Leaders in Decision Medicine™

oncoReveal™ Myeloid
USER MANUAL

UM-0033 version 4.0
For Research Use Only. Not for use in diagnostic procedures.

oncoReveal™ Myeloid Panel User Manual

Revision History
Version 1.0 – 3.0: Initial Release; Edits for clarity & consistency
Version 4.0: Edits to branding, formatting; edits to improve clarity

For Research Use Only

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oncoReveal™ Myeloid Panel User Manual

Table of Contents
1.

PRODUCT INTRODUCTION.............................................................................. 3

2.

PRODUCT DESCRIPTION .................................................................................. 33.

GENERAL LABORATORY GUIDELINES .......................................................... 44.

REAGENTS & EQUIPMENT ............................................................................... 5
4.1

Kit Components ...........................................................................................................................5

4.2

Indexing Kits..................................................................................................................................5

4.3

User-Supplied Reagents ............................................................................................................5

4.4

Other Consumables ................................................................................................................... 6

4.5

Equipment .......................................................................................................................................7

5.

WORKFLOW......................................................................................................... 8

6.

LIBRARY PREPARATION PROTOCOL ............................................................ 9
6.1

Gene-Specific PCR (GS-PCR) & Purification ............................................................... 9

6.2

Index PCR & Purification...................................................................................................... 15

6.3

Quantitation and Normalization of Purified Libraries ............................................ 207.

SEQUENCING ..................................................................................................... 23

8.

TROUBLESHOOTING ....................................................................................... 27

9.

APPENDIX A: INDEX SEQUENCES.................................................................. 30

10. APPENDIX B: ANALYSIS USING PIVAT ....................................................... 31

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oncoReveal™ Myeloid Panel User Manual

1. Product Introduction
The oncoReveal™ Myeloid is a robust NGS assay that targets numerous gene regions of interest for researchers looking
to explore the genetic sequences of myeloid cancers.

2. Product Description
The oncoReveal™ Myeloid panel utilizes our SLIMamp® (stem-loop inhibition mediated amplification) technology
with a single-tube multiplex PCR reaction. Pairs of DNA oligos designed for each region of interest, or hotspot, are usedin the first round of gene-specific PCR (GS-PCR) and the products are subsequently purified via size selection. After
purification, a second round of PCR adds index adaptors and P5 & P7 sequences to each library for sample tracking andsequencing. The final libraries are further purified and can be sequenced on the Illumina sequencing platform.
The panel requires a dual barcode indexing kit, available in low or high throughput, and the resulting libraries aredesigned for sequencing on the Illumina platform using a paired-end read length of 2x150 or up to 275 (2x275). The
workflow of this panel can be performed and loaded onto the sequencing instrument within one day. In addition, theprotocol also contains numerous stopping points for users who have time limitations.

Limitations
The oncoReveal™ Myeloid Panel can detect internal tandem duplications (ITDs) in the FLT3 gene. An ITD of up to 126
bases can be detected using paired end sequencing at read lengths of 175 bp (2x175).

26 Cycles

6+ Cycles

Figure 1. Overview of the oncoReveal™ Myeloid Panel library preparation.

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oncoReveal™ Myeloid Panel User Manual

3. General Laboratory Guidelines
The following steps are recommended to improve consistency and reduce contamination:•Work areas: To reduce the risk of contamination from PCR amplicons, supplies should not be moved from one area
to another. Separate storage areas (refrigerator, freezer) should also be designated for pre- and post-PCR products.

•

Lab cleanliness: To further reduce the possibility of contamination, work areas should be cleaned between
experiments with laboratory cleaning solution (70% alcohol or freshly-made 10% hypochlorite solution). A periodic
cleaning of the floor is also recommended.

•

Floor: Items that have fallen to the floor are assumed to be contaminated and should be discarded. If a sample tube
or non-consumable item has fallen to the floor and remained sealed, the outer surface of the item should bethoroughly cleaned before use using 70% alcohol or freshly-made 10% hypochlorite solution. Gloves should bechanged after handling a contaminated item.

•

Aliquot reagents: Frozen reagents should be aliquoted into smaller volumes to reduce freeze/thaw cycles. To reduce
the risk of stock contamination it is recommended to aliquot from the stock and work from the aliquots. In cases ofcontamination, the use of aliquots can also help to determine the source more quickly and easily.

•

Multichannel pipettes: Multichannel pipettes should be used to maintain consistency and efficiency acrossnumerous samples.

•

Pipette tips: Tips should be changed between each sample to prevent cross-contamination. Any tips that may havebecome contaminated due to contact with gloves, the lab bench, tube exteriors, etc., should be discarded.

•

Open containers and lids: To prevent possible contamination from the air, tubes should be kept closed when notdirectly in use, and plates, troughs, and similar reservoirs should be covered with seals or lint-free laboratory wipes.
Additionally, reaching over open containers should be avoided.

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oncoReveal™ Myeloid Panel User Manual

4. Reagents & Equipment
This section describes the necessary equipment, reagents, and consumables needed before performing the protocol.
All reagents should be used in designated pre-PCR or post-PCR areas to prevent amplicon contamination. Each area
designated for pre- and post-PCR should have dedicated equipment, reagents, and supplies (including gloves, lab coats,etc.) to prevent contamination.

