Thermo Scientific Ion 520 User manual

For Research Use Only. Not for use in diagnostic procedures.
Ion 520™& Ion 530™ExT Kit – Chef
USER GUIDE
for use with:
Ion Chef™System
Ion S5™System
Ion S5™XL System
Ion GeneStudio™S5 System
Ion GeneStudio™S5 Plus System
Ion GeneStudio™S5 Prime System
Catalog Number A30670
Publication Number MAN0015805
Revision F.0

Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, California 92008 USA
For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. 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.
Revision history: Pub. No. MAN0015805
Revision Date Description
F.0 24 November 2021 •Updated “Start the Ion Chef™run” to update the recommended concentration in
“Dilute the sample library” on page 25.
•Updated “Start the Ion Chef™run” to update the volume of library in “Dilute the
control library” on page 25.
E.0 25 June 2019 •Updated for Torrent Suite™Software 5.12.
•Separated "Prepare the libraries and consumables" into two topics: “Prepare the
consumables” and “Add the diluted libraries to the Ion Chef™Library Sample
Tube”.
•Updated “Start the Ion Chef™run” to include the Run Options screen for
step‑by‑step Ion Chef™run setup.
•Updated the guidance for storing loaded chips before sequencing in “Unload the
chip for sequencing”.
•Added a note in “Start the Ion Chef™run” and “Unload the chip for sequencing”
to NOT remove any residual liquid from chip wells after the Ion Chef™run.
•Removed Ion Chef™System maintenance procedures from “Maintain the
Ion Chef™System”.
•Updated “Maintain the Ion Chef™System” to add references to the Ion Chef™
System maintenance procedures in Ion Chef™Instrument User Guide.
•Updated “Related documentation” to include the Ion Chef™Instrument User
Guide.
D.0 12 December 2018 •Updated for Ion GeneStudio™S5 Series Systems.
•Updated for Torrent Suite™Software 5.10; Planned Run creation topics
reorganized for ease of use.
•Clarification made to dilute barcoded library pools, if used.
•Added a new troubleshooting topic: “Planned Run status does not advance to
"Planned"”.
•Removed reference to quality control of ISPs using the Attune™NxT Acoustic
Focusing Cytometer.
C.0 22 February 2017 Volume of Ion S5™ExT Calibration Standard provided in ordered item (Cat. No. A30738)
changed from 24 µL to 16 µL.
B.0 26 January 2017 •Number of flows recommended for the instrument reset run (formerly "purge and
clean") procedure changed from 500 to 200, and recommended time to perform
the procedure after sequencer initialization changed from 27 hours to 24 hours
(see “Perform an instrument reset run with an initialized sequencer that is loaded
with an unused Reagents cartridge” on page 66)
•Web links updated
A.0 13 October 2016 New user guide with detailed instructions for use of the Ion 520™& Ion 530™ExT Kit –
Chef with the Ion Chef™and the Ion S5™/Ion S5™XL Systems.
Important Licensing Information: This product may be covered by one or more Limited Use Label Licenses. By use of this product,
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. Guava and
easyCyte are trademarks of Guava Technologies, Inc. Agilent and Bioanalyzer are trademarks of Agilent Technologies, Inc.
©2021 Thermo Fisher Scientific Inc. All rights reserved.

Contents
■CHAPTER 1 Product information .................................................. 6
Product description ............................................................. 6
Library compatibility ......................................................... 6
Ion GeneStudio™S5 Series instrument reference ............................... 6
Software compatibility ....................................................... 7
Kit contents and storage ......................................................... 7
Kit summary ............................................................... 7
Kit components ............................................................ 7
Related products ................................................................ 8
Compatible Ion Chip™kits ................................................... 8
Ion S5™ExT Calibration Standard ............................................. 8
Ion S5™Controls Kit Plus .................................................... 9
E. coli DH10B Control 600 Library ............................................. 9
Required materials not supplied ................................................... 9
About the Ion Chef™System ..................................................... 10
Ion Chef™Instrument components ........................................... 10
Interior hardware and consumables .......................................... 11
About the Ion Chef™System touchscreen interface ............................ 12
Workflow ..................................................................... 13
■CHAPTER 2 Before you begin .................................................... 14
Precautions ................................................................... 14
Avoid nucleic acid contamination ............................................ 14
Avoid chip damage ........................................................ 14
Protection by equipment .................................................... 14
Guidelines for using Ion Chef™consumables and reagents .......................... 15
Prepare the Ion Chef™Instrument for use ......................................... 15
■CHAPTER 3 Create a Planned Run .............................................. 17
About Planned Runs ........................................................... 17
Create a Planned Run .......................................................... 18
Planned Run workflow key fields ................................................. 22
Ion 520™& Ion 530™ExT Kit − Chef User Guide 3

