Ion AmpliSeq™ Exome RDY Library Preparation User Guide (Pub

Ion AmpliSeq™ Exome RDY Library Preparation User Guide (Pub
USER GUIDE
Ion AmpliSeq™ Exome RDY Library Preparation
for use with:
Ion AmpliSeq™ Exome RDY Kit
Ion AmpliSeq™ Exome S5™ RDY Kit
Catalog Numbers A27192 , A27193, A29854, and A29855
Publication number MAN0010084
Revision C.0
For Research Use Only. Not for use in diagnostic procedures.
The information in this document is subject to change without notice.
DISCLAIMER
TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT,
PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT.
Important Licensing Information
These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all
applicable Limited Use Label Licenses.
Trademarks
All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a trademark of Roche Molecular Systems,
Inc., used under permission and license. Agilent and Bioanalyzer are trademarks of Agilent Technologies, Inc. Agencourt and AMPure are trademarks of
Beckman Coulter, Inc. NanoDrop is a trademark of NanoDrop Technologies, LLC.
© 2015 Thermo Fisher Scientific Inc. All rights reserved.
2
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Contents
About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Changes from previous version . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Purpose of this guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
■ CHAPTER 1
Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Ion AmpliSeq™ Exome and Exome S5™ RDY Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Shared Kit Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Kit-specific components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
(Optional) Ion Library Equalizer™ Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
(Required) Ion Xpress™ Barcode Adapters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Required materials and equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Procedure overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Procedure guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
■ CHAPTER 2
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Input DNA requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Quantify DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Amount and volume of DNA needed per target amplification reaction . . . . . . . . . . . . . . . . . . . 13
Amplify targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Combine PCR products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Partially digest primer sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Ligate barcode adapters to the amplicons and purify . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Barcoded libraries only: Combine and dilute adapters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Set up and run the ligation reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Purify the unamplified library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
Option 1: Equalize the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Before starting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Amplify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Wash the Equalizer™ Beads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Add Equalizer™ Capture to the amplified library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Add the Equalizer™ Beads and wash . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Elute the equalized library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
(Optional) Combine equalized libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
CONFIDENTIAL — For Developer Access Use Only — Do Not Distribute
3
Contents
Store equalized libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Option 2: Quantify the unamplified library by qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Elute the unamplified library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Quantify the library by qPCR and calculate the dilution factor . . . . . . . . . . . . . . . . . . . . . . . . . .
(Optional) Combine amplicon libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Store libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20
20
20
21
21
Option 3: Quantify the amplified library with the Qubit™ 2.0 Fluorometer or Agilent™ 2100
Bioanalyzer™ instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Amplify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Purify the amplified library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Qubit™ 2.0 Fluorometer: Quantify the library and calculate the dilution factor . . . . . . . . . . . .
Agilent™ Bioanalyzer™: Quantify the library and calculate the dilution factor . . . . . . . . . . . . . .
(Optional) Combine amplicon libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Store libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22
22
23
25
25
26
26
■ CHAPTER 3
Combining Ion AmpliSeq™ libraries . . . . . . . . . . . . . . . . . 27
Ion Chip capacities for Ion AmpliSeq™ libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
■ APPENDIX A
Tips and Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . 28
Tips and modifications to the standard workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Modifications to the standard workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
■ APPENDIX B
Supplemental information
. . . . . . . . . . . . . . . . . . . . . . . 35
Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Install the hg19 reference on your Torrent Server . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Import the BED files into your Torrent Server . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Coverage Analysis Plugin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Torrent Variant Caller Plugin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Copy number variation analysis using Ion Reporter™ Software . . . . . . . . . . . . . . . . . . . . . . . . .
■ APPENDIX C
35
35
36
36
40
42
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
■ APPENDIX D Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . 46
Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Limited Product Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
About This Guide
CAUTION! ABBREVIATED SAFETY ALERTS. Hazard symbols and hazard
types specified in procedures may be abbreviated in this document. For the
complete safety information, see the “Safety” appendix in this document.
IMPORTANT! Before using this product, read and understand the information the
“Safety” appendix in this document.
Changes from previous version
Revision
Date
Description
C.0
07 September 2015
• Added new Ion AmpliSeq Exome S5™ RDY Kit
information
B.0
24 Februrary 2015
• Updated product catalog numbers
• Corrected typo in “Input DNA requirements”
A.0
27 March 2014
• New user guide
Purpose of this guide
The Ion AmpliSeq™ Exome RDY Library Preparation Guide provides reference
information for the products listed on the following page.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
5
1
Product information
Product description
This guide covers the following products:
• Ion AmpliSeq™ Exome RDY Kit, 4x2 (Cat. no. A27193) and Ion AmpliSeq™
Exome RDY Kit, 1x8 (Cat. no. A27192)
• Ion AmpliSeq™ Exome S5™ RDY Kit, 4x2 (Cat. no. A29855) and Ion AmpliSeq™
Exome RDY Kit, 1x8 (Cat. no. A29854)
• Ion Library Equalizer™ Kit (Cat. no. 4482298)
• Ion Xpress™ Barcode Adapters 1–96 Kits (various cat. nos.)
Ion AmpliSeq™ Exome RDY Kit contains reagents for the rapid preparation of 8
exome libraries for sequencing using the Ion Proton™ System. This kit includes Exome
RDY plates that have 12 primer pools dried into 2 rows of 4 plates (Cat. no. A27193) or
8 rows of one plate (Cat. no. A27192) to minimize pipetting steps and facilitate
automation. The plates are bundled with the Ion AmpliSeq™ Library Kit Plus to
increase uniformity, robustness, and performance with the Ion Equalizer™ Kit. This kit
also includes the Ion PI™ Chip Kit v3.
Ion AmpliSeq™ Exome S5™ RDY Kit contains all of the same reagents as the Ion
AmpliSeq™ Exome RDY Kits, but instead includes the Ion 540™ Chip for use with the
Ion S5™ or S5™ XL Systems.
Ion Xpress™ Barcode Adapters 1–96 Kits enable the preparation of barcoded libraries
using the above-mentioned library kit. Multiple barcoded libraries can be combined
and loaded onto a single Ion Chip to minimize the sequencing run time and cost.
Ion Library Equalizer™ Kit provides an optional, streamlined method for normalizing
library concentration without the need for quantitation.
6
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Chapter 1 Product information
Ion AmpliSeq™ Exome and Exome S5™ RDY Kits
1
Ion AmpliSeq™ Exome and Exome S5™ RDY Kits
The Ion AmpliSeq™ Exome (Cat. nos. A27192 and A27193) and Exome S5™ (Cat. nos.
A29854 and A29855) RDY Kits provide primers, library reagents, and chips for
preparing and running 8 exome libraries.
The Ion AmpliSeq™ Exome RDY Kits are for use with the Ion Proton™ System, and
include the Ion PI™ Chip Kit v3 (Cat. no. A26771).
The Ion Exome S5™ RDY Kits are for use with the Ion S5™ or S5™ XL Systems, and
include the Ion 540™ Chip Kit (Cat. no. A27766).
