2024年2月8日发(作者：)
Procedure & Checklist – Preparing HiFi SMRTbell®
Libraries using the SMRTbell Express Template Prep
Kit 2.0
This procedure describes the construction of HiFi SMRTbell libraries for de novo assembly and variant
detection applications using the SMRTbell Express Template Prep Kit 2.0 and recommended HiFi sequencing
conditions using PacBio’s new Sequel® II Binding Kit 2.2. A minimum input amount of 5 µg of high-molecular
weight genomic DNA is recommended for generating HiFi library yields sufficient for running multiple SMRT®
Cells on the Sequel II or Sequel IIe System (Sequel II Systems). Note that final HiFi library construction yields
will be dependent on the specific size-selection method employed.
We recommend fragmenting the gDNA so that the target size distribution mode is between 15 kb - 18 kb. To
reduce the presence of fragments >30 kb, PacBio recommends a 2-cycle shearing method on the Megaruptor 3
system. Generally, a narrower fragment size distribution results in more uniform and higher-quality HiFi data.
Details regarding DNA shearing conditions (e.g., buffers and DNA sample concentration) are described in the
“DNA Requirements for Shearing” section.
Required
Equipment
Femto Pulse
Megaruptor 3
PippinHT
BluePippin
SageELF
Vendor
Agilent
Technologies
Diagenode
Sage Science
Sage Science
Sage Science
Throughput
Process up to 11 samples per run
Batch process up to 88 samples
Shear up to 8 samples at a time
Maximum of 20 samples per instrument run
Maximum of 4 samples per instrument run
Maximum of 2 samples per instrument run
Run Time
85 mins
40 mins
(for 1 cycle of shearing)
2 hrs
4.5 hrs
4.5 hrs
Table 1: Recommended equipment for HiFi SMRTbell library construction for de novo assembly and variant
detection applications.
Page 1 PN 101-853-100 Version 05 (August 2021)
Required Materials
DNA Sizing
Femto Pulse
Agilent Technologies, Inc. P-0003-0817
ThermoFisher Scientific Q33238
ThermoFisher Scientific Q33230
DNA Quantitation
Qubit™ Fluorometer
Qubit 1X dsDNA HS Assay Kit
DNA Shearing
Megaruptor 3 System
Megaruptor 3 Shearing Kit
Diagenode B06010003
Diagenode E07010003
PacBio 100-938-900
PacBio 100-265-900
PacBio 101-932-600
PacBio 102-067-400
Any MLS
Rainin 30389241
USA Scientific, TempAssure
1402-4708
Rainin, 17013810
V&P Scientific, Inc, VP 772F4-1
Any MLS
SMRTbell Library Preparation
SMRTbell® Express Template Prep Kit 2.0
AMPure® PB Beads
SMRTbell® Enzyme Clean Up Kit 2.0 (New*)
Sequencing Primer v5 (New*)
100% Ethanol, Molecular Biology Grade
Wide Orifice Tips (Tips LTS W-O 200UL Fltr RT-L200WFLR)
Lo-Bind 0.2 mL tube strips
Multi-channel Pipette
Magnetic separation rack
Thermal Cycler that is 100 µL and 8-tube strip compatible
Size-selection (One of the following systems)
PippinHT System
0.75% Agarose Gel Cassettes, Marker 75E
BluePippin System
0.75% Agarose Cassettes, Marker S1
SageELF System
0.75% Agarose Cassettes
Sage Science HTP0001
Sage Science HPE7510
Sage Science BLU0001
Sage Sciences BLF7510
Sage Science ELF0001
Sage Science ELD7510
Sequencing
Sequel® II Binding Kit 2.2 (New*)
Sequel® II Sequencing Kit 2.0
SMRT® Cell 8M Tray
PacBio 101-894-200
PacBio 101-820-200
PacBio 101-389-001
* To obtain a copy of the previous version of this Procedure & Checklist that specifies use of SMRTbell Enzyme Clean Up Kit (PN
101-746-400) and Sequencing Primer v2 (PN 101-847-900), contact ****************.
Page 2 PN 101-853-100 Version 05 (August 2021)
HiFi Library Construction Workflow
PacBio recommends that gDNA samples be resuspended in an appropriate buffer (e.g., Qiagen Elution
Buffer) before proceeding with DNA shearing.
