MiSeq System Denature And Dilute Libraries Guide (15039740) Mi Seq (15039740 V03)
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MiSeq System
Denature and Dilute Libraries Guide
Overview
Protocol A: Standard Normalization Method
Protocol B: Bead-Based Normalization Method
Denature and Dilute PhiX Control
Supplemental Information
Next Steps
Revision History
Technical Assistance
Document # 15039740 v03
December 2017
For Research Use Only. Not for use in diagnostic procedures.
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ILLUMINA PROPRIETARY
MiSeq System Denature and Dilute Libraries Guide
This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for
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Document # 15039740 v03
For Research Use Only. Not for use in diagnostic procedures.
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MiSeq System Denature and Dilute Libraries Guide
Overview
This guide explains steps to denature and dilute prepared libraries for sequencing on the Illumina ® MiSeq ®
system.
This guide also includes instructions for preparing a PhiX library for use as a sequencing control.
Loading Volume and Concentration
This procedure denatures and dilutes libraries to a final volume of 600 µl. The recommended loading
concentration varies depending on the version of MiSeq Reagent Kit used for the sequencing run. In practice,
loading concentration can vary depending on library preparation and quantification methods.
Chemistry
Recommended Final Loading Concentration
MiSeq Reagent Kit v3
Supports 6–20 pM loading concentration.
Requires at least a 4 nM library before diluting and denaturing.
MiSeq Reagent Kit v2
Supports 6–10 pM loading concentration.
Protocol Variations
Follow the appropriate denature and dilute protocol depending on the normalization procedure used during
library prep.
u
Standard normalization—Libraries are normalized using standard library quantification and quality control
procedures recommended in the library prep documentation. For these libraries, follow Protocol A. See
Protocol A: Standard Normalization Method on page 4.
u
Bead-based normalization—Libraries are normalized using a bead-based procedure described in the
library prep documentation for methods that support bead-based normalization. For these libraries,
follow Protocol B. See Protocol B: Bead-Based Normalization Method on page 6.
Consumables and Equipment
Consumables
The following consumables are required to prepare DNA libraries for sequencing on the MiSeq.
Consumable
Supplier
HT1 (Hybridization Buffer), thawed and prechilled
Illumina, Provided in the MiSeq Reagent Kit
Illumina PhiX Control, Catalog # FC-110-3001
Illumina (Optional)
1.0 N NaOH, molecular biology grade
General lab supplier
Tris-Cl 10 mM, pH 8.5 with 0.1% Tween 20
General lab supplier
Equipment
The following equipment is used to denature libraries that have been normalized using a bead-based
method.
Equipment
Supplier
Hybex Microsample Incubator
SciGene, catalog # 1057-30-O (115 V), or equivalent
SciGene, catalog # 1057-30-2 (230 V), or equivalent
Block for 1.5 ml microcentrifuge tubes
SciGene, catalog # 1057-34-0, or equivalent
Document # 15039740 v03
For Research Use Only. Not for use in diagnostic procedures.
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MiSeq System Denature and Dilute Libraries Guide
Best Practices
u
Always prepare freshly diluted NaOH for denaturing libraries for cluster generation. This step is essential
to the denaturation process.
u
To prevent small pipetting errors from affecting the final NaOH concentration, prepare at least 1 ml of
freshly diluted NaOH.
u
For best results, begin thawing the reagent cartridge before denaturing and diluting libraries. For
instructions, see the MiSeq System User Guide (part # 15027617).
About Low Diversity Libraries
Low diversity libraries are libraries where a significant number of the reads have the same sequence. This lack
of variation shifts the base composition because the reads are no longer random.
For example, low diversity can occur with some expression studies with > 25% of one type of transcript, lowplexity amplicon pools, adapter dimer, or bisulfite sequencing. A higher concentration spike-in of PhiX helps
balance the overall lack of sequence diversity.
NOTE
For low diversity libraries, dilute your PhiX control library to the same concentration as your denatured library.
Protocol A: Standard Normalization Method
Use protocol A to denature and dilute libraries that have been normalized using standard library quantification
and quality control procedures recommended in the library prep documentation.