■CHAPTER 4 Start the Ion Chef™run ............................................. 24
Materials required .............................................................. 24
Dilute the library ............................................................... 24
Guidelines for library dilution ................................................ 25
Dilute the sample library .................................................... 25
Dilute the control library .................................................... 25
Prepare the consumables ....................................................... 25
Add the diluted libraries to the Ion Chef™Library Sample Tube ....................... 26
Load the Ion Chef™System ..................................................... 27
Load the tip racks and PCR Plate ............................................ 28
Load the Reagents and Solutions cartridges .................................. 29
Load the Recovery Tubes and Enrichment Cartridge v2 ......................... 31
Load the Chip-loading centrifuge ............................................ 33
Confirm that consumables are correctly installed .............................. 35
Ion Chef™pre-run checklist ................................................. 36
Start the Ion Chef™run ......................................................... 37
Unload the chip for sequencing .................................................. 42
■CHAPTER 5 Initialize the sequencer ............................................ 45
Ion S5™/Ion GeneStudio™S5 Systems component positions ........................ 45
Before you begin .............................................................. 46
When a manual cleaning of the sequencer is required .............................. 46
Initialize the sequencer ......................................................... 47
■CHAPTER 6 Start the sequencing run ........................................... 49
Chip handling guidelines ........................................................ 49
Start the sequencing run ........................................................ 49
Maintain the sequencer ......................................................... 52
Materials required .......................................................... 52
Clean or decontaminate the sequencer ....................................... 52
Reagent consumables disposal .................................................. 52
CO2scrubber removal and disposal .......................................... 52
Recycle Ion S5™ExT Wash Solution and Ion S5™Cleaning Solution bottles ....... 53
■CHAPTER 7 Clean the Ion Chef™System ...................................... 54
About the cleaning protocol ..................................................... 54
Materials required .............................................................. 54
Clean the Ion Chef™Instrument .................................................. 54
Remove and dispose of used consumables ................................... 55
Inspect and clean the Recovery centrifuges and buckets ....................... 57
Start the cleaning ......................................................... 58
Contents
4Ion 520™& Ion 530™ExT Kit − Chef User Guide

■APPENDIX A Troubleshooting .................................................... 60
Ion Chef™System setup and operation ........................................... 60
Ion S5™/Ion GeneStudio™S5 Systems setup and operation ......................... 63
Instrument alarms and events ............................................... 63
Initialization—General errors ................................................ 64
■APPENDIX B Supplemental procedures ........................................ 65
Perform a manual cleaning of the sequencer ...................................... 65
Perform an instrument reset run with an initialized sequencer that is loaded with
an unused Reagents cartridge ................................................. 66
Quality control of Ion Sphere™Particles .......................................... 67
Quality control using the Guava™easyCyte 5 Benchtop Flow Cytometer .......... 67
Maintain the Ion Chef™System .................................................. 67
■APPENDIX C Safety ............................................................... 68
Symbols on this instrument ..................................................... 68
Conformity symbols ........................................................ 69
Location of safety labels on this instrument ........................................ 70
Safety information for instruments not manufactured by Thermo Fisher Scientific ...... 70
Instrument safety .............................................................. 70
General ................................................................... 70
Physical injury ............................................................. 70
Electrical safety ........................................................... 71
Cleaning and decontamination .............................................. 71
Ultraviolet (UV) Safety ...................................................... 71
Safety and electromagnetic compatibility (EMC) standards .......................... 71
Safety compliance ......................................................... 72
EMC ..................................................................... 72
Environmental design ...................................................... 72
Instrument safety — Ion S5™/Ion GeneStudio™S5 Systems ......................... 73
Chemical safety ................................................................ 73
Biological hazard safety ......................................................... 74
■Documentation and support ....................................................... 75
Related documentation ......................................................... 75
Customer and technical support ................................................. 76
Limited product warranty ........................................................ 76
Contents
Ion 520™& Ion 530™ExT Kit − Chef User Guide 5