DNA Shearing
Remove Single-Strand Overhangs
DNA Damage Repair
DNA End Repair/A-tailing
Adapter Ligation
1X AMPure PB Bead Purification
Nuclease Treatment
1X AMPure PB Bead Purification
Size-Selection
1X AMPure PB Bead Purification, QC
Primer Annealing, Polymerase Binding
Sequencing
Figure 1: Workflow for preparing HiFi libraries using the SMRTbell Express Template Prep Kit 2.0.
Page 3 PN 101-853-100 Version 05 (August 2021)
Reagent Handling
Several reagents in the SMRTbell Express Template Prep Kit 2.0 (shown in Table 2 below) are sensitive to
temperature and vortexing. We recommend to:
• Never leave reagents at room temperature.
• Always work on ice when preparing master mixes.
• Finger-tap followed by a quick spin prior to use.
Reagent
DNA Prep Additive
DNA Prep Enzyme
DNA Damage Repair Mix v2
End Prep Mix
Overhang Adapter v3
Ligation Mix
Ligation Additive
Ligation Enhancer
SMRTbell Enzyme Clean Up Mix
SMRTbell Enzyme Cleanup Buffer 2.0
Table 2: Temperature sensitive reagents
Where Used
Remove single-strand overhangs
Remove single-strand overhangs
DNA Damage Repair
End-Repair/A-tailing
Ligation
Ligation
Ligation
Ligation
Nuclease Treatment
Nuclease Treatment
Genomic DNA (gDNA) Quality Evaluation
This procedure requires high-quality, high-molecular weight input gDNA with a majority of the DNA
fragments >50 kb as determined by pulsed-field gel or capillary electrophoresis. Any of the three
commercially available systems listed in Table 4 below may be used to evaluate gDNA quality, but the
Femto Pulse system is highly recommended for high-throughput library construction due to its ability to
rapidly process multiple samples in a single run using very low amounts (<1 ng) of DNA per sample.
Links to recommended procedures for each system are also provided in the table. Examples of gDNA
quality assessment using Bio-Rad’s CHEF Mapper (2A) and Agilent Technologies’ Femto Pulse (2B) are
shown in Figure 2. Lanes A3 and B1 correspond to high-quality gDNA samples that are suitable for HiFi
library construction using this procedure. Lanes A4 and B2 show degraded gDNA samples that not
suitable for use in this procedure.
Method
Femto Pulse
Bio-Rad CHEF Mapper XA Pulsed
Field Electrophoresis System
Sage Science Pippin Pulse
Procedure
Agilent Technologies, Inc.
Procedure & Checklist - Using the BIO-RAD® CHEF
Mapper®
XA Pulsed Field Electrophoresis System
Procedure & Checklist - Using the Sage Science Pippin
Pulse Electrophoresis Power Supply System
Table 3. gDNA Quality Evaluation Methods and Procedures.
Page 4 PN 101-853-100 Version 05 (August 2021)
A
1
2
3
4
B
80 kb----------
1
2
3
Lane 1: HMW gDNA
Lane 2: Degraded gDNA
Lane 3: 165 kb ladder
Lane 1: 8 kb - 48 kb Ladder (Bio-Rad)
Lane 2: 5 kb ladder (Bio-Rad)
Lane 3: HMW gDNA
Lane 4: Degraded gDNA
48 kb-
165.5
20 kb-
10 kb-
33 kb
21 kb
17.7 kb
10 kb
1.3 kb
50 kb42 kb1 bp
Figure 2: Evaluation of high-molecular weight gDNA quality using two DNA sizing analysis systems. A) Bio-Rad
CHEF Mapper and B) Agilent Technologies’ Femto Pulse.
Page 5 PN 101-853-100 Version 05 (August 2021)
DNA Requirements for
Shearing
Before shearing, ensure that the genomic DNA is in an appropriate buffer (e.g., Qiagen Elution Buffer,
10 mM Tris-Cl, pH 8.5 or PacBio EB buffer). If you are unsure of the buffer composition or if the
gDNA is not in Elution Buffer, perform a 1X AMPure PB bead purification followed by elution with Elution
Buffer or an equivalent low salt buffer (i.e., 10 mM Tris-Cl, pH 8.5- 9.0).
PacBio highly recommends Diagenode’s Megaruptor 3 system for shearing gDNA. The Megaruptor 3
system allows up to 8 gDNA samples to be processed simultaneously with a consistent fragment size
distribution across multiple hydropore-syringes. Furthermore, the Megaruptor 3 system generates a
narrower size distribution than the g-TUBE device (Covaris).
