Short Read
Eliminator(SRE), DNA
shearing, andcleanup for the
Hamilton
Microlab Prep
systemGuide & overview© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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Page 2

Table of contents
Introduction................................................................................................................................................................ 3
Overview .................................................................................................................................................................... 3
Workflow overview ..................................................................................................................................................... 4
Required materials and equipment.............................................................................................................................. 5
Before you begin ........................................................................................................................................................ 7
Genomic DNA (gDNA) QC and input amount recommendations............................................................................................... 7
Reagent handling .................................................................................................................................................................................. 7
Instrument.............................................................................................................................................................................................. 7
Safety precautions ............................................................................................................................................................................... 7
Workflow steps .......................................................................................................................................................... 8
1. Short Read Eliminator (SRE) .......................................................................................................................................................... 8
2. DNA Shearing .................................................................................................................................................................................. 15
3: 1X Post-shear cleanup .................................................................................................................................................................. 20
Appendix .................................................................................................................................................................. 27
Installation (New User) ...................................................................................................................................................................... 27
Troubleshooting.................................................................................................................................................................................. 27© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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Page 3Introduction
This Guide & overview describes the sample, reagent, and consumable preparation for Short Read Eliminator (SRE),
DNA shearing, and post-shear cleanup on the Hamilton Microlab Prep (MLP) Liquid Handling system. This Guide &overview is meant to be used with the HiFi prep kit 96, HiFi plex prep kit 96, or SMRTbell® prep kit 3.0 library protocols.

Overview
Overview
Samples per run recommended

8, 16, 2424-sample workflow

Prep time off-deckInstrument run time

Total time per workflow

SRE

10 minutes

3 hours

3 hours 10 minutes

DNA Shearing

0 minutes

22 minutes

22 minutes

Post-shear Cleanup

20 minutes

1 hour 10 minutes

1 hour 30 minutes

8-sample workflow

Prep time off-deckInstrument run time

Total time per workflow

SRE

10 minutes

2 hours 40 minutes

2 hours 50 minutes

DNA Shearing

0 minutes

7 minutes

7 minutes

Post-shear Cleanup

20 minutes

40 minutes

1 hour© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
103-424-100 REV01 MAR2024

Total
Time

5 hours

Total
Time

4 hours

Page 4

Workflow overview
1

Input gDNA quality control

2

SRE on gDNA & Post-SRE
quantification QC
Safe stop3

DNA shearing
Optional post-DNA shearing Femto QC
Safe stop

4

1X Post-shearing cleanup (WGS)4

1X Post-shearing cleanup(multiplex)© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
103-424-100 REV01 MAR2024

Page 5Required materials and equipment
DNA sizing QC
Femto Pulse system

Agilent Technologies, Inc. M5330AA

Femto Pulse gDNA 165kb analysis kit

Agilent Technologies, Inc. FP-1002-0275

DNA quantitation QC (one or more of the following may be used)
Qubit 4 fluorometer

ThermoFisher Scientific, Q33238

Qubit Flex Fluorometer

ThermoFisher Scientific, Q33327

Qubit 1X dsDNA HS assay kit

ThermoFisher Scientific, Q33230

Centrifugation
Plate Centrifuge with 2250 g force capability

Any major lab supplier (MLS)

MicroAmp Clear Adhesive Film

ThermoFisher Scientific 00146104

Incubation
ALPS 50 V-Manual Heat Sealer

Thermo Scientific, AB-1443A

Easy Pierce Heat Sealing Foil

Thermo Scientifc, AB-0757

C1000 Touch Thermal Cycler with 96-Well Reaction Module1

Bio-Rad, 184-1100

Reagents
SRE
SMRTbell® cleanup beads

PacBio®, 102-208-300 (24 rxns)
PacBio®, 103-124-500 (96 rxns)
PacBio®, 103-306-300, or included inlibrary prep kit

Buffer LTE (low TE buffer)

Included in all library prep and bead kits

200 Proof ethanol, molecular biology or ACS grade

Any major lab supplier (MLS)

