MiSeqDx Universal Kit 1.0 Reference Guide (15039610 B)

MiSeqDx Universal Kit 1.0 Reference Guide (15039610 B)

MiSeqDx

®

Universal Kit 1.0

Reference Guide

FOR IN VITRO DIAGNOSTIC USE

Introduction

Getting Started

Protocol Workflow

MiSeqDx Sample Sheet Preparation

Hybridization of Oligonucleotide Pool

Removal of Unbound Oligonucleotides

Extension-Ligation of Bound Oligonucleotides

PCR Amplification

PCR Clean-Up

Library Normalization

Library Pooling

What's Next

Technical Assistance

23

24

27

30

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14

18

20

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6

33

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ILLUMINA PROPRIETARY

Part # 15039610 Rev. B

February 2015

This document and its contents are proprietary to Illumina, Inc. and its affiliates ("Illumina"), and are intended solely for the contractual use of its customer in connection with the use of the product(s) described herein and for no other purpose. This document and its contents shall not be used or distributed for any other purpose and/or otherwise communicated, disclosed, or reproduced in any way whatsoever without the prior written consent of Illumina. Illumina does not convey any license under its patent, trademark, copyright, or common-law rights nor similar rights of any third parties by this document.

The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product(s) described herein. All of the contents of this document must be fully read and understood prior to using such product(s).

FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN

MAY RESULT IN DAMAGE TO THE PRODUCT(S), INJURY TO PERSONS, INCLUDING TO USERS OR OTHERS, AND

DAMAGE TO OTHER PROPERTY.

ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT(S)

DESCRIBED HEREIN (INCLUDING PARTS THEREOF OR SOFTWARE) OR ANY USE OF SUCH PRODUCT(S) OUTSIDE

THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION

WITH CUSTOMER'S ACQUISITION OF SUCH PRODUCT(S).

FOR IN  VITRO  DIAGNOSTIC USE

© 2015 Illumina, Inc. All rights reserved.

Illumina, Genetic Energy, MiSeqDx, Powered by Illumina, the pumpkin orange color, and the streaming bases design are trademarks of Illumina, Inc. and/or its affiliate(s) in the U.S. and/or other countries. All other names, logos, and other trademarks are the property of their respective owners.

AMPure, Beckman, and Beckman Coulter are trademarks or registered trademarks of Beckman Coulter, Inc.

Revision History

Part #

15036910

15036910

Revision

B

A

Date

February

2015

February

2014

Description of Change

• Throughout—Updated MSDS (now SDS) support site web address.

• Sample Throughput—Added limitation of 48 samples per sheet to sample sheet preparation note.

• Removal of Unbound Oligos—Removed note regarding keeping spare filter plates on hand.

• Inspect the Reagent Cartridge—Added recommendation to visually inspect reagents in positions 2 and 4 (in addition to 1).

• Initial Release

Introduction

Intended Use

The Illumina MiSeqDx Universal Kit 1.0 is a set of reagents and consumables used in the processing of human genomic DNA samples derived from peripheral whole blood, and in the subsequent targeted sequencing of the resulting sample libraries. Usersupplied analyte specific reagents are required for the preparation of libraries targeting specific genomic regions of interest. The MiSeqDx Universal Kit is intended for use with the MiSeqDx instrument.

About this Guide

This reference guide provides more detailed instructions, technique tips, and helpful hints to newly trained users to help guide proper execution of the MiSeqDx Universal Kit protocol. This is intended as a supplement and is not meant to replace the package insert.

How Does the Kit Work?

One pair of custom oligonucleotides is designed for each amplicon. Hybridization of these oligonucleotides to genomic DNA occurs in a 96-well plate, followed by extension and ligation to form DNA templates consisting of the regions of interest flanked by universal primer sequences. Using indexed primers supplied with the kit, DNA templates are then amplified by PCR, pooled into a single tube, and sequenced on the

MiSeqDx instrument.

A

Hybridization of custom oligonucleotide probes

B

Extension and ligation

C

Addition of indices and sequencing adapters by PCR

D

Final amplicon ready for sequencing with MiSeqDx

MiSeqDx Universal Kit 1.0 Reference Guide

4

Process Overview

The Illumina MiSeqDx Universal Kit 1.0 process can be summarized into the following steps:

Create a Sample Sheet

First, prepare a sample sheet, which will be used by the MiSeqDx to identify each sample and its corresponding index. To prepare the sample sheet, use the Illumina Worklist

Manager, a wizard-based application for recording the sample ID, indices, and other parameters applicable to the 96-well plate. The sample sheet is also used as a guide for setting up the plate during the assay workflow.

Prepare Libraries

Prepare the libraries using the protocol detailed in this user guide.

Sequence Samples on the MiSeqDx

The MiSeqDx Universal Kit must be sequenced on a MiSeqDx instrument using a paired-end 150 cycle run with dual indexing. For instructions for performing a sequencing run on the MiSeqDx, see the MiSeqDx Instrument Reference Guide (part #

15038353).

Automated Sequencing and Data Analysis

MiSeq Reporter processes the base calls generated by the MiSeqDx instrument. It is an on-instrument software, which is built in to the instrument's processes. MiSeq Reporter produces information about alignment and structural variants. For more information about this software, see the MiSeq Software Reporter User Guide (part # 15038356).

Tracking Tools

Illumina provides the following tools for sample tracking and guidance in the lab:

} The Lab Tracking Form can be used to record information such as operator name, sample and index information, start and stop times, reagent lot numbers, and barcodes.

}

The Illumina Worklist Manager is used to create the sample sheet using a wizardbased application. The Illumina Worklist Manager provides a feature for recording parameters for the sample plate, such as sample ID, dual indices, and other features applicable to the run.

NOTE

You can download the above Illumina MiSeqDx Universal Kit 1.0 documents from the

Illumina website. Go to the Illumina MiSeqDx Universal Kit 1.0 support page and click the Documentation & Literature tab.

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Part # 15039610 Rev. B

Getting Started

This section describes the Illumina MiSeqDx Universal Kit 1.0 kit contents, consumables and equipment used, DNA input recommendations, and best practices to apply during the protocol.

MiSeqDx Universal Kit Contents

The Illumina MiSeqDx Universal Kit 1.0 contains the components listed in

Table 1

through

Table 8 . Store the kit components at the specified temperature and in designated

pre- and post-amplification areas.

Because the pre- and post-amp reagents are shipped together, it is important to unpack the reagents in the pre-amp lab area, and then move the post-amp reagents to the proper post-amp storage area.

MiSeqDx Universal Kit, Box 1

Table 1 Box 1A Pre-Amp Reagents

Component Quantity

Fill

Volume

1 tube Hybridization

Buffer

Extension-

Ligation Mix

Index Primers

A (A501) - H

(A508)

1 tube

1 tube per primer

Active Ingredients

4.32 ml

Buffered aqueous solution containing salts and formamide

4.8 ml

Buffered aqueous solution containing proprietary blend of DNA polymerases, DNA ligase, and dNTPs

192 µl

PCR primers with index sequences and sequencing adapters

Index Primers 1

(A701) - 12

(A712)

PCR

Polymerase

PCR Master

Mix

1 tube per primer

1 tube

1 tube

128 µl

PCR primers with index sequences and sequencing adapters

56 µl

2.8 ml

Proprietary DNA polymerase

Buffered aqueous solution containing salts and dNTPs

Storage

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

Table 2 Box 1B Post-Amp Reagents

Component Quantity

Fill

Volume

Library

Normalization

Diluent

1 tube

1 tube

4.6 ml

Active Ingredients

Buffered aqueous solution containing salts, 2-Mercaptoethanol, and formamide

4.5 ml

Buffered aqueous solution Library

Dilution Buffer

PhiX Internal

Control

1 tube

10 µl

Buffered aqueous solution containing

PhiX genomic DNA

Storage

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

MiSeqDx Universal Kit, Box 2

Table 3 Box 2 Post-Amp Reagents

Component

MiSeqDx Reagent

Cartridge

Quantity Contents

2 cartridges Single-use cartridge that contains cluster generation and sequencing reagents for use with the MiSeqDx, including formamide, 2-