Shared kit
components
Ion AmpliSeq™ Library Kit Plus
Component
Cap color
5X Ion AmpliSeq™ HiFi Mix
Number of
tubes
Volume
per tube
Storage
–30°C to –10°C
Red
1 tube
130 µL
FuPa Reagent
Brown
1 tube
60 µL
Switch Solution
Yellow
1 tube
130 µL
DNA Ligase
Blue
1 tube
60 µL
25X Library Amp Primers
White
1 tube
60 µL
1X Library Amp Mix
Black
1 tube
1.5 mL
Low TE
Clear
1 tube
1.7 mL
Room temp
(15°C to 30°C)
Ion AmpliSeq™ Exome RDY Panels
Component
Ion AmpliSeq™ Exome RDY Panel, 1x2
Ion
AmpliSeq™
Exome RDY Panel, 1x8
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Quantity
Storage
4 plates (8 libraries)
Room temp
(15°C to 30°C)
1 plate (8 libraries)
7
1
Chapter 1 Product information
(Optional) Ion Library Equalizer™ Kit
Kit-specific
components
Ion PI™ Chip Kit v3 (included in Cat. nos. A27192 and A27193)
Component
Quantity
Storage
8 chips
Room temp
(15°C to 30°C)
Ion PI™ Chip
Ion 540™ Chip Kit (included in Cat. nos. A29854 and A29855)
Component
Quantity
Storage
8 chips
Room temp
(15°C to 30°C)
Ion 540™ Chip
(Optional) Ion Library Equalizer™ Kit
The Ion Library Equalizer™ Kit contains reagents for 96 reactions:
Ion Library Equalizer™ Kit (Cat. no. 4482298)
Component
Cap color
Quantity
Volume
Equalizer™ Primers
Pink
1 tube
200 µL
Equalizer™ Capture
Purple
1 tube
1 mL
Equalizer™
Elution Buffer
Clear
1 bottle
10 mL
Equalizer™
Beads
Orange
1 tube
300 µL
Equalizer™
Wash Buffer
Clear
1 bottle
35 mL
Storage
2°C to 8°C
Room temp
(15°C to 30°C)
(Required) Ion Xpress™ Barcode Adapters
Ion Xpress™ Barcode Adapters Kits are required for preparing barcoded libraries.
Each kit includes reagents sufficient for preparing up to 40 Ion AmpliSeq™ libraries
per barcode (40 × 16 libraries).
• Ion Xpress™ Barcode Adapters 1–16 Kit (Cat. no. 4471250)
• Ion Xpress™ Barcode Adapters 17–32 Kit (Cat. no. 4474009)
• Ion Xpress™ Barcode Adapters 33–48 Kit (Cat. no. 4474518)
• Ion Xpress™ Barcode Adapters 49–64 Kit (Cat. no. 4474519)
• Ion Xpress™ Barcode Adapters 65–80 Kit (Cat. no. 4474520)
• Ion Xpress™ Barcode Adapters 81–96 Kit (Cat. no. 4474521)
• Ion Xpress™ Barcode Adapters 1-96 (Cat. no. 4474517)
8
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Chapter 1 Product information
Required materials and equipment
1
Required materials and equipment
Unless otherwise indicated, all materials are available through thermofisher.com.
MLS: Fisher Scientific (www.fisherscientific.com) or other major laboratory supplier.
Description
One of the following:
• GeneAmp™ PCR System 9700 Single or Dual 96-well
Thermal Cycler
Source
See web product
pages
• AB™ 2720 Thermal Cycler
• Veriti™ 96-well Thermal Cycler
• Proflex™ 96-well PCR System
MicroAmp™ Optical 96-well Reaction Plates (Optionaldepending on library preparation method)
N8010560
4306737 (with
barcode)
MicroAmp™ Clear Adhesive Film
4306311
MicroAmp™ Optical Film Compression Pad
4312639
Agencourt™ AMPure™ XP Kit
Beckman Coulter,
A63880 or A63881
DynaMag™-96 Side, or other plate magnet
12331D
Nuclease-Free Water
AM9932
Absolute ethanol
MLS
Pipettors, 2–200 μL, and low-retention filtered pipette tips
MLS
Choose one option for library quantitation and normalization:
• Ion Library Quantitation Kit
4468802
• Qubit™ 2.0 Fluorometer and Qubit™ dsDNA HS Assay Kit
Q32866, Q32851
• Agilent™ 2100 Bioanalyzer™ instrument and Agilent™ High
Sensitivity DNA Kit
Agilent, G2939AA,
5067-4626
Recommended for DNA quantitation: TaqMan™ RNase P
Detection Reagents Kit
4316831
Optional for DNA quantitation: Qubit™ 2.0 Fluorometer and
Qubit™ dsDNA HS Assay Kit
Q32866, Q32851
Ion Xpress™ Barcode Adapters
See web product
pages
(Optional): Real-time PCR instrument (e.g., Applied
Biosystems™ 7900HT, 7500, StepOne™, StepOnePlus™,
ViiA™ 7 Systems, or QuantStudio™ 12K Flex Real-Time PCR
System)
See web product
pages
(Optional): MagMax™ DNA Multi-Sample Kit
4413020
(Optional): PureLink™ Genomic DNA Mini Kit
K182000
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
9
1
Chapter 1 Product information
Procedure overview
Procedure overview
Amplify target regions from 50-100 ng of genomic DNA (gDNA) in the Ion
AmpliSeq™ Exome RDY plates and the Ion AmpliSeq™ HiFi Mix. Treat amplicons
with FuPa Reagent to partially digest the primers and phosphorylate the amplicons.
The amplicons are then ligated to Ion Xpress™ Barcode Adapters and purified.
Libraries can be normalized without the need for quantitation or dilution using the Ion
Library Equalizer™ Kit. Alternatively, libraries may be quantified by qPCR without
amplification. If you intend to quantify libraries with a method that does not
specifically detect amplifiable molecules, such as the Qubit™ 2.0 Fluorometer or
Agilent™ 2100 Bioanalyzer™ instrument, library amplification and purification are
required before quantitation.
Up to 3 barcoded exome libraries may be combined prior to template preparation and
sequencing.
10
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Chapter 1 Product information
Procedure guidelines
1
Procedure guidelines
• Thaw components that contain enzymes—such as 5X AmpliSeq™ HiFi Mix, FuPa
Reagent, DNA Ligase and 1X Library Amp Mix—on ice, and keep on ice during
the procedure. All other components may be thawed at room temperature. Gently
vortex and spin down all reagents before use.
• If there is visible precipitate in the Switch Solution or the tube cap after thawing,
vortex or pipet up and down at room temperature to resuspend.
• Use good laboratory practices to minimize cross-contamination of products. If
possible, perform PCR setup in an area or room that is separate from template
preparation. Always change pipette tips between samples.
• Use a calibrated thermal cycler specified in “Required materials and equipment”
on page 9.
• Pipet viscous solutions slowly and ensure complete mixing by vigorous vortexing
or pipetting up and down several times.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
11
2
Methods
Workflow
Amplify targets
(page 13)
Genomic DNA
Amplify targets using
Ion AmpliSeq™ Primer Pool
Combine PCR products
Partially digest primer sequences
(page 14)
Ligate barcode adapters to the amplicons and purify
(page 15)
Partially digest primer sequences
Adapters
A
P1
OR
Barcode Adapters
Option 1:
Equalize the
library
(page 17)
Option 2:
Quantify the
unamplified
library by
qPCR
(page 20)
Option 3: Quantify
the amplified
library with the
Qubit™ 2.0
Fluorometer or
Agilent™ 2100
Bioanalyzer™
instrument
(page 22)
X
P1
Ligate adapters
A
P1
Nonbarcoded library
X
OR
P1
Barcoded library
Combine libraries (optional) and proceed to template
preparation.
12
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Chapter 2 Methods
Input DNA requirements
2
Input DNA requirements
Quantify DNA
We recommend the TaqMan™ RNase P Detection Reagents Kit (Cat. no. 4316831) for
quantifying amplifiable DNA (see http://ioncommunity.thermofisher.com/docs/
DOC-7431). The Qubit™ dsDNA HS Assay Kit (Cat. no. Q32851 or Q32854) may also
be used. Methods such as densitometry (e.g., NanoDrop™ Spectrophotometers) are not
recommended, because they do not discriminate between DNA and RNA and thus are
extremely sensitive to small fragments of hydrolyzed RNA. This can lead to gross
overestimation of the concentration of sample DNA, underseeding of the target
amplification reaction, low-quality libraries, and low library yields.
Amount and
volume of DNA
needed per target
amplification
reaction
For each exome library, use 50–100 ng of genomic DNA (gDNA). The maximum
volume of DNA is 56 µL.
Amplify targets
1. For each sample, prepare a master mix:
Component
Volume
5X Ion AmpliSeq™ HiFi Mix (red cap)
14 µL
50–100 ng gDNA (non-FFPE)*
Y µL
Nuclease-free Water
(56 – Y) µL
Total
70 µL
* Where DNA is not limiting, we recommend 100 ng. For library
amplification followed by Qubit™ quantitation, we recommend 50 ng.