Nuclease-free water, molecular biology grade

Any MLS

Hard Shell 96 PCR full skirt 200 μL plate

Bio-Rad HSP9601

Abgene 96 Well 0.8 mL Polypropylene Deepwell Plate

ThermoFisher Scientific, AB0859

300 μL CO-RE II Tips (Filtered, Conductive)

Hamilton, 235903

1000 μL CO-RE II Tips (Filtered, Conductive)

Hamilton, 235905

60mL Reagent Trough (Black)

Hamilton, 56694-03

Alpaqua Magnum FLX magnet

Alpaqua, ALPQ-0008© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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Instrument
Hamilton Microlab Prep - PacBio® configuration
8 channels + 2 independent channels
7 position deck
Hamilton 5 × 60 mL reagent reservoir rack
HHS
2 tip stands

PacBio®, 103-283-600

1Any off-deck heater with 96-well plate adapter block© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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Before you begin
Genomic DNA (gDNA) QC and input amount recommendations
Please see the respective HiFi prep kit 96, HiFi plex prep kit 96, or SMRTbell prep kit 3.0 protocols for genomic DNA
quality and input recommendations.
Note: The maximum mass supported per well for shearing is 3 µg (10 ng/µL in 300 µL of low TE buffer/Buffer LTE).

Reagent handling
Room temperature is defined as any temperature in the range of 18–25°C for this protocol.
Bring SMRTbell cleanup beads to room temperature for 30 minutes (10 mL) – 1.5 hours/overnight (HT kits) prior touse. Vortex SMRTbell cleanup beads immediately before adding to the reagent trough.
Bring Elution Buffer to room temperature for 30 minutes prior to use.
Buffer SRE and buffer LTE should be at room temperature. Quick spin buffer SRE tube in microcentrifuge to collectliquid at bottom prior to use.

Instrument
Microlab Prep software version 3.0.4 or higher is required. See Appendix for how to check software version.
See Appendix for method and custom liquid class installation.

Safety precautions
Refer to the Safety Data Sheet (SDS) for information on reagent hazards and protocols for safe handling, use, storage,
and disposal.

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Page 8Workflow steps
1. Short Read Eliminator (SRE)
Short read eliminator (SRE) will progressively deplete fragments up to 25 kb from genomic DNA samples. This meansthat depletion will decrease as fragment sizes approach 25 kb. The depletion occurs in an unbiased manner andresults in improved sample quality for HiFi sequencing with most fragments under 10 kb removed.
Important: Use SRE on genomic DNA only. Attempting to use SRE on HiFi libraries (post library construction) will resultin poor recoveries and potential loss of the entire library.
1.

Prepare the sample plate for SRE.
Bring DNA samples to a concentration of 40 to 100 ng/µL in a total volume of 50 µL using Buffer LTE. The DNA
input requirement into shearing is ≤3 µg so gDNA input into SRE will depend on expected recovery. Manually
pipette exactly 50 µL of gDNA sample into a 96-well full-skirted PCR 200 µL plate (PN: HSP9601) starting withposition A1. Proceed to fill the plate by column (see Figure 1). Seal with an adhesive film and spin down the plate
to collect liquid at the bottom of the wells and to remove any bubbles present.

Note: The MLP runs in mutliples of 8. If your sample count is not in multiples of 8, fill the remaining wells in thecolumn with 50 µL of blanks (Buffer LTE or water).

Figure 1. Sample input plate example for SRE for 24 samples (s1–s24). Prepare in a 96-well PCR 200 µL plate (PN: HSP9601).

2.

Prepare the reagent plates for the SRE method.
• Manually pipette 55 µL of Buffer SRE into a 96-well full-skirted PCR 200 µL plate (PN: HSP9601) in well
positions aligning with your sample plate. Seal with an adhesive seal and spin down the plate in a centrifuge tocollect liquid at the bottom of the well and remove any bubbles present.
• Manually pipette 310 µL of Buffer LTE into a 96-well deepwell plate (PN: AB0859) in well positions aligning withyour sample plate. Seal with an adhesive film and spin down the plate in a centrifuge to collect liquid at thebottom and remove any bubbles present.