Shearing Using Diagenode’s Megaruptor 3 System
To maximize HiFi yield per SMRT Cell, PacBio recommends fragmenting the gDNA to a size distribution
mode between 15 kb – 18 kb for human whole genome sequencing. Libraries with a size distribution
mode larger than 20 kb are not recommended for HiFi sequencing. Recommended library insert size
distributions to use for different WGS applications are summarized in Table 4 below.
Application
Human Variant Detection
Human de Novo
Plant/Animal de Novo
Recommended Library Insert Size
15 – 18 kb
15 – 18 kb
15 – 20 kb
Table 4: Library size recommendations for Human variant detection and de novo assembly.
To shear gDNA on the Megaruptor 3 system, use a two-cycle shear method, which requires running a
second round of shearing immediately following the first fragmentation step in the same hydropore-syringe. The recommended concentration is 83.3 ng/µL (5 µg of input DNA in 60 µL Elution Buffer).
The DNA shearing guidelines below have been tested by PacBio on the Megaruptor 3 system only. The
response of individual gDNA samples to the shearing recommendations described below may differ;
therefore, performing a small-scale test shear is highly recommended, including the Megaruptor 3
system.
For the Megaruptor and Megaruptor 2 systems, shearing optimization is necessary before proceeding
with this Procedure & Checklist. The shearing procedure described in the “Shearing Using Diagenode’s
Megaruptor 3 system” section below is not compatible with the Megaruptor or Megaruptor 2 systems.
For Megaruptor and Megaruptor 2 systems, follow Diagenode’s DNA shearing recommendations
described in their manual. For additional guidance, contact Technical Support or your local FAS.
The g-TUBE device generates a broader DNA fragment size-distribution compared to the Megaruptor 3
system. Note that HiFi read quality and overall HiFi data yield may be reduced due to the residual
presence of large DNA fragments generated by g-TUBEs. For additional guidance, contact
Technical
Support or your local FAS.
Page 6 PN 101-853-100 Version 05 (August 2021)
STEP
1
2
Shear DNA
Dilute 5 µg of high-molecular weight gDNA in 1X Elution Buffer to a concentration
of 83.3 ng/µL in a final volume of 60 µL.
Shear the gDNA sample with a disposable shearing syringe using the following
recommended settings for the Megaruptor 3 system software. See Figure 3 for
examples of human DNA samples sheared using the following 2-cycle shear
method below (use the same hydropore-syringe device for both shearing cycles).
Notes
Shearing Cycle
Cycle 1
Cycle 2
Megaruptor 3 Speed Setting
31*
32*
*Adjust speed setting parameters if the size distribution mode is larger than 20 kb.
Always perform test shears before scaling up for production workflows.
Recover the sheared DNA into a Lo-Bind tube strip. The typical sample recovery
volume range is 45.0 µL - 53.0 µL.
Note: If the input gDNA sizing QC indicates the sample is already fragmented, 1
cycle of shearing may be sufficient.
3
Proceed to the “Remove Single-strand Overhangs” section.
Figure 3: Examples of human genomic DNA samples sheared to a target 15 kb - 18 kb size distribution mode using a 2-cycle shear method on the Megaruptor 3 system.
Prepare SMRTbell Libraries
Always work on ice throughout the library construction process. To process multiple samples at a time, the
following equipment are required:
• Lo-Bind tube strips
• Multi-channel pipette
• Wide-bore tips
• Magnetic rack compatible with tube strips
• Thermocycler compatible with tube strips
Page 7 PN 101-853-100 Version 05 (August 2021)
Remove Single-Strand Overhangs
The sample volume recovered from the Megaruptor 3 system after shearing is used directly in the single-strand overhang digestion step. Before proceeding, ensure that the sheared DNA is in Elution Buffer or an
equivalent low salt buffer (i.e., 10 mM Tris-Cl, pH 8.5- 9.0). In this step, DNA Prep Additive is diluted first
followed by digestion. Scale up the reaction volumes for digestion if working with multiple samples.
1. Prepare the DNA Prep Additive. The DNA Prep Additive is diluted with Enzyme Dilution Buffer to
a total volume of 5 µL. This amount is sufficient for processing 1 to 4 samples. The volume may
not be sufficient for 5 samples due to pipetting errors. We recommend scaling up the dilution
volume based on the number of samples to be processed (example: prepare 2X volume for 8
samples and 4X volume for 16 samples).
Note: The diluted DNA Prep Additive should be used immediately and should not be stored.