2. Mix thoroughly.
3. Remove the seal from the Ion AmpliSeq™ Exome RDY plate.
4. For each sample, use a low volume pipettor to carefully dispense 5-µL aliquots of
master mix into a single row (12 wells) of the plate without changing the tip.
Note: A blue dye has been added to the primers to help identify wells that contain
dry primers. All rows in the 1x8 format (Part. no. 4489838) contain primers; only
rows “C” and “F” in the 1x2 format (Part. no. 4489840) contain primers.
5. Apply a MicroAmp™ clear adhesive film (Cat. no. 4306311), ensure a tight seal,
and briefly centrifuge the plate to collect droplets.
6. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermal
cycler, and run the following program for 5 µL volume.
IMPORTANT! Use of the recommended plates, seals, compression pads, and a
Thermo Fisher Scientific thermal cycler are critical for best performance.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
13
2
Chapter 2 Methods
Combine PCR products
Stage
STOPPING POINT
Step
Temp
Time
Hold
Activate
enzyme
99°C
2 min
Cycle
Denature
99°C
15 sec
(10 cycles)
Anneal/extend
60°C
16 min
Hold
—
10°C
Hold
PCR products may be stored at 10°C overnight. For longer periods,
store at –20°C.
Combine PCR products
1. Briefly centrifuge the plate to collect droplets. Carefully remove the plate seal and
combine the 12 PCRs for each sample (row) by transferring the material from
wells 1–5 and 7–12 into the column 6 well, without changing the tip.
Combine PCRs
Amplify
Partially digest primer sequences
1. Add 6 µL of FuPa Reagent (brown cap) to each combined PCR product. The total
volume is approximately 60 µL.
2. Seal the plate with MicroAmp® adhesive film, vortex thoroughly, and spin down
to collect droplets. Alternatively, mix by pipetting at least half the total volume up
and down at least 5 times prior to sealing the plate.
3. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermal
cycler, and run the following program:
14
Temperature
Time
50°C
10 min
55°C
10 min
60°C
20 min
10°C
Hold (for up to 1 hour)
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Chapter 2 Methods
Ligate barcode adapters to the amplicons and purify
2
Ligate barcode adapters to the amplicons and purify
Barcoded libraries
only: Combine and
dilute adapters
For each barcode X chosen, prepare a mix of Ion P1 Adapter and Ion Xpress™
Barcode X at a final dilution of 1:4. Use 6 µL of this barcode adapter mix for the Ion
AmpliSeq™ Adapters in step 2 below. Diluted adapters should be stored at –20°C.
For example, combine the volumes indicated in the following table. Scale volumes as
necessary.
IMPORTANT! When handling barcoded adapters, be careful to avoid crosscontamination by changing gloves frequently and opening one tube at a time.
Set up and run the
ligation reaction
1. Carefully remove the plate seal and add 6 µL of diluted barcode adapters to each
well of digested amplicons:
Example barcode adapter mix for up to 4 reactions
Component
Volume
Ion P1 Adapter
1.5 µL
Ion Xpress™ Barcode X*
1.5 µL
Nuclease-free Water
3 µL
Total
6 µL
* X = barcode chosen
2. Add 12 µL of Switch Solution to each well. If there is visible precipitate in the Switch
Solution, vortex or pipet up and down at room temperature to resuspend.
3. Add 6 µL of DNA Ligase to each well to bring the total reaction volume to
approximately 80 µL.
Note: Do not combine DNA Ligase and barcode adapters prior to adding to
reaction mixture.
4. Seal the plate with MicroAmp™ adhesive film, vortex thoroughly and briefly
centrifuge to collect droplets. Alternatively, mix by pipetting at least half the total
volume up and down at least 5 times prior to sealing the plate.
5. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermal
cycler, and run the following program:
STOPPING POINT
Temperature
Time
22°C
30 min
72°C
10 min
10°C
Hold (for up to 1 hour)
Samples may be stored at –20°C.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
15
2
Chapter 2 Methods
Ligate barcode adapters to the amplicons and purify
Purify the
unamplified library
IMPORTANT! Bring AMPure™ XP reagent to room temperature and vortex thoroughly
to disperse the beads before use. Pipet solution slowly.
Use freshly prepared 70% ethanol for the next steps. Combine 230 µL of ethanol with
100 µL of nuclease-free water per sample.
Do NOT substitute a Dynabeads®-based purification reagent for the Agencourt®
AMPure® XP reagent.
1. Carefully remove the plate seal and add 80 µL (~1X sample volume) of
Agencourt™ AMPure™ XP Reagent to each library and pipet up and down 5
times to thoroughly mix the bead suspension with the DNA.
2. Incubate the mixture for 5 minutes at room temperature.
3. Place the plate in a magnetic rack such as the DynaMag™-96 Side Magnet (Cat.
no. 12331D) and incubate for 2 minutes or until solution clears. Carefully remove
and discard the supernatant without disturbing the pellet.
4. Add 150 µL of freshly prepared 70% ethanol and move the plate side to side in the
magnet to wash the beads, then remove and discard the supernatant without
disturbing the pellet.
Note: If your magnet does not have two positions for shifting the beads, remove
the plate from the magnet and gently pipet up and down five times (with the
pipettor set at 100 µL), then return the plate to the magnet and incubate for 2
minutes or until the solution clears.
5. Repeat step 4 for a second wash.
6. Ensure that all ethanol droplets are removed from the wells. Keeping the plate in
the magnet, air-dry the beads at room temperature for 5 minutes. Do not overdry.
Note: Residual ethanol drops will inhibit library amplification. If necessary, spin
down the plate and remove residual ethanol, prior to air-drying the beads.
7. Proceed immediately to one of the following:
• “Option 1: Equalize the library” below or
• “Option 2: Quantify the unamplified library by qPCR” on page 20 or
• “Option 3: Quantify the amplified library with the Qubit™ 2.0 Fluorometer
or Agilent™ 2100 Bioanalyzer™ instrument” on page 22.
Note: Since qPCR typically provides the best sensitivity and barcode
balance, it is the recommended quantitation method. The Ion Library
Equalizer™ Kit offers the greatest convenience, but can result in sample loss
if library yield is too low. Qubit® 2.0 fluorometry is the most economical, but
lacks specificity. Agilent® 2100 Bioanalyzer® quantitation generates the most
information for troubleshooting, but lacks accuracy due to the 1 µL sample
volume.
16
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Chapter 2 Methods
Option 1: Equalize the library
2
Option 1: Equalize the library
The Ion Library Equalizer™ Kit (Cat. no. 4482298) provides a method for normalizing
library concentration at ~100 pM without the need for quantitation. First amplify the
Ion AmpliSeq™ library, then capture the library on Equalizer™ Beads and dilute.
Before starting
Amplify the library
Warm all the reagents in the Ion Library Equalizer™ Kit to room temperature. Vortex
and briefly spin down all reagents before use.
1. Remove the plate from the magnet and add 50 µL of 1X Library Amp Mix and 2 µL
of 25X Library Amp Primers to each bead pellet. The Library Amp Mix and Library
Amp Primers may be combined before addition.
Note: 25X Library Amp Primers are compatible with the Ion Library Equalizer™
Kit. You may use either the 25X Library AMP Primers or the Equalizer™ Primers
to amplify the library.
Note: Library amplification takes place in the presence of the AMPure™ XP
beads.
2. Place a MicroAmp™ Compression Pad on the plate, load the plate in the thermal
cycler, and run the following program. During cycling, wash the Equalizer™
Beads, if necessary.
Stage
Temperature
Time
Hold
98°C
2 min
7 cycles
98°C
15 sec
64°C
1 min
10°C
Hold (for up to 1 hour)
Hold
Wash the
Equalizer™ Beads†
Note: Beads for multiple reactions may be prepared in bulk, and can be stored in
Equalizer™ Wash Buffer at 4°C for up to 6 months until use. After 6 months, beads
should be rewashed with an equal volume of Equalizer™ wash buffer.
