuge to collect liquid at bottom prior to use.
Keep all temperature-sensitive reagents on ice.

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102-359-000 REV03 DEC2023

Page 6

Temperature-sensitive reagents
Step Used
Repair & A-tailing

Adapter ligation

Nuclease treatment

Tube

Reagent

Blue

End repair mix

Green

DNA repair mix

OrangeSMRTbell adapter

Yellow

Ligation mix

RedLigation enhancer

Light green

Nuclease mix

Bring SMRTbell cleanup beads and Qubit 1X dsDNA HS reagents to room temperature for 30–60 minutes prior to use.
Pipette-mix all bead binding and elution steps until beads are distributed evenly in solution.
Pipette-mix all SMRTbell prep reactions by pipetting up and down 10 times.
Samples can be stored at 4°C at all safe stopping points listed in the protocol.

© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-359-000 REV03 DEC2023

Page 7

Thermocycler programs
Program thermocycler(s) prior to beginning the protocol for the first time.
Repair and A-tailing, adapter ligation, and nuclease treatment thermocycler steps can be combined into a single
program and paused in between prep treatments if preferred.
Set the lid temperature to 75°C for all programs. If the lid temperature is not programmable, it is acceptable to leave at
95–105°C.

1. Repair & A-tailing
Step

Time

Temperature1

30 min

37°C

2

5 min

65°C

3

Hold

4°C

2. Adapter ligation
Step

Time

Temperature1

30 min

20°C

2

Hold

4°C

3. Nuclease treatment
Step

Time

Temperature1

15 min

37°C

2

Hold

4°C

© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-359-000 REV03 DEC2023

Page 8Procedure and checklist
1. Input DNA quality control & cleanup
Prior to library preparation, evaluate the quantity and size distribution of input DNA to determine whether it is suitable
for the protocol.

a StepInstructions
DNA QC

1.1

Measure DNA concentration of each sample with a Qubit fluorometer using the 1X dsDNA HS kit following
manufacturer’s instructions.

1.2

Recommended: measure the DNA size distribution with the appropriate sizing technology following the
manufacturer’s instructions.
• Amplicons £10 kb: Agilent 2100 Bioanalyzer, TapeStation, or Fragment Analyzer.
• Amplicons ³10 kb: Agilent FEMTO Pulse system.

1.3

Proceed to the next step if sample concentration and quality is acceptable.

1.4

Add the appropriate mass of each sample to a 0.2 mL PCR strip tube. If volume exceeds 100 µL, then use a
1.5 mL DNA LoBind tube instead.
Cleanup with 1.3X SMRTbell cleanup beads

1.5

Add 1.3X volume per volume (v/v) of resuspended, room-temperature SMRTbell cleanup beads to each tube.

1.6

Pipette-mix the beads until evenly distributed.

1.7

Quick-spin the tube strip in a microcentrifuge to collect liquid.

1.8

Leave at room temperature for 10 minutes to allow DNA to bind beads.

1.9

Place tube strip in a magnetic separation rack until beads separate fully from the solution.

1.10

Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.

1.11

Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into each tube. After
30 seconds, pipette off the 80% ethanol and discard.

1.12

Repeat the previous step.

1.13

Remove residual 80% ethanol:• Remove the tube strip from the magnetic separation rack.
• Quick-spin the tube strip in a microcentrifuge.
• Place the tube strip back in a magnetic separation rack until beads separate fully from the solution.
• Pipette off residual 80% ethanol and discard.

1.14

Remove the tube strip from the magnetic rack. Immediately add 47 µL of low TE buffer to each tube andresuspend the beads by pipetting 10 times or until evenly distributed.

1.15

Quick-spin the tube strip in a microcentrifuge to collect liquid.

1.16

Leave at room temperature for 5 minutes to elute DNA.

1.17

Place the tube strip in a magnetic separation rack until beads separate fully from the solution.

1.18

Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new
0.2 mL PCR tube strip. Discard the old tube strip with beads.
SAFE STOPPING POINT – Store at 4°C© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-359-000 REV03 DEC2023

Page 9

2. Repair & A-tailinga StepInstructions
Add the following components in the order and volume listed below to a new microcentrifuge tube.
Adjust component volumes for the number of samples being prepared, plus 10% overage. For individual
preps, add components directly to the sample from the previous step at the specified volumes and skip
RM1 steps (Steps 2.2 to 2.4).

2.1

Reaction mix 1 (RM1)

a

Tube

Component

Volume per sample

PurpleRepair buffer

8 µL

Blue

End repair mix

4 µL

Green

DNA repair mix

2 µL

Total volume

14 µL

2.2

Pipette-mix RM1.

2.3

Quick-spin RM1 in a microcentrifuge to collect liquid.

2.4

Add 14 µL of the RM1 to each sample. The total reaction volume should be 60 µL.