4.1 Kit Components
oncoReveal™ Myeloid Panel Part No.: HDA-MY-1001-24
Reagent
Gene-Specific PCR Master Mix (2x)Myeloid Oligo Pool
GC Rescue G
Exonuclease I
GC Rescue D
Indexing PCR Master Mix (2x)Use
Gene-Specific PCR
Gene-Specific PCR
Gene-Specific PCR
Gene-Specific PCR
Indexing PCR
Indexing PCR

Area Use
Pre-PCR
Pre-PCR
Pre-PCR
Pre-PCR
Pre-PCR
Pre-PCR

Storage-25° to -15°C-25° to -15°C-25° to -15°C-25° to -15°C-25° to -15°C-25° to -15°C

4.2 Indexing Kits
Reagent

Part Number

Use

Area Use

Storage

Pillar Custom Indexing Primers Kit A, indices PI5018, PI701-4 (32 combinations - 96 reactions)

IDX-PI-1001-96

Indexing
PCR

Pre-PCR-25° to -15°C

Pillar Custom Indexing Primers Kit D, indices PI501-8,
PI701-12 (96 combinations - 192 reactions)

IDX-PI-1004-192

Indexing
PCR

Pre-PCR-25° to -15°C

Only one index kit is needed per assay. Multiple options are available to meet a variety of throughput needs.

4.3 User-Supplied Reagents
Reagent
10N NaOH or 1N NaOH

Area Use
Post-PCR

AMPure XP Beads

Post-PCR

Ethanol, 200 proof for molecular biology
Nuclease-free water
Qubit dsDNA High Sensitivity Assay kit
Qubit dsDNA Broad Range Assay kit1
Agarose gel, 2% (optional) 1
DNA molecular weight markers (optional) 1
TapeStation or equivalent (optional) 1
10 mM Tris-HCl w/ 0.1% Tween-20, pH 8.5 (optional)
PhiX Control v3
200 mM Tris-HCl, pH 7.0 (optional) 2

Post-PCR
Post-PCR
Post-PCR
Post-PCR
Post-PCR
Post-PCR
Post-PCR
Post-PCR
Post-PCR
Post-PCR

Supplier
General lab supplier
Beckman Coulter, Cat# A63881 or
A63880General lab supplier
General lab supplier
Invitrogen, Cat# Q32851 or Q32854
Invitrogen, Cat# Q32850 or Q32853
General lab supplier
General lab supplier
Agilent Technologies
Teknova, Cat# T7724
Illumina, Cat# FC-110-3001
General lab supplier

The Qubit dsDNA High Sensitivity Assay kit is the primary DNA quantitation assay used throughout this protocol. Additional DNA
quantification can optionally be performed using the Qubit dsDNA Broad Range Assay kit, an agarose gel, or TapeStation (or equivalent).
1

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oncoReveal™ Myeloid Panel User Manual
Sensitivity DNA Analysis kit.
The 200 mM Tris-HCl, pH 7.0 reagent is only required for denaturing libraries for sequencing on the Illumina NextSeq or MiniSeq. If sequencing
on the MiSeq this reagent is not needed.
2

4.4 Other Consumables
Compatible Sequencing Reagents
Sequencing Reagent Kit
MiSeq™ Reagent kit v2 (300 cycles)‡MiSeq™ Reagent kit v3 (600 cycles)‡NextSeq™ 500/550 Mid Output v2.5 kit (300 cycles)‡NextSeq™ 1000/2000 P1 Reagents (300 cycles)
NextSeq™ 1000/2000 P1 Reagents (600 cycles)
NextSeq™ 1000/2000 P2 Reagents v3 (300 cycles)
NextSeq™ 1000/2000 P2 300M Reagents (600 cycles)
NextSeq™ 2000 P3 Reagents (300 cycles)

Supplier
Illumina
Illumina
Illumina
Illumina
Illumina
Illumina
Illumina
Illumina

Catalog No.
MS-102-2002
MS-102-3003
20024905
20050264
20075294
20046813
20075295
20040561‡ Sequencers the oncoReveal™ Myeloid Panel has been validated on. However, libraries generated using this panel are compatible with all Illumina
sequencing reagents.

General Laboratory Consumables
In addition to the consumables listed below other general laboratory supplies needed to carry out the protocol include
gloves, prechilled benchtop coolers, tube racks, etc.
Consumable
1.5 mL microcentrifuge tubes

Area Use
Pre- and Post-PCR

96-well PCR plates, 0.2 mL

Pre- and Post-PCR

Microplate sealing film

Pre- and Post-PCR

Conical tubes, 15 mL
Conical tubes, 50 mL
Low retention, aerosol filter pipette tips

Pre- and Post-PCR
Post-PCR
Pre- and Post-PCR

Solution basin (trough or reservoir)

Pre- and Post-PCR

Qubit Assay tubes

Post-PCR

For Research Use Only

Supplier
General lab supplier
Corning Life Sciences, Cat# 6551 orequivalentCorning Life Sciences, Cat# PCR-TS orequivalent
General lab supplier
General lab supplier
General lab supplier
Fisher Scientific, Cat# 13-681-506 orequivalent
Invitrogen, Cat# Q32856

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oncoReveal™ Myeloid Panel User Manual

4.5 Equipment
Equipment
Centrifuge adapted for PCR plates, tabletop
Gel electrophoresis apparatus (optional)*

Area Use
Pre- and Post-PCR
Post-PCR

Supplier
General lab supplier
General lab supplier

TapeStation or equivalent*
Magnetic stand for 96 wells
Microfuge
Thermal cycler, heated lid capability
Pipettes, 0.5-1000 μL capabilities
Qubit Fluorometer
Vortex mixer

Post-PCR
Post-PCR
Pre- and Post-PCR
Post-PCR
Pre- and Post-PCR
Post-PCR
Pre- and Post-PCR

Agilent Technologies
Life Technologies, Cat# 12331D or 12027
General lab supplier
General lab supplier
General lab supplier
Invitrogen, Cat# Q33216/Q33218
General lab supplier* The Qubit dsDNA High Sensitivity kit is the primary DNA quantitation assay used throughout this protocol. Additional DNA quantification can
optionally be performed using an agarose gel.