Product information
■Product description .................................................................... 6
■Kit contents and storage ............................................................... 7
■Related products ...................................................................... 8
■Required materials not supplied ......................................................... 9
■About the Ion Chef™System ........................................................... 10
■Workflow ............................................................................ 13
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product description
The Ion 520™& Ion 530™ExT Kit – Chef (Cat. No. A30670) contains all the reagents and materials that
are required to perform the following steps in the sequencing workflow.
•Prepare enriched, template-positive Ion Sphere™Particles (ISPs) and load them onto an Ion 520™
Chip or Ion 530™Chip using the Ion Chef™Instrument.
•Sequence the loaded chips on an Ion S5™Sequencer, Ion S5™XL Sequencer, or Ion GeneStudio™
S5 Series Sequencer.
The Ion Chef™Instrument uses automated isothermal amplification technology with the Ion 520™&
Ion 530™ExT Kit – Chef to prepare 400 to 600‑base‑read templates with greatly reduced run times
compared to emulsion PCR-based templating. Each kit contains all the supplies that are required
to load and sequence 4 chips. The kit supports 4 single-chip Ion Chef™runs and 4 sequencer
initializations (1 sequencing run/sequencer initialization).
Library compatibility
The Ion 520™& Ion 530™ExT Kit – Chef can be used with up to 400 to 600-base-read libraries of any
type prepared using any available Ion Torrent™-branded library kit.
Ion GeneStudio™S5 Series instrument reference
In this document, Ion GeneStudio™S5 Series Sequencer or Ion GeneStudio™S5 Series System refers
generically to the following systems, unless otherwise specified.
•Ion GeneStudio™S5 System (Cat. No. A38194)
•Ion GeneStudio™S5 Plus System (Cat. No. A38195)
•Ion GeneStudio™S5 Prime System (Cat. No. A38196)
1
6Ion 520™& Ion 530™ExT Kit − Chef User Guide

Software compatibility
The Ion 520™& Ion 530™ExT Kit – Chef is compatible with Torrent Suite™Software v5.2.1 and later. Be
sure to update your Ion Torrent™Server to the latest available version of Torrent Suite™Software before
using this kit.
Kit contents and storage
IMPORTANT!
·Do not substitute components from any other Ion sequencing kits. We verified this protocol using
these specific materials. Substitution can adversely aect system performance.
·Store all consumables and cartridges under the recommended conditions and in an upright position.
Do NOT store the Ion S5™ExT Sequencing Reagents (Part No. A30522) on dry ice or in a closed
environment where dry ice is present.
Each Ion 520™& Ion 530™ExT Kit –
Chef contains all the supplies that are required to prepare and
sequence 4 Ion 520™Chips or Ion 530™Chips. Cat. No. A30670 supports 4 single-chip Ion Chef™
runs and 4 sequencer initializations (1 sequencing run/sequencer initialization) for 400 to 600‑base‑read
libraries.
On arrival, inspect all consumables and contact Technical Support if any of the products have been
damaged during shipping.
Kit summary
Component Part No. Quantity per kit
Ion S5™ExT Chef Supplies A30673 4 boxes
Ion S5™Chef Solutions A27754 1 box
Ion S5™ExT Chef Reagents A30672 1 box
Ion S5™ExT Sequencing Solutions A30521 1 box
Ion S5™ExT Sequencing Reagents A30522 1 box
Kit components
Contents Amount / box Storage
Ion S5™ExT Chef Supplies (Part No. A30673)
Chip Adapter 1 15°C to 30°C
Enrichment Cartridge v2 1
Ion S5™ExT Tip Cartridge 1
PCR Plate and Frame Seal v2 1 each
Chapter 1 Product information
Kit contents and storage 1
Ion 520™& Ion 530™ExT Kit − Chef User Guide 7

