Thermo Fisher Scientific Ion 510, Ion 520, Ion 530 User Manual
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef
USER GUIDE
Instructions for automated template preparation, chip loading, and sequencing
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 Numbers A34461, A34019
Publication Number MAN0016854
Revision F.0
For Research Use Only. Not for use in diagnostic procedures.
Life Technologies Corporation | 5781 Van Allen Way | Carlsbad, CA 92008
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. MAN0016854
Revision Date Description
F.0 29 October 2020
•
Included chip compatibility information in “Ion S5™ Calibration Standard”
on page 10.
•
Added new troubleshooting items: “Chip Check fails” on page 67 and
“Reagent Check fails” on page 67.
•
Updated requirement for Ion AmpliSeq™ panels with amplicons greater
than 325 bp.
•
Added recommendation for chips used for initialization.
•
Updated run time information.
E.0 25 June 2019
D.0 05 December 2018
•
Updated for Torrent Suite™ Software 5.12.
•
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 chips for sequencing”.
•
Added a note in “Start the Ion Chef™ run” and “Unload the chips for
sequencing” to NOT remove any residual liquid from chip wells after the
Ion Chef™ run.
•
Added a note regarding templated ISP range specification in “Ion
Sphere™ Assay troubleshooting table”.
•
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.
•
Updated for Ion GeneStudio™ S5 Systems.
•
Clarification made to dilute barcoded library pools, if used.
•
Updated for the Qubit™ 4 Fluorometer.
•
References to ISP quality control user guides updated to the Ion Sphere
Quality Control Kit User Guide (Pub. No. MAN0017531).
•
Added a new troubleshooting topic: “Planned Run status does not
advance to "Planned"”.
•
Updated for Torrent Suite™ Software 5.10; Planned Run creation topics
reorganized for ease of use.
•
Ion S5™ Controls Kit replaced with Ion S5™ Controls Kit Plus.
•
Removed reference to quality control of ISPs using the Attune™ NxT
Acoustic Focusing Cytometer.
™
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. Guava is a
trademark of Merck KGaA. Agencourt and Ampure are trademarks of Beckman Coulter, Inc. Bioanalyzer is a trademark of Agilient
Technologies, Inc. Axygen™ and MAXYMum Recovery™are trademarks of Corning, Inc.
©2020 Thermo Fisher Scientific Inc. All rights reserved.
Contents
■
CHAPTER 1 Product information .................................................. 7
Product description ............................................................. 7
Library compatibility ......................................................... 7
Mixing chip types ........................................................... 7
Sequencer compatibility ..................................................... 8
Software compatibility ....................................................... 8
Kit contents and storage ......................................................... 8
Kit summary ............................................................... 9
Ion Chef™ reagents and materials ............................................. 9
Ion S5™ sequencing reagents and materials .................................... 9
Related products .............................................................. 10
Compatible Ion Chip™ kits .................................................. 10
Ion S5™ Calibration Standard ................................................ 10
Ion S5™ Controls Kit Plus ................................................... 10
Required materials not supplied ................................................. 11
About the Ion Chef™ System ..................................................... 12
Ion Chef™ Instrument components ........................................... 12
Interior hardware and consumables .......................................... 13
About the Ion Chef™ System touchscreen interface ............................ 14
Workflow ..................................................................... 15
■
■
Ion 510
CHAPTER 2 Before you begin .................................................... 16
Precautions ................................................................... 16
Avoid nucleic acid contamination ............................................ 16
Avoid chip damage ........................................................ 16
Protection by equipment .................................................... 16
Guidelines for using Ion Chef™ reagents and consumables .......................... 16
Prepare the Ion Chef™ Instrument for use ......................................... 17
CHAPTER 3 Create a Planned Run .............................................. 18
About Planned Runs ........................................................... 18
Create a Planned Run .......................................................... 19
Planned Run workflow key fields ................................................. 23
™
& Ion 520™ & Ion 530™ Kit – Chef User Guide
3
Contents
■
CHAPTER 4 Start the Ion Chef™ run ............................................. 25
Materials required .............................................................. 25
Dilute the libraries .............................................................. 25
Guidelines for library dilution ................................................ 26
Dilute the sample libraries ................................................... 26
Dilute the control library (if used) ............................................. 27
Prepare the consumables ....................................................... 27
Add the diluted libraries to the Ion Chef™ Library Sample Tube ....................... 28
Load the Ion Chef™ System ..................................................... 28
Load the pipette tip racks and PCR Plate ..................................... 29
Load the Reagents and Solutions cartridges .................................. 31
Load the Recovery Tubes and Enrichment Cartridge v2 ......................... 33
Load the Chip-loading centrifuge ............................................ 34
Confirm that consumables are correctly installed .............................. 36
Single chip loading workflow ................................................ 37
Ion Chef™ pre-run checklist ................................................. 39
Start the Ion Chef™ run ......................................................... 40