Mercaptoethanol, and < 2% DMSO

Storage

-25°C to -15°C

MiSeqDx Universal Kit 1.0 Reference Guide

6

MiSeqDx Universal Kit, Box 3

Table 4 Box 3A Pre-Amp Reagents

Component Quantity

Fill

Volume

Stringent Wash

Buffer

Universal Wash

Buffer

1 bottle

1 tube

24 ml

Active Ingredients

Buffered aqueous solution containing salts, 2-Mercaptoethanol and formamide

4.8 ml

Buffered aqueous solution containing salts

Storage

2°C to 8°C

2°C to 8°C

Table 5 Box 3B Post-Amp Reagents

Component Quantity

Fill

Volume

PCR Clean-Up

Beads

Library

Normalization

Wash

Library Beads

MiSeqDx Flow

Cell

1 tube

2 tubes

1 tube

2 containers

5 ml

1 flow cell

Active Ingredients

Buffered aqueous solution containing solid phase paramagnetic beads and polyethylene glycol

4.8 ml

Buffered aqueous solution containing salts, 2-Mercaptoethanol and formamide

1.2 ml

Buffered aqueous solution containing solid phase paramagnetic beads

Glass substrate with covalently bound oligonucleotides

Storage

2°C to 8°C

2°C to 8°C

2°C to 8°C

2°C to 8°C

MiSeqDx Universal Kit, Box 4

Table 6 Box 4 Post-Amp Reagents

Component Quantity

Fill

Volume

MiSeqDx SBS

Solution (PR2)

Active Ingredients

2 bottles

353.1 ml Buffered aqueous solution

Storage

2°C to 8°C

MiSeqDx Universal Kit, Box 5

Table 7 Box 5 Pre-Amp Reagents

Component Quantity

Fill

Volume

Filter Plate 2 plates

N/A

Active Ingredients

Polypropylene microtiter plate with a modified polyethersulfone membrane

Storage

15°C to 30°C

Table 8 Box 5 Post-Amp Reagents

Component Quantity

Fill

Volume

Elution Buffer

Library

Storage Buffer

1 tube

1 tube

Active Ingredients

4.8 ml

Buffered aqueous solution

3.5 ml

Buffered aqueous solution

Storage

15°C to 30°C

15°C to 30°C

Equipment and Materials

Equipment and Materials Provided, Sold Separately

1 MiSeqDx Instrument, Catalog # DX-410-1001

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Part # 15039610 Rev. B

2 TruSeq Index Plate Fixture Kit, Catalog # FC-130-1005

3 TruSeq Index Plate Fixture & Collar Kit, Catalog # FC-130-1007

4 Index Adapter Replacement Caps, Catalog #DX-502-1003

Equipment and Materials Required, Not Provided

Pre-Amp Equipment and Materials

1 Heat Block—One heat block for a 96 well plate is required. The heat block must meet the following performance specifications. Heat blocks with heated lids are acceptable for use.

• Temperature Range: Ambient +5°C to 99°C

• Temperature Regulation: ±0.1°C at 37°C; ±0.4°C at 60°C

2 Sample Incubator—One incubator (hybridization oven) is required. The incubator must meet the following performance specifications.

• Temperature Range: 10°C to 100°C

• Temperature Regulation: ±0.2°C

3 Tabletop Centrifuge—One tabletop centrifuge capable of maintaining 20°C is required. (A separate centrifuge is required in the post-amp area.) Any plate centrifuge that attains the designated speeds of the protocol (280 to 2400 × g) is acceptable.

4 Precision Pipettes—One set of precision pipettes is required. (A separate set is required in the post-amp area.) The use of precision pipettes is required to ensure accurate reagent and sample delivery. Single-channel or multi-channel pipettes can be used if they are calibrated regularly and are accurate within 5% of stated volume.

5 Consumables—The following consumables are required.

• 96-well skirted PCR plates, 0.2 ml, polypropylene, or equivalent

• 96-well storage plates, 0.8 ml (MIDI plates)

• Solution basin, PVC, DNase, RNase-free (trough)

• Adhesive aluminum foil seal

• Appropriate PCR plate seal

• Aerosol resistant pipette tips

Post-Amp Equipment and Materials

1 Thermal Cycler—One thermal cycler is required. The thermal cycler must have a heated lid and meet the following performance specifications:

• Temperature Control Range: 4°C to 99°C

• Control Accuracy: ±0.25°C from 35°C to 99°C

2 Microplate Shaker—One microplate shaker is required in the post-amp lab area. The plate shaker must meet the following performance specifications:

• Max Mixing Speed: 3000 rpm

• Mixing Speed Range: 200 to 3000 rpm

3 Tabletop Centrifuge—One tabletop centrifuge capable of maintaining 20°C is required. (A separate centrifuge is required in the pre-amp area.) Any plate centrifuge that attains the designated speeds of the protocol (280 to 2400 × g) is acceptable.

MiSeqDx Universal Kit 1.0 Reference Guide

8

4 Heat Block—One heat block for tubes is required. The heat block must meet the following performance specifications.

• Temperature Range: Ambient +5°C to 99°C

• Temperature Regulation: ±0.1°C at 37°C; ±0.4°C at 60°C

5 Magnetic Stand—One magnetic stand for a 96 well plate is required. Better performance is seen when the magnets are on the side of the stand and not on the bottom.

6 Precision Pipettes—One set of precision pipettes is required. (A separate set is required in the pre-amp area.) The use of precision pipettes is required to ensure accurate reagent and sample delivery. Single-channel or multi-channel pipettes can be used if they are calibrated regularly and are accurate within 5% of stated volume.

7 Consumables—The following consumables are required.

• 96-well skirted PCR plates, 0.2 ml, polypropylene, or equivalent

• 96-well storage plates, 0.8 ml (MIDI plates)

NOTE

Make sure that the 96-well plate is fit compatible with the magnetic stand.

• Conical tubes, 15 ml

• Eppendorf microcentrifuge tubes (screw-top recommended)

• PCR eight-tube strips

• Solution basins, PVC, DNase, RNase-free (trough)

• Adhesive aluminum foil seals

• Adhesive plate seals

• Aerosol resistant pipette tips

Prevent PCR Product Contamination

The PCR process is commonly used in the laboratory to amplify specific DNA sequences.

Unless proper laboratory hygiene is used, PCR products can contaminate reagents, instrumentation, and genomic DNA samples, causing inaccurate and unreliable results.

PCR product contamination can shut down lab processes and significantly delay normal operations.

Make sure that the lab is set up appropriately to reduce the risk of PCR product contamination:

}

Physically Separate Pre- and Post-Amp Areas

• Physically separate laboratory space where pre-amp processes are performed

(DNA extraction, quantification, and normalization) from the laboratory space where post-amp processes are performed.

• Never use the same sink to wash pre-amp and post-amp troughs.

• Never share the same water purification system for pre-amp and post-amp processes.

• Store all supplies used in the protocols in the pre-amp area, and transfer to the post-amp area as needed.

}

Use Dedicated Equipment and Supplies

• Dedicate separate full sets of equipment and supplies (pipettes, centrifuges, oven, heat block, etc.) to pre-amp and post-amp lab processes, and never share between processes.

• Dedicate separate storage areas (freezers and refrigerators) to pre-amp and postamp consumables.

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Part # 15039610 Rev. B

Because the pre- and post-amplification reagents are shipped together, it is important to unpack the reagents in the pre-amp lab area, and then move the post-amp reagents to the proper post-amp storage area.

Pre- and Post-Amp Lab Procedures

To prevent PCR product contamination, it is important to establish lab procedures and follow best practices. Illumina recommends daily and weekly cleaning of lab areas using

0.5% Sodium Hypochlorite (10% Bleach).