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oncoReveal™ Myeloid Panel User Manual

5. Workflow
The following chart demonstrates the workflow for performing the oncoReveal™ Myeloid Panel library preparation.
Gene-Specific PCR
Hands-on time: 30 - 40 min.
Total time: 2.75 - 3 hours

Pre-PCR
Primer Digestion
Hands-on time: 10 - 15 min.
Total time: 45 - 50 min.

Post-PCR

Stopping Point (Optional)

Purify PCR ProductHands-on time:3 – 5 hours

Hands-on time: 30 - 45 min.
Total time: 30 - 45 min.

Total time:
6.5 – 8.5 hours

Indexing PCR
Hands-on time: 20 - 40 min.
Total time: 50 - 70 min.

Indexing PCR plate is set up in
Pre-PCR area.

Purify Libraries
Hands-on time: 30 - 45 min.
Total time: 30 - 45 min.

Quantify and Load Libraries
Hands-on time: 1 - 1.5 hours
Total time: 1.5 - 2 hours
Figure 2. Library preparation workflow for oncoReveal™ Myeloid Panel. The workflow can be completed within one day but contains multiple
optional stopping points for users with time constraints.

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oncoReveal™ Myeloid Panel User Manual
SymbolDescription
Information: Information that follows this symbol is important and may require action.
Optional Stopping Point: A point in the workflow at which work may be safely paused, andsamples can be stored appropriately.
Caution: Information that follows this symbol is critical to the workflow. Information following
this symbol should not be skipped or ignored.

Symbols used throughout this protocol and their associated meaning.

6. Library Preparation Protocol
Hands-on Time3 – 5 hours

Total Time

6.5 – 8.5 hours

6.1 Gene-Specific PCR (GS-PCR) & Purification
Hands-on Time

30 – 40 min.

Total Time2.75 – 3 hours

Amplify Genomic DNA Targets

For this portion of the protocol prepare a prechilled benchtop cooler and keep the gene-specific PCR Master Mix (GSPCR MMX), the custom oligo pool, and GC Rescue G in the cooler.
1.

Prepare Gene-Specific PCR Mix 1: Vortex and centrifuge the GS-PCR MMX reagent and the oligo pool before use.
For each PCR reaction, the volume of each component is listed below.
Important: The gene-specific PCR master mix reagent is viscous. Ensure the mix is fully homogenized before adding otherreaction components. Vortexing is recommended and will not adversely affect enzyme activity.

GS-PCR Mix 1
Reagent
Gene-Specific PCR Master Mix
Myeloid Oligo Pool
GC Rescue G
Subtotal
2.

Volume (μL)
12.5
7.0
1.25
20.75

Transfer: Transfer 20.75 μL of GS-PCR Mix 1 to each sample well in a PCR plate, strip tube, or PCR tube.

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oncoReveal™ Myeloid Panel User Manual3.

Dilute input DNA: Add 4.25 μL of DNA (diluted if necessary) to the assigned sample well in the GS-PCR plate. Add
4.25 μL of nuclease-free water to the no-template control well.
The recommended input range is 20 – 60 ng of genomic DNA.
Final GS-PCR Mix
Reagent
GS-PCR Mix 1
DNA (or water)
Total

Volume (μL)
20.75
4.25
25.0

* The DNA concentration can be determined using the Qubit
dsDNA HS Assay Kit or the Qubit dsDNA BR Assay Kit.

4.

Seal and mix: Carefully seal the reactions and vortex for 10 – 15 seconds.

5.

Spin: Briefly centrifuge the reactions to remove any air bubbles from the bottom of the wells and to spin down
droplets from the seal or side walls.

6.

Perform GS-PCR: Perform the following program with the heated lid on:
Temperature
95°C
98°C
72°C
98°C
58°C
60°C
64°C
72°C
95°C
66°C
8°C

GS-PCR Program
Time
Number of Cycles
15 min
1
1 min
3
1 min
1 min
2 min
4 min
5
1 min
1 min
30 sec
18
3 min
Hold
1

Do not leave reactions on the thermocycler overnight at 8C. Once cycling is complete thereactions can be stored at -20C.
Optional Stopping Point: The gene-specific PCR reactions may be stored at -20C after cycling is complete.

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oncoReveal™ Myeloid Panel User ManualGene-Specific Primer Digestion
Hands-on Time

10 – 15 min.

Total Time

45 – 50 min.

For this portion of the protocol prepare a prechilled benchtop cooler. The exonuclease should be kept in the cooler until
it is needed. Keep the sample reactions at ambient temperature.
1.

Briefly centrifuge the gene-specific PCR product to remove droplets from the side walls. Carefully remove the sealor caps.

2.

Dilute Exonuclease: Invert the Exonuclease I to mix and centrifuge briefly to remove any droplets from the lid.
Prepare the Exonuclease Dilution as indicated below, pipette to mix and place on in the cooler.
Exonuclease Dilution(per reaction)
Reagent
Exonuclease I
Exonuclease I Buffer (or nuclease-free water)
Total

Volume (μL)
3.0
2.0
5.0

Important: The exonuclease solution is viscous and requires careful attention when pipetting. Ensure the dilution is mixed
thoroughly.

3.

Add Exonuclease: Add 5 μL of the diluted exonuclease to each well containing gene-specific PCR product. Pipetteup and down to mix.

4.

Seal and mix: Carefully seal the reactions. Pulse vortex the reactions on a medium setting for 5 – 10
seconds.

5.

Spin: Briefly centrifuge the reactions to remove any air bubbles from the bottom of the wells and to spin down
droplets from the seal or side walls.

6.

Perform the digestion: Perform the following program with the heated lid on:
GS Primer Digestion Program
Temperature
Time
Number of Cycles
20 min1
37C
10 min
1
80C
Hold
1
8C
Do not leave reactions on the thermocycler overnight at 8C. Once cycling is complete thereactions can be stored at -20C.

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Optional Stopping Point: The reactions may be stored at -20C after primer digestion is complete.