Unload the chips for sequencing ................................................. 45
■
CHAPTER 5 Initialize the sequencer ............................................ 48
Ion S5™/Ion GeneStudio™ S5 Systems component positions ........................ 48
Before you begin .............................................................. 49
When a manual cleaning of the sequencer is required .............................. 49
Initialize the sequencer ......................................................... 50
■
CHAPTER 6 Start the sequencing run ........................................... 52
Place chip in chip clamp ........................................................ 52
Start the sequencing run ....................................................... 53
Maintain the sequencer ......................................................... 55
Materials required .......................................................... 55
Clean or decontaminate the sequencer ....................................... 55
Reagent consumables disposal .................................................. 55
CO2 scrubber removal and disposal .......................................... 55
Recycle Ion S5™ Wash Solution and Ion S5™ Cleaning Solution bottles ........... 56
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Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
■
CHAPTER 7 Clean the Ion Chef™ System ...................................... 57
About the cleaning protocol ..................................................... 57
Materials required .............................................................. 57
Clean the Ion Chef™ Instrument .................................................. 57
Remove and dispose of used consumables ................................... 58
Inspect and clean the Recovery centrifuges and buckets ....................... 59
Start the cleaning ......................................................... 61
■
APPENDIX A Troubleshooting .................................................... 63
View troubleshooting and FAQs online ............................................ 63
Ion Chef™ System setup and operation ........................................... 63
Ion S5™/Ion GeneStudio™ S5 Systems setup and operation ......................... 66
Instrument alarms and events ............................................... 66
Initialization—General errors ................................................ 67
Ion Sphere™ Assay troubleshooting table ......................................... 69
Contents
■
APPENDIX B Supplemental procedures ........................................ 70
Perform a manual cleaning of the sequencer ...................................... 70
Perform an instrument reset run with an initialized sequencer that is loaded with
an unused Reagents cartridge ................................................. 71
Quality control of Ion 510™ ISPs, Ion 520™ ISPs, or Ion 530™ ISPs ................... 73
Quality control using the Ion Sphere™ Assay on the Qubit™ Fluorometer .......... 73
Acceptance criteria for unenriched Ion 510™ ISPs, Ion 520™ ISPs, or Ion
530™ ISPs .............................................................. 73
Quality control using the Guava™ easyCyte 5 Benchtop Flow Cytometer .......... 73
Maintain the Ion Chef™ System .................................................. 75
■
APPENDIX C Safety ............................................................... 76
Symbols on this instrument ..................................................... 76
Conformity symbols ........................................................ 77
Location of safety labels on this instrument ........................................ 78
Safety information for instruments not manufactured by Thermo Fisher Scientific ...... 78
Instrument safety .............................................................. 78
General ................................................................... 78
Physical injury ............................................................. 78
Electrical safety ........................................................... 79
Cleaning and decontamination .............................................. 79
Ultraviolet (UV) Safety ...................................................... 79
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
5
Contents
■
Safety and electromagnetic compatibility (EMC) standards .......................... 79
Safety compliance ......................................................... 80
EMC ..................................................................... 80
Environmental design ...................................................... 80
Instrument safety — Ion S5™/Ion GeneStudio™ S5 Systems ......................... 81
Chemical safety ................................................................ 81
Biological hazard safety ......................................................... 82
Documentation and support ....................................................... 83
Related documentation ......................................................... 83
Customer and technical support ................................................. 83
Limited product warranty ........................................................ 83
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Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
1
Product description ................................................................... 7
■
Kit contents and storage ............................................................... 8
■
Related products ..................................................................... 10
■
Required materials not supplied ........................................................ 11
■
About the Ion Chef™ System ........................................................... 12
■
Workflow ............................................................................ 15
■
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.
Product description
Product information
The Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef (Cat. Nos. A34461, A34019) is used for automated
template preparation and sequencing of 400 base–read Next Generation sequencing libraries using the
Ion Chef™ System and the Ion S5™ System, Ion S5™ XL System, or Ion GeneStudio™ S5 System. The kit
contains prepackaged single-use template and sequencing reagent cartridges with integrated sample
tracking.