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Page 93.

Load the deck and begin the SRE method.
On the Hamilton Microlab Prep home touch screen, select the “PacBio SRE” method. If it is not a favorite on thehome screen, tap “Browse” and search for “PacBio SRE.”

Select “Run” at the bottom right-hand corner to continue.

4.

Select the number of samples to process for each plate.
This must be done for every plate on the deck. To select the sample positions on each plate, tap on themagnifying glass next to the plate to open the selection menu.

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Select the appropriate sample wells by tapping, or touch and dragging in multiples of 8. Tap “Done” after sample
count is selected for each plate.

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Select “Next” to continue.

5.

Place the consumambles on the deck as indicated in the figure below. Unseal all plates after spinning down.
•
•
•
•
•
•
•

6.

Position 2: Tip stand & 300 µL tips
Position 3: Tip stand & 300 µL tips
Position 4: Abgene 96 Deepwell plate 0.8 mL (Empty)
Position 5: 96 well PCR full skirt 200 µL plate (Empty)
Position 6: Abgene 96 Deepwell plate 0.8 mL (310 µL of Buffer LTE)
Position 7: 96 well PCR full skirt 200 µL plate (50 µL of Sample)
Position 8: 96 well PCR full skirt 200 µL plate (55 µL of Buffer SRE)

Verify the deck.

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Page 12The deck with the required labware will display. Tap “Detect”. When “detect” is selected, the Microlab Prep
captures a photo of the current deck.

If a “Missing”, “Wrong Labware”, or “Confirm Labware” warning appears, tap on it to review the required labware
for the position.

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Page 13

Tap “Confirm” when the highlighted positions match the tips in the rack on-deck or when the plate is present.

7.

Resolve all deck verification errors to proceed. Tap “Start” to begin the SRE run.

8.

A prompt displays a description of the SRE method and guidance on MLP. Tap “Resume” to continue. At thispoint, the automation will begin. Buffer SRE will be added to the sample plate.

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9.

Seal the sample plate. When prompted, remove the sample plate in position 7 to foil heat seal. Return the sealed
plate to position 7 and tap “Resume” to continue. The sample plate will shake to mix on deck.

10. Incubate at 50°C for 1 hour. When prompted, take the sealed sample plate off of postion 7 to incubate off-deck.
When the incubation is complete, spin down, carefully unseal the foil seal and return the sample plate to position
7. Tap “Resume” to continue. The sample will be transferred to a deep well plate for centrifugation.

11. Centrifugation. When prompted, remove the deepwell plate containing the samples in position 4. Seal the platewith an adhesive seal. Centrifuge at 2250 rcf (max 3220 rcf) for 1 hour at room temperature. If using a centrifuge
with temperature control (i.e., cooling function), turn this function off by specifying a target temperature set
point higher than ambient room temperature (e.g., 29°C). Once the spin cycle is complete, unseal and
immediately place the deepwell plate back into position 4. Tap “Resume” to continue.

12. Method completion and QC. Once the SRE protocol is complete, remove the deepwell plate in position 4 toquantify the samples using the Qubit 1x dsDNA HS assay. Seal the supernatant plate in position 5 and discard allused consumables and tip waste. The supernatant plate can be disposed of once it is confirmed that sample
loss did not occur. If recovery is lower than 50%, vortex your sample plate and re-quant. If it is still below 50%© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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recovery, refer to the troubleshooting section in the Appendix. Continue to DNA Shearing or store samples at 4Cfor up to 2 weeks.