Gel Image After Gene-Specific PCR
The following image is an example of samples after gene-specific PCR and before primer digestion on a 2% agarose gel.

Figure 3. Analysis of GS-PCR product on 2% agarose gel. Lane 1 is genomic DNA after GSPCR. Lane 2 is a no-template control.

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oncoReveal™ Myeloid Panel User Manual

Purify the Gene-Specific PCR ProductHands-on Time

30 – 45 min.

Total Time

30 – 45 min.

Before beginning the purification:•

Remove AMPure XP beads from 4°C and incubate at room temperature for at least 30 minutes before use.
Caution: Ensure that the AMPure bead solution reaches room temperature before performing thepurification. The temperature of the bead solution can have adverse effects on the purification process.

•If gene-specific PCR products were stored at -20°C, allow to thaw at room temperature and then proceed
immediately to purification.

•

Prepare 70% ethanol by mixing three parts of water with seven parts of absolute ethanol in a conical tube,
invert tube a few times to mix and dispense sufficient volume to a disposable trough for convenient dispensing
using a multichannel pipette.
Caution: Fresh 70% ethanol is required for optimal results.

Gene-Specific Product Purification
1.

If condensation has formed or if reactions were stored at -20°C briefly centrifuge the reactions upon thawing.
Carefully remove the seal.

2.

Mix Beads: Vortex AMPure XP beads thoroughly until all beads are well-dispersed.
Caution: Ensure that the AMPure bead solution is homogenous. A non-uniform distribution can have
unpredictable effects on the purification process.

3.

Add water to sample: Add 20 μL of nuclease-free water to each well containing GS-PCR product to bring thereaction volume to 50 μL.

4.

Add beads: Add 60 μL of AMPure beads (equivalent to a 1.2x bead ratio) to each well. Pipette the mixture up anddown 10 times. If bubbles form on the bottom of the wells, briefly centrifuge the samples and mix again.

5.

Bind GS-PCR product to beads: Incubate for 5 minutes at room temperature.

6.

Separate beads containing GS-PCR product: Place the PCR plate on a magnetic rack until the solution appears
clear, which can take up to 5 minutes.

7.

Remove supernatant: Carefully remove the supernatant from each well without disturbing the beads.

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8.

Wash beads: With the PCR plate still on the magnetic rack add 150 μL of freshly prepared 70% ethanol to each wellwithout disturbing the beads. Incubate at room temperature for 30 seconds, and then remove the supernatant fromeach well.
Important: Do not allow the ethanol mixture to remain open to the air. The ethanol concentration will change over time,
affecting the washing of the beads. Pour only enough solution for each wash.

9.

Second wash: Repeat step 8 for a second 70% ethanol wash. Remove the supernatant from each well. The unused
solution of ethanol can be used to purify the libraries after the indexing PCR is completed.

10. Remove remaining ethanol wash: Centrifuge for 10–15 seconds, place the PCR plate back on the magnetic rack,and use a 10 or 20 μL tip to remove any trace amounts of ethanol from each well.
11. Dry beads: Keep the samples on the magnetic rack and let the beads air dry at room temperature for 2 – 5 minutes.
Important: To mitigate product loss, do not allow the beads to over-dry. The beads have sufficiently dried when the beadmass has small cracks in the middle. If large cracks have appeared among the entire bead ring or they are flaky, they areover-dried. Beads that are too dry may be difficult to resuspend.

12. Resuspend beads: Remove the samples from the magnetic rack and resuspend the dried beads in each well using 32
μL of nuclease-free water. Gently pipette the suspension up and down 10 times. If bubbles form on the bottom of
the wells, briefly centrifuge and mix again.
TIP: After resuspending the beads, cover the reactions and prepare for the indexing PCR step using the Indexing PCR Master Mix. The
Indexing PCR Master Mix should be handled in the pre-PCR area. Alternatively, the purified gene-specific PCR product may be stored at 20°C after elution.

13. Incubate and Elute: Incubate at room temperature for 5 minutes.
14. Separate GS-PCR product from beads: Place the reactions on a magnetic rack at room temperature until thesolution appears clear. Transfer 30 μL of clear supernatant from each well to a new PCR plate.
Optional Stopping Point: The purified GS-PCR products can be stored at -20°C.

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6.2 Index PCR & Purification
Indexing PCR: Amplify the Libraries
Hands-on Time

20 – 40 min.

Total Time

50 – 70 min.

For this portion of the protocol prepare a prechilled benchtop cooler. The Indexing PCR Master Mix should be kept in
the cooler until it is needed.
1.

Prepare Indexing Primer Mix: In a new PCR plate add 4 μL each of the appropriate forward and reverse indexing
primers to each sample well that will be used.
Indexing Primer Mix(per reaction)
Reagent
Volume (μL)
Pi700 Pillar Index
4.0
Pi500 Pillar Index
4.0
Subtotal
8.02.

Prepare Indexing PCR Mix 1: Briefly vortex and centrifuge the Indexing PCR Master Mix before use. Prepare theIndexing PCR Mix 1 by combining the Indexing PCR Master Mix, GC Rescue D, and water with sufficient overage.
Indexing PCR Mix 1(per reaction)
Reagent
Indexing PCR Master Mix (2x)
GC Rescue D
Nuclease-free water
Subtotal

3.

Volume (μL)
25.0
5.0
6.0
36.0

Add Indexing PCR Mix 1 to Indexing Primer Mix: Transfer 36 μL of the Indexing PCR Mix 1 to each sample wellof the PCR plate in step 1 that contains Indexing Primer Mix. To prevent cross-contamination of indices, be sure tochange tips between each well.
Indexing PCR Mix 2(per reaction)
Reagent
Volume (μL)
Indexing Primer Mix
8.0
Indexing PCR Mix 1
36.0
Subtotal
44.0

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If the purified GS-PCR products were stored at -20°C after the GS-PCR purification, ensure that they have been
thawed at room temperature before proceeding.
4.