Key features of the kit include:
•
Automated workflow for sequencing of 8 loaded chips with complete run traceability.
•
Maximum flexibility with compatibility for the Ion 510™ Chip, Ion 520™ Chip, and Ion 530™ Chip.
Library compatibility
The Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef can be used with up to 400-base-read libraries of any
type prepared using any available Ion Torrent™-branded library kit.
Mixing chip types
You can perform templating runs with two
chips. However, you cannot mix template sizes (200‑base and 400‑base template run lengths) in a
single run.
IMPORTANT! To perform templating runs with mixed chip types, you must update the Torrent Suite
Software to version 5.6 or later (see “Software compatibility”).
dierent types of chips, using any combination of the three
™
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
7
Chapter 1 Product information
1
Kit contents and storage
Sequencer compatibility
Ion GeneStudio™ S5 Series Sequencer or Ion GeneStudio™ S5 Series System refers generically to
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)
Software compatibility
The Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef is compatible with Torrent Suite™ Software 5.4 and later.
Be sure to update your Torrent Suite™ Software to the latest available version before using this kit. For
more information, see the Torrent Suite™ Software User Guide for your version of the software.
Kit contents and storage
IMPORTANT!
Do not substitute components from any other sequencing kits. We have verified this protocol using
·
these specific materials. Substitution can adversely aect system performance.
Store all consumables and cartridges under the recommended conditions and in an upright position.
·
Do NOT store the Ion S5™ Sequencing Reagents (Part No. A27768) on dry ice or in a closed
environment where dry ice is present.
™
Each Ion 510
and sequence 8 Ion 510™ Chips, Ion 520™ Chips, or Ion 530™ Chips.
•
Catalog No. A34461 supports 4 Ion Chef™ runs (2 chips/Ion Chef™ run) and 4 sequencer
initializations (2 sequencing run/sequencer initialization). Select A34461 for templating and
sequencing of 200‑base–read libraries only.
•
Catalog No. A34019 supports 4 Ion Chef™ runs (2 chips/Ion Chef™ run) and 8 sequencer
initializations (1 sequencing run/sequencer initialization). Select A34019 for templating and
sequencing of 400‑base‑read libraries.
On arrival, inspect all consumables and contact Technical Support if any of the products have been
damaged during shipping.
& Ion 520™ & Ion 530™ Kit – Chef contains all the supplies that are required to prepare
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Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
Kit summary
Chapter 1 Product information
Kit contents and storage
1
Component
Ion S5™ Chef Supplies A27755 4 boxes 4 boxes
Ion S5™ Chef Solutions A27754 1 box 1 box
Ion 510™ & Ion 520™ & Ion 530™ Chef Reagents A34018 1 box 1 box
Ion S5™ Sequencing Solutions A27767 1 box 2 boxes
Ion S5™ Sequencing Reagents A27768 1 box 2 boxes
Ion Chef™ reagents and materials
Contents
Ion S5™ Chef Supplies (Part No. A27755)
Chip Adapter 2 15°C to 30°C
Enrichment Cartridge v2 1
Tip Cartridge v2 1
Quantity per kit
Part No.
A34461 A34019
Amount / box Storage
PCR Plate 1
Frame Seal v2 1
Recovery Station Disposable Lid v2 2
Recovery Tube v2 12
Ion S5™ Chef Solutions (Part No. A27754)
Ion S5™ Chef Solutions 4 cartridges 15°C to 30°C
Ion 510™ & Ion 520™ & Ion 530™ Chef Reagents (Part No. A34018)
Ion 510™ & Ion 520™ & Ion 530™ Chef Reagents 4 cartridges –30°C to –10°C
Ion S5™ sequencing reagents and materials
Contents
Ion S5™ Sequencing Solutions (Part No. A27767)
Ion S5™ Wash Solution 4 × 1.5 L 15°C to 30°C
Ion S5™ Cleaning Solution
Amount / box Storage
250 mL
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
9
Chapter 1 Product information
1
Related products
Contents
Ion S5™ Sequencing Reagents (Part No. A27768)
Ion S5™ Sequencing Reagents 4 cartridges –30°C to –10°C
[1]
Cartridges ship at 2°C to 8°C. Store as indicated, do not store on dry ice.