CAUTION

To prevent sample or reagent degradation, make sure that all vapors from the cleaning solution have fully dissipated before beginning any processes.

Daily Cleaning of Pre-Amp Area

A daily cleaning of the pre-amp area using a 0.5% Sodium Hypochlorite (10% Bleach) solution helps to eliminate PCR product that has entered the pre-amp area.

Identify pre-amp areas that pose the highest risk of contamination, and clean these areas with a 0.5% Sodium Hypochlorite (10% Bleach) solution before beginning any pre-amp processes. High-risk areas might include, but are not limited to, the following items:

}

Bench tops

} Door handles

}

Refrigerator/freezer door handles

}

Computer mouse

}

Keyboards

Daily Cleaning of Post-Amp Area

Reducing the amount of PCR product in the post-amp area helps reduce the risk of contamination in the pre-amp area. Daily cleaning of the post-amp area using a 0.5%

Sodium Hypochlorite (10% Bleach) solution helps achieve this.

Identify post-amp areas that pose the highest risk of contamination, and clean these areas with a 0.5% Sodium Hypochlorite (10% Bleach) solution daily. High-risk areas might include, but are not limited to, the following items:

} Thermal cyclers

} Bench space used to process amplified DNA

}

Door handles

}

Refrigerator/freezer door handles

} Computer mouse

} Keyboards

Weekly Cleaning of All Lab Areas

Once a week, perform a thorough cleaning of the pre-amp and post-amp areas using

0.5% Sodium Hypochlorite (10% Bleach).

}

Clean all bench tops and laboratory surfaces.

} Clean all instruments that are not cleaned daily.

} Thoroughly mop lab floors.

}

Make sure that personnel responsible for weekly cleaning are properly trained on prevention of PCR product contamination.

MiSeqDx Universal Kit 1.0 Reference Guide

10

Items Fallen to the Floor

The floor is contaminated with PCR product transferred on the shoes of individuals coming from the post-amp area; therefore, anything falling to the floor must be treated as contaminated.

}

Disposable items that have fallen to the floor, such as empty tubes, pipette tips, gloves, lab coat hangers, must be discarded.

}

Non-disposable items that have fallen to the floor, such as a pipette or an important sample container, must be immediately and thoroughly cleaned with a 0.5% Sodium

Hypochlorite (10% Bleach) solution to remove PCR product contamination.

} Clean any lab surface that has come in contact with the contaminated item.

Individuals handling anything that has fallen to the floor, disposable or nondisposable, must discard their lab gloves and put on a new pair.

Precautions

Adhere to the following recommendations when preparing libraries for sequencing using this protocol.

Ensuring Consistency

}

Use multi-channel pipettes—To ensure consistency across samples, use a multichannel pipette where possible. Calibrate pipettes periodically.

} Consistency for smaller sample preparations—Each reagent tube supplied with the kit contains sufficient volume to generate results using manual pipettes and reagent troughs following standard laboratory techniques. To ensure accurate reagent volumes, single pipette reagent into each well or pipette from a PCR 8-tube strip.

Handling Magnetic Beads

} Use at room temperature—Prior to use, allow the beads to reach room temperature prior to use.

}

Vortex until well-suspended—Immediately prior to use, vortex the beads until they are well-suspended and the color appears homogeneous.

} Mix samples thoroughly—After adding the beads to the samples, mix thoroughly by pipetting up and down ten times. Illumina also recommends using a shaker to thoroughly mix samples.

}

Allow maximum binding—For best results, incubate the bead/sample mixtures at room temperature for the entire duration indicated in the protocol.

}

Slowly aspirate cleared solution—After placing the plate on the magnetic stand, wait for the solution to clear before proceeding. Keep the plate on the magnetic stand when slowly aspirating cleared solution, taking care not to disturb the separated beads.

Avoiding Cross-Contamination

} Change tips between dispensing reagents and samples—Always use fresh pipette tips between dispensing reagents and samples.

}

Mix plates as directed—Mix samples with a multi-channel pipette and centrifuge the plate when indicated. Do not vortex the plates.

}

Use aerosol-resistant tips—Using aerosol-resistant pipette tips reduces the risk of amplicon carry-over and sample-to-sample cross-contamination.

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Part # 15039610 Rev. B

Washing with 80% Ethanol During the PCR Clean-Up Step

}

Prepare fresh 80% ethanol—Always prepare fresh 80% ethanol for wash steps.

Ethanol can absorb water from the air impacting the results.

} Remove all ethanol from wells—Make sure that all ethanol is removed from the bottom of the wells, as it may contain residual contaminants. Use a P20 multichannel pipette to remove residual ethanol and accelerate drying.

}

Allow complete evaporation—Allow at least ten minutes of drying time off the magnetic stand at room temperature for complete evaporation. Residual ethanol can impact the performance of subsequent reactions.

DNA Input Requirements

}

The Illumina MiSeqDx Universal Kit 1.0 protocol requires 250 ng of genomic DNA.

Illumina strongly recommends quantifying the starting genomic material.

} Input DNA Quantitation—Quantify the starting genomic material using UV spectrophotometer methods based on A260/A280 OD readings.

}

Assessing DNA Quality—Absorbance measurements at 260 nm are commonly used to quantify DNA. The ratio of absorbance at 260 nm to absorbance at 280 nm is used as an indication of sample purity. This protocol is optimized for DNA with absorbance ratio values greater than 1.5.

Quality Controls

}

Good laboratory practices mandate that a positive control DNA sample and a negative (no-template) control sample are included in every run.

} The positive control DNA sample should be a well-characterized sample with known variants in the region of interest.

Acronyms

Acronym

AMP

CLP

COP

DAL

FPU

HYB

LNP

NTC

PAL

POS

SGP

Table 9 Illumina MiSeqDx Universal Kit 1.0 Acronyms

Definition

Amplification Plate

CLean-up Plate

Custom Oligo Pool

Diluted Amplicon Library

Filter Plate Unit

HYBridization Plate

Library Normalization Plate

Negative Template Control

Pooled Amplicon Library

Positive Control

StoraGe Plate

MiSeqDx Universal Kit 1.0 Reference Guide

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Protocol Workflow

The following diagram illustrates the Illumina MiSeqDx Universal Kit 1.0 workflow. Safe stopping points are marked between steps.

Figure 1 Illumina MiSeqDx Universal Kit 1.0 Workflow

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Part # 15039610 Rev. B

MiSeqDx Sample Sheet Preparation

1 From the Illumina Worklist Manager Welcome screen, select Create Worklist. The

Enter Run Parameters screen opens.

Figure 2 Illumina Worklist Manager, Enter Run Parameters screen

2 In the Test Type field, select MiSeqDx Universal.

3 In the Worklist Name field, enter a name for the sample sheet. This is a required field.

• If the alpha-numeric reagent cartridge barcode ID is used for the sample sheet name, the MiSeq Operating Software will find the sample sheet automatically.

(The barcode ID is located on the reagent cartridge label directly below the barcode.)

• If any other name is used for the sample sheet, the Browse button in the MiSeq

Operating Software can be used to locate the appropriate sample sheet.

4 [Optional] Enter a description to identify the run.

5 Make sure that the date matches the start date of the run. The current date appears by default.

6 Select Next. The Enter Sample Information screen opens.

Figure 3 Illumina Worklist Manager, Enter Sample Information screen

MiSeqDx Universal Kit 1.0 Reference Guide

14

Enter Sample Information

1 From the Table tab or the Plate tab, enter the following information for each well containing sample: a Sample ID—Enter a unique sample ID. The sample ID is used to track the sample from preparation through sequencing and analysis. The ID is usually a barcode; however, any value is acceptable.

b Index 1 and Index 2—Specify the index adapter that will be used for each Index

Read. Illumina recommends using combinations that result in at least one A or

C base (red) and at least one G or T base (green) at every cycle.