2. DNA Shearing
Important:
A mean fragment size between 15 to 20 kb is recommended for this protocol. In addition, the distribution of fragment
sizes should be narrow and generally between 10 to 30 kb. Fragments that are too short produce less yield per read,and fragments that are too long may result in lower read accuracy and are less likely to produce HiFi reads. Deviatingfrom the concentration and automation settings is not recommended and will result in undersheared DNA.
These shearing parameters are not universal and are specific for only the Hamilton Microlab Prep system, or assayready workstations like the NGS STAR, STARlet, and STAR V systems

1.

Input DNA quality control
Adjust the DNA concentration to ≤10 ng/µL if necessary (e.g., if more than 3 µg of gDNA was recovered fromSRE) or if bypassing SRE. Use Buffer LTE to dilute simples in a 96-well deepwell plate (PN: AB0859). Ensure all
samples are 300 µL and ≤10 ng/µL per sample. Using a DNA concentration >10 ng/µL or a volume greater orless than 300 µL will result in inefficient shearing. Parameters for shearing on the Microlab Prep, or Hamilton
assay-ready workstations are listed in the table below.
Table 1: The pipette tip shearing parameters are part of the custom liquid class that is imported with the DNA shearing method.

2.

Parameter

Setting

DNA concentration

 10 ng/µL

Volume of Buffer LTE

300 µL

Number of mixes

300 cycles

Pipette mixing speed

500 µL/sec

Mix volume

83% volume

Liquid following, cLLD

On

Pipette tip

300 CO-RE II tips (filtered, conductive)

Prepare sample plate for shearing© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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If SRE has been run, the samples are in the output 96-well deepwell plate and this step can be skipped.
If beginning at this method, pipette 300 µL of sample in the 96-well deepwell plate (PN: AB0859) starting withposition A1. Proceed to fill the plate with samples by column. Seal with an adhesive seal, vortex and spin down
the plate to collect liquid at the bottom and to remove any bubbles present. Remove the seal when ready toplace on-deck.
3.

Load the deck and begin shearing method
When at the Microlab Prep home touch screen, select the “PacBio DNA Shearing” method on the MLP. If it is nota favorite on the home screen, tap “Browse” then search for “PacBio DNA Shearing.

Tap “Run” at the bottom right-hand corner to continue.

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Select your wells by tapping on the magnifying glass icon and highlighting the wells to match the input sample
plate.

Select the appropriate sample wells by holding down and dragging in multiples of 8. Tap “Done” after theappropriate sample count is selected.

Select “Next” to continue.

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4.

Load the deck and begin the DNA shearing method.

Place the consumables on deck as indicated in the figure below. Position 4 should contain a tip stand and 300µL tips. Position 8 should contain a 96-well deepwell plate (PN: AB0859) with samples (300 µL at 10ng/µL)

5.

Verify the deck.
The deck with the required labware will be displayed. When “detect” is selected, the Microlab Prep captures aphoto of the current deck.

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Page 19

If “Missing”, “wrong labware”, or “Confirm Labware” warning appears, tap on it to review the required labware forthe position. Confirm if the labware is correct.

Confirm if the labware is correct.

Resolve all deck verification errors to proceed. Tap “Start” to begin DNA Shearing.

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Page 20

6.

Method complete.
At the end of DNA Shearing, remove the 96-well deep well (PN: AB0859) output plate in position 8. Perform
optional QC using the Femto Pulse to check size distribution (range of 15–20 kb). If your samples did not shear
to the expected range, see Appendix. Continue to the PacBio Post-shearing Cleanup process.

3: 1X Post-shear cleanup1.

Prepare sample plate for post-shear cleanup
If the DNA shearing method has been run, the samples are in the 96-well deepwell output plate and this step canbe skipped.
If beginning here, manually pipette 300 µL of sample diluted with Buffer LTE into a 96-well deepwell plate (PN:
AB0859) targeting ≤3 µg input. Start at position A1 then proceed to fill the plate with samples in a column-based
down direction.
Seal, vortex then spin down the plate to collect liquid at the bottom and to remove any bubbles present. Remove
the seal when ready to place on-deck.

2.