IMPORTANT! Do not store the Ion S5
closed environment containing dry ice.
Related products
Compatible Ion Chip™ kits
Description
Ion 510™ Chip Kit (2 × 4‑pack) A34292 8 chips 15°C to 30°C
Ion 520™ Chip Kit (2 × 4‑pack) A27762 8 chips
Ion 530™ Chip Kit (2 × 4‑pack) A27764 8 chips
Amount / box Storage
™
Sequencing Reagents (Part No. A27768) on dry ice or in a
Catalog No. Quantity Storage
[1]
Ion S5™ Calibration Standard
For de novo sequencing applications, the Ion S5™ Calibration Standard can be added to diluted libraries
to increase base-calling accuracy. The Ion S5™ Calibration Standard (Cat. No. A27988) is ordered
separately.
Use the Ion S5™ Calibration Standard with Ion 520™ Chip or Ion 530™ Chip workflows only. The
standard has not been validated for use with the Ion 510™ Chip workflow.
Contents
Ion S5™ Calibration Standard 80 µL –30°C to –10°C
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
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
Amount Storage
Amount Storage
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Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
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
Chapter 1 Product information
Required materials not supplied
1
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)
[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.
[2]
MLS
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
11
Chapter 1 Product information
1
About the Ion Chef™ System
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 includes a complete set of cartridge-based consumables and reagents
that enable a user to load two chips in approximately 13 hours with less than 15 minutes of hands-on
time. The Ion Chef™ System features network integration with the 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.
1 2 3
4
Door – Provides access to the interior of the
1
instrument. The door is locked in the closed position
during operation.
Micropipettor – A mechanical positive-displacement
2
pipettor that performs all fluid transfers during sample
and chip preparation.
Robotic arm – Enables fluid transfer by the
3
Micropipettor. The arm also contains an optical sensor
that reads the barcodes of instrument reagents and
consumables.
Touchscreen – Provides access to all instrument
4
functions for operation, maintenance, and
troubleshooting.
5 6 7
8
Power button – Power switch for the Ion Chef
5
Instrument, where the states are on (illuminated) and
o.
Power port – A 100–240 VAC port that provides power
6
to the Ion Chef™ Instrument.
Ethernet port – An RJ45 port that provides Ethernet
7
(100 Mbit) communication with the Ion Chef
Instrument.
USB port – Provides USB communication with the
8
Ion Chef™ Instrument. Used to update the instrument
firmware and to transfer data during service or
maintenance.
™
™
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Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
Interior hardware and consumables
A
B
C
D
E
F
G
H
1 2 3 456 7 8 9 10 11
12
5
3
8
12
6
4
7
530
CCBD02226
530
CCBD02226
9
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.
Chapter 1 Product information
About the Ion Chef™ System
1
Waste pipette tip rack – The position of the rack containing waste (used) pipette tips.
1
Automated heated cover – Transfers the plate cover to the PCR reaction plate within the sample block. During
2
thermal cycling, the heated cover applies compression to seal the reaction plate and heats the cover to prevent
condensation.
New pipette tips – The position of the rack containing unused pipette tips.
3
Thermal cycler sample block – Performs thermal cycling of the sequencing reactions on a 96-well PCR reaction
4
plate.
Reagents station – The position on the instrument deck of the diluted libraries, NaOH, and the Ion 510™ & Ion 520
5
™
& Ion 530™ Chef Reagents cartridge.
Solutions station – The position on the instrument deck of the Ion S5™ Chef Solutions cartridge, which is maintained
6
at room temperature.
Recovery centrifuges – Twin stations that perform centrifugation of the ISPs during the recovery phase of template
7
preparation.
Enrichment station – The position of the rack containing consumables for enrichment of the template-positive ISPs.
8
Chip-loading centrifuge – Performs centrifugation of sequencing chips that have been mounted to chip-loading
9
adapters and loaded with template-positive ISPs.
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
13
Chapter 1 Product information
1
About the Ion Chef™ System
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
Set up run button – Set up the Ion Chef™ template preparation and chip-loading routine. Choose Step by Step to
1
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.
Open Door button.
2
Notifications button – View notifications about instrument status during and between runs.
3
Quick Start button – Proceed directly to the Quick Start instrument setup mode. User verifies the loading of a new
4
pipette tip cartridge and an empty pipette tip rack to hold waste tips generated during the run, before proceeding to
Deck Scan.