Reagent
Enzyme Dilution Buffer
DNA Prep Additive
Total Volume
Tube Cap Color
1 to 4 Samples
4.0 μL
1.0 μL
5.0 μL
Up to 8 Samples
8.0 μL
2.0 μL
10.0 μL
Notes
2. Prepare the digestion by following the reaction table below. For multiple samples, prepare a
master mix, followed by addition of 10.0 μL master mix to each sheared DNA sample.
Reagent (Reaction Mix 1)
DNA Prep Buffer
NAD
Diluted DNA Prep Additive (see step 1)
DNA Prep Enzyme
Total Volume
Tube Cap
Color
1 Sample
7.0 μL
1.0 μL
1.0 μL
1.0 μL
10.0 μL
N Samples
7.0 x N x 1.2*
1.0 x N x 1.2*
1.0 x N x 1.2*
1.0 x N x 1.2*
Notes
3.
4.
5.
6.
7.
*includes 20% overage for pipetting errors
Add 10.0 µL of the above master mix to the tube-strips containing 45.0 µL - 53.0 µL of sheared
DNA. The total volume in this step is 55.0 µL - 63.0 µL.
Using a multi-channel pipette, mix the reaction wells by pipetting up and down 10 times with wide-orifice pipette tips.
Spin down the contents of the tube strips with a quick spin in a microfuge.
Incubate at 37°C for 15 minutes, then return the reaction to 4°C.
Proceed to the next step.
Page 8 PN 101-853-100 Version 05 (August 2021)
Repair DNA Damage
To each Reaction Mix 1, add 2.0 µL of DNA Damage Repair Mix v2.
Reagent (Reaction Mix 2)
Reaction Mix 1
Tube Cap Color
Volume
Notes
55.0 μL - 63.0 μL
2.0 μL
DNA Damage Repair Mix v2
Total Volume
57.0 μL - 65.0 μL
End-Repair/A-tailing
To each Reaction Mix 2, add 3.0 µL of End Prep Mix.
1. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips.
2. Spin down the contents of the tube strips with a quick spin in a microfuge.
3. Incubate at 37°C for 30 minutes, then return the reaction to 4°C.
4. Proceed to the next step.
Reagent (Reaction Mix 3)
Reaction Mix 2
End Prep Mix
Total Volume
Tube Cap Color
Volume
57.0 μL - 65.0 μL
3.0 μL
Notes
60.0 μL - 68.0μL
1. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips.
2. Spin down the contents of the tube strips with a quick spin in a microfuge.
3. Incubate at 20°C for 10 minutes.
4. Incubate at 65°C for 30 minutes, then return the reaction to 4°C.
5. Proceed to the next step.
Adapter Ligation
In this step, 5.0 µL of Overhang Adapter is added to each Reaction Mix 3 (from the previous step). Then, 32.0
µL of the ligase master mix is added to each Reaction Mix 3/Adapter Mix for incubation. Always work on ice.
1. To each Reaction Mix 3, add 5.0 µL of Overhang Adapter.
Reagent
Reaction Mix 3
Overhang Adapter v3
Total Volume
Tube Cap Color
Volume
60.0 μL - 68.0 μL
5.0 μL
Notes
65.0 μL - 73.0 μL
2. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips. Leave the tube
strips on ice.
Page 9 PN 101-853-100 Version 05 (August 2021)
3. Prepare a Master Mix containing Ligation Enhancer, Ligation Additive and Ligation Mix using the table
below:
Reagent (Reaction Mix 4)
Ligation Mix
Ligation Additive
Ligation Enhancer
Total Volume
Tube Cap
Color
1 sample
30.0 μL
1.0 μL
1.0 μL
32.0 μL
N Samples
30.0 x N x 1.2*
1.0 x N x 1.2*
1.0 x N x 1.2*
Notes
*includes 20% overage for pipetting errors
4. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips. It is important to mix
well.
5. To the Reaction Mix 3/Adapter Mix, add 32.0 µL of the Ligase Master Mix. The total volume in this step is
97.0 µL - 105.0 µL.
6. Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips. It is important to mix
well.
7. Incubate at 20°C for 1 hour. Optional: The Ligation reaction may also be left at 20°C overnight.
8. Proceed to the next step.
Page 10 PN 101-853-100 Version 05 (August 2021)
Purify SMRTbell Library Using 1.0X AMPure® PB Beads
STEP
AMPure PB Bead Purification
Add 1.0X volume of AMPure PB beads to the ligation reaction.
