MiSeq System Denature And Dilute Libraries Guide (15039740) Mi Seq (15039740 V03)

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MiSeq System

Denature and Dilute Libraries Guide

Overview 3

Protocol A: Standard Normalization Method 4

Protocol B: Bead-Based Normalization Method 6

Denature and Dilute PhiX Control 7

Supplemental Information 8

Next Steps 9

Revision History 9

Technical Assistance 10

Document # 15039740 v03

December 2017

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For Research Use Only. Not for use in diagnostic procedures.

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Document # 15039740 v03

For Research Use Only. Not for use in diagnostic procedures.

2

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.

uStandard 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.

uBead-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.

3

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.

uTo prevent small pipetting errors from affecting the final NaOH concentration, prepare at least 1 ml of

freshly diluted NaOH.

uFor 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, low-

plexity 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 NexteraTM 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.

uLaboratory-grade water (800µl)

uStock 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.

4

MiSeq System Denature and Dilute Libraries Guide

2 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.

u4nM library (5µl)

u0.2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.

u2nM library (5µl)

u0.2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.

5

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.

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

Table 1 General Amplicon Recommendations

2 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.

u10nM PhiX library (2µl)

u10 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.

u4nM PhiX library (5µl)

u0.2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.

uDenatured PhiX library (10µl)

uPrechilled 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.

u20 pM denatured PhiX library (375 µl)

uPrechilled 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)

Denatured and diluted PhiX 6 µl 30µl

Denatured and diluted library (from protocol A

or protocol B)

594µl 570µl

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

Library Pool Prechilled HT1 Chemistry

24 µl 576 µl MiSeq Reagent Kit v3 and v2

Table 2 Nextera XT DNA

NOTE

24 µl is a suggested starting volume for Nextera XT DNA.

Library Pool Prechilled HT1 Chemistry

20 µl 580 µl MiSeq Reagent Kit v3

6µl 594 µl MiSeq Reagent Kit v2

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

Document # 15039740 v03

For Research Use Only. Not for use in diagnostic procedures.

8

MiSeq System Denature and Dilute Libraries Guide

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

Table 5 TruSeq Custom Amplicon Low Input Kit

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

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9

MiSeq System Denature and Dilute Libraries Guide

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support.illumina.com, select a product, then select Documentation & Literature.

Document # 15039740 v03

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MiSeq System Denature and Dilute Libraries Guide

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Document # 15039740 v03