Add purified GS-PCR product: Aliquot 6 μL of purified GS-PCR product into the appropriate wells containing
Indexing PCR Mix 2.
Indexing PCR Final Mix(per reaction)
Reagent
Indexing PCR Mix 2
Purified Gene-Specific PCR product
Total

Volume (μL)
44.0
6.0
50.0

5.

Seal and mix: Carefully seal the reactions and vortex for 10-15 seconds.

6.

Spin: Briefly centrifuge the reactions to remove any air bubbles from the bottom of the wells and to spin down
droplets from the seal or side walls.

7.

Perform Indexing PCR: Perform the following program with the heated lid on.
Temperature
95°C
95°C
66°C
72°C
72°C
8°C

Indexing PCR Program
Time
Number of Cycles
2 min
1
30 sec30 sec
6*
1 min
5 min
1
Hold
1

* Additional Indexing PCR cycles can be performed if final library yield is low or
initial DNA input is below recommended minimum.

Optional Stopping Point: The indexed libraries can be stored at -20C after cycling is complete.

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oncoReveal™ Myeloid Panel User ManualIndexing PCR: Purify the Libraries
Hands-on Time

30 – 45 min.

Total Time

30 – 45 min.

Before beginning the purification:•

Keep AMPure XP beads at room temperature while the Indexing PCR is being performed unless reactions will
be stored at -20°C after the program is complete.
Caution: Ensure that the AMPure bead solution reaches room temperature before performing thepurification. The temperature of the bead solution can have adverse effects on the purification process.

•If the indexed libraries were stored at -20°C remove them from the freezer to thaw thoroughly to ambient
temperature before purification. After samples have thawed, briefly centrifuge to remove any droplets from theside walls.

Library Purification

1.

Mix beads: Vortex AMPure XP beads thoroughly until all beads are well dispersed.
Caution: Ensure that the AMPure bead solution is homogenous. A non-uniform distribution can have
unpredictable effects on the purification process.

2.

Add beads: Add 50 μL beads (equivalent to a 1.0x bead ratio) to each well. Pipette the mixture up and down 10
times. If bubbles form on the bottom of the wells, briefly centrifuge and mix again.

3.

Bind libraries to beads: Incubate for 5 minutes at room temperature.

4.

Separate libraries on beads: Place the PCR plate on a magnetic rack until the solution appears clear, which can takeup to 5 minutes.

5.

Remove supernatant: Carefully remove the supernatant from each well without disturbing the beads.

6.

Wash beads: With the PCR plate still on the magnetic rack add 150 μL of freshly prepared 70% ethanol to each wellwithout disturbing the beads. Incubate at room temperature for 30 seconds, and then remove the supernatant fromeach well.
Caution: Do not allow the ethanol mixture to remain open to the air. The ethanol concentration will changeover time, affecting the washing of the beads. Pour only enough solution for each wash.

7.

Second wash: Repeat step 6 for a second 70% ethanol wash. Remove the supernatant from each well.

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8.

Remove remaining ethanol wash: Centrifuge for 10 – 15 seconds, place the PCR plate back on the magnetic rack,and use a 10 or 20 μL tip to remove any trace amounts of ethanol from each well.

9.

Dry beads: Let the beads air dry at room temperature for 2 – 5 minutes.
Important: To mitigate product loss, do not allow the beads to over-dry. The beads have sufficiently dried when the beadmass has small cracks in the middle. If large cracks have appeared among the entire bead ring or they are flaky, they areover-dried. Beads that are too dry may be difficult to resuspend.

10. Resuspend beads: Remove the PCR plate from the magnetic rack and resuspend the dried beads in each well using
32 μL of nuclease-free water. Gently pipette the bead suspension up and down 10 times. If bubbles form on thebottom of the wells briefly centrifuge and mix again.
11. Elute libraries: Incubate at room temperature for 5 minutes.
12. Separate libraries from beads: Place the bead suspensions on the magnetic rack at room temperature until thesolution appears clear. Transfer 30 μL of clear supernatant to a new plate.
TIP: During the incubation and magnetic separation of the beads, cover the samples with microplate sealing film and prepare the solutions
needed for quantitation performed in the next section.

13. Quantification: Analyze an aliquot of each library per the instructions in the next section.
Optional Stopping Point: The purified libraries can be stored at 4°C for up to 3 days, or at -20°C for longerterm storage.

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Final Library Gel Image
The following image is an example of final libraries after both rounds of PCR and purification on a 2% agarose gel.

Figure 4. Gel analysis of final library material. Lane 1 is a genomic DNA
sample after indexing and purification. Lane 2 is a no-template control.

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6.3 Quantitation and Normalization of Purified Libraries
Prior to sequencing, libraries must be quantified, normalized, and then pooled together. The following section describes
how to quantify libraries using the Qubit system. Other library quantification methods, such as qPCR quantification
kits or Bioanalyzer, may be used according to the manufacturer’s protocol.

Qubit Quantitation
Hands-on Time

30 – 45 min.

Total Time

30 – 45 min.

1.

Prepare Qubit working solution: Dilute the Qubit dsDNA HS reagent 1:200 in Qubit dsDNA HS buffer. Vortex
briefly to mix the Qubit working solution.
For example, 2000 μL is sufficient buffer for 10 readings (8 samples + 2 standards). Combine 1990 μL of Qubit dsDNA HS buffer and 10 μL HS
reagent.
Important: Fluorescent dyes are sensitive to light. Protect the Qubit working solution from light.

2.

Label tubes: Set up 0.5 mL Qubit tubes for standards and samples. Label the tube lids.

3.