NOTE

Please refer to

Sample Throughput and Index Representation on page 15

for assistance in choosing the appropriate indexes.

c Manifest—Specify the name of the manifest file that contains information about the samples in that particular well. Do not include the file extension as part of the name.

2 [Optional] To record more detailed information about the samples, enter a sample name and description.

3 [Optional] To identify controls on the plate, select Negative or Positive from the

Control drop-down menu.

4 Go to the Plate Graphic tab and use the Copy to Clipboard or Print option to capture an image of the sample plate.

Figure 4 Illumina Worklist Manager, Plate Graphic tab

5 Select Finish.

Sample Throughput and Index Representation

For the Illumina MiSeqDx Universal Kit 1.0, the sample throughput per MiSeqDx run can be between 8 to 48 samples. The indexing primers used during PCR amplification must be chosen based on desired final sample throughput to ensure diversity in index sequence.

NOTE

For maximum throughput efficiency, perform library preparation for up to 96 samples, and then divide the samples into two sequencing runs with a maximum of 48 samples each. Create separate sample sheets for each set of 48 samples as the MiSeqDx can only sequence 48 samples at a time.

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Part # 15039610 Rev. B

MiSeqDx uses a green LED to sequence G/T bases and a red LED to sequence A/C bases.

At each cycle at least one of two nucleotides for each color channel needs to be read to ensure proper registration. It is important to maintain color balance for each base of the index read being sequenced, otherwise registration failure could occur during sequencing of the Index Read.

See Table 10

for choosing index primer combinations for 48 or 96 sample runs.

Table 10 Index Primer Combinations for 48-Sample or 96-Sample Sequencing Runs

Rows A–H Columns 1–6 Columns 7-12

Index Primer A (A501)

Index Primer B (A502)

Index Primer C (A503)

Index Primer D (A504)

Index Primer E (A505)

Index Primer F (A506)

Index Primer G (A507)

Index Primer H (A508)

Index Primer 1 (A701)

Index Primer 2 (A702)

Index Primer 3 (A703)

Index Primer 4 (A704)

Index Primer 5 (A705)

Index Primer 10 (A710)

--

--

Index Primer 6 (A706)

Index Primer 7 (A707)

Index Primer 8 (A708)

Index Primer 9 (A709)

Index Primer 11 (A711)

Index Primer 12 (A712)

--

--

If sequencing fewer than the 48 samples in a sequencing run, select the appropriate indexes based on their sequences to maintain color balance in the green and red channels. See

Table 12

and

Table 13 . At a minimum, runs with 8 to 48 samples must

include the indexing primer combinations identified in

Table 11

.

To accurately process smaller runs, at least eight samples must be present. If six unique samples (excluding the positive and negative controls) are not available, it is acceptable to fill the run with sample replicates or any human genomic DNA sample. See

Table 11

for the minimal set of color-balanced indexes to use for 8-sample sequencing runs.

Table 11 Index Primer Combinations for 8-Sample Sequencing Runs

Index Primer C

(A503)

Index Primer 1

(A701)

Sample 1

Index Primer 2

(A702)

Sample 2

Index Primer D

(A504)

Sample 4 Sample 5

Index Primer E

(A505)

Sample 7 Sample 8

Index Primer 10

(A710)

Sample 3

Sample 6

--

Index Primer Sequences

Table 12 Sequences for Index Primers A (A501) - H (A508)

Index Primer Sequence

Index Primer A (A501)

Index Primer B (A502)

Index Primer C (A503)

Index Primer D (A504)

Index Primer E (A505)

Index Primer F (A506)

TGAACCTT

TGCTAAGT

TGTTCTCT

TAAGACAC

CTAATCGA

CTAGAACA

MiSeqDx Universal Kit 1.0 Reference Guide

16

Index Primer

Index Primer G (A507)

Index Primer H (A508)

Sequence

TAAGTTCC

TAGACCTA

Table 13 Sequences for Index Primers 1 (A701) - 12 (A712)

Index Primer Sequence

Index Primer 1 (A701)

Index Primer 2 (A702)

Index Primer 3 (A703)

Index Primer 4 (A704)

ATCACGAC

ACAGTGGT

CAGATCCA

ACAAACGG

Index Primer 5 (A705)

Index Primer 6 (A706)

Index Primer 7 (A707)

Index Primer 8 (A708)

Index Primer 9 (A709)

Index Primer 10 (A710)

Index Primer 11 (A711)

Index Primer 12 (A712)

ACCCAGCA

AACCCCTC

CCCAACCT

CACCACAC

GAAACCCA

TGTGACCA

AGGGTCAA

AGGAGTGG

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Part # 15039610 Rev. B

Hybridization of Oligonucleotide Pool

During this step, the user-supplied custom oligonucleotide pool containing upstream and downstream oligonucleotides specific to the region of interest is hybridized to genomic DNA samples.

WARNING

This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region.

For more information, see the SDSs for this kit, at support.illumina.com/sds.html.

Estimated Time

} Total duration: 1 hour 35 minutes

}

Hands-on: 15 minutes

Consumables

Item

Hybridization Buffer

Custom oligo pool

Quantity

1 tube

5 µl per sample well

5 µl

Storage

-25° to -15°C

Userdetermined

-25° to -15°C

Supplied By

Illumina

User

User Genomic DNA (50 ng/µl recommended)

96-well skirted PCR plate

Adhesive aluminum foil seal

Sterile troughs

1 plate

2 seals

As needed

15° to 30°C

15° to 30°C

15° to 30°C

User

User

User

Preparation

1 Remove the custom oligo pool, Hybridization Buffer, genomic DNA samples, and positive control sample from -25°C to -15°C storage and thaw at room temperature.

2 Vortex the custom oligo pool and Hybridization Buffer vigorously to make sure that all precipitates have completely dissolved, then briefly centrifuge the tubes to collect liquid.

NOTE

Before using Hybridization Buffer, hold the tube in front of a light and visually inspect to make sure that all precipitates have completely dissolved.

3 Set a 96-well heat block to 95°C.

4 Pre-heat an incubator to 37°C to prepare for the extension-ligation step.

MiSeqDx Universal Kit 1.0 Reference Guide

18

5 Create the sample plate according to the Plate Graphic printed from Illumina

Worklist Manager. Verify the location of positive and negative controls match.

Illumina recommends processing samples in batches no smaller than eight.

NOTE

Use of controls enables Illumina Technical Support to provide effective troubleshooting assistance. Illumina Technical Support will not provide assistance unless these control reactions were included in the run.

Procedure

1 Label a new 96-well PCR plate "HYB_Plate_ID".

2 Add 5 µl of sample or control at 50 ng/µl (250 ng total) to the appropriate wells in the HYB plate. Follow the generated plate layout for correct well selection.

NOTE

Verify that DNA sample layout and the positions of positive and negative controls match the plate graphic.

3 Using a multi-channel pipette, add 5 µl of the custom oligo pool to all wells containing genomic DNA. Change tips after each column to avoid crosscontamination.

4 Using a multi-channel pipette, add 40 µl of Hybridization Buffer to each sample in the HYB plate. Gently pipette up and down 3–5 times to mix. Change tips after each column to avoid cross-contamination.

NOTE

Ensure any crystals or precipitate in Hybridization Buffer have dissolved.

NOTE

Do not mix the custom oligo pool and Hybridization Buffer for storage. If combined, the custom oligo pool becomes unstable, even when stored frozen.

5 Seal the HYB plate with adhesive aluminum foil and secure the seal with a rubber roller or sealing wedge.

6 Centrifuge at 1000 × g at 20°C for 1 minute.

7 Place the HYB plate in the pre-heated block at 95°C and incubate for 1 minute.

8 Reduce the temperature of the pre-heated block to 40°C and continue incubating until the heat block reaches 40°C. Ramp down time takes approximately 80 minutes.