Contents Amount / box Storage
Recovery Station Disposable Lid v2 15°C to 30°C2
Recovery Tube 12
Ion S5™Chef Solutions (Part No. A27754)
Ion S5™Chef Solutions 4 cartridges 15°C to 30°C
Ion S5™ExT Chef Reagents (Part No. A30672)
Ion S5™ExT Chef Reagents 4 cartridges –30°C to –10°C
Ion S5™ExT Sequencing Solutions (Part No. A30521)
Ion S5™ExT Wash Solution 4 × 1.5 L 15°C to 30°C
Ion S5™Cleaning Solution 250 mL
Ion S5™ExT Sequencing Reagents (Part No. A30522)
Ion S5™ExT Sequencing Reagents 4 cartridges –30°C to –10°C
IMPORTANT! Do not store the Ion S5™ExT Sequencing Reagents (Part No. A30522) on dry ice or in a
closed environment containing dry ice.
Related products
Compatible Ion Chip™kits
The following Ion Chip™kits, ordered separately, are compatible with the Ion 520™& Ion 530™ExT Kit –
Chef.
Description Cat. No. Quantity Storage
Ion 520™Chip Kit (4-pack) A27761 4 chips 15°C to 30°C
Ion 530™Chip Kit (4-pack) A27763 4 chips
Ion S5™ExT Calibration Standard
For de novo sequencing applications that do not include a reference BAM file, the Ion S5™ExT
Calibration Standard can be added to libraries to increase base-calling accuracy. The Ion S5™ExT
Calibration Standard (Cat. No. A30738) is ordered separately.
Contents Amount Storage
Ion S5™ExT Calibration Standard 16 µL –30°C to –10°C
Chapter 1 Product information
Related products
1
8Ion 520™& Ion 530™ExT Kit − Chef User Guide

Ion S5™Controls Kit Plus
The Ion S5™Controls Kit Plus (Cat. No. A30729) is ordered separately. See the Ion S5™Controls Kit Plus
Product Information Sheet (Pub. No. MAN0016206) for details.
Contents Amount Storage
Human CEPH Genomic DNA Control (red cap) 30 µL −30°C to −10°C
Human CEPH Control 200 Library (yellow cap) 12 µL
E. coli DH10B Control 400 Library (orange cap) 16 µL
E. coli DH10B Control 600 Library
The E. coli DH10B Control 600 Library includes sucient material for 4 templating runs on the
Ion Chef™System. The E. coli DH10B Control 600 Library (Cat. No. A32470) is ordered separately.
Contents Amount Storage
E. coli DH10B Control 600 Library (lilac cap) 20 µL –30°C to –10°C
Required materials not supplied
Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that
the material is available from fisherscientific.com or another major laboratory supplier.
Item Source
Ion Chef™System 4484177
Microcentrifuge[1] MLS
2‑, 20‑, 200‑, and 1,000‑µL pipettes and appropriate filtered tips MLS
Microcentrifuge tubes, 1.5-mL or 1.7-mL MLS
Vortex mixer with a rubber platform MLS
Gloves, powder-free nitrile MLS
Ice buckets and ice —
Nuclease-free water, molecular biology grade MLS
Isopropyl alcohol, 70% solution MLS
Wipes, disposable lint-free MLS
(Optional) Uninterruptible Power Supply (UPS) [2] MLS
[1] Must fit standard 0.2- and 1.5‑mL microcentrifuge tubes and generate 21,000 × g. To convert the RPMs of your centrifuge to RCF in
units of gravity, see tools.thermofisher.com/content/sfs/brochures/TR0040-Centrifuge-speed.pdf.
[2] For laboratories that experience frequent power outages or line voltage fluctuations, we recommend that you use an uninterruptible
power supply that is compatible with 2500 W output or higher.
Chapter 1 Product information
Required materials not supplied 1
Ion 520™& Ion 530™ExT Kit − Chef User Guide 9