Follow the steps most appropriate for your library and the version of MiSeq Reagent Kit you are using.
Loading concentration can also vary depending on library type and quantification methods.
For the Nextera TM DNA Flex Library Prep Kit, see dilute and denature directions in the Nextera DNA Flex
Library Prep Reference Guide (document # 1000000025416).
For the TruSight ® Cardio Sequencing Kit, see dilute and denature directions in the TruSight Cardio
Sequencing Kit Reference Guide (document # 15063774).
Chemistry
Compatible Denature and Dilute Steps
MiSeq Reagent Kit v3
4 nM library—Results in a 6–20 pM loading concentration.
MiSeq Reagent Kit v2
4 nM library—Results in a 6–20 pM loading concentration.
2 nM library—Results in a 6–10 pM loading concentration.
The denaturation steps described in this guide make sure that the concentration of NaOH is not more than
0.001 (1 mM) in the final solution after diluting with HT1. Higher concentrations of NaOH in the library inhibit
library hybridization to the flow cell and decrease cluster density.
Prepare Reagents
Prepare a Fresh Dilution of NaOH
1
Combine the following volumes in a microcentrifuge tube.
u Laboratory-grade water (800 µl)
u Stock 1.0 N NaOH (200 µl)
The result is 1 ml of 0.2 N NaOH.
Document # 15039740 v03
For Research Use Only. Not for use in diagnostic procedures.
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MiSeq System Denature and Dilute Libraries Guide
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Invert the tube several times to mix.
NOTE
Use the fresh dilution within 12 hours.
Prepare HT1
1
Remove HT1 from -25°C to -15°C storage and thaw at room temperature.
2
Store at 2°C to 8°C until you are ready to dilute denatured libraries.
Denature a 4 nM Library
1
Combine the following volumes in a microcentrifuge tube.
u 4 nM library (5 µl)
u 0.2 N NaOH (5 µl)
2
Vortex briefly and then centrifuge at 280 × g for 1 minute.
3
Incubate at room temperature for 5 minutes.
4
Add 990 µl prechilled HT1 to the tube containing denatured library.
The result is 1 ml of a 20 pM denatured library.
Dilute Denatured 20 pM Library
1
Dilute to the desired concentration using the following volumes.
Concentration
6 pM
8 pM
10 pM
12 pM
15 pM
20 pM
20 pM library
180 µl
240 µl
300 µl
360 µl
450 µl
600 µl
Prechilled HT1
420 µl
360 µl
300 µl
240 µl
150 µl
0 µl
2
Invert to mix and then pulse centrifuge.
3
To add a PhiX control, proceed to Denature and Dilute PhiX Control on page 7. Otherwise, see Next
Steps on page 9.
Denature a 2 nM Library
1
Combine the following volumes in a microcentrifuge tube.
u 2 nM library (5 µl)
u 0.2 N NaOH (5 µl)
2
Vortex briefly and then centrifuge at 280 × g for 1 minute.
3
Incubate at room temperature for 5 minutes.
4
Add 990 µl prechilled HT1 to the tube containing denatured library.
The result is 1 ml of a 10 pM denatured library.
Dilute Denatured 10 pM Library
1
Dilute to the desired concentration using the following volumes.
Document # 15039740 v03
For Research Use Only. Not for use in diagnostic procedures.
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MiSeq System Denature and Dilute Libraries Guide
Concentration
6 pM
8 pM
10 pM
10 pM library
360 µl
480 µl
600 µl
Prechilled HT1
240 µl
120 µl
0 µl
2
Invert to mix and then pulse centrifuge.
3
To add a PhiX control, proceed to Denature and Dilute PhiX Control on page 7. Otherwise, see Next
Steps on page 9.
Protocol B: Bead-Based Normalization Method
Use protocol B to denature and dilute libraries that have been normalized and pooled using a bead-based
procedure described in the library prep documentation for methods that support bead-based normalization.