With a multi-channel pipette, mix by pipetting 10X – 15X with wide bore tips.
Allow the DNA to bind to beads on the bench top
at room temperature for 20 minutes.
Quickly spin down the tube strip (for 1 second) to collect beads.
Place the tube strip in a magnetic rack to collect the beads to the side.
Notes
1
2
3
4
5
6
7
Slowly pipette off the cleared supernatant. Avoid disturbing the beads. Optional: save the
supernatant in another tube strip in case of poor DNA recovery.
Wash the beads with freshly prepared 80% ethanol.
Note that 80% ethanol is hygroscopic and should be prepared FRESH to achieve
optimal results. Also, 80% ethanol should be stored in a tightly capped polypropylene
tube for no more than 3 days.
– Fill the tubes with 200 μL of 80% ethanol. Slowly dispense the 80% ethanol on
the side of the tubes. Ensure the bead pellet is covered with ethanol solution.
– After 30 seconds, pipette and remove the 80% ethanol.
8
9
Repeat step 7.
Remove residual 80% ethanol.
– Remove tube strip from the magnetic rack and quickly spin. Both beads and any
residual 80% ethanol will be at the bottom of each tube.
– Place the tube strip back on magnetic rack.
– Pipette off any remaining 80% ethanol.
Remove the tube strip from the magnetic rack.
10
11
12
13
Add 15.0 μL Elution Buffer to elute the DNA.
Pipette mix 10X – 15X until beads are uniformly re-suspended
– Elute the DNA
at
37°C for 15 minutes.
– Quickly spin.
– Let beads separate fully in the magnetic rack. Then without disturbing the
beads, transfer supernatant to a new DNA Lo-Bind tube strip.
Optional: Take 1.0 μL of eluted sample and measure the DNA concentration using the
dsDNA HS Assay Kit with a Qubit fluorometer. The same sample aliquot used to
measure DNA concentration on the Qubit system may be used for DNA sizing QC on
the Femto Pulse system. Dilute the sample to 250 pg/ μL with the Femto Pulse Dilution
Buffer (0.25X TE).
Proceed to the Nuclease Treatment section below.
Page 11 PN 101-853-100 Version 05 (August 2021)
Nuclease Treatment of SMRTbell Library
To each library sample, add the nuclease mix to remove damaged SMRTbell templates.
1. Prepare a Master Mix of the Enzyme Cleanup Mix and Buffer.
Tube Cap
Color
1 sample
7.0 μL
7.0 μL
41.0 μL
55.0 μL
Reagent (Reaction Mix 5)
SMRTbell Enzyme Clean Up Mix
SMRTbell Enzyme Clean Up Buffer
2.0
Molecular Biology Grade Water
Total Volume
N samples
7.0 x N x 1.2*
7.0 x N x 1.2*
41.0 x N x 1.2*
Notes
*includes 20% overage for pipetting errors
2.
3.
4.
5.
6.
7.
8.
Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips. It is important to mix
well.
Spin down the contents of the tube strips with a quick spin in a microfuge.
To each 15.0 μL of sample, add 55.0 μL of Nuclease Master Mix. The total reaction volume at this step is
70.0 µL.
Mix the reaction well by pipetting up and down 10 times with wide-orifice pipette tips. It is important to mix
well.
Incubate at 37°C for 30 mins and store on ice immediately.
Spin down the contents of tube strips with a quick spin in a microfuge.
Proceed directly to the AMPure PB bead purification step below immediately. Do not store samples at this
stage. Do not let samples sit for long periods of time. Always work on ice.
Page 12 PN 101-853-100 Version 05 (August 2021)
Purify SMRTbell Library Using 1.0X AMPure® PB Beads
STEP
AMPure PB Bead Purification
Bring the volume of each sample to 100 μL with Elution Buffer. Add 1.0X volume of
AMPure PB beads to the nuclease-treated ligation reaction.
With a multi-channel pipette, mix by pipetting 10X - 15X with wide bore tips.
Allow the DNA to bind to beads on the bench top at room temperature for 20 minutes.
Quickly spin down the tube strip (for 1 second) to collect beads.
Place the tube strip in a magnetic rack to collect the beads to the side.
Notes
1
2
3
4
5
6
7
Slowly pipette off the cleared supernatant. Avoid disturbing the beads. Optional: save the
supernatant in another tube strip in case of poor DNA recovery.
Wash the beads with freshly prepared 80% ethanol.