NOTE

During incubation, the heat block temperature gradually decreases from 95°C to

40°C. This process typically takes 80 minutes. This gradual cooling is critical for proper hybridization; therefore, PCR thermal cyclers with active cooling (e.g., Peltier, thermoelectric cooled) are not recommended for this process.

SAFE STOPPING POINT

After the heat block reaches 40°C, the HYB plate is stable holding at 40°C for 2 hours.

19

Part # 15039610 Rev. B

Removal of Unbound Oligonucleotides

This process removes unbound oligonucleotides from genomic DNA using a filter capable of size selection. Two wash steps using Stringent Wash Buffer ensure complete removal of unbound oligonucleotides. A third wash step using Universal Wash Buffer removes residual Stringent Wash Buffer and prepares samples for the extension-ligation step.

WARNING

This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region.

For more information, see the SDSs for this kit, at support.illumina.com/sds.html.

Estimated Time

} Total duration: 20 minutes

}

Hands-on: 20 minutes

Consumables

Item

Extension-Ligation Mix

Stringent Wash Buffer

Universal Wash Buffer

Filter Plate

Adapter collar

MIDI plate

Troughs

Quantity

1 tube

1 bottle

1 tube

1 plate

1 plate

1 plate

As needed

Storage

-25°C to -15°C

2°C to 8°C

2°C to 8°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

Supplied By

Illumina

Illumina

Illumina

Illumina

Illumina

User

User

Preparation

1 Remove Extension-Ligation Mix from -25°C to -15°C storage and thaw at room temperature.

Extension-Ligation Mix is used in the Extension-Ligation step and takes approximately 20 minutes to thaw.

2 Remove Stringent Wash Buffer and Universal Wash Buffer from 2°C to 8°C storage and set aside at room temperature.

3 Assemble the filter plate assembly unit (FPU) in the following order (from top to bottom):

MiSeqDx Universal Kit 1.0 Reference Guide

20

Figure 5 Filter Plate Unit Assembly

A

Lid

B

Filter plate

C

Adapter collar

D

MIDI plate

4 Label the filter plate "FPU_Plate_ID". Plate ID should match the ID used for the HYB plate.

5 Pre-wash the filter plate membrane as follows: a Using a multi-channel pipette, add 45 µl of Stringent Wash Buffer to each well.

b Cover the FPU plate with the filter plate lid and keep it covered during each centrifugation step.

c Centrifuge the FPU at 2400 × g at 20°C for 5 minutes.

Procedure

1 After the hybridization is complete, confirm the heat block has cooled to 40˚C. While the HYB plate is still in the heat block, reinforce the seal using a rubber roller or sealing wedge. If 40˚C is not reached within 80 minutes, continue incubating until the heat block has cooled to 40˚C.

2 Remove the HYB plate from the heat block and centrifuge at 1000 × g at 20°C for 1 minute to collect condensation.

3 Using a multi-channel pipette set to 60 µl, transfer the entire volume of each sample onto the center of the corresponding pre-washed wells of the filter plate. Change tips after each column to avoid cross-contamination.

4 Cover the filter plate with the lid and centrifuge at 2400 × g at 20°C for 5 minutes.

5 Wash the filter plate as follows:

21

Part # 15039610 Rev. B

a Using a multi-channel pipette, add 45 µl of Stringent Wash Buffer to each sample well.

Changing tips between columns is not required if you use care to avoid crosscontamination.

b Cover the filter plate with the lid and centrifuge at 2400 × g at 20°C for 5 minutes.

6 Repeat the wash as follows: a Using a multi-channel pipette, add 45 µl of Stringent Wash Buffer to each sample well.

Changing tips between columns is not required if you use care to avoid crosscontamination.

b Cover the filter plate with the lid and centrifuge at 2400 × g at 20°C for 5 minutes.

c If the wash buffer does not drain completely, centrifuge the filter plate again at

2400 × g at 20°C for 5 minutes.

7 Discard all the flow-through (containing formamide) collected up to this point in an appropriate hazardous waste container, then reassemble the FPU. The same MIDI plate can be re-used for the rest of the pre-amplification process.

8 Using a multi-channel pipette, add 45 µl of Universal Wash Buffer to each sample well.

Changing tips between columns is not required if you use care to avoid crosscontamination.

9 Cover the filter plate with the lid and centrifuge at 2400 × g at 20°C for 10 minutes.

NOTE

Make sure that all liquid has drained after centrifugation. Repeat centrifugation if necessary. Residual wash buffer may inhibit subsequent enzymatic reactions.

MiSeqDx Universal Kit 1.0 Reference Guide

22

Extension-Ligation of Bound Oligonucleotides

This process connects the hybridized upstream and downstream oligonucleotides.

A DNA polymerase extends from the upstream oligonucleotide through the targeted region, followed by ligation to the 5’ end of the downstream oligonucleotide using a

DNA ligase. This results in the formation of products containing the targeted regions of interest flanked by sequences required for amplification.

Estimated Time

} Total duration: 50 minutes

}

Hands-on: 5 minutes

Consumables

Item

Extension-Ligation Mix

Adhesive aluminum foil seal

Troughs

Quantity

1 tube

1 seal

As needed

Storage

-25°C to -15°C

15°C to 30°C

15°C to 30°C

Supplied By

Illumina

User

User

Procedure

1 Using a multi-channel pipette, add 45 µl of Extension-Ligation Mix to each sample well of the filter plate. The Extension-Ligation reaction takes place on the filter plate membrane.

Changing tips between columns is not required if you use care to avoid crosscontamination.

2 Seal the filter plate with adhesive aluminum foil, and then cover with the lid to secure the foil during incubation.

3 Incubate the entire FPU assembly in the pre-heated 37°C incubator for 45 minutes.

4 While the FPU plate is incubating, prepare the AMP (Amplification Plate) as described in the following section.

23

Part # 15039610 Rev. B

PCR Amplification

In this step, the extension-ligation products are amplified using primers that add index sequences for sample multiplexing, as well as common adapters required for cluster generation.

Estimated Time

} Total duration: ~90 minutes

}

Hands-on: 30 minutes

Consumables

Item

PCR Master Mix

Index Primers A (A501) - H

(A508)

Index Primers 1 (A701) - 12

(A712)

PCR Polymerase

Appropriate PCR plate seal

0.05 N NaOH, freshly prepared

96-well skirted PCR plate

Troughs

Quantity

1 tube

1 tube per primer

1 tube per primer

1 tube

1

As needed

1 plate

As needed

Storage

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

-25°C to -15°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

Supplied By

Illumina

Illumina

Illumina

Illumina

User

User

User

User

Preparation

1 Prepare fresh 0.05 N NaOH by adding 25 µl of 10 N NaOH to 4975 µl of sterile water.

2 Determine the index primers to be used according to the plate graphic printout from

Illumina Worklist Manager.

3 Remove PCR Master Mix and the appropriate index primers from -25°C to -15°C storage and thaw on a bench at room temperature.

Allow approximately 20 minutes to thaw the reagents.

4 After the index primers are completely thawed, vortex each tube to mix and briefly centrifuge the tubes in a microcentrifuge. Use 1.7 ml Eppendorf tubes as adapters for the microcentrifuge.

5 Arrange the primers in a rack using the following arrangements: a Arrange Index Primers A (A501) - H (A508) primer tubes (white caps, clear solution) vertically, aligned with rows A through H.

b Arrange Index Primers 1 (A701) - 12 (A712) primer tubes (orange caps, yellow solution) horizontally, aligned with columns 1 through 12.

MiSeqDx Universal Kit 1.0 Reference Guide

24

Figure 6 Index Plate Fixture

A

Index Primers A (A501) - H (A508) (white caps)

B

Index Primers 1 (A701) - 12 (A712) (orange caps)

C

AMP plate

6 Label a new 96-well PCR plate "AMP" (Amplification Plate).

7 Add index primers to the AMP plate as follows: a Using a multi-channel pipette, add 4 µl of the selected index primers [A (A501) –

H (A508)] (clear solution) to the appropriate well in a column of the AMP plate.