1. Bring the Equalizer™ Beads to room temperature and mix thoroughly.
2. For each reaction, pipet 3 µL of beads into a clean strip tube or plate well and add
6 µL/reaction of Equalizer™ Wash Buffer.
3. Place the strip tube/plate in a magnetic rack for 3 minutes or until the solution is
completely clear.
4. Carefully remove and discard the supernatant without disturbing the pellet.
5. Remove the tube/plate from the magnet, add 6 µL/reaction of Equalizer™ Wash
Buffer, and pipet up and down to resuspend.
† If not previously performed.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
17
2
Chapter 2 Methods
Option 1: Equalize the library
Add Equalizer™
Capture to the
amplified library
1. After thermal cycling, remove the seal from the plate and add 10 µL of
Equalizer™ Capture to each library amplification reaction.
2. Seal the plate with MicroAmp® adhesive film, vortex thoroughly, and spin down
to collect droplets. Alternatively, mix by pipetting at least half the total volume up
and down at least 5 times prior to sealing the plate.
3. Incubate at room temperature for 5 minutes.
Add the Equalizer™
Beads and wash
1. Mix the washed Equalizer™ Beads by gentle vortexing or pipetting up and down.
2. Add 6 µL of washed beads to each plate well containing the capture reaction.
3. Set the pipette volume to 40 µL and pipet the mixture up and down at least
5 times to mix thoroughly.
4. Incubate at room temperature for 5 minutes.
5. Place the plate in the magnet and incubate for 2 minutes or until the solution is
clear.
6. Carefully remove and discard the supernatant without disturbing the pellet, and
add 150 µL of Equalizer™ Wash Buffer to each reaction.
7. Move the plate side to side in the magnet to wash the beads.
Note: If your magnet does not have two positions for shifting the beads, remove
the plate from the magnet and gently pipet up and down five times (with the
pipettor set to at least half the total volume), then return the plate to the magnet
and incubate for 2 minutes or until the solution clears.
8. With the plate still in the magnet, carefully remove and discard the supernatant
without disturbing the pellet, and add 150 µL of Equalizer™ Wash Buffer to each
reaction.
9. Repeat the bead wash as described in step 7.
10. With the plate still in the magnet, carefully remove and discard the supernatant.
Note: Ensure that as much wash buffer as possible is removed without disturbing
the pellet.
Elute the equalized
library
1. Remove the plate from the magnet and add 100 µL of Equalizer™ Elution Buffer
to each pellet. Seal the plate with MicroAmp® adhesive film, vortex thoroughly,
and spin down to collect droplets. Alternatively, mix by pipetting at least half the
total volume up and down at least 5 times prior to sealing the plate.
Note: Spin with enough force to collect droplets, but not pellet beads. If beads are
pelleted, vortex again and spin more gently.
2. Elute the library by incubating in a thermal cycler at 32°C for 5 minutes.
3. Place the plate in the magnet and incubate at room temperature for 5 minutes or
until the solution is clear.
4. The supernatant contains the equalized library. Proceed to template preparation,
or combine or store libraries as described below.
Note: The final concentration of each equalized library is ~100 pM.
18
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Chapter 2 Methods
Option 1: Equalize the library
2
(Optional) Combine
equalized libraries
Up to 3 barcoded exome libraries can be combined and run on a single Ion PI™ chip,
depending on the coverage depth desired.
Store equalized
libraries
Libraries may be stored at 4–8°C for up to 1 month. For longer term storage, store at
–20°C.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
19
2
Chapter 2 Methods
Option 2: Quantify the unamplified library by qPCR
Option 2: Quantify the unamplified library by qPCR
Elute the unamplified Ion AmpliSeq™ library, then determine the concentration by
qPCR with the Ion Library Quantitation Kit (Cat. no. 4468802). After quantitation,
determine the dilution factor that results in a concentration of ~100 pM.
Note: The Ion Library Quantitation Kit may also be used to quantify libraries that have
been amplified using the procedure described in “Amplify the library” on page 22.
Elute the
unamplified library
1. Remove the plate containing the Ion AmpliSeq™ library from the magnet, and
add 50 µL of Low TE to the pellet to disperse the beads. Seal the plate with
MicroAmp® adhesive film, vortex thoroughly, and spin down to collect droplets.
Alternatively, mix by pipetting at least half the total volume up and down at least
5 times prior to sealing the plate.
2. Place the plate in the magnet for at least 2 minutes. The supernatant containts the
library. Prepare a 100-fold dilution by removing 5 µL and combining with 495 µL
of Nuclease-free Water for quantitation.
Quantify the library
by qPCR and
calculate the
dilution factor
Determine the concentration of each Ion AmpliSeq™ library by qPCR with the Ion
Library Quantitation Kit using the steps below. Each sample, standard, and negative
control should be analyzed in duplicate 20-µL reactions.
Unamplified libraries typically have yields of 100–500 pM.
1. Prepare three 10-fold serial dilutions of the E. coli DH10B Ion Control Library
(~68 pM; from the Ion Library Quantitation Kit) at 6.8 pM, 0.68 pM, and 0.068 pM.
Mark these as standards and use these concentrations in the qPCR instrument
software.
2. Prepare reaction mixtures. For each sample, control, and standard, combine 20 µL
of 2X TaqMan™ MasterMix and 2 µL of 20X Ion TaqMan™ Assay and mix
thoroughly. Dispense 11-µL aliquots into the wells of a PCR plate.
3. Add 9 µL of the diluted (1:100) Ion AmpliSeq™ library or 9 µL of each control
dilution to each well (two wells per sample as noted before), for a total reaction
volume of 20 µL.
4. Program your real-time instrument as follows:
• Enter the concentrations of the control library standards.
• Use ROX™ Reference Dye as the passive reference dye.
• Select a reaction volume of 20 µL.
• Select FAM™ dye/MGB as the TaqMan™ probe reporter/quencher.
• The Ion Library TaqMan™ qPCR Mix can be used on a variety of
instruments, as listed below. The fast cycling program was developed using
the StepOnePlus™ System in Fast mode.
20
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Chapter 2 Methods
Option 2: Quantify the unamplified library by qPCR
Real-Time PCR System
7900 HT System
7900 HT Fast System (Fast 96Well, Standard 96-Well, or 384Well Block Modules)
Stage
Temp
Time
Hold (optional)
50°C
2 min
Hold
95°C
20 sec
Cycle (40 cycles)
95°C
1 sec
60°C
20 sec
Hold (optional)
50°C
2 min
Hold
95°C
20 sec
Cycle (40 cycles)
95°C
3 sec
60°C
30 sec
ViiA™ 7 System
2
StepOne™ System
StepOnePlus™ System
7500 Fast System
7500 System
5. Following qPCR, calculate the average concentration of the undiluted Ion
AmpliSeq™ library by multiplying the concentration determined with qPCR by
100.
6. Based on the calculated library concentration, determine the dilution that results
in a concentration of ~100 pM.
For example:
• The undiluted library concentration is 300 pM.
• The dilution factor is 300 pM/100 pM = 3.
• Therefore, 10 µL of library mixed with 20 µL of Low TE (1:3 dilution) yields
approximately 100 pM.
7. Dilute library to ~100 pM as described and proceed to combining libraries or
template preparation.
(Optional) Combine
amplicon libraries
Up to 3 barcoded exome libraries can be combined and run on a single Ion PI™ chip,
depending on the coverage depth desired.
Store libraries
Libraries may be stored at 4–8°C for up to 1 month. For longer term storage, store at
–20°C.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
21
2
Chapter 2 Methods
Option 3: Quantify the amplified library with the Qubit™ 2.0 Fluorometer or Agilent™ 2100 Bioanalyzer™ instrument
Option 3: Quantify the amplified library with the Qubit™ 2.0
Fluorometer or Agilent™ 2100 Bioanalyzer™ instrument
Ion AmpliSeq™ libraries must be amplified prior to quantitation to enrich amplifiable
material and obtain sufficient material for accurate quantitation. Amplify the library
using 1X Library Amp Mix, then purify each library. Quantify the library using the
Qubit™ 2.0 Fluorometer or the Agilent™ 2100 Bioanalyzer™ instrument.