Settings button – Advance to a screen providing the following options:
5
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
•
14
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
Workflow
The following workflow shows how to prepare and load samples using the Ion 510™ & Ion 520™ &
Ion 530™ Kit – Chef and Ion Chef™ System for sequencing on an Ion S5™ Sequencer, Ion S5™ XL
Sequencer, or Ion GeneStudio™ S5 Series Sequencer.
Chapter 1 Product information
Workflow
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef Workflow
Create a Planned Run (page 19)
Create a planned run in Torrent Suite™ Software by entering run parameters for templating and
sequencing your samples.
Dilute the library samples (page 25)
If needed, dilute sample and control libraries. Thaw the Reagents cartridge at room temperature
for 45 minutes before use.
Load the Ion Chef™ Instrument (page 28)
1
Load the Ion Chef™ Instrument with sequencing chips, reagents, and sample libraries.
Start the Ion Chef™ run (page 40)
Start the Ion Chef™ run by following the step-by-step instructions on the Ion Chef™ Instrument
home touchscreen.
Unload the Ion Chef™ Instrument (page 45)
After the Ion Chef™ Run completes, unload the prepared sequencing chips from the Ion Chef
Instrument.
™
Load the prepared sequencing chip into the sequencer and begin the sequencing run
(page 52)
Load the prepared sequencing chips on a sequencer and follow the instructions on the
touchscreen to start the sequencing run.
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
15
2
Precautions .......................................................................... 16
■
Guidelines for using Ion Chef™ reagents and consumables ................................ 16
■
Prepare the Ion Chef™ Instrument for use ............................................... 17
■
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
Before you begin
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).
Guidelines for using Ion Chef™ reagents and consumables
•
Store all consumables and cartridges under the recommended conditions and in an upright
position.
•
Inspect all consumables and cartridges for damage on arrival and again before use.
•
Hold sequencing chips by gently gripping them by their edges.
•
When the instrument is not in use, remove all consumables and reagents from the deck and close
the instrument door.
16
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
Prepare the Ion Chef™ Instrument for use
•
Except for the new Tip Cartridge v2, do not reuse any of the consumables or reagents. After each
run, the empty Tip Cartridge v2 is transferred to the waste tip station.
IMPORTANT! All 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 aect the performance of the instrument and chips.
•
Remove and sequence chips within 1 hour after the instrument finishes loading them. If you cannot
sequence a loaded chip immediately, store it in 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.
Chapter 2 Before you begin
2
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 510™ & Ion 520™ & Ion 530™ Chef Reagents cartridge at room temperature for
45 minutes before use.
•
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.
™
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
17
3
About Planned Runs .................................................................. 18
■
Create a Planned Run ................................................................. 19
■
Planned Run workflow key fields ....................................................... 23
■
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.
Create a Planned Run
IMPORTANT! For more information on creating a Planned Run in Torrent Suite
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.
™
Software, including
18
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
Create a Planned Run
Chapter 3
Create a Planned Run
Create a Planned Run
3
IMPORTANT! If you are using the Ion 510
were prepared using Oncomine™ panels, see the corresponding Oncomine™ Assay User Guide for
assay-specific instructions on creating a Planned Run in Torrent Suite™ Software.
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
AmpliSeq DNA) from the left navigation menu, then click one of the following options:
•
To plan a new run using the Generic Sequencing template for the selected application, click
Plan New Run.
•
To plan a new run using a specific Planned Run template, in the row of the template, click
(Actions)4Plan Run.
™
& Ion 520™ & Ion 530™ Kit – Chef with libraries that
3.
In the Create Plan workflow bar, review the Ion Reporter 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 from 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.
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
19
Chapter 3 Create a Planned Run
3
Create a Planned Run
e.
Select IonChef for Template Kit, then select Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef
from the Template Kit dropdown list.
f.
Select Ion S5 Sequencing Kit from the Sequencing Kit dropdown list.
g.
Enter the appropriate number of flows in the Flows field.
Note:
If you are using Ion AmpliSeq™ On‑Demand panels, set the flows to 550. Due to the
·
recommended flows setting of 550, you cannot perform more than one sequencing run
per initialization.
If you are using Ion AmpliSeq™ panels with amplicons longer than 325 bp, increase the
·
number of flows to 650. A file with information about amplicon length can be downloaded
from AmpliSeq.com. See the Ion AmpliSeq™ Designer Help (Pub. No. MAN0018937).