Note that 80% ethanol is hygroscopic and should be prepared FRESH to achieve
optimal results. Also, 80% ethanol should be stored in a tightly capped polypropylene
tube for no more than 3 days.
– Fill the tubes with 200 μL of 80% ethanol. Slowly dispense the 80% ethanol on
the side of the tubes. Ensure the bead pellet is covered with ethanol solution.
– After 30 seconds, pipette and remove the 80% ethanol.
Repeat step 7.
Remove residual 80% ethanol.
– Remove tube strip from the magnetic rack and quickly spin. Both beads and any
residual 80% ethanol will be at the bottom of each tube.
– Place the tube strip back on magnetic rack.
– Pipette off any remaining 80% ethanol.
Remove the tube strip from the magnetic rack.
Add 21.0 μL Elution Buffer to elute the DNA.
– Pipette mix 10X – 15X until beads are uniformly re-suspend.
– Elute the DNA
at
37°C for 15 minutes.
– Quickly spin.
– Let beads separate fully in the magnetic rack. Then without disturbing the
beads, transfer supernatant to a new DNA Lo-Bind tube strip.
Take 1.0 μL of eluted sample and measure the DNA concentration using the dsDNA
HS Assay Kit with a Qubit fluorometer. The same sample aliquot used to measure
DNA concentration on the Qubit system may be used for DNA sizing QC on the
Femto Pulse system. Dilute the sample to 250 pg/ μL with the Femto Pulse Dilution
Buffer (0.25X TE).
Proceed to size-selection or store the samples at -20°C for future use.
8
9
10
11
12
13
Page 13 PN 101-853-100 Version 05 (August 2021)
Size Selection of SMRTbell Libraries
For high-throughput whole genome sequencing applications, PacBio highly recommends the PippinHT
system (Sage Science) for size-selection of SMRTbell libraries for HiFi sequencing. Typical recovery yields
are 35% - 50% and are highly dependent on the size distribution of the starting SMRTbell library.
Size Selection Using the PippinHT System
Verify that your PippinHT system software is up to date and follow the procedure below to remove SMRTbell
templates <10 kb using the PippinHT system. For the latest PippinHT System Operations Manual and guidance
on size-selection protocols, contact Sage Science ().
STEP
1
2
PippinHT Size Selection
Prepare ~1.5 µg of SMRTbell libraries in a final volume of 20.0 μL of Elution Buffer for
each PippinHT lane.
Bring the Loading Solution to room temperature, and then add 5.0 μL of the Loading
Solution to the 20.0 μL DNA sample. For loading multiple lanes with the same sample,
scale up the volumes proportionally. The Loading Solution is viscous, so pipet slowly to
ensure complete transfer into the DNA sample.
– Pipette mix using wide-bore pipette tips to mix.
– Spin briefly to collect the contents at the bottom of the tube.
Follow the manufacturer’s recommendations to set up a run protocol.
– Select the “6-10kb High Pass Marker 75E” Cassette Definition File.
– Using the “Range” selection mode, enter a desired “BPstart” value of 10000
and a BP End value of 50000. Be sure to assign a marker lane.
Load the 75E marker and samples into the gel cassette and start the run. Run time is
approximately 1 hr 15 mins.
To maximize recovery of eluted DNA, wait at least 45 minutes after the run terminates
before removing the sample from the elution chamber.
– Wash the elution well with 30 µL PippinHT 0.1% Tween20 (supplied with the
cassettes). Wait 1 minute and add the recovered wash liquid to the eluted
sample.
– Wash the elution well with 30 µL of Elution Buffer and add the recovered wash
liquid to the eluted sample.
– The total volume after the pooling recovered washes with the original eluted
sample is ~ 90 µL.
Proceed to the AMPure PB bead purification step.
Notes
3
4
5
6
Page 14 PN 101-853-100 Version 05 (August 2021)
Sage Science’s BluePippin system may also be used for size-selection of HiFi SMRTbell libraries. Verify that
your BluePippin system software is up to date and follow the procedure below to remove SMRTbell templates
<10 kb using the BluePippin system. Typical recovery yields are highly dependent on the size distribution of
the starting SMRTbell library. For the latest BluePippin system User Manual and guidance on size-selection
protocols, contact Sage Science ().
Size Selection Using the BluePippin System
STEP
1
2
BluePippin Size Selection
Prepare ~1.5 µg of SMRTbell library in a final volume of 30 μL of Elution Buffer for
each BluePippin lane.