Note: The Ion Library Quantitation Kit may also be used to quantify libraries that have
been amplified using the procedure described in “Amplify the library” on page 22.
After quantitation, determine the dilution factor that results in a concentration of
~100 pM (22 ng/mL for the Qubit™ 2.0 Fluorometer).
Amplify the library
1. Remove the plate containing the Ion AmpliSeq™ library from the magnet and add
50 µL of 1X Library Amp Mix and 2 µL of 25X Library Amp Primers to each bead
pellet. The Library Amp Mix and primers may be combined before addition.
2. Seal the plate with MicroAmp® adhesive film, vortex thoroughly, and spin down
to collect droplets. Alternatively, mix by pipetting at least half the total volume up
and down at least 5 times prior to sealing the plate.
Note: Library amplification takes place in the presence of the AMPure™ XP
beads.
3. Place a MicroAmp™ Compression Pad on the plate, load in the thermal cycler,
and run the following program.
Stage
Temperature
Time
Hold
98°C
2 min
5 cycles
98°C
15 sec
64°C
1 min
10°C
Hold
Hold
4. Proceed to “Purify the amplified library” below.
STOPPING POINT
22
Samples may be stored at –20°C.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Chapter 2 Methods
Option 3: Quantify the amplified library with the Qubit™ 2.0 Fluorometer or Agilent™ 2100 Bioanalyzer™ instrument
Purify the amplified
library
2
Perform a two-round purification process with Agencourt™ AMPure™ XP Reagent.
• First round at 0.5X bead-to-sample-volume ratio: High molecular-weight DNA is
bound to beads, while amplicons and primers remain in solution. Save the
supernatant.
• Second round at 1.2X bead-to-original-sample-volume ratio: Amplicons are
bound to beads, and primers remain in solution. Save the bead pellet, and elute
the amplicons from the beads.
IMPORTANT! Bring AMPure™ XP reagent to room temperature and vortex thoroughly
to disperse the beads before use. Pipet solution slowly.
Use freshly prepared 70% ethanol for the next steps. Combine 230 µL of ethanol with
100 µL of nuclease-free water per sample.
Do NOT substitute a Dynabeads®-based purification reagent for the Agencourt®
AMPure® XP reagent.
First-round purification
1. Add 25 µL (0.5X sample volume) of Agencourt™ AMPure™ XP Reagent to each
plate well containing ~50 µL of sample. Pipet up and down 5 times to thoroughly
mix the bead suspension with the DNA, and seal the plate with MicroAmp®
adhesive film.
2. Incubate the mixture for 5 minutes at room temperature.
3. Place the plate in a magnet such as the DynaMag™-96 Side Magnet for at least
5 minutes or until the solution is completely clear.
4. Carefully transfer the supernatant from each well to a single well of a new 96-well
PCR plate, without disturbing the pellet.
IMPORTANT! The supernatant contains the final library. Do not discard!
Second-round purification
1. To the supernatant from step 4 above, add 60 µL (1.2X original sample volume) of
Agencourt™ AMPure™ XP Reagent. Pipet up and down 5 times to thoroughly
mix the bead suspension with the DNA, and seal the plate with MicroAmp®
adhesive film.
2. Incubate the mixture for 5 minutes at room temperature.
3. Place the plate in the magnet for 3 minutes or until the solution is clear. Carefully
remove and discard the supernatant without disturbing the pellet.
IMPORTANT! The amplicons are bound to the beads. Save the bead pellet.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
23
2
Chapter 2 Methods
Option 3: Quantify the amplified library with the Qubit™ 2.0 Fluorometer or Agilent™ 2100 Bioanalyzer™ instrument
4. Add 150 µL of freshly prepared 70% ethanol to each well and move the plate side
to side in the magnet to wash the beads. Remove and discard the supernatant
without disturbing the pellet.
Note: If your magnet does not have two positions for shifting the beads, remove
the plate from the magnet and gently pipet up and down five times (with the
pipettor set at 100 µL). Return the plate to the magnet and incubate for 2 minutes
or until the solution clears.
5. Repeat step 4 for a second wash.
6. Ensure that all ethanol droplets are removed from the wells. Keeping the plate in
the magnet, air-dry the beads at room temperature for 5 minutes. Do not overdry.
7. Remove the plate from the magnet and add 50 µL of Low TE to the pellet to
disperse the beads. Seal the plate with MicroAmp® adhesive film, vortex
thoroughly, and spin down to collect droplets. Alternatively, mix by setting a
pipettor to 40 µL and pipet the mixture up and down at least 5 times prior to
sealing the plate.
IMPORTANT! The supernatant contains the desired amplicons. Do not discard!
8. Place the plate in the magnet for at least 2 minutes and analyze an aliquot of the
supernatant as described in:
• “Qubit™ 2.0 Fluorometer: Quantify the library and calculate the dilution
factor” or
• “Agilent™ Bioanalyzer™: Quantify the library and calculate the dilution
factor”.
24
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Chapter 2 Methods
Option 3: Quantify the amplified library with the Qubit™ 2.0 Fluorometer or Agilent™ 2100 Bioanalyzer™ instrument
Qubit™ 2.0
Fluorometer:
Quantify the library
and calculate the
dilution factor
2
Analyze 10 µL of each amplified library using the Qubit™ 2.0 Fluorometer (Cat. no.
Q32866) and the Qubit™ dsDNA HS Assay Kit. Amplified libraries typically have
concentrations of 300–3000 ng/mL. For more information, see the Qubit™ dsDNA HS
Assay Kits User Guide.
1. Determine the amplified library concentration:
a. Make a 1:200 working dilution of Qubit™ dsDNA HS reagent using the
Qubit™ dsDNA HS Buffer.
b. Combine 10 µL of the amplified Ion AmpliSeq™ library with 190 µL of dye
reagent, mix well, and incubate for at least 2 minutes.
c. Prepare each Qubit™ standard as directed in the user guide.
d. Measure the concentration on the Qubit™ 2.0 Fluorometer.
e. Calculate the concentration of the undiluted library by multiplying by 20.
This can be calculated automatically using the “Calculate Stock
Concentration” button and inputting 10 µL as the sample volume.
2. Based on the calculated library concentration, determine the dilution that results
in a concentration of ~100 pM (22 ng/mL).
For example:
• The library concentration is 660 ng/mL.
• The dilution factor is 660 ng/mL divided by 22 ng/mL = 30.
• Therefore, 10 µL of library mixed with 290 µL of Low TE (1:30 dilution)
yields approximately 22 ng/mL (~100 pM).
3. Dilute library to ~100 pM as described and proceed to combining libraries or
template preparation.
Agilent™
Bioanalyzer™:
Quantify the library
and calculate the
dilution factor
Analyze 1 µL of amplified library on the Agilent™ Bioanalyzer™ instrument with the
Agilent™ High Sensitivity DNA Kit (Cat. no. 5067-4626). Amplicon libraries should
have multiple peaks in the 125–300 bp size range. Amplified libraries typically have
concentrations of 1,000–10,000 pM. If the library concentration is over 20,000 pM,
dilute the library 1:10 and repeat the quantitation to obtain a more accurate
measurement.
1. Determine the molar concentration of the amplified library using the
Bioanalyzer® software. Ensure that the upper and lower marker peaks are
identified and assigned correctly. Follow the manufacturer’s instructions to
perform a region analysis (smear analysis). Briefly:
a. Select the Data icon in the Contexts panel, and view the electropherogram of
the sample to be quantified.
b. Select the Region Table tab below, and create a region spanning the desired
amplicon peaks. Correct the baseline if needed.
c. The molarity is automatically calculated and displayed in the table in pmol/L
(pM).