Prepare standards: Transfer 190 μL of Qubit working solution into two tubes for standard 1 and standard 2, andthen add 10 μL of each standard to the corresponding tube.
Caution: New standard dilutions should be prepared with the libraries to be quantified. Do not re-use
standard dilutions from previous experiments.

4.

Prepare samples: Transfer 198 μL of Qubit working solution to each 0.5 mL tube, and then add 2 μL of each library
to its corresponding Qubit tube (1:100 dilution).

5.

Mix and spin: Vortex to mix and then centrifuge the tubes briefly.

6.

Incubate the tubes at room temperature for 2 minutes.

7.

Measure concentration: Measure the concentration of each library on the Qubit 2.0 Fluorometer per the
manufacturer’s instructions. Use the dsDNA High Sensitivity assay to read standards 1 and 2 followed by thelibraries.
If any concentration is above the linear range of the instrument, prepare a new dilution by combining 199 μL Qubit
working solution and 1 μL library (1:200 dilution). Repeat steps 5–7.

8.

Calculate concentration: 1 ng/μL of library is equal to 5 nM. Example calculation is below. Adjust dilution factor
accordingly.
2 μL of library + 198 μL Qubit working solution:

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𝑄𝑢𝑏𝑖𝑡 𝑟𝑒𝑎𝑑𝑖𝑛𝑔
1,000

𝑛𝑔
𝑚𝐿 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 100 𝑥 𝑐𝑜𝑛𝑣𝑒𝑟𝑠𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 5 = 𝑛𝑀

Optional Stopping Point: Once libraries have been quantified either proceed with normalization and poolingor else keep them stored at 4°C for up to 3 days. Store libraries at -20°C for long-term storage.

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Normalization and Pooling
Hands-on Time

30 – 45 min.

Total Time

30 – 45 min.

If sequencing on the MiniSeq, libraries should be normalized to 1 nM prior to pooling. For allother Illumina sequencing platforms, libraries should be normalized to 5 nM prior to pooling.
1.

Normalize libraries to 5 nM: Dilute an aliquot (e.g., 4 μL) of each sample library to 5 nM using nuclease-free wateror 10 mM Tris-HCl with 0.1% Tween-20, pH 8.5. An example calculation is as follows:2.

Mix and spin: Mix the 5 nM libraries thoroughly by vortexing and then centrifuge briefly.

3.

Prepare library pool: Label a new 1.5 mL microtube for the library pool. Prepare an equimolar 5 nM mixture oflibraries by combining each library at equal volume (e.g., mixing 5 μL of each 5 nM library). Gently pipette theentire solution up and down 10 times to mix thoroughly. Quickly vortex the pool and then briefly centrifuge.

4.

Quantify library pool (recommended): It is recommended that the library pool be quantified using Qubit oranother library quantitation method (qPCR) to ensure the pool is at 5 nM (± 10%) to prevent poor sequencing
performance. If the final dilution is not 5 nM (±10%), adjust the dilution for loading the sequencer accordingly toobtain the desired concentration.
Optional Stopping Point: The normalized libraries can be stored at 4°C overnight for sequencing the nextday. For longer storage, the normalized libraries can be stored at -20°C.

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7. Sequencing
Prepare Diluted Libraries for Sequencing
Hands-on Time

30 – 40 min.

Total Time

30 – 40 min.

The libraries generated using this protocol can be multiplexed and sequenced on Illumina® sequencers. Table 1 outlines
the sequencing parameters and the recommended per-sample sequencing coverage.
The number of samples that can be multiplexed together is dependent on several factors, among them are the estimatedthroughput of the flowcell and sequencing platform, the desired sequencing depth, as well as the number of uniqueindex combinations available.
The estimated maximum number of samples that can be multiplexed on a single flowcell using each kit is displayed inTable 2. Select the appropriate sequencing kit based on the number of samples to be sequenced.

Sequencing Configuration

Index

Recommended Coverage

Recommended Reads Per
Sample2 x 150orup to 2 x 275

Dual(8 bp each)

Mean: 2500X
Min: 500X>3.83 million PE reads

Table 1. Sequencing recommendations.

Sequencing Flowcell

Estimated Instrument Output(million PE reads)

Estimated Maximum Sequencing Batch
Size

MiSeq v230

7MiSeq v3

50

11

NextSeq 500/550 Mid Output

260

61

Table 2. Multiplexing recommendations for Illumina sequencers. Calculations assume ≥ 90% effective on-target rate after read mapping andminimum segment coverage > 20% of the mean coverage.

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For this portion of the protocol prepare prechilled benchtop cooler.

Sequencing Using Illumina MiSeq™ (v2 or v3) Reagents
MiSeq recommended final library pool loading concentration: 10 pM
MiSeq recommended PhiX spike-in concentration: 20 pM
The following steps can be found in greater detail in Illumina’s “MiSeq System: Denature & Dilute Libraries Guide” (Doc#15039740).
1.

Normalize: Dilute libraries to 5 nM, as demonstrated in the previous section “Quantification and Normalization of
Purified Libraries.”2.

Prepare 0.2 N NaOH: Label a new 1.5 mL microtube for 0.2 N NaOH. Prepare the NaOH by combining 800 μL of
nuclease-free water with 200 μL of 1 N NaOH. Vortex the solution to mix.
NOTE: Alternatively, prepare a 1 N NaOH solution by combining 500 μL of 10 N NaOH with 4.5 mL of nuclease-free water. Vortex thesolution to mix. If 1 N NaOH has not been prepared within the last week from a 10 N solution, prepare a new 1 N NaOH solution.

3.

Denature the library pool: Label a new 1.5 mL microtube for the denatured 25 pM library pool.
a.
b.
c.
d.
e.

Denature the 5 nM library pool by combining 5 μL of the library pool and 5 μL of the freshly prepared 0.2
N NaOH.
Vortex thoroughly for 10 seconds and centrifuge for 1 minute.
Let the solution stand at room temperature for 5 minutes.
Add 990 μL of Illumina’s HT1 hybridization buffer to the denatured library pool.
Invert the mixture several times, centrifuge briefly, and place on in the cooler.