Bring the Loading Solution to room temperature, and then add 10 μL of the Loading
Solution to the 30 μL DNA sample. The Loading Solution is viscous, so pipet slowly to
ensure complete transfer into the DNA sample.
– Pipette mix using wide-bore pipette tips to mix.
– Spin briefly to collect the contents at the bottom of the tube.
Follow the manufacturer’s recommendations to set up a run protocol.
– Select the “0.75%DF Marker S1 High-Pass 6-10kb vs3” Cassette
Definition File.
– Using the “Range” selection mode, enter a desired “BPstart” value of 10000
and a “BP End” value of 50000.
– Be sure to assign a marker lane.
Load S1 marker and samples into the BluePippin gel cassette and start the run. Run
time is approximately 4.5 hours.
To maximize recovery of eluted DNA, wait at least 45 minutes after the run terminates
before removing the sample from the elution chamber.
– Collect the eluate (~40 µL) into a 1.5 mL DNA LoBind tube or 0.2 mL PCR
tube strips.
• Wash the elution well with 40 µL BluePippin 0.1% Tween20 (supplied with the
cassettes).
• Wash the elution well with 40 µL of Elution Buffer and add the recovered wash
liquid to the eluted sample. The total volume after the pooling recovered
washes with the original eluted sample is ~ 120 µL.
Proceed to the AMPure PB Bead purification step.
Notes
3
4
5
6
Page 15 PN 101-853-100 Version 05 (August 2021)
Size Selection Using the SageELF System
Sage Science’s SageELF system may also be used to fractionate SMRTbell libraries for HiFi whole genome
sequencing applications. Verify that your SageELF system software is up to date and follow the size selection
procedure below. For the latest SageELF User Manual and guidance on size-selection protocols, contact
Sage Science ().
STEP
1
2
3
SageELF Size Selection
Follow the SageELF manufacturer’s instructions to calibrate the instrument. A new
calibration is recommended before each run.
Inspect the gel cassette (using Sage Science’s SageELF instructions).
– Ensure that the buffer wells are full.
– Ensure that there is no separation of the gel from the cassette.
Prepare the gel cassette:
– While the cassette is sealed, remove all bubbles from the elution buffer
chamber by tilting the cassette and tapping it until all air bubbles move
into the buffer chamber.
– Hold the cassette firmly on the bench top and carefully remove the plastic
seals on the cassette.
– Remove the buffer from the elution well and fill with 30 μL of fresh
Electrophoresis Buffer.
• Keep the pipette down the center of the well and avoid creating a vacuum
in the well.
• The bottom of the well is okay to touch.
•
If the well “bubbles” over when adding the buffer to the well, remove buffer
and try again.
– Cover the elution wells with a clear adhesive tape and verify that it is tightly
sealed.
– Remove the buffer from the sample well and fill with 70 μL of fresh
Electrophoresis Buffer. Do not touch the sides and bottom of the sample well.
– Carefully place the gel cassette in the SageELF system.
– Verify that the buffer in the “moat” on both sides of the cassette that connects
the electrode reservoirs is at the correct level: add additional electrophoresis
buffer to fill the moat completely, then remove 1 mL from one side of the
moat.
– Close the lid and perform a Current Test.
Prepare samples for loading.
– Add 30 μL of sample containing >1 μg of size-fractionated HiFi library to a
clean tube.
– Add 10 μL of Sage Science’s Marker 75. Mix well and do a quick spin down.
Load samples:
– Remove 40 μL of buffer from the sample well.
– Load all 40 μL of the sample prepared in step 4 into the sample well.
– If necessary, top off well with additional Electrophoresis Buffer. Do not overfill
the well.
Notes
4
5
Page 16 PN 101-853-100 Version 05 (August 2021)
6
Set up the run Protocol:
– In the “Protocol Editor” tab, click on the “New Protocol” button.
– Select the “0.75% 1-18kb v2” in the cassette definition menu.
– Select “size-based” for separation mode.
– Enter 3450 in the “Target Value” field and move the bar slider to select
well #12.
– Save as new protocol.
– On the Main screen, clear previous run data, select cassette description,
cassette definition and protocol, enter sample ID(s).
– Select in the Nest Selector the cartridge that will be run.
Start the run.
7
8
9
10
11
Once the run is complete, (approximately 4.5 hours), collect 30 μL of the respective
fractions from the elution wells. Fractions of interest are typically ~11 kb, ~13 kb,
~15 kb, ~17 kb.