Sequencing type Number of flows
200‑base‑read 500
400‑base‑read 850
IMPORTANT! Do not exceed 1,200 total flows for one Ion S5
cartridge. For 200‑base‑read sequencing, you can run two chips per sequencer initialization.
For 400‑base‑read sequencing, you can run only one chip per sequencer initialization.
h.
In the Advanced Settings pane, select Customize, then select the templating protocol from
the Templating Protocol dropdown list.
Note:
Select Chef Protocol ‑ 200 for libraries with <300-base average read length.
·
Select Chef Protocol ‑ 400 for libraries with ≥300‑base average read length.
·
For Ion AmpliSeq™ On‑Demand panels, ensure that Chef Protocol ‑ 200 is selected.
·
i.
Select or edit the remaining optional information fields appropriately for your run.
™
Sequencing Reagents
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 are adding Ion S5™ Calibration
Standard to the Ion Chef™ Library Sample Tubes. Selecting this option and adding the Ion
S5™ Calibration Standard to your library allows greater accuracy of base-calling in libraries for
which a reference BAM file does not exist.
20
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
j.
Click Next.
Chapter 3 Create a Planned Run
Create a Planned Run
3
5.
Review the Plugins and Projects steps, 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.
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, then enter the sample name and chip barcode for each sample.
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.
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
21
Chapter 3
3
Create a Planned Run
Create a Planned Run
e.
Enter a sample name for each barcode in the appropriate Sample Name (required) fields.
22
7.
After you have completed your selections, click Plan Run at the bottom right of the Plan step
screen to save the run.
The run is listed in the Planned Runs screen under the name that you specified and is automatically
used by the Ion Chef™ System when the associated sample is loaded.
If you are performing a dual‑chip run, repeat step 2−step 7 to create a Planned Run for the second chip.
You can copy the first Planned Run in the Planned Runs screen, then edit as needed.
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
Planned Run workflow key fields
Chapter 3 Create a Planned Run
Planned Run workflow key fields
3
Step or field name
Ion Reporter If the Ion Reporter™ Software is installed and enabled, and you want to analyze the
run data using the software, select the Ion Reporter Account, then select the Ion
Reporter™ workflow from the Existing Workflow dropdown list.
Research Application Select the Research Application and Target Technique that represent your
sequencing experiment.
Instrument Select Ion GeneStudio™ S5 System.
Chip Type Select the Ion 510™ Chip, Ion 520™ Chip, or Ion 530™ Chip.
Library Kit Type Select the kit used to prepare the library.
Template Kit Select Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef.
Sequencing Kit Select Ion S5 Sequencing Kit.
Barcode Set (optional) If you are using barcodes with:
•
DNA libraries – Select the appropriate barcode set used.
•
RNA libraries prepared using the Ion Total RNA-Seq Kit v2 – Select the
IonXpressRNA barcode set, which contains all 16 barcodes in the Ion Xpress
RNA‑Seq Barcode 1–16 Kit.
If you are not using barcodes with:
•
DNA libraries – Leave the Barcode Set field blank.
•
RNA libraries prepared using the Ion Total RNA-Seq Kit v2 – Select
RNA_Barcode_None from the dropdown list. This will ensure that the proper
trimming is performed on the resulting sequence when the RNA library does
not have a barcode.
Description
™
Flows Enter the appropriate number of flows for the read length (for example, 500 flows
for 200‑base‑read sequencing and 850 flows for 400‑base‑read sequencing).
Plugins Select the appropriate plugins for your application.
Projects Select or add a project to group your run data. You can include runs in multiple
projects, and remove runs from a project at any time.
Run Plan Name Enter a name for the run.
Reference Library Select a reference library uploaded to the Torrent Suite™ Software, if any.
BED files Select the Target Regions or HotSpot Regions BED file in the Torrent Suite
Software, if any.
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
™
23
Chapter 3 Create a Planned Run
3
Planned Run workflow key fields
(continued)
Step or field name
Description
Sample Tube Label (Required) Enter or scan the barcode of the Ion Chef™ Library Sample Tube that you will use to
load your sample into the Ion Chef™ Instrument.
IMPORTANT! Each chip that is prepared in an Ion Chef
Planned Run. You must scan or enter the barcode of the appropriate Ion Chef
™
run requires its own
™
Library Sample Tube for each Planned Run that is created for each individual chip.
Chip Barcode Enter or scan the chip barcode.
Sample Name (Required) Did you select a Barcode Set in the Kits step?