2. Based on the calculated library concentration, determine the dilution that results
in a concentration of ~100 pM.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
25
2
Chapter 2 Methods
Option 3: Quantify the amplified library with the Qubit™ 2.0 Fluorometer or Agilent™ 2100 Bioanalyzer™ instrument
For example:
• The library concentration is 3000 pM.
• The dilution factor is 3000 pM/100 pM = 30.
• Therefore, 10 µL of library mixed with 290 µL of Low TE (1:30 dilution)
yields approximately 100 pM.
3. Dilute library to ~100 pM as described and proceed to combining libraries or
template preparation.
(Optional) Combine
amplicon libraries
Up to 3 barcoded exome libraries can be combined and run on a single Ion PI™ chip,
depending on the coverage depth desired.
Store libraries
Libraries may be stored at 4–8°C for up to 1 month. For longer term storage, store at
–20°C.
26
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
3
Ion Chip capacities
for Ion AmpliSeq™
libraries
Combining Ion AmpliSeq™ libraries
Up to 3 barcoded exome libraries can be combined and run on a single Ion PI™ Chip,
depending on the coverage and depth desired. The number of exome libraries that can
be accommodated in a single sequencing run depends on the size of the chip, the
ability to reliably quantify and combine barcoded libraries, and the coverage required.
As the number of libraries on a single chip increases, the average coverage depth
decreases. This relationship is shown in the following table. The numbers below
presume high-accuracy quantitation, and should serve as a guide for approximate
capacities. We suggest determining real limits empirically.
Multiple chips may be run to increase coverage depth. Run data may be combined by
using the CombineAlignments plugin. For detailed instructions on running the
CombineAlignments plugin, see “Running the Installed Plugins” in the Torrent Browser
Analysis Report Guide.
No. of exomes per
Ion PI™ chip
Approximate average
read depth*
1
250X
2
125X
3
83X
* Actual read depth per sample may vary as a result of
barcode balance and chip loading.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
27
A
Tips and Troubleshooting
Tips and modifications to the standard workflow
Tips
• Plate seals can be firmly applied using the applicator in the MicroAmp® Optical
Adhesive Film Kit. Plate seals can be removed with much less effort when hot.
Try removing seals right after taking the plate out of thermal cycler.
• Combine and dilute barcode adapters in large batches and aliquot into 96-well
plates.
• If library yield is below 100 pM, libraries may still be sequenced by using a
proportionally larger volume into a combined library or into template
preparation.
Modifications to the
standard workflow
The following modifications to the standard protocol are designed to allow advanced
users to successfully modify and customize the standard Ion AmpliSeq™ protocol.
These modifications are unsupported and in some cases may decrease performance.
Limited Samples
• DNA from high quality FFPE tissue may be used with Ion AmpliSeq™ Exome.
Uniformity and representation of longer amplicons may decrease.
• When using the Qubit® 2.0 or Bioanalyzer® instrument, amplified libraries with
undetectable product may still be quantified with qPCR (“Option 2: Quantify the
unamplified library by qPCR” on page 23).
Shortcuts
• When using the Bioanalyzer® instrument for quantitation (but not the Qubit® 2.0
Fluorometer), a single round of purification at 1.7X volume (85 µL) may be
substituted for the two-round purification following library amplification
(“Purify the amplified library” on page 26). High molecular weight material will
not interfere with sequencing, but be sure the markers are assigned correctly.
• When using qPCR quantitation, careful removal of ethanol after the final wash
eliminates the need for drying AMPure® XP beads. However, residual ethanol
may inhibit the library amplification reaction.
28
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Appendix A Tips and Troubleshooting
Troubleshooting
A
Troubleshooting
If troubleshooting does not resolve the problem, contact Technical Support.
Library Yield and Quantitation
Observation
Possible cause
Recommended action
Unwashed beads
Be sure to wash Equalizer™ Beads prior to use
Residual salt after wash
Carefully remove all wash solution prior to elution
Less than 100 ng of input DNA
Add more DNA or increase target amplification
cycles to 11 or 12 (“Amplify targets” on page 16)
Inefficient PCR, digestion, or
ligation
Ensure proper dispensing and mixing of viscous
components at each step
Library discarded during
purification of the amplified
library
Be sure to save the supernatant during first-round
purification, and save the bead pellet during the
second round (“Purify the amplified library” on
page 26)
Over-drying of AMPure® XP
beads
Do not dry the AMPure® XP beads more than
5 minutes
Residual ethanol drops inhibiting
library amplification
Carefully remove all drops, using an additional spin
and removal step, if necessary
Mis-quantitation of input DNA
Requantify input DNA using the TaqMan® RNase P
Detection Reagents Kit
AMPure® beads inhibiting library
amplification
Transfer library off of beads prior to amplification
Mis-quantitation of input DNA
Quantify input DNA using the TaqMan® RNase P
Detection Reagents Kit
More than 100 ng of input DNA
Add less DNA or decrease target amplification
cycles to 9 (“Amplify targets” on page 16)
High library yield on the
Bioanalyzer® instrument
Marker mis-assignment
(Figure 1)
Ensure that markers are assigned correctly
High molecular weight
material on the Bioanalyzer®
instrument (Figure 2) or
High molecular weight DNA was
not removed during purification
of the amplified library (does not
interfere with sequencing)
Carefully remove less supernatant in the firstround (0.5X) purification (“First-round purification”
on page 26) and be sure not to disturb bead pellet
Low library yield with the Ion
Equalizer™ Kit
Low library yield—general
High library yield
High library yield on the Qubit®
Fluorometer
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Increase AMPure® XP Reagent volume from 25 to
35 µL (0.5X to 0.7X) in the first-round purification
(“First-round purification” on page 26)
29
A
Appendix A Tips and Troubleshooting
Troubleshooting
Bias in amplicon representation
Observation
Loss of short amplicons
Possible cause
Poor purification
Recommended action
Vortex AMPure® XP Reagent thoroughly before use, and be
sure to dispense the full volume
In unamplified library purification (“Purify the unamplified
library” on page 19), increase AMPure® XP Reagent volume
from 80 to 96 µL (1X to 1.2X)
In amplified library purification (“Purify the amplified library”
on page 26), increase AMPure® XP Reagent volume in
second round from 60 to 70 µL (1.2X to 1.4X)
Denaturation of digested
amplicon
Verify use of the 60°C/20 minute temperature incubation
during the primer digestion step (“Partially digest primer
sequences” on page 18)
Low quality DNA
FFPE DNA is not recommended for Ion AmpliSeq™ Exome
RDY
Too few nucleotide flows
Use at least 500 flows to sequence through exome amplicons
Denaturation of digested
amplicon
Verify use of the 60°C/20 minute temperature incubation
during the primer digestion step
Unknown
Amplicons with >80% AT often exhibit low representation
Loss of GC-rich amplicons
Inadequate denaturation
Use a calibrated thermal cycler
(see Figure 5)
Inefficient library
amplification
Do not amplify the library (not required for qPCR
quantitation)
Inadequate amplification
Increase the anneal/extend temperature of the target
amplification reaction from 60°C to 62°C for the first two
cycles of the target amplification reaction and increase cycle
number if necessary
Unknown
Amplicons with >80% GC often exhibit low representation
Uneven pool
representation
Inaccurate pipetting
Thoroughly mix target amplification master mix and dispense
equal volumes into each well
(see Figure 6)
Inaccurate PCR product
combination
Carefully combine all target amplification products without
changing tips. Centrifuge plate and transfer residual
droplets, if needed.
Loss of long amplicons
Loss of AT-rich amplicons
(see Figure 4)
30
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Appendix A Tips and Troubleshooting
Troubleshooting
A
Other
Observation
Adapter dimers on the
Bioanalyzer® instrument
at 90–105 bp (see
Figure 3,)
Possible cause
Inefficient purification
In unamplified library purification (“Purify the unamplified
library” on page 19), decrease AMPure® XP Reagent
volume from 80 to 60 µL (1X to 0.75X)
In amplified library purification (“Purify the amplified
library” on page 26), decrease AMPure® XP Reagent
volume in second round from 60 to 50µL (1.2X to 1X)
or
Adapter dimers during
sequencing
Recommended action
Adapter dimer formation
Do not combine Adapters, DNA ligase, and Switch
solution prior to addition
Use a 65°C temperature incubation instead of 60°C
during the primer digestion step (“Partially digest primer
sequences” on page 18).