4.

Dilute library pool to 10 pM: Label a new 1.5 mL microtube for the 10 pM library mix. Combine 240 μL of the 25
pM library pool with 360 μL of Illumina’s HT1 hybridization buffer. Adjust the volumes as needed for libraries thatare over or under 25 pM. Invert the mixture several times, centrifuge briefly, and place in the cooler.

5.

Combine library mix and PhiX control: Label a new 1.5 mL microtube for the mixture that will be loaded onto thesequencing cartridge. Combine 594 μL of the 10 pM library mix with 6 μL of 20 pM PhiX library control. Briefly
vortex, centrifuge, and place in the cooler.

6.

Load MiSeq cartridge: Using a clean 1000 μL tip puncture the foil cap above the sample loading well on the MiSeq
cartridge. Load the 600 μL library pool and PhiX mixture into the reagent cartridge, ensuring that the solution hasreached the bottom of the well.

7.

Run the MiSeq: Sequence the libraries on the MiSeq per the manufacturer’s instructions using a paired-end read
length of 150 (2x150) or a paired-end read length of up to 275 (2x275), and two indexing reads of 8 cycles each. See“MiSeq System User Guide” (part #15027617).

8.

Store diluted libraries and mixtures at -20C for long-term storage.

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Sequencing Using Illumina’s NextSeq™ 500/550 ReagentsMid Output recommended final library pool loading concentration: 1.8 pM
Mid Output recommended PhiX spike-in concentration: 1.8 pM
1.

Normalize: Dilute libraries to 5 nM, as demonstrated in the previous section “Normalization and Pooling.”

2.

Prepare 0.2 N NaOH: Label a new 1.5 mL microtube for 0.2 N NaOH. Prepare the NaOH by combining 800 μL
nuclease-free water with 200 μL of 1 N NaOH. Vortex the solution to mix.
NOTE: Alternatively, prepare a 1 N NaOH solution by combining 500 μL of 10 N NaOH with 4.5 mL of nuclease-free water. Vortex thesolution to mix. If 1 N NaOH has not been prepared within the last week from a 10 N solution, prepare a new 1 N NaOH solution.

3.

Denature the library pool: Label a new 1.5 mL microtube for the denatured 25 pM library pool.
a.
b.
c.
d.
e.
f.
g.

Denature the 5 nM library pool by combining 5 μL of the library pool and 5 μL of the freshly prepared 0.2
N NaOH.
Vortex thoroughly for 10 seconds and centrifuge for 1 minute.
Let the solution stand at room temperature for 5 minutes.
Add 5 μL of 200 mM Tris-HCl, pH 7.0.
Vortex briefly and centrifuge for 1 minute.
Add 985 μL of Illumina’s HT1 hybridization buffer to the denatured library mix.
Vortex briefly and centrifuge for 1 minute.

4.

Dilute 25 pM library pool to 1.8 pM: Dilute the denatured library to 1.8 pM in 1400 μL by combining 101 μL of the 25
pM denatured library mix with 1299 μL of Illumina’s HT1 hybridization buffer. Invert to mix and centrifuge briefly.

5.

Combine library mix and PhiX control: Label a new 1.5 mL microtube for the mixture that will be loaded. Combine
1287 μL of the 1.8 pM library mix with 13 μL of a 1.8 pM PhiX library control. Briefly vortex, centrifuge, and place inthe cooler.

6.

Load NextSeq cartridge: Using a clean 1000 μL tip, puncture the foil cap above the sample loading well on the
NextSeq cartridge. Load 1300 μL library mix and PhiX mixture into the cartridge and ensure the solution has reached
the bottom of the cartridge well.

7.

Run the NextSeq: Sequence the libraries on the NextSeq per the manufacturer’s instructions using a paired-end read
length of 150 (2x150) and two indexing reads of 8 cycles each. See “NextSeq System User Guide” (part #15046563 or
15069765).

8.

Store diluted libraries and mixtures at -20C for long-term storage.

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Preparing a Sample Sheet for Sequencing
TIP: Prepare the sample sheet prior to loading the MiSeq or NextSeq cartridge. If an error has been made during the indexing PCR where samples
have the same indices, it can be remedied before loading the samples on the sequencer.

The available Pillar indexing primers and their barcode sequences are listed in Appendix A. For the i5 indexing primers,
indexing on the NextSeq requires the reverse complement of the barcode sequence. The correct barcode sequences forsequencing on the MiSeq and the NextSeq are provided in Appendix A. Additionally, the Pillar sample sheet generator
will automatically populate the correct barcode sequence when the indexing primer is selected.
In Appendix A, note that indexing primers highlighted in yellow have the same barcode sequences as Illumina TruSeq
Custom Amplicon (TSCA) indices.
In the Pillar sample sheet generator, prepare a sample sheet that contains the information for the samples that are being
loaded. Ensure that the appropriate sample sheet is being made for the MiSeq or the NextSeq.
1.

Open the Pillar sample sheet generator and enter user input in the shaded cells. Cells that are shaded blue are
required and cells that are shaded grey are optional.

2.

Enter the “Sample_ID” for each sample. Each Sample_ID must be unique and contain only alphanumericcharacters, dashes (-), and underscores (_). All other characters are not allowed. To check that the Sample_ID meetsall requirements click “Reset Sample_ID color” and then click “Check Sample_ID”.

3.

If text is green, the Sample_ID is acceptable. If text is red, Sample_ID is not acceptable. Change Sample_IDaccordingly and repeat step 2 until all text is green.

4.

Next, enter indices into appropriate fields. Index sequences will be populated once the index_ID is entered.

5.