Changing tips between columns is not required.

b To avoid index cross-contamination, discard the original white caps and apply new white caps.

c Using a multi-channel pipette, add 4 µl of the selected index primers [1 (A701) –

12 (A712)] (yellow solution) to the appropriate row of the AMP plate. Tips must

be changed after each row to avoid index cross-contamination.

d To avoid index cross-contamination, discard the original orange caps and apply new orange caps. Remove all the index primer tubes from the working area.

8 Prepare the PCR Master Mix/PCR Polymerase PCR working solution as follows: a For 96 samples, add 56 µl of PCR Polymerase to 2.8 ml of PCR Master Mix.

b Invert the prepared PCR working solution 20 times to mix.

You will add this working solution to the AMP plate in the next section. The PCR working solution is stable at room temperature for 10 minutes.

NOTE

Always add PCR Polymerase to PCR Master Mix just prior to use. Never store the combined PCR working solution.

Procedure

1 When the 45-minute extension-ligation reaction is complete, remove the FPU from the incubator. Remove the aluminum foil seal and replace with the filter plate lid.

Removing the aluminum foil seal before centrifugation is recommended to ensure the reaction supernatant will drain into the waste plate effectively.

25

Part # 15039610 Rev. B

2 Centrifuge the FPU at 2400 × g at 20°C for 2 minutes.

3 Using a multi-channel pipette, add 25 µl of 0.05 N NaOH to each sample well on the filter plate. Ensuring that pipette tips come in contact with the membrane, pipette the

NaOH up and down 5–6 times. Tips must be changed after each column.

4 Cover and incubate the filter plate at room temperature for 5 minutes.

5 While the filter plate is incubating, use a multi-channel pipette to transfer 22 µl of the PCR working solution to each well of the AMP plate containing index primers.

Change tips between samples.

6 Transfer samples eluted from the filter to the AMP plate as follows: a Set a multi-channel P20 pipette to 20 µl.

b Pipette the samples in the first column of the filter plate up and down 5–6 times.

c Transfer 20 µl from the filter plate to the corresponding column of the AMP plate.

d Gently pipette up and down 5–6 times to thoroughly combine the DNA with the

PCR working solution.

NOTE

Slightly tilt the FPU plate to ensure complete aspiration and to avoid air bubbles.

e Transfer the remaining columns from the filter plate to the AMP plate in a similar manner. Tips must be changed after each column to avoid index and

sample cross-contamination.

f After all the samples have been transferred, the waste collection MIDI plate of the FPU can be discarded. The metal adapter collar should be cleaned and put away for future use.

7 Cover the AMP plate with the appropriate plate seal and secure with a rubber roller.

8 Centrifuge at 1000 × g at 20°C for 1 minute.

9 Transfer the AMP plate to the post-amplification area.

10 Perform PCR using the following program on a thermal cycler:

• 95°C for 3 minutes

• 25 cycles of:

— 95°C for 30 seconds

— 62°C for 30 seconds

— 72°C for 60 seconds

• 72°C for 5 minutes

• Hold at 10°C

SAFE STOPPING POINT

If not proceeding immediately to PCR Clean-Up, the AMP plate can remain on the thermal cycler overnight, or can be stored at 2°C to 8°C up to 48 hours.

MiSeqDx Universal Kit 1.0 Reference Guide

26

PCR Clean-Up

This process uses PCR Clean-Up Beads to purify the PCR products from the other reaction components.

Estimated Time

} Total duration: 50 minutes

}

Hands-on: 20 minutes

Consumables

Item

Elution Buffer

PCR Clean-Up Beads

80% Ethanol, freshly-prepared

96-well MIDI plates

Adhesive plate seal

Troughs

Quantity

1 tube

400 µl per 8 samples

5 ml per 8 samples

2

As needed

As needed

Storage

15°C to 30°C

2°C to 8°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

Supplied By

Illumina

Illumina

User

User

User

User

Preparation

NOTE

Please review the Precautions section at the beginning of this protocol regarding the handling of magnetic beads and washing with 80% ethanol during the PCR clean-up.

1 Bring the PCR Clean-Up Beads to room temperature.

2 Prepare fresh 80% ethanol from absolute ethanol.

NOTE

Always prepare fresh 80% ethanol for wash steps. Ethanol can absorb water from the air impacting the results.

Procedure

1 Centrifuge the AMP plate at 1000 × g at 20°C for 1 minute to collect condensation.

2 Label a new MIDI plate "CLP_Plate_ID" (CLean-up Plate).

3 Invert PCR Clean-Up Beads 10 times. Vortex vigorously and then invert again 10 times.

4 Visually inspect solution to ensure that beads are resuspended well.

5 Using a multi-channel pipette, add 45 µl of PCR Clean-Up Beads to each well of the

CLP plate.

6 Using a multi-channel pipette set to 60 µl, transfer the entire PCR product from the

AMP plate to the CLP plate. Change tips between samples.

7 Seal the CLP plate with an adhesive plate seal.

8 Shake the CLP plate on a microplate shaker at 1800 rpm for 2 minutes.

27

Part # 15039610 Rev. B

9 Incubate at room temperature (15°C to 30°C) without shaking for 10 minutes.

10 Place the plate on a magnetic stand for a minimum of 2 minutes or until the supernatant is clear.

11 With the CLP plate on the magnetic stand and a multi-channel pipette set to 100 µl, carefully remove and discard the supernatant. Change tips between samples.

NOTE

If any beads are inadvertently aspirated into the tips, dispense the beads back to the plate and let the plate rest on the magnet for 2 minutes and confirm that the supernatant has cleared.

12 With the CLP plate on the magnetic stand, wash the beads with freshly prepared

80% ethanol as follows: a Using a multi-channel pipette, add 200 µl of freshly prepared 80% ethanol to each sample well. Changing tips is not required if you use care to avoid crosscontamination. You do not need to resuspend the beads at this time.

b Incubate the plate on the magnetic stand for a minimum of 30 seconds or until the supernatant is clear.

c Carefully remove and discard the supernatant.

13 With the CLP plate on the magnetic stand, perform a second ethanol wash as follows: a Using a multi-channel pipette, add 200 µl of freshly prepared 80% ethanol to each sample well.

b Incubate the plate on the magnetic stand for a minimum of 30 seconds or until the supernatant appears clear.

c Carefully remove and discard the supernatant.

14 Use a P20 multi-channel pipette set to 20 µl to remove excess ethanol.

15 Remove the CLP plate from the magnetic stand and allow the beads to air-dry for 10 minutes.

16 Using a multi-channel pipette, add 30 µl of Elution Buffer to each sample and then vortex briefly.

Changing tips is not required if you use care to avoid cross-contamination.

17 Seal the plate with an adhesive plate seal.

18 Shake the CLP plate on a microplate shaker at 1800 rpm for 2 minutes.

NOTE

Ensure all samples are completely resuspended. If there are samples in which the beads are not completely resuspended, gently pipette up and down to resuspend the beads and repeat the previous two steps.

19 Incubate at room temperature (15°C to 30°C) for 2 minutes.

20 Place the CLP plate on the magnetic stand for a minimum of 2 minutes or until the supernatant is clear.

21 Label a new MIDI plate "LNP_Plate_ID" (Library Normalization Plate).

MiSeqDx Universal Kit 1.0 Reference Guide

28

22 Using a P20 multi-channel pipette and fine tips, carefully transfer 20 µl of the supernatant from the CLP plate to the LNP plate. Change tips between samples to avoid cross-contamination.

NOTE

If any beads are inadvertently aspirated into the tips, dispense the beads back to the plate and let the plate rest on the magnet for 2 minutes and confirm that the supernatant has cleared.

23 [Optional] Transfer the remaining 10 µl of supernatant from the CLP plate to a new plate and label the plate with a run name and date. Store this plate at -25°C to -15°C until completion of the sequencing run and data analysis. The cleaned up PCR products can be used for troubleshooting efforts in the event of sample failures.