About the Ion Chef™System
The Ion Chef™System (Cat. No. 4484177) provides automated, high-throughput template preparation
and chip loading for use with an Ion S5™Sequencer, Ion S5™XL Sequencer, or Ion GeneStudio™S5
Series Sequencer. The system uses a set of cartridge-based consumables and reagents that enables
a user to load an Ion 520™Chip, or an Ion 530™Chip in about 7 hours with less than 15 minutes
of hands-on time. The Ion Chef™System features network integration with Torrent Suite™Software to
enable sample and reagent traceability throughout the chip preparation workflow.
Ion Chef™Instrument components
The following figure illustrates the major external and internal features of the Ion Chef™Instrument.
4
1 2 3
5 6 7
8
1Door – Provides access to the interior of the
instrument. The door is locked in the closed position
during operation.
2Micropipettor – A mechanical positive-displacement
pipettor that performs all fluid transfers during sample
and chip preparation.
3Robotic arm – Enables fluid transfer by the
Micropipettor. The arm also contains an optical sensor
that reads the barcodes of instrument reagents and
consumables.
4Touchscreen – Provides access to all instrument
functions for operation, maintenance, and
troubleshooting.
5Power button – Power switch for the Ion Chef™
Instrument, where the states are on (illuminated) and
o.
6Power port – A 100–240 VAC port that provides power
to the Ion Chef™Instrument.
7Ethernet port – An RJ45 port that provides Ethernet
(100 Mbit) communication with the Ion Chef™
Instrument.
8USB port – Provides USB communication with the
Ion Chef™Instrument. Used to update the instrument
firmware and to transfer data during service or
maintenance.
Chapter 1 Product information
About the Ion Chef™System
1
10 Ion 520™& Ion 530™ExT Kit − Chef User Guide

Interior hardware and consumables
The following figure illustrates the interior of the Ion Chef™Instrument and describes the stations
involved in the preparation of chips for sequencing on an Ion S5™Sequencer, Ion S5™XL Sequencer, or
Ion GeneStudio™S5 Series Sequencer.
A
B
C
D
E
F
G
H
1 2 3 4 56 7 8 9 10 11 12
530 DABD02226
Ion Chef™
S5 Series
Chip Balance
1
5
2 3
4
6
7
9 8
1New pipette tips – The position of the rack containing unused pipette tips.
2Automated heated cover – Transfers the plate cover to the PCR reaction plate within the sample block. During
thermal cycling, the heated cover applies compression to seal the reaction plate and heats the cover to prevent
condensation.
3Waste pipette tip rack – The position of the rack containing waste (used) pipette tips.
4Thermal cycler sample block – Performs thermal cycling of the sequencing reactions on a 96-well PCR reaction
plate.
5Reagents station – The position on the instrument deck of the diluted libraries, NaOH, and the Ion S5™ExT Chef
Reagents cartridge.
6Solutions station – The position on the instrument deck of the Ion S5™Chef Solutions cartridge, which is maintained
at room temperature.
7Recovery centrifuges – Twin stations that perform centrifugation of the ISPs during the recovery phase of template
preparation.
8Enrichment station – The position of the rack containing consumables for the enrichment of the template-positive
ISPs.
9Chip-loading centrifuge – Performs centrifugation of the sequencing chip that has been mounted to a chip-loading
adapter and loaded with template-positive ISPs, and a chip balance loaded opposite to the sequencing chip.
Chapter 1 Product information
About the Ion Chef™System 1
Ion 520™& Ion 530™ExT Kit − Chef User Guide 11

About the Ion Chef™System touchscreen interface
The Ion Chef™System features a simple interface for loading chips, cleaning the instrument, and
performing system maintenance and configuration tasks.
2
1
3 4 5
1Set up run button – Set up the Ion Chef™template preparation and chip-loading routine. Choose Step by Step to
have the instrument lead you stepwise through installation of reagents and consumables, or choose Quick Start to
proceed if you have already installed the consumables.
2Open Door button.
3Notifications button – View notifications about instrument status during and between runs.
4Quick Start button – Proceed directly to the Quick Start instrument setup mode. User verifies the loading of a new
pipette tip cartridge and an empty pipette tip rack to hold waste tips generated during the run, before proceeding to
Deck Scan.
5Settings button – Advance to a screen providing the following options:
•Notifications: view notifications about instrument status during and between runs
•Instrument Settings: view current settings and network configuration, set instrument name, adjust time zone
•Service tools: access screens for service-related maintenance and instrument diagnostics
•Torrent Server: add and manage Ion Torrent™Server connections
•Clean Ion Chef: proceed directly to the instrument cleaning routine
•Check for updates: check availability of system software updates
Chapter 1 Product information
About the Ion Chef™System
1
12 Ion 520™& Ion 530™ExT Kit − Chef User Guide