Load deck and begin 1X Post-shear cleanup method
There are two 1X Post-shear cleanup methods to chose depending on the library preparation protocol
performing. Reference the table below for guidance on which 1X Post-shear cleanup you will select to run.
Note: To determine the post-shear cleanup method to use, please see companion documentation.
Table 2: PacBio 1X post-shear cleanup protcols with their associated library prep protocols. The difference between the post-shear
methods is the eluate volume.

Library preparation protocol

1X post-shear cleanup protocol

Eluate volume (µL)

HiFi prep (WGS)

1X post-shear cleanup (WGS)50

HiFi plex prep (mulitplex)

1X post-shear cleanup (multiplex)

25.5© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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When at the home touch screen, select one of the PacBio 1X post-shear cleanup methods on the MLP. If it is not afavorite on the home screen, tap “Browse” then search for 1X post-shear cleanup method you’re selecting to run.

Select “Run” at the bottom right-hand corner to continue.

3.

Select sample count for each plate in post-shear cleanup
Select your wells by tapping the magnifiying glass, then highlight the wells to match your sample count.
• Position 6: 96-well deepwell sample plate (300 µL)• Position 7: 96-well PCR plate (Empty)© 2024 PacBio. All rights reserved. Research use only. Not for use in diagnostic procedures.
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Select your wells by tapping the magnifying glass, then highlight the wells to match the input sample plate.
Select the appropriate sample wells by holding down and dragging in multiples of 8. Tap “Done” after theappropriate sample count is selected.

Tap “Next” to continue.

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4.

Enter reagent volumes for the 60 mL troughs

Note: Before loading the reagent troughs into the reservoir rack, make sure to remove the middle insert from theblack 60 mL reservoir.

Enter the volume for SMRTbell beads, Elution buffer, and 80% ethanol required. Reference the following table todetermine how much reagent volume is needed in each 60mL trough based on sample count.
Note: User must enter reagent volumes manually for every run. The volumes will automatically display the reagent
volumes from the previous run, so these must be modified if the sample count is different. Select “Next” tocontinue.
Table 3: Reagent volumes for 1X post-shear cleanup

5.

Number of Samples

8

16

24

SMRTbell bead volume (µL)

4,400

6,800

9,200

Elution buffer volume (µL)

2,400

2,800

3,200

80% ethanol volume (µL)

3,600

5,200

6,800

Load the deck and begin post-shear cleanup method.

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Place the consumables on deck as indicated in the figure below. To place the 1000 µL tips on-deck, remove thebase plate in position 2. Then place the tip stand in position 2. The 1000 µL tips will dip below the deck whensecured in the tip stand.
•
•
•
•
•
•
•

6.

Position 2: Tip stand & 1000 µL tips (base plate removed)
Position 3: Tip stand & 300 µL tips
Position 4: Abgene 96 Deepwell plate 0.8 mL (Empty)
Position5: Alpaqua Magnum FLX magnet plate
Position 6: Abgene 96 Deepwell plate 0.8 mL with samples (300 uL)
Position 7: 96 well PCR full skirt 200 µL plate (Empty)
Position 8: 60 mL Reagent Reservoir (three 60 mL troughs)

Verify the deck.
The deck with the required labware will appear. Tap “Detect”. When “detect” is selected, the Microlab Prep
captures a photo of the current deck.

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If a “Missing”, “Wrong Labware”, or “Confirm Labware” warning appears, tap on it to review the required labware
for the position.

Tap “Confirm” if correct.

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Resolve all deck verification errors to proceed. Then tap “Start” to begin the run.

7.

Method Complete.
Once complete, the samples are ready to continue with library prep. Seal and store DNA at 4°C for up to 2 weeks
until ready to proceed to library preparation.

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Appendix
Installation (New User)
Please contact your PacBio FAS to import Microlab Prep methods and liquid class files or visit Hamilton Microlab Prep
Support for more information.
1.

Plug in the USB drive with the desired files to import• Protocol file type: “.mlppe”• Custom Liquid Class file: “.mlplc”

Note: “.mlppe” is a protocol file “.mlplc” is a liquid class file
2. Tap “Browse” to open the File Manager
3.