2.5

Pipette-mix each sample.

2.6

Quick-spin the strip tube in a microcentrifuge to collect liquid.

2.7

Run the repair & A-tailing thermocycler program.

2.8

Proceed to the next step of the protocol.

© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-359-000 REV03 DEC2023

Page 10

3. Adapter ligation & cleanupa StepInstructions
Adapter ligation

3.1

Add 4 µL of SMRTbell adapter (primer-indexed amplicon pools) or indexed SMRTbell adapter (non-indexed
amplicons) to each sample tube from the previous step.
Add the following components in the order and volume listed below to a microfuge tube. Adjust
component volumes for the number of samples being prepared, plus 10% overage. For individual preps,
add components directly to each sample from the previous step in the order and volume listed below, thenskip RM2 steps (steps 3.3 to 3.5).

3.2

Reaction mix 2 (RM2)

a

Tube

Component

Volume per sample

Yellow

Ligation mix

30 µL

Red

Ligation enhancer

1 µL

Total volume

31 µL

3.3

Pipette-mix RM2.

3.4

Quick-spin RM2 in a microcentrifuge to collect liquid.

3.5

Add 31 µL of RM2 to each sample from previous step. The total volume should be 95 µL.

3.6

Pipette-mix each sample.

3.7

Quick-spin the strip tube in a microcentrifuge to collect liquid.

3.8

Run the adapter ligation thermocycler program.
Cleanup with 1.3X SMRTbell cleanup beads

3.9

Add 124 µL of resuspended, room-temperature SMRTbell cleanup beads to each sample.

3.10

Pipette-mix the beads until evenly distributed.

3.11

Quick-spin the tube strip in a microcentrifuge to collect all liquid from the sides of the tubes.

3.12

Leave at room temperature for 10 minutes to allow DNA to bind beads.

3.13

Place the tube strip in a magnetic separation rack until beads separate fully from the solution.

3.14

Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.

3.15

Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into each tube.
After 30 seconds, pipette off the 80% ethanol and discard.

3.16

Repeat the previous step.

© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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Page 11

3.17

Remove residual 80% ethanol:• Remove the tube strip from the magnetic separation rack.
• Quick spin the tube strip in a microcentrifuge.
• Place the tube strip back in a magnetic separation rack until beads separate fully from the solution.
• Pipette off residual 80% ethanol and discard.

3.18

Remove the tube strip from the magnetic rack. Immediately add 40 µL of elution buffer to each tube andresuspend the beads by pipetting 10 times or until evenly distributed.

3.19

Quick-spin the tube strip in a microcentrifuge.

3.20

Leave at room temperature for 5 minutes to elute DNA.

3.21

Place the tube strip in a magnetic separation rack until beads separate fully from the solution.

3.22

Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a newtube strip. Discard old tube strip with beads.

3.23

Proceed to the next step of the protocol.
SAFE STOPPING POINT – Store at 4°C

4. Nuclease treatment & cleanupa StepInstructions
Nuclease treatment
Add the following components in the order and volume listed below to a microfuge tube. Adjust component
volumes for the number of samples being prepared, plus 10% overage. For individual preps, add
components directly to each sample from the previous step in the order and volume listed below, then skip
RM3 steps (steps 4.2 to 4.4).

4.1

Reaction mix 3 (RM3)

a

Tube

Component

Volume per sample

Light purple

Nuclease buffer

5 µL

Light green

Nuclease mix

5 µL

Total volume

10 µL

4.2

Pipette-mix RM3.

4.3

Quick-spin RM3 in a microcentrifuge to collect liquid.

4.4

Add 10 µL of RM3 to each sample. Total volume should equal 50 µL.

4.5

Pipette-mix each sample.

4.6

Quick-spin the strip tube in a microcentrifuge to collect liquid.

4.7

Run the nuclease treatment thermocycler program.

© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-359-000 REV03 DEC2023

Page 12Cleanup with 1.3X SMRTbell cleanup beads
4.8

Add 65 µL of resuspended, room-temperature SMRTbell cleanup beads to each sample.

4.9

Pipette-mix the beads until evenly distributed.

4.10

Quick-spin the tube strip in a microcentrifuge to collect all liquid from the sides of the tubes.

4.11

Leave at room temperature for 10 minutes to allow DNA to bind beads.

4.12

Place the tube strip in a magnetic separation rack until beads separate fully from the solution.

4.13

Slowly pipette off the cleared supernatant without disturbing the beads. Discard the supernatant.

4.14

Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into each tube.
After 30 seconds, pipette off the 80% ethanol and discard.

4.15

Repeat the previous step.

4.16

Remove residual 80% ethanol:• Remove the tube strip from the magnetic separation rack.
• Quick-spin the tube strip in a microcentrifuge.
• Place the tube strip back in a magnetic separation rack until beads separate fully from the solution.
• Pipette off residual 80% ethanol and discard.