Check that the index combination for each sample is unique. If “Check_index_uniqueness” column is green, thenall index combinations are unique. If the column is red for a sample, then the index combination is not unique. Do
not load samples together in the same run that have the same index combination.

6.

Once all requirements for the sample sheet are met, export the sample sheet as a comma-separated values (.csv) fileby clicking “Export”.

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8. Troubleshooting
Low Yield of Gene-Specific Product
Potential Cause

Solution

DNA quantity or quality

The recommended input for the assay is 20–60 ng of genomic DNA. Higher quantities
may be necessary for low- or poor-quality samples.

Improper cycling

Check that the cycling protocol performed is the appropriate protocol for genespecific amplification.

Low Indexing Efficiency
Potential Cause

Solution
Incomplete AMPure purification or loss of gene-specific product will affect theindexing PCR reaction. The purified gene-specific product can be checked on an
agarose gel to ensure the product was not lost, and that the clean-up was sufficient toremove excess primers.

Improper AMPure
purification

The AMPure bead ratio and ethanol concentration affect the PCR clean-up. Ensurethat the correct AMPure bead concentration was used for clean-up, and fresh 70%
ethanol was used for the wash.
Leftover ethanol from the wash steps can hinder the PCR reaction. Remove as muchof the ethanol during the final wash step with a pipette and dry the beads to ensure
the residual ethanol has evaporated.

Partial primer digestion

Poor digestion of the gene-specific primers can hinder the indexing efficiency of theindexing PCR reaction. Check the primer digestion using an agarose gel.

Incomplete deactivation of
exonuclease

The inactivation of the nuclease and AMPure purification is necessary before
performing indexing PCR. Leftover active exonuclease can digest the indexing PCRprimers, reducing the yield of the indexing PCR reaction.

Low Yield of Final Library
Potential Cause

Solution

DNA quantity or quality

The recommended input for the assay is 20–60 ng of genomic DNA. Higher quantities
may be necessary for low- or poor-quality samples.

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Run the product from the gene-specific PCR on an agarose gel to check the yield.
Incomplete AMPure purification or loss of product will affect the final yield. The
purified product can be checked on an agarose gel to ensure that the product was not
lost during PCR clean-up.
Improper AMPure
purification
The AMPure bead ratio and ethanol concentration affect the PCR clean-up. Ensurethat the correct AMPure bead concentration was used for clean-up, and fresh 70%
ethanol was used for the wash.

Amplicon Contamination in No-Template Control
Potential Cause

Solution
Make sure to change tips between samples and avoid waving tips over tubes or plates.
Poor sealing or residual liquid in tips can cause contamination of nearby samples. If
possible, leave adjacent wells empty between samples.

Cross-contamination

Workspaces and equipment for pre-PCR and post-PCR should be separated toprevent amplicon contamination.
Periodically clean the workspace, floor, equipment, and instrumentation with alaboratory cleaning solution to break down amplicons on surfaces.
Recommended cleaning solutions are 10% bleach, 70% isopropanol, or 70% ethanol.

Sequencing Performance
Potential Cause

Solution
Confirm that the appropriate loading concentration was used for the applicablesequencing instrument.

Improper normalizationand pooling of libraries

Improper libraryquantitation

For Research Use Only

Check the 5 nM (or 1 nM if sequencing on the MiniSeq) library mix using Qubit or
RT-PCR. Dilute the denatured library mix as needed to adjust for the difference inconcentration.
Improper library quantitation may result in artificially high or low yields, which
affects downstream normalization.

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Re-quantitate the final libraries and/or the normalized libraries to check for the
expected values.
Changing the ratio of AMPure beads affects the purification of the products. Notably,
the presence of primer dimers can cause an underestimation of total quantity, causing
over-clustering.
Improper AMPure
purification

The final libraries can be checked on an agarose gel for the proper product size andpresence of primer dimers.
The AMPure bead ratio and ethanol concentration affect the PCR clean-up. Ensurethat the correct AMPure bead concentration was used for clean-up, and fresh 70%
ethanol was used for the wash.

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9. Appendix A: Index Sequences
Indexing primers highlighted in yellow have the same barcode sequences as Illumina TruSeq Custom
Amplicon (TSCA) indices.

Index ID
Pi701
Pi702
Pi703
Pi704
Pi705
Pi706
Pi707
Pi708
Pi709
Pi710
Pi711
Pi712
Pi713
Pi714
Pi715
Pi716

Index ID
Pi501
Pi502
Pi503
Pi504
Pi505
Pi506
Pi507
Pi508
Pi509
Pi510
Pi511
Pi512
Pi513
Pi514
Pi515
Pi516

For Research Use Only

i7 Index Sequences
Index Barcode Sequence
ATTACTCG
TCCGGAGACGCTCATTGAGATTCCATTCAGAAGAATTCGTCTGAAGCTTAATGCGC
ATCACGACACAGTGGTCAGATCCA
ACAAACGGGAAACCCATGTGACCA
AGGGTCAAAGGAGTGG
i5 Index Sequences
Index Barcode Sequence for MiSeq
TATAGCCTATAGAGGC
CCTATCCTGGCTCTGAAGGCGAAGTAATCTTACAGGACGTGTACTGACTGAACCTTTGCTAAGTTGTTCTCTTAAGACACCTAATCGACTAGAACATAAGTTCCTAGACCTA

Index Barcode Sequence for NextSeq
AGGCTATAGCCTCTATAGGATAGGTCAGAGCC
CTTCGCCTTAAGATTAACGTCCTG
GTCAGTACAAGGTTCAACTTAGCAAGAGAACAGTGTCTTATCGATTAGTGTTCTAG
GGAACTTATAGGTCTA

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10. Appendix B: Analysis Using PiVAT
See the PiVAT User Manual (Doc. No. UM-0073) for detailed instructions on initializing the analysis & understanding theoutput in PiVAT®.

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