24 If stopping at this point, seal the LNP plate with an adhesive plate seal, and then centrifuge at 1000 × g at 20°C for 1 minute to ensure all the supernatant is at the bottom of the well.

SAFE STOPPING POINT

After PCR Clean-Up, the plate is stable for up to 3 hours at 2°C to 8°C.

29

Part # 15039610 Rev. B

Library Normalization

This process normalizes the quantity of each library to ensure equal library representation in the pooled sample.

Estimated Time

} Total duration: 1 hour 20 minutes

}

Hands-on: 30 minutes

Consumables

Item

Library Normalization Diluent

Library Beads

Library Normalization Wash

Library Storage Buffer

0.1 N NaOH, freshly prepared

96-well skirted PCR plate

15 ml conical tube

Adhesive plate seal

Quantity

1 tube

1 tube

2 tubes

1 tube

2 ml per 48 samples

1 plate

1 tube

As needed

Storage

-25°C to -15°C

2°C to 8°C

2°C to 8°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

Supplied By

User

User

User

User

Illumina

Illumina

Illumina

Illumina

WARNING

This set of reagents contains formamide, an aliphatic amide that is a probable reproductive toxin. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Dispose of containers and any unused contents in accordance with the governmental safety standards for your region.

For more information, see the SDSs for this kit, at support.illumina.com/sds.html.

Preparation

1 Prepare fresh 0.1 N NaOH by adding 30 µl of 10 N NaOH to 2970 µl of

RNase/DNase-free water.

2 Remove Library Normalization Diluent from -25°C to -15°C storage and bring to room temperature. Use a 20°C to 25°C water bath as needed.

NOTE

Library Normalization Diluent might form visible precipitates or crystals. Before use, vortex vigorously, and then hold the tube in front of a light and visually inspect to make sure that all precipitate has completely dissolved.

3 Remove Library Beads and Library Normalization Wash from 2°C to 8°C storage and bring to room temperature.

Use a 20°C to 25°C water bath as needed.

4 Vortex Library Beads vigorously for 1 minute with intermittent inversion until the beads are resuspended and no pellet is found at the bottom of the tube when the tube is inverted.

MiSeqDx Universal Kit 1.0 Reference Guide

30

Procedure

1 For 96 samples, add 4.4 ml of Library Normalization Diluent to a fresh 15 ml conical tube. If processing < 24 samples, use a fresh 1.5 ml tube.

2 Use a P1000 pipette set to 1000 µl to resuspend Library Beads thoroughly by pipetting up and down 10 times.

NOTE

It is extremely critical to completely resuspend the library bead pellet at the bottom of the tube. The use of a P1000 ensures that the beads are homogeneously resuspended and that there is no bead mass at the bottom of the tube. This is essential for achieving consistent cluster density on the flow cell.

3 For 96 samples, pipette 800 µl of Library Beads to the tube containing Library

Normalization Diluent. Mix well by inverting the tube 15–20 times.

NOTE

A P1000 set to 1000 µl is required to resuspend the beads completely in step 2. Mix only the specified amounts of Library Normalization Diluent and Library Beads. You must store the remaining Library Normalization Diluent and Library Beads separately at their respective recommended temperatures. To preserve stability,

Library Beads should never be frozen or mixed with Library Normalization Diluent if not used immediately.

4 Using a multi-channel pipette, add 45 µl of the combined Library Normalization

Diluent/Library Beads working solution to each well of the LNP plate containing libraries. Changing tips between columns is not required if you use care to avoid cross-contamination.

5 Seal the LNP plate with an adhesive plate seal.

6 Shake the LNP plate on a microplate shaker at 1,800 rpm for 30 minutes.

NOTE

This 30-minute incubation is critical for proper library normalization. Incubation times of greater or less than 30 minutes may affect library representation and cluster density.

7 Place the plate on a magnetic stand for a minimum of 2 minutes or until the supernatant is clear.

8 With the LNP plate on the magnetic stand, using a multi-channel pipette set to 80 µl, carefully remove and discard the supernatant in an appropriate hazardous waste container.

NOTE

If any beads are inadvertently aspirated into the tips, dispense the beads back to the plate and let the plate rest for 2 minutes or until the supernatant has cleared.

9 Remove the LNP plate from the magnetic stand and wash the beads with Library

Normalization Wash as follows: a Using a multi-channel pipette, add 45 µl of Library Normalization Wash to each sample well.

Changing tips between columns is not required if you use care to avoid crosscontamination.

b Seal the LNP plate with an adhesive plate seal.

c Shake the LNP plate on a microplate shaker at 1,800 rpm for 5 minutes.

d Place the plate on the magnetic stand for a minimum of 2 minutes or until the supernatant is clear.

31

Part # 15039610 Rev. B

e Carefully remove and discard the supernatant in an appropriate hazardous waste container.

10 Remove the LNP plate from the magnetic stand and repeat the wash with Library

Normalization Wash as follows: a Using a multi-channel pipette, add 45 µl of Library Normalization Wash to each well.

Changing tips between columns is not required if you use care to avoid crosscontamination.

b Seal the LNP plate with an adhesive plate seal.

c Shake the LNP plate on a microplate shaker at 1,800 rpm for 5 minutes.

d Place the plate on the magnetic stand for a minimum of 2 minutes.

e Carefully remove and discard the supernatant in an appropriate hazardous waste container.

11 Use a P20 multi-channel pipette set to 20 µl to remove excess Library Normalization

Wash.

12 Remove the LNP plate from the magnetic stand and add 30 µl of 0.1 N NaOH to each well to elute the sample.

13 Seal the LNP plate with an adhesive plate seal.

14 Shake the LNP plate on a microplate shaker at 1,800 rpm for 5 minutes.

15 During the 5 minute elution, label a new 96-well PCR plate "SGP_Plate_ID" (StoraGe

Plate).

16 Add 30 µl Library Storage Buffer to each well to be used in the SGP plate.

17 After the 5-minute elution, ensure all samples in the LNP plate are completely resuspended. If the samples are not completely resuspended, gently pipette those samples up and down or lightly tap the plate on the bench to resuspend the beads, then shake for another 5 minutes.

18 Place the LNP plate on the magnetic stand for a minimum of 2 minutes.

19 Using a multi-channel pipette set to 30 µl, transfer the supernatant from the LNP plate to the SGP plate. Gently pipette up and down 5 times to mix.

NOTE

If any beads are inadvertently aspirated into the tips, dispense the beads back to the plate and let the plate rest on the magnet for 2 minutes and confirm that the supernatant has cleared.

20 Seal the SGP plate with an adhesive plate seal and then centrifuge at 1,000 × g at

20°C for 1 minute.

SAFE STOPPING POINT

If not proceeding immediately to Library Pooling and subsequent sequencing on the

MiSeqDx, store the sealed SGP plate at -25°C to -15°C for up to 3 days.

MiSeqDx Universal Kit 1.0 Reference Guide

32

Library Pooling

In preparation for cluster generation and sequencing, equal volumes of normalized library are combined, diluted in hybridization buffer, and heat denatured prior to sequencing on the MiSeqDx. PhiX is used as an internal control for sequencing.

Estimated Time

} Total duration: 10 minutes

}

Hands-on: 10 minutes

Consumables

Item

Library Dilution Buffer

10 nM PhiX Internal Control

0.1 N NaOH, freshly prepared

1X TE Buffer

Eppendorf tubes (screwcap recommended)

PCR eight-tube strip

2.5 L Ice bucket

1

1

Quantity

1 tube

2 µl

1 ml

8 µl

2 tubes

Storage

-25°C to -15°C

-25°C to -15°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

15°C to 30°C

Supplied By

Illumina

Illumina

User

User

User

User

User

Prepare for Library Pooling

1 Set a heat block suitable for 1.5 ml centrifuge tubes to 96°C.

2 In an ice bucket, prepare an ice-water bath. Chill the Library Dilution Buffer in the ice-water bath.

3 Begin thawing the MiSeqDx reagent cartridge.

Prepare a Fresh Dilution of NaOH

CAUTION

Using freshly diluted NaOH is essential in order to completely denature samples for cluster generation on the MiSeqDx.