•
No – Enter the sample name for each sample loaded onto each chip in
the Sample Name (required) column, then enter or scan the corresponding
Ion Chef™ Library Sample Tube barcode and the chip barcode in the Sample
Tube Label and Chip Barcode columns respectively.
•
Yes – Enter the sample name for each sample in the Sample Name (required)
column, then select the barcode that is associated with each sample from the
dropdown list in the Barcode column.
Did you select the Ion Reporter Account and the associated Ion Reporter
™
workflow?
•
No – For each sample name listed, make the appropriate selections in each
column in the table.
•
Yes – For each sample name listed, also make the appropriate selections in the
Ion Reporter Workflow, Relation, Gender, and IR Set ID columns.
Monitoring Thresholds Set thresholds for Bead Loading, Usable Sequence, and Key Signal. In the Torrent
Suite™ Software Monitor4Runs in Progress screen, an alert is displayed if the
values for a run fall below the selected thresholds.
24
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
Start the Ion Chef™ run
4
Materials required .................................................................... 25
■
Dilute the libraries .................................................................... 25
■
Prepare the consumables ............................................................. 27
■
Add the diluted libraries to the Ion Chef™ Library Sample Tube ............................. 28
■
Load the Ion Chef™ System ........................................................... 28
■
Start the Ion Chef™ run ............................................................... 40
■
Unload the chips for sequencing ....................................................... 45
■
This chapter describes how to perform the following procedures:
•
Set up the Ion Chef™ Instrument for use by diluting the libraries, and loading the instrument with all
of the required reagents and consumables
•
Start an Ion Chef™ run
•
Unload the chips for sequencing
Materials required
•
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef (Cat. Nos. A34461, A34019)
•
Ion 510™ Chip, Ion 520™ Chip, or Ion 530™ Chip (1 or 2)
•
Library or combined library stock solution (1 or 2)
•
Molecular-biology grade nuclease-free water
•
P200 pipettor and filtered tips
Dilute the libraries
Sample libraries are quantified at the end of the library preparation workflow and are typically diluted
to a concentration of 100 pM. The Ion Library Equalizer™ Kit procedure also normalizes library
concentration to 100 pM. Barcoded libraries are typically mixed in an equimolar ratio to create a
combined library. Libraries or combined libraries can require further dilution before use in Ion Chef
template runs.
™
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
25
Chapter 4 Start the Ion Chef™ run
4
Dilute the libraries
Guidelines for library dilution
•
The optimal input concentration for a given library preparation depends on the library and chip
type, and often needs to be empirically determined starting from the recommended concentration.
•
The recommendations for diluting your sample libraries represent optimal input concentrations for
control libraries.
•
Recommendations are based on qPCR quantification. If libraries are quantified with an Agilent
2100 Bioanalyzer™ instrument, a higher calculated concentration may need to be used for
equivalent input.
•
Prepare a fresh dilution of each library or combined library before use with the Ion Chef™ System,
and use the library dilutions within 48 hours.
•
Barcoded libraries are typically combined (or pooled) before templating to allow multiple libraries
to be sequenced on a single chip. We recommend that you combine equal volumes of individual
barcoded libraries at the same concentration (for example, 100 pM), then dilute the combined
library to the final concentration recommended in the following table.
•
Follow the recommendations of your library preparation user guide if it recommends a specific final
library concentration for an Ion Chef™ run.
™
Dilute the sample libraries
IMPORTANT! Before proceeding, dilute the two libraries or combined libraries to the optimal input
concentration. The quality of your sequencing data relies greatly on achieving the correct concentration
of starting library.
Dilute the individual or combined 200-bp or 400-bp stock libraries according to the following table.
If needed, use polyclonality and low-quality filter results from a sequencing run performed with ISPs
templated at the starting concentration, then titrate up or down to achieve optimal concentrations.
IMPORTANT!
in a single templating run.
Library length
[1]
Library length is average insert length plus adapter length.
Note: For the Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef, input for all sizes is identical.
Do NOT mix template sizes (for example, 200‑base and 400‑base template run lengths)
[1]
<300 bp 25–60 pM 600–900 × 10
≥300 bp 25–60 pM 600–900 × 10
Recommended
concentration
Molecules per 25-µL
input volume
6
6
Templating size in
Planned Run setup
200
400
26
Ion 510™ & Ion 520™ & Ion 530™ Kit – Chef User Guide
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