Workflow
The following workflow illustrates how to prepare and a load sample using the Ion 520™& Ion 530™ExT
Kit – Chef, and Ion Chef™System for sequencing on an Ion S5™Sequencer, Ion S5™XL Sequencer, or
Ion GeneStudio™S5 Series Sequencer.
Prepare and load a sample
Create a planned run (see page 18)
Dilute the sample library (see page 25)
Prepare the consumables (see page 25)
Load the Ion Chef™System (see page 27)
Start the Ion Chef™run (see page 37)
Unload the chip for sequencing (see page 42)
Initialize the sequencer (see page 47)
Start the sequencing run (see page 49)
Chapter 1 Product information
Workflow 1
Ion 520™& Ion 530™ExT Kit − Chef User Guide 13

Before you begin
■Precautions .......................................................................... 14
■Guidelines for using Ion Chef™consumables and reagents ................................ 15
■Prepare the Ion Chef™Instrument for use ............................................... 15
Precautions
Avoid nucleic acid contamination
IMPORTANT! A primary source of contamination is spurious DNA fragments from previous sample
processing steps. Do not introduce amplified DNA into the library preparation laboratory or work area.
Avoid chip damage
IMPORTANT! To avoid possible damage to the chip due to electrostatic discharge, ground yourself
before picking up a chip or placing a chip on a surface such as a lab bench. For example, touch the
metal trim on the chip compartment before inserting or removing a chip from the chip clamp.
Protection by equipment
WARNING! The protection that is provided by the equipment can be impaired if the instrument is
operated outside the environment and use specifications, the user provides inadequate maintenance,
or the equipment is used in a manner that is not specified by the manufacturer (Thermo Fisher
Scientific).
2
14 Ion 520™& Ion 530™ExT Kit − Chef User Guide

Guidelines for using Ion Chef™consumables and reagents
To ensure the proper function of the Ion Chef™System, handle the associated consumables and
reagents according to the following guidelines:
•Store all consumables and cartridges under the recommended conditions and in an upright
position.
•Inspect all consumables and cartridges for damage upon arrival and again before use.
•Hold sequencing chips by gently gripping them by their edges. Do not wear gloves when handling
chips.
•When the instrument is not in use, remove all consumables and reagents from the deck and close
the instrument door.
•Except for the new Ion S5™ExT Tip Cartridge, do not reuse any of the consumables or reagents.
After each run, transfer the empty Ion S5™ExT Tip Cartridge to the waste tip station for use as the
waste container for the next run.
IMPORTANT! All Ion Chef™Instrument components are single-use only.
•Use only Ion Torrent™kits and supplies with the Ion Chef™Instrument. The use of third-party
reagents and supplies can adversely aect the performance of the Ion Chef™Instrument and chips
prepared.
•Remove and sequence chips within 1 hour after the Ion Chef™System finishes loading them. If you
cannot sequence a loaded chip immediately, store it within a chip storage container at 4°C until
you are ready to run it (up to 6–8 hours maximum).
IMPORTANT! If you choose to store a loaded chip, remove the chip from 4°C storage (but keep
it in the chip storage container) at least 20 minutes before running it, allowing the chip to warm to
room temperature.
Prepare the Ion Chef™Instrument for use
•Ensure that the Ion Chef™Instrument has been cleaned following the previous run. If not, clean the
instrument before loading it with consumables.
Note: For more information on the cleaning procedure, see Chapter 7, “Clean the Ion Chef™
System”.
•Inspect the empty compartments of the Reagents and Solutions stations for condensation.
Condensate can collect in these compartments, depending on temperature and humidity
conditions. Before loading consumables into the instrument, wipe the compartments dry with a
laboratory wipe or absorbent cloth, if needed.
•Thaw the Ion S5™ExT Chef Reagents cartridge at room temperature for 45 minutes before use.
Chapter 2 Before you begin
Guidelines for using Ion Chef™consumables and reagents 2
Ion 520™& Ion 530™ExT Kit − Chef User Guide 15

•Ensure that the Ion Chef™Instrument has a connection to the Ion Torrent™Server. On the
instrument home touchscreen, tap Settings4Ion Torrent™Server to view the connection status.
Note: If the instrument is not connected, see the Ion Chef™Instrument Network Connection User
Guide (Pub. No. MAN0013444) for instructions on how to configure a direct or indirect network
connection.
Chapter 2 Before you begin
Prepare the Ion Chef™Instrument for use
2
16 Ion 520™& Ion 530™ExT Kit − Chef User Guide