Bead-based normalization procedures can be variable. The actual volume of library varies depending upon
library type and experience. Loading concentration can also vary depending on library type and
quantification methods.
For TruSight HLA Sequencing Kits, see dilute and denature directions in the TruSight HLA v1 Sequencing Kit
Reference Guide (document # 15056536) or TruSight HLA v2 Sequencing Kit Reference Guide (document #
1000000010159).
Prepare HT1
1
Remove HT1 from -25°C to -15°C storage and thaw at room temperature.
2
Store at 2°C to 8°C until you are ready to dilute denatured libraries.
Prepare Incubator
1
Preheat the incubator to 98°C.
Dilute Library to Loading Concentration
1
Combine the following volumes of pooled libraries and prechilled HT1 in a microcentrifuge tube.
The total volume is 600 µl. If cluster density results are too high or low, adjust the dilution ratio.
Check BBN Loading Concentration Exceptions on page 8 to see if your kit requires loading volumes that
are different from general amplicon recommendations.
Table 1 General Amplicon Recommendations
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Library Pool
Prechilled HT1
Chemistry
6 µl
594 µl
MiSeq Reagent Kit v3 or v2
7 µl
593 µl
MiSeq Reagent Kit v3 or v2
8 µl
592 µl
MiSeq Reagent Kit v3 or v2
9 µl
591 µl
MiSeq Reagent Kit v3 or v2
10 µl
590 µl
MiSeq Reagent Kit v3 or v2
Vortex briefly and then centrifuge at 280 × g for 1 minute.
Denature Diluted Library
1
Place the tube on the preheated incubator for 2 minutes.
Document # 15039740 v03
For Research Use Only. Not for use in diagnostic procedures.
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MiSeq System Denature and Dilute Libraries Guide
2
Immediately cool on ice.
3
Leave on ice for 5 minutes.
4
To add a PhiX control, proceed to Denature and Dilute PhiX Control on page 7. Otherwise, see Next
Steps on page 9.
Denature and Dilute PhiX Control
Use the following procedure to denature and dilute a PhiX library for use as a sequencing control.
Follow the steps appropriate for the version of MiSeq reagent kit you are using.
Chemistry
Final PhiX Concentration
MiSeq Reagent Kit v3
Dilute the denatured PhiX control to 20 pM, which produces an optimal cluster
density using v3 reagents.
MiSeq Reagent Kit v2
Dilute the denatured PhiX control to 12.5 pM, which produces an optimal cluster
density using v2 reagents.
Dilute PhiX to 4 nM
1
Combine the following volumes in a microcentrifuge tube.
u 10 nM PhiX library (2 µl)
u 10 mM Tris-Cl, pH 8.5 with 0.1% Tween 20 (3 µl)
2
If not prepared within the last 12 hours, prepare a fresh dilution of 0.2 N NaOH.
Denature PhiX Control
1
Combine the following volumes in a microcentrifuge tube.
u 4 nM PhiX library (5 µl)
u 0.2 N NaOH (5 µl)
2
Vortex briefly to mix.
3
Centrifuge at 280 × g for 1 minute.
4
Incubate at room temperature for 5 minutes.
Dilute Denatured PhiX to 20 pM
1
Add prechilled HT1 to the denatured PhiX library.
u Denatured PhiX library (10 µl)
u Prechilled HT1 (990 µl)
The result is 1 ml of a 20 pM PhiX library.
2
Invert to mix.
NOTE
You can store the denatured 20 pM PhiX library up to 3 weeks at -15°C to -25°C. After 3 weeks, cluster
numbers tend to decrease.
Dilute Denatured PhiX to 12.5 pM
If you are using MiSeq Reagent Kit v3, no further dilution is required.
Document # 15039740 v03
For Research Use Only. Not for use in diagnostic procedures.
7
MiSeq System Denature and Dilute Libraries Guide
1
Add prechilled HT1 to the denatured PhiX library.
u 20 pM denatured PhiX library (375 µl)
u Prechilled HT1 (225 µl)
The result is 600 µl of a 12.5 pM PhiX library.
2
Invert to mix.