Adapter concentration too high
Ensure that barcode adapters are diluted properly
Lower-than-expected
number of on-target reads
Unknown
Increase the number of target amplification cycles by 10
to 12, and/or increase the anneal/extend temperature of
the target amplification reaction from 60°C to 62°C for the
first two cycles of the target amplification reaction
Uneven barcode
representation
Inaccurate library quantitation
Use the Ion Library Quantitation Kit for the most specific
and accurate library quantitation
Inaccurate library combination
Dilute libraries to 100 pM, then combine equal volumes
Overseeding of library
Decrease amount of library added to the template
preparation reaction by 50%
Library mis-quantitation
Ensure that library was quantified correctly
Other
Check the appropriate template preparation user guide
for more information.
Underseeding of library
Double the volume of library used in template prep
High polyclonal ISPs
(>40%)
High low quality ISPs
(>15%)
Use a fresh dilution of library prepared in a low-bind tube
Library mis-quantitation
Ensure that library was quantified correctly
Other
Check the appropriate template preparation user guide
for more information.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
31
A
Appendix A Tips and Troubleshooting
Troubleshooting
Figure 1: Marker mis-assignment. Marker mis-assignment on the Agilent® 2100
Bioanalyzer® instrument. When the upper marker is assigned to the incorrect peak,
library yield may be overestimated by more than 10-fold.
Figure 2: High molecular weight material in an Ion AmpliSeq™ Exome library. High
molecular weight material on Bioanalyzer® instrument. DNA outside the region
window will not interfere with template preparation or sequencing, but may lead to an
overestimation of library concentration when using the Qubit® 2.0 Fluorometer for
library quantitation.
32
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Appendix A Tips and Troubleshooting
Troubleshooting
A
Figure 3: Adapter dimers. Standard adapters produce peaks at ~43 and ~53 bp, while
adapter dimers run at ~90 bp. Barcode adapters run at ~53 bp, and barcode adapter
dimers run at ~105 bp.
Figure 4: Example of loss of AT-rich amplicons. Within the Coverage Analysis
Plugin, amplicon representation is plotted by GC content for the Ion AmpliSeq™
Exome Panel. Amplicons with 23-50% GC content show an excess failure rate (less
than 20% of the mean read depth).
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
33
A
Appendix A Tips and Troubleshooting
Troubleshooting
Figure 5: Example of loss of GC-rich amplicons. Within the Coverage Analysis
Plugin, amplicon representation is plotted by GC content for the Ion AmpliSeq™
Exome Panel. Amplicons with 50-80% GC content show an excess failure rate (less
than 20% of the mean read depth).
Figure 6: Example of pool imbalance. Within the Coverage Analysis Plugin, mean
read depth per primer pool is plotted for Ion AmpliSeq™ Exome. In this example, pool
1 has approximately half the reads of other pools.
34
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
B
Supplemental information
Data analysis
Visit the Ion Community at http://ioncommunity.lifetechnologies.com and select
Products Systems and Software  Torrent Suite to access the latest user guides and
information for Torrent Suite software.
Torrent Suite is required for sequence analysis of libraries prepared with Ion
AmpliSeq™ Primer Pools. The software includes the Torrent Variant Caller Plugin,
which can be used to call single nucleotide polymorphism (SNP) and insertion/
deletion (InDel) variants within the genomic regions covered by the Ion AmpliSeq™
Primer Pools.
To enable variant calling:
• Install the hg19 human genome reference on your Torrent Server, if it is not
already installed.
• Upload the BED files for the Ion AmpliSeq™ Primer Pool target regions to your
Torrent Server, or use the analysis plan available at AmpliSeq.com.
Install the hg19
reference on your
Torrent Server
Brief instructions for installing the hg19 reference on your Torrent Server are provided
below. Detailed instructions are provided under “Adding a Reference Sequence” in the
Torrent Browser User Interface Guide.
1. Download the FASTA file from http://updates.iontorrent.com/reference/
hg19.zip.
2. In the Torrent Browser, click the Settings button on the right side of the screen and
select References.
3. Click on Add Reference Sequence and enter the information in the fields. In the
Short Name field, the short form of the genome name must be entered as hg19.
• The genome index creation will take a few hours.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
35
B
Appendix B Supplemental information
Data analysis
Import the BED
files into your
Torrent Server
Browser Extensible Data (BED) files supply chromosome regions and restrict analysis
to the regions in the designated Ion AmpliSeq™ panel.
These two types of BED files are frequently used:
• Designed BED file - Specifies the amplified regions that are used with targeted
sequencing.
• Hotspot BED file - Specifies regions of known mutations, for example from
COSMIC or dbSNP databases or from customer-defined regions. A hotspot file is
optional. Positions in a hotspot file will always be present in the VCF file and in
the variant table in Torrent Suite™ software and Ion Reporter™. They may have a
variant present, or they may be reference, or no-call. All alleles present at this
position, with the number of reads of each allele, will always be shown at Hotspot
positions. In addition, Hotspots can be called with different thresholds (for
example, lower allele frequency).
To import and download the BED files you require, follow the steps listed in “Create a
Template with AmpliSeq.com Import” in the Torrent Browser User Interface Guide.
Coverage Analysis
Plugin
The Coverage Analysis Plugin provides statistics and graphs describing the level of
sequence coverage produced for targeted genomic regions.
You can run the Coverage Analysis Plugin automatically or manually.
Option 1: Include the Coverage Analysis Plugin in a run plan
To run the Coverage Analysis Plugin automatically, select the Coverage Analysis
plugin during template setup. Refer to the “Plan Tab” and “Templates” sections of the
Torrent Browser User Interface Guide for information about how to set up a template and
create a planned run.
Option 2: Manually launch the Coverage Analysis Plugin
To run the Coverage Analysis Plugin manually:
1. Open your run report on the Torrent Browser.
2. Scroll to the Plugin Summary area. Click Select plugins to run. The Select a
plugin popup appears:
3. Select CoverageAnalysis to launch the Coverage Analysis Plugin interface.
36
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Appendix B Supplemental information
Data analysis
B
4. Select a Library Type: either “Ion AmpliSeq” or “Ion AmpliSeq Exome.”
5. Select your designed BED file in the “Targeted Regions” pull-down menu.
6. Check the boxes for the remaining options if desired:
• Barcode-specific Targets - If you are running multiple Ion AmpliSeq™
libraries created from different primer pools, you can select barcode-specific
target BED files to analyze against.
• Use Only Uniquely Mapped Reads - If you would like the plugin to
examine only reads that map preferably to a single location, check this box.
7. When you are satisfied with your selections, click Submit.
Coverage Analysis Report
The run report will include basic run metrics, but more detail can be found by clicking
on the coverageAnalysis.html link, including options to download a barcode summary
report, and an amplicon coverage matrix.
More detailed information can be obtained for each barcoded library by clicking the
Barcode ID.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
37
B
Appendix B Supplemental information
Data analysis
At the top of the Coverage Analysis Report you will find a table that shows amplicon
statistics on the left side and base statistics on the right side. All metrics are defined
with flyover explanations. The plugin run options may be reviewed as flyover help on
the report title “Coverage Analysis Report.”
The Representation Plots (which are revealed by clicking on the triangle in the blue
bar) allow you to visualize amplicon representation by GC content and length. If the
designed BED file defines multiple primer pools, a mean target depth per primer pool
representation plot is also available.
In the example below, pool 1 is under represented:
The Amplicon Coverage Chart shows amplicons binned by representation, low to
high, with a variety of overlays. You can zoom into this graph to see how many
amplicons are in each bin, and click on a bar to get more information about that group
of amplicons.