Create a Planned Run
■About Planned Runs .................................................................. 17
■Create a Planned Run ................................................................. 18
■Planned Run workflow key fields ....................................................... 22
About Planned Runs
Planned Runs are digital instructions that are created in Torrent Suite™Software for controlling the
template preparation and sequencing instruments. Planned Runs contain settings such as number
of flows, kit types, barcodes, sample information, and reference files (if any). Planned Runs are also
used to track samples, chips, and reagents throughout the workflow, from template preparation on the
Ion Chef™Instrument through sequencing on an Ion S5™Sequencer, Ion S5™XL Sequencer, or Ion
GeneStudio™S5 Series Sequencer and subsequent data analysis. Each chip that is prepared in an
Ion Chef™run requires its own Planned Run.
IMPORTANT! For more information on creating a Planned Run in Torrent Suite™Software, including
a complete description of each field in the Create Plan workflow bar, see the Torrent Suite™Software
Help, available by clicking the Help button in the software.
3
Ion 520™& Ion 530™ExT Kit − Chef User Guide 17

Create a Planned Run
1. Sign in to the Torrent Suite™Software.
2. In the Plan tab, in the Templates screen, select the application that you want to run (such as
Whole Genome) from the left navigation menu, then click one of the following options:
•Plan New Run—to plan a new run using the Generic Sequencing template for the selected
application.
•(Actions)4Plan Run in the row of the template—to plan a new run using a specific
Planned Run template.
3. In the Create Plan workflow bar, review the IonReporter and Research Application steps, then
make selections appropriate to your run. Click Next.
4. In the Kits step, make the following selections:
a. Select Ion GeneStudio™S5 System from the Instrument dropdown list.
b. Select the appropriate chip type in the Chip Type dropdown list.
c. Select the library kit used to prepare your libraries from the Library Kit Type dropdown list.
d. (Optional) For barcoded libraries, select the barcode set used during library preparation from
the Barcode Set dropdown list.
e. Select IonChef for Template Kit, then select Ion 520 & Ion 530 ExT Kit-Chef from the
Template Kit dropdown list.
f. Select Ion S5 ExT Sequencing Kit from the Sequencing Kit dropdown list.
Chapter 3 Create a Planned Run
Create a Planned Run
3
18 Ion 520™& Ion 530™ExT Kit − Chef User Guide

g. Enter the appropriate number of flows in the Flows field.
Sequencing type Number of flows
400‑base‑read 850
600‑base‑read 1,350
IMPORTANT! Do not exceed 1,400 total flows for one Ion S5™ExT Sequencing Reagents
cartridge.
h. Select or edit the remaining optional information fields appropriately for your run.
IMPORTANT! For de novo sequencing applications that do not include a reference BAM
file, select Enable Calibration Standard from the Base Calibration Mode dropdown list in
the Advanced Settings pane. Select this option only if you will add Ion S5™ExT Calibration
Standard to the Ion Chef™Library Sample Tube. Selecting this option and adding the Ion S5™
ExT Calibration Standard to your library allows greater accuracy of base-calling in libraries for
which a reference BAM file does not exist.
i. Click Next.
5. Review the Plugins and Projects screens, then make selections appropriate to your run.
6. In the Plan step, enter or make the following selections:
a. Enter a Run Plan Name, then select Reference and BED files appropriate to your run.
Chapter 3 Create a Planned Run
Create a Planned Run 3
Ion 520™& Ion 530™ExT Kit − Chef User Guide 19

b. Enter the number of barcodes you are using in your combined library in the Number of
barcodes field, then click to the right of this field. Edit the auto-populated list of barcodes
that appears, if needed.
Note: If you did not use barcode adapters in library preparation and did not select a Barcode
Set in the Kits step, fields appear in the Plan step where you enter the number of chips that
are used (enter 1 for Ion Chef™ExT runs), then enter the sample name and chip barcode.
c. Scan or enter the barcode of the Ion Chef™Library Sample Tube into the Sample Tube Label
field.
d. Scan or enter the chip barcode into the Chip Barcode field.
Chapter 3 Create a Planned Run
Create a Planned Run
3
20 Ion 520™& Ion 530™ExT Kit − Chef User Guide