Combine Library and PhiX Control
For most libraries, use a low-concentration PhiX control spike-in of 1% as a sequencing control. For low
diversity libraries, increase the PhiX control spike-in to at least 5%.
1
Combine the following volumes of denatured PhiX control and denatured library.
Most Libraries
(1% Spike-In)
Low-Diversity Libraries
(≥ 5% Spike-In)
6 µl
30 µl
594 µl
570 µl
Denatured and diluted PhiX
Denatured and diluted library (from protocol A
or protocol B)
2
Set aside on ice until you are ready to load it onto the reagent cartridge.
NOTE
Actual PhiX percentage varies depending upon the quality and quantity of the library pool.
Supplemental Information
BBN Loading Concentration Exceptions
Table 2 Nextera XT DNA
Library Pool
Prechilled HT1
Chemistry
24 µl
576 µl
MiSeq Reagent Kit v3 and v2
NOTE
24 µl is a suggested starting volume for Nextera XT DNA.
Table 3 TruSight Myeloid Sequencing Panel
Library Pool
Prechilled HT1
Chemistry
20 µl
580 µl
MiSeq Reagent Kit v3
6 µl
594 µl
MiSeq Reagent Kit v2
Table 4 TruSeq® Custom Amplicon v1.5
Library Pool
Prechilled HT1
Chemistry
20 µl
580 µl
MiSeq Reagent Kit v3
6 µl
594 µl
MiSeq Reagent Kit v2
Document # 15039740 v03
For Research Use Only. Not for use in diagnostic procedures.
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MiSeq System Denature and Dilute Libraries Guide
Table 5 TruSeq Custom Amplicon Low Input Kit
Library Pool
Prechilled HT1
Chemistry
7 µl
593 µl
MiSeq Reagent Kit v3 or v2
8 µl
592 µl
MiSeq Reagent Kit v3 or v2
9 µl
591 µl
MiSeq Reagent Kit v3 or v2
10 µl
590 µl
MiSeq Reagent Kit v3 or v2
Next Steps
After denaturing and diluting your libraries and preparing the optional PhiX control, you are ready to load
libraries onto the reagent cartridge and set up the sequencing run. See the MiSeq System User Guide (part #
15027617).
Revision History
Document
Date
Description of Change
Document # 15039740 v03
December
2017
Added recommendation in Protocol A to reference the Nextera DNA Flex
Library Prep Reference Guide when working with the Nextera DNA Flex Library
Prep Kit.
Document # 15039740 v02
February
2017
Added loading concentration recommendations for TruSeq Myeloid
Sequencing Panel, TruSeq Custom Amplicon v1.5, and TruSeq Custom
Amplicon Low Input Sequencing Kit.
Document # 15039740 v01
January
2016
Added procedure for denaturing and diluting libraries that have been
normalized using a bead-based procedure. Organized procedures as Protocol
A and Protocol B.
Part # 15039740 Rev. D
November
2013
Added recommendation for low diversity libraries to dilute PhiX control libraries
to the same concentration as denatured sample libraries.
Part # 15039740 Rev. C
August
2013
Added recommendation to use molecular biology grade NaOH.
Added recommended library denaturation and PhiX control protocols for use
with MiSeq Reagent Kit v3.
Removed loading samples library information. That information is now in the
MiSeq System User Guide (part # 15027617) .
Part # 15039740 Rev. B
March
2013
Reduced PhiX recommendations for low diversity libraries from ≥ 25% to ≥ 5%.
This change is possible when using RTA 1.17.28, or later, released with
MCS v2.2.
Corrected the resulting NaOH concentration for denatured 10 pM library to 1
mM.
Updated instructions for combining prepared libraries and PhiX control to total
600 µl.
Part # 15039740 Rev. A
January
2013
Initial release.
Document # 15039740 v03
For Research Use Only. Not for use in diagnostic procedures.
9
MiSeq System Denature and Dilute Libraries Guide
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Document # 15039740 v03
For Research Use Only. Not for use in diagnostic procedures.
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Document # 15039740 v03
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