4.17

Remove the tube strip from the magnetic rack. Immediately add 15 µL of elution buffer to each tube andresuspend the beads by pipetting 10 times or until evenly distributed.

4.18

Quick-spin the tube strip in a microcentrifuge.

4.19

Leave at room temperature for 5 minutes to elute DNA.

4.20

Place the tube strip in a magnetic separation rack until beads separate fully from the solution.

4.21

Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a newtube strip. Discard old tube strip with beads.

4.22

Take a 1 µL aliquot from each tube and dilute with 9 µL of elution buffer or water. Measure DNA
concentration with a Qubit Fluorometer using the 1x dsDNA HS kit. Calculate the total mass.

4.23

For primer-indexed samples that don’t require any additional pooling, proceed to the SMRT® Link Sample
Setup to prepare sample(s) for sequencing. The protocol is complete. Store SMRTbell libraries at 4°C if
sequencing within the week. Long-term storage should be at -20°C. Minimize freeze-thaw cycles whenhandling SMRTbell libraries.
For samples indexed with SMRTbell adapters that require pooling, proceed to the next section for
instructions on pooling and concentrating.

© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-359-000 REV03 DEC2023

Page 13

5. Pooling & concentrating samples with indexed SMRTbell adapters✓

Step

Instructions
Pooling

5.1

Combine an equal mass sampled indexed with SMRTbell adapters together into a single pool using a 1.5 mL
DNA LoBind Tube. Proceed to the next step to concentrate the pool. Alternatively, samples can be pooled inequal molar portions for better sequence coverage balance.
The total SMRTbell library mass should be ³100 ng when amplicon sizes are less than 10 kb, and ³300 ng
when greater or equal to 10 kb.
Concentrate with 1.3X SMRTbell cleanup beads

5.2

Add 1.3X v/v SMRTbell cleanup beads to each pool.

5.3

Pipette-mix the beads until evenly distributed.

5.4

Quick-spin the tube in a microcentrifuge to collect all liquid.

5.5

Leave at room temperature for 10 minutes to allow DNA to bind beads.

5.6

Place the tube in a magnetic separation rack until beads separate fully from the solution.

5.7

Slowly pipette off the cleared supernatant without disturbing the beads.

5.8

Slowly dispense 200 µL, or enough to cover the beads, of freshly prepared 80% ethanol into each tube. After
30 seconds, pipette off the 80% ethanol and discard.

5.9

Repeat the previous step.

5.10

Remove residual 80% ethanol:• Remove the tube from the magnetic separation rack.
• Quick-spin the tube in a microcentrifuge.
• Place the tube back in a magnetic separation rack until beads separate fully from the solution.
• Pipette off residual 80% ethanol and discard.

5.11

Remove the tube from the magnetic rack. Immediately add 15 µL of elution buffer to each tube andresuspend the beads by pipetting 10 times or until evenly distributed.

5.12

Quick-spin the tube in a microcentrifuge to collect liquid.

5.13

Leave at room temperature for 5 minutes to elute DNA.

5.14

Place the tube in a magnetic separation rack until beads separate fully from the solution.

5.15

Slowly pipette off the cleared supernatant without disturbing the beads. Transfer supernatant to a new tube.
Discard old tube with beads.

5.16

Take a 1 µL aliquot from each tube and dilute with 9 µL of elution buffer or water. Measure DNA
concentration with a Qubit fluorometer using the 1x dsDNA HS kit.

5.17

Proceed to SMRT Link Sample Setup to prepare sample(s) for sequencing. Store SMRTbell libraries at 4°C if
sequencing within the week. Long-term storage should be at -20°C. Minimize freeze-thaw cycles whenhandling SMRTbell libraries.
PROTOCOL COMPLETE© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-359-000 REV03 DEC2023

Page 14

Revision history (description)

Version

Date

Initial release01

Apr 2022

Clarify minimum DNA input requirements and best practices

02

Sep 2022

Updated to include recommendations for the Revio system03

Dec 2023

Research use only. Not for use in diagnostic procedures. © 2023 Pacific Biosciences of California, Inc. (“PacBio”). All rights reserved.
Information in this document is subject to change without notice. PacBio assumes no responsibility for any errors or omissions in thisdocument. Certain notices, terms, conditions and/or use restrictions may pertain to your use of PacBio products and/or third-party
products. Refer to the applicable PacBio terms and conditions of sale and to the applicable license terms at pacb.com/license. Pacific
Biosciences, the PacBio logo, PacBio, Circulomics, Omniome, SMRT, SMRTbell, Iso-Seq, Sequel, Nanobind, SBB, Revio, Onso, Apton, andKinnex are trademarks of PacBio.
.
© 2023 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
102-359-000 REV03 DEC2023