To denature the samples, prepare 1 ml of 0.1 N NaOH. Preparing a volume of 1 ml prevents small pipetting errors from affecting the final NaOH concentration.

1 Combine the following volumes in a microcentrifuge tube:

• DNase, RNase-free water (900 µl)

• Stock 1.0 N NaOH (100 µl)

2 Invert the tube several times to mix.

Denature and Dilute PhiX Internal Control

1 Combine the following volumes to dilute the PhiX Internal Control library to 2 nM:

• 10 nM PhiX Internal Control library (2 µl)

33

Part # 15039610 Rev. B

• 1X TE Buffer (8 µl)

2 Combine the following volumes of 2 nM PhiX Internal Control library and 0.1 N

NaOH in a microcentrifuge tube to result in a 1 nM PhiX Internal Control library:

• 2 nM PhiX Internal Control library (10 µl)

• 0.1 N NaOH (10 µl)

3 Vortex briefly to mix the 1 nM PhiX Internal Control library solution.

4 Centrifuge the template solution at 280 × g at 20°C for 1 minute.

5 Incubate for 4.5 minutes at room temperature to denature the PhiX Internal Control library into single strands.

6 Add the following volume of pre-chilled Library Dilution Buffer to the tube containing denatured PhiX Internal Control library to result in a 20 pM PhiX Internal

Control library.

• Denatured PhiX Internal Control library (20 µl)

• Pre-chilled Library Dilution Buffer (980 µl)

NOTE

You can store the denatured 20 pM PhiX Internal Control library up to 3 weeks at -

25°C to -15°C as single-use aliquots. After 3 weeks, cluster numbers tend to decrease.

Prepare the Reagent Cartridge

The following instructions describe how to thaw the reagent cartridge using a room temperature water bath. This method requires approximately one hour.

1 Remove the reagent cartridge from -25°C to -15°C storage.

2 Place the reagent cartridge in a water bath containing enough room temperature deionized water to submerge the base of the reagent cartridge up to the water line printed on the reagent cartridge. Do not allow the water to exceed the maximum water line.

Figure 7 Maximum Water Line

3 Allow the reagent cartridge to thaw in the room temperature water bath for approximately 1 hour or until thawed.

4 Remove the cartridge from the water bath and gently tap it on the bench to dislodge water from the base of the cartridge. Dry the base of the cartridge. Make sure that no water has splashed on the top of the reagent cartridge.

Inspect the Reagent Cartridge

1 Invert the reagent cartridge ten times to mix the thawed reagents, and then visually inspect that all positions are thawed.

NOTE

It is critical that the reagents in the cartridge are thoroughly thawed and mixed to ensure proper sequencing.

MiSeqDx Universal Kit 1.0 Reference Guide

34

2 Visually inspect the reagents in positions 1, 2, and 4 to make sure that they are fully mixed and free of precipitates.

3 Gently tap the cartridge on the bench to reduce air bubbles in the reagents.

NOTE

The MiSeqDx sipper tubes go to the bottom of each reservoir to aspirate the reagents, so it is important that the reservoirs are free of air bubbles.

4 Place the reagent cartridge on ice or set aside at 2°C to 8°C (up to 6 hours) until ready to set up the run. For best results, proceed directly to loading the sample and setting up the run.

Prepare Samples for Sequencing

1 Bring Library Dilution Buffer to room temperature. Vortex Library Dilution Buffer and ensure that all precipitates have dissolved completely.

2 If the SGP plate was stored frozen, thaw the SGP plate at room temperature.

3 Centrifuge the SGP plate at 1000 × g at 20°C for 1 minute to collect condensation.

4 Label a fresh Eppendorf tube "PAL_Plate_ID" (Pooled Amplicon Library).

5 Determine the samples to be pooled for sequencing. A maximum of 48 samples can be pooled for sequencing.

6 If the SGP plate was stored frozen, using a P200 multi-channel pipette set to 40 µl, mix each library to be sequenced by pipetting up and down 3–5 times. Change tips between samples.

7 Transfer 5 µl of each library to be sequenced from the SGP plate, column by column, to a PCR eight-tube strip. Seal SGP with an adhesive plate seal and set aside.

NOTE

After use, store the sealed SGP plate at -25°C to -15°C. The sealed SGP plate is stable for up to 3 days.

8 Combine and transfer the contents of the PCR eight-tube strip into the PAL tube. Mix the PAL tube thoroughly.

9 Label a fresh Eppendorf tube "DAL_Plate_ID" (Diluted Amplicon Library).

10 Add 585 µl of Library Dilution Buffer to the DAL tube.

11 Add 6 µl of 20 pM PhiX Internal Control to the DAL tube. Using the same tip, pipette up and down 3–5 times to rinse the tip and ensure complete transfer.

12 Transfer 9 µl of PAL to the DAL tube containing Library Dilution Buffer. Using the same tip, pipette up and down 3–5 times to rinse the tip and ensure complete transfer.

13 Mix DAL by vortexing the tube at top speed.

NOTE

If you would like to save the remaining PAL for future use, store the PAL tube at -

25°C to -15°C. Stored PAL is good for three days.

The diluted library DAL should be freshly prepared and used immediately for

MiSeqDx loading. Storage of the DAL will result in a significant reduction of cluster density.

14 Centrifuge the DAL tube at 1000 × g at 20°C for 1 minute to collect contents.

15 Using a heat block, incubate the DAL tube at 96°C for 2 minutes.

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Part # 15039610 Rev. B

16 After the incubation, invert the DAL tube 1–2 times to mix, then immediately place in the ice-water bath.

17 Keep the DAL tube in the ice-water bath for 5 minutes.

NOTE

You must perform the heat denaturation step immediately before loading DAL into the MiSeqDx reagent cartridge to ensure efficient template loading on the MiSeqDx flow cell.

MiSeqDx Universal Kit 1.0 Reference Guide

36

What's Next

After the amplicon library has been pooled with the diluted and denatured PhiX, the libraries are ready to be loaded onto the MiSeqDx Reagent Cartridge in the designated reservoir labeled Load Samples. The sequencing run is then set up using the MiSeq

Operating Software interface. See the MiSeqDx Instrument Reference Guide (part # 15038353) for instructions.

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Part # 15039610 Rev. B

Technical Assistance

For technical assistance, contact Illumina Technical Support.

Table 14 Illumina General Contact Information

Website

www.illumina.com

Email

[email protected]

Table 15 Illumina Customer Support Telephone Numbers

Region

North America

Australia

Austria

Belgium

Denmark

Finland

France

Germany

Ireland

Contact Number

1.800.809.4566

1.800.775.688

0800.296575

0800.81102

80882346

0800.918363

0800.911850

0800.180.8994

1.800.812949

Region

Italy

Netherlands

New Zealand

Norway

Spain

Sweden

Switzerland

United Kingdom

Other countries

Contact Number

800.874909

0800.0223859

0800.451.650

800.16836

900.812168

020790181

0800.563118

0800.917.0041

+44.1799.534000

Safety Data Sheets

Safety data sheets (SDSs) are available on the Illumina website at support.illumina.com/sds.html

.

Product Documentation

Product documentation in PDF is available for download from the Illumina website. Go to support.illumina.com, select a product, then click Documentation & Literature.

MiSeqDx Universal Kit 1.0 Reference Guide

Illumina

5200 Illumina Way

San Diego, California 92122 U.S.A.

+1.800.809.ILMN (4566)

+1.858.202.4566 (outside North America) [email protected]

www.illumina.com

Emergo Europe

Molenstraat 15

2513 BH The Hague

The Netherlands

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