38
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Appendix B Supplemental information
Data analysis
B
By clicking on the magnifying glass in the upper right corner of the Amplicon
Coverage Chart, you can change and filter what is shown on the chart by number of
reads, chromosome, or gene.
The Reference Coverage Chart shows the strand-specific coverage in red and green for
each amplicon. The example below shows the coverage for each exon of the TP53 gene:
Finally, at the bottom of the Coverage Analysis Report you will find links to download
the data in this report as well as the BAM and BAI files required for IGV.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
39
B
Appendix B Supplemental information
Data analysis
Torrent Variant
Caller Plugin
For detailed instructions on running the Torrent Variant Caller, refer to "Running the
Torrent Variant Caller Plugin" in the Torrent Browser Analysis Report Guide. The Torrent
Variant Caller (TVC) Plugin calls single-nucleotide polymorphisms (SNPs), multinucleotide polymorphisms (MNPs), insertions, and deletions in a sample across a
reference or within a targeted subset of that reference.
This plugin provides optimized pre-set parameters for many experiment types, but is
also very customizable. After you find a parameter combination that works well on
your data and that has the balance of specificity and sensitivity that you want, you can
save that parameter set and reuse it over and over in your research. This is supported
on both manual launches of the plugin and in automatic launches through the run plan
template wizard.
Note: The TVC plugin run uses the same target regions file and hotspots file as the
main Torrent Suite™ Software analysis (if those files are present in the main analysis).
Through the run plan wizard there is no capability in the TVC configuration to change
the target regions file or hotspots file. You can use a different target regions file and
hotspots file with a manual TVC launch from a completed run report.
Parameters
TVC provides several ways of handling its parameter options:
You can import parameter settings that are optimized for fixed panels and community
panels from AmpliSeq.com (click on Panel Files—TVC settings are .json files).
• You can select one of TVC default pre-set parameter groups or customize
them in the TVC UI. The parameters used in a TVC run can also be
downloaded and modified, or reused in future TVC runs.
TVC's default parameters setting groups are organized according to these attributes:
• Variant frequency - Somatic settings are optimized to detect low frequency
variants. Germ-line settings are optimized for high frequency settings.
• Sequencing instrument - The Ion PGM™ or the Ion Proton™ sequencer.
Parameter defaults are different for Ion Proton™ data than for Ion PGM™
data.
• Stringency - High stringency settings are optimized to minimize false
positives. Low stringency settings minimize false negatives.
Run the Torrent Variant Caller Plugin
There are two ways to run the TVC Plugin:
• Automatically, by preconfiguring the plugin during the run planning or
• Manually, allowing you to run the plugin at any time from a completed run
report.
40
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Appendix B Supplemental information
Data analysis
B
1. When you select the Plugins chevron in the template or Run Plan Wizard and
enable the variantCaller checkbox, a Configure link appears:
2. Click the Configure link to open the variantCaller configuration popup:
3. Select one of the pre-defined parameter settings or upload your own custom
settings file (see Ampliseq.com for the best settings for the panel you are using).
Torrent Variant Caller Plugin output
The TVC plugin output includes the following reports and sections:
• Run report plugin summary
• Variant Caller Report
• Variant Caller Report summary section
• Variant Caller Report variant calls table
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
41
B
Appendix B Supplemental information
Data analysis
Copy number
variation analysis
using Ion
Reporter™
Software
Ion Reporter™ Software comprises a suite of bioinformatics tools that streamlines and
simplifies data analysis, annotation, and reporting of Ion semiconductor sequencing
data. Ion Reporter™ Software helps you to interpret DNA variants faster and more
consistently.
The software allows you to integrate your variants with comprehensive public and
curated annotations, along with your own lab-specific content. Additionally, analysis
modules and parameters are configurable, allowing you to customize workflows as
needed.
All the steps, from data import to annotating variants, are automated in the Ion
Reporter™ Software workflow. Key features include the following:
• Detection of SNPs and Indels
• Detection of Copy Number Variants (CNVs) on Fixed Ion AmpliSeq™ panels
and on most Custom Ion AmpliSeq™ panels
• Tumor : normal analyses
• Multi-sample analyses, including:
– Tumor : normal workflow to detect somatic variants present only in a
Tumor samples and not in its matched Normal sample.
– Genetic Disease Trio workflow, to identify, annotate, and filter variants
of special interest in Trio analysis (Sample, Father and Mother)
See http://ioncommunity.lifetechnologies.com/community/products/ion_reporter for
more information about Ion Reporter™ Software.
42
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Appendix B Supplemental information
Data analysis
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
B
43
C
Safety
Chemical safety
WARNING! GENERAL CHEMICAL HANDLING. To minimize hazards,
ensure laboratory personnel read and practice the general safety guidelines for
chemical usage, storage, and waste provided below, and consult the relevant
SDS for specific precautions and instructions:
·Read and understand the Safety Data Sheets (SDSs) provided by the chemical
manufacturer before you store, handle, or work with any chemicals or
hazardous materials. To obtain SDSs, see the “Documentation and Support”
section in this document.
·Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing).
·Minimize the inhalation of chemicals. Do not leave chemical containers open.
Use only with adequate ventilation (for example, fume hood).
·Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the
manufacturer's cleanup procedures as recommended in the SDS.
·Handle chemical wastes in a fume hood.
·Ensure use of primary and secondary waste containers. (A primary waste
container holds the immediate waste. A secondary container contains spills
or leaks from the primary container. Both containers must be compatible
with the waste material and meet federal, state, and local requirements for
container storage.)
·After emptying a waste container, seal it with the cap provided.
·Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
·Ensure that the waste is stored, transferred, transported, and disposed of
according to all local, state/provincial, and/or national regulations.
·IMPORTANT! Radioactive or biohazardous materials may require special
handling, and disposal limitations may apply.
44
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
Appendix C Safety
Biological hazard safety
C
Biological hazard safety
WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,
infectious agents, and blood of humans and other animals have the potential to
transmit infectious diseases. Follow all applicable local, state/provincial, and/or
national regulations. Wear appropriate protective equipment, which includes
but is not limited to: protective eyewear, face shield, clothing/lab coat, and
gloves. All work should be conducted in properly equipped facilities using the
appropriate safety equipment (for example, physical containment devices).
Individuals should be trained according to applicable regulatory and company/
institution requirements before working with potentially infectious materials.
Read and follow the applicable guidelines and/or regulatory requirements in
the following:
In the U.S.:
·U.S. Department of Health and Human Services guidelines published in
Biosafety in Microbiological and Biomedical Laboratories found at:
www.cdc.gov/biosafety
·Occupational Safety and Health Standards, Bloodborne Pathogens
(29 CFR§1910.1030), found at: www.access.gpo.gov/nara/cfr/waisidx_01/
29cfr1910a_01.html
·Your company’s/institution’s Biosafety Program protocols for working with/
handling potentially infectious materials.
·Additional information about biohazard guidelines is available at:
www.cdc.gov
In the EU:
Check local guidelines and legislation on biohazard and biosafety precaution
and refer to the best practices published in the World Health Organization
(WHO) Laboratory Biosafety Manual, third edition, found at: www.who.int/
csr/resources/publications/biosafety/WHO_CDS_CSR_LYO_2004_11/en/
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
45
D
Documentation and Support
Customer and technical support
Visit thermofisher.com/support for the latest in services and support, including:
• Worldwide contact telephone numbers
• Product support, including:
– Product FAQs
– Software, patches, and updates
• Order and web support
• Product documentation including:
– User guides, manuals, and protocols
– Certificates of Analysis
– Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers,
contact the manufacturer.
Limited Product Warranty
Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth
in the Life Technologies' General Terms and Conditions of Sale found on Life
Technologies’ website at www.lifetechnologies.com/termsandconditions. If you have
any questions, please contact Life Technologies at www.lifetechnologies.com/support.
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
46
Appendix D Documentation and Support
Limited Product Warranty
Ion AmpliSeq™ Exome RDY Library Preparation User Guide
D
47
For support visit thermofisher.com/support or email techsupport@lifetech.com
thermofisher.com
2 September 2015
49
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