Check the sizes of all 12 fractions by loading on a Femto Pulse. To determine the
average library size, perform a smear analysis by selecting the region of interest
by defining the start and end points of the fractions.
Pool together fractions that have an average library size 10 – 20 kb.
Proceed to the AMPure PB Bead purification step.
Page 17 PN 101-853-100 Version 05 (August 2021)
Purify Size-Selected HiFi Library Fractions with 1.0X AMPure® PB Beads
STEP
1
AMPure PB Bead Purification
For samples with volumes <100 µL, bring to 100 µL with 1X Elution Buffer (EB) before
performing AMPure PB bead purification.
Add 1.0X volume of AMPure PB beads to the size-selected libraries.
With a multi-channel pipette, mix by pipetting 10X – 15X with wide bore tips.
Allow the DNA to bind to beads on the bench top
at room temperature for 20 minutes.
Quickly spin down the tube strip (for 1 second) to collect beads.
Place the tube strip in a magnetic bead rack to collect the beads to the side.
Slowly pipette off the cleared supernatant. Avoid disturbing the beads. Optional: save the
supernatant in another tube strip in case of poor DNA recovery.
Wash the beads with freshly prepared 80% ethanol.
Note that 80% ethanol is hygroscopic and should be prepared FRESH to achieve optimal
results. Also, 80% ethanol should be stored in a tightly capped polypropylene tube for no
more than 3 days.
– Fill the tubes with 200 μL of 80% ethanol. Slowly dispense the 80% ethanol on
the side of the tubes. Ensure the bead pellet is covered with ethanol solution.
– After 30 seconds, pipette and remove the 80% ethanol
Repeat step 7.
Remove residual 80% ethanol.
– Remove tube strip from the magnetic rack and quickly spin. Both beads and any
residual 80% ethanol will be at the bottom of each tube.
– Place the tube strip back on magnetic rack.
– Pipette off any remaining 80% ethanol.
Remove the tube strip from the magnetic rack.
Notes
2
3
4
5
6
7
8
9
10
11
12
13
Sequencing Preparation
Add 11.0 μL Elution Buffer to elute the DNA. Pipette mix 10X – 15X until beads are
uniformly re-suspend.
– Elute the DNA
at
37°C for 15 minutes.
– Quickly spin.
– Let beads separate fully in the magnetic rack. Then without disturbing the
beads, transfer supernatant to a new DNA Lo-Bind tube strip.
Take 1.0 μL of eluted sample and measure the DNA concentration using the dsDNA HS
Assay Kit with a Qubit fluorometer. The same sample aliquot used to measure DNA
concentration on the Qubit system may be used for DNA sizing QC on the Femto Pulse
system. Dilute the sample to 250 pg/μL with the Femto Pulse Dilution Buffer (0.25X TE).
Proceed to the next step or store the final SMRTbell libraries at -20ºC for future use.
See Quick Reference Card - Loading and Pre-Extension Recommendations for Sequel II/IIe Systems.
Revision History (Description)
Initial release.
Internal revision with no content change (not uploaded to website).
On page 1, changed “HiFi reads” to just “Reads”. On page 12, under Repair DNA Damage,
corrected “remove single strand overhangs” to “repair DNA damage”. On page 13, corrected
“remove single strand overhangs” to “adapter ligation”.
Updated for SMRTbell Enzyme Clean Up Kit 2.0 and Sequencing Primer v5.
Version Date
01 September 2019
02
03
04
05
December 2019
January 2020
April 2021
August 2021
Removed SMRT Link Sample Setup and Run Design tables. Added reference to QRC.
For Research Use Only. Not for use in diagnostic procedures. © Copyright 2020 - 2021, Pacific Biosciences of California, Inc. All rights reserved. Information in this
document is subject to change without notice. Pacific Biosciences assumes no responsibility for any errors or omissions in this document. Certain notices, terms,
conditions and/o r use restrictions may pertain to your use of Pacific Biosciences products and/or third p arty products. Please refer to the
applicable Pacific
Biosciences Terms and Conditions of S ale and to the applicable license terms at
/lice . Pacific Biosciences, the Pacific
Biosciences logo, PacBio, S MRT, SMRTbell, Iso-Seq and Sequel are trademarks of Pacific Biosciences. Femto Pulse and Fragment Analyzer are trademarks of Agilent
Technologies. All other trademarks are the sole property of their respective owners.
Page 18 PN 101-853-100 Version 05 (August 2021)
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