In the Protocol tab, select the protocol and tap the three-line menu
select “Import”(located next to the search bar), then4.

In the Liquid tab, scroll to find the liquid class and select the three -line menu, (located next to the search bar),then select “Import”

Troubleshooting
Issue

Installation issues

Instrument error or insufficientvolumeLow to no recovery at the endof SRE run

Possible Cause

Corrective Action

Software not up to date

From the home page, tap the settings
button that looks like a gear. In the
Software tab, tap “Configuration”, thenopen the “Version" tab. The version will be
associated with the Microlab Prep Core
Installer.

1. Not enough volume in the
consumable

1. Fill the consumable to therecommended amount specified in theloading dialog

2. No blanks were inserted into
the remaining wells of the lastcolumn. Liquid level detection isused.
1. Not heating effectively during
off-deck incubation

2. Insert a blank of water or TE buffer into
the wells to fill out the column
1. Ensure plate is heat sealed for the 50°C
incubation and the plate is spun down
before removing the seal.
Ensure the off-deck heating temperature is
at 50°C and the appropriate plate adapter
is used.

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2. Input volume or SRE Buffer
volume not accurate

3. Incorrect centrifuge settings

2. Sample input volume in Buffer LTE
buffer must be 50 µL per well. Buffer SRE
must be 55 µL per well.
3. Ensure that plate centrifuge is set at>2250 rcf (max 3220 rcf) at 29°C or room
temperature for 60 minutes.
4. (a) Ensure the Prep is leaving behind 8–
10 µL of supernatant after removal. It is
possible the DNA pellet is aspirated in the
tip during supernatant removal. Quantsupernant plate to see if the DNA pellet
was dispensed into it. If so, the sample
can be recovered by transferring into a
1.5mL DNA Lo-Bind tube andrecentrifuging as outlined in the HiFi prep
kit 96 protocol steps 1.5–1.10.

4. Not enough supernatant left
behind(b) Ensure after centrifugation the plate is
not disturbed and immediately placed on
deck.
(c) The coordinates for the channels could
have adjusted: Re-calibrate yourinstrument. Clean the stop discs and orings with DI water and lint free cloth
before starting and use a set of new tips
when prompted to add them on the deck.
•

5. Sample lost in supernatant

Sample Volume discarded towaste during DNA Shearing

Custom Liquid class for shearing
not imported

DNA not shearing to expectedfragment range

1. Jelly-like HMW gDNA

From the Home screen, tap
Settings -> Instrument tab ->System Calibration -> Tap
Pipetting Tools Calibration -> Run
5. DNA pellet aspirated in the tip during
supernatant removal. Quant thesupernatant plate. If sample is present insupernatant plate, the sample can berecovered by transferring ~100 µL in a 1.5
mL DNA Lo-Bind tube and recentrifuging
as outlined in the HiFi prep kit 96 protocol
steps 1.5–1.10.
Ensure the custom liquid class wasimported by following the installation
procedure in Appendix.
1. HMW gDNA sometimes contains dense
clumps. Vortex sample to fully
homogenize and re-QC to ensure quants
are ≤10 ng/µL per well in the plate. Then

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re-shear by running the DNA Shearing
protocol again.
2. Ensure fluorescence is used forquanting. HMW gDNA may not behomogenized. Vigorously vortex sample
right before quanting. If inhomogenous, it
may be necessary to take multiple quants
to ensure that values are within 20%.

2. Initial quant of gDNA not
accurate

If experiencing mechanical pipetting errors or frequent instrument errors, reach out to Hamilton Support team via
Hamilton support ticket submission: https://robotics.hamiltoncompany.com/benchtopticket

Revision history (description)

Version

Date

Initial release

01

March 2024

Research use only. Not for use in diagnostic procedures. © 2024 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,
Kinnex, and PureTarget are trademarks of PacBio.
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