Fluidigm Access Array Bedienungsanleitung
PN 100-5024 E2
Access Array System for the Ion Torrent
PGM Sequencing System
USER GUIDE
For Use Only with Access Array Reagents
Access Array System for the Ion Torrent PGM Sequencing System: User Guide
2
For technical support visit fluidigm.com/support.
Japan +81 3 3662 2150 | tech[email protected]
China (excluding Hong Kong) +86 21 3255 8368 | techsupportchina@fluidigm.com
For Research Use Only. Not for use in diagnostic procedures.
Information in this publication is subject to change without
notice. It is Fluidigm policy to improve products as new
techniques and components become available. Therefore,
Fluidigm reserves the right to change specifications at any
time. Every effort has been made to avoid errors in the text,
diagrams, illustrations, figures, and screen captures. However,
Fluidigm assumes no responsibility for any errors or omissions.
In no event shall Fluidigm be liable for any damages in
connection with or arising from the use of this publication.
Patent and Limited License Information
Fluidigm products are covered by issued and pending patents
in the United States and other countries. Patent and limited
license information is available at fluidigm.com/legalnotices.
Limited Use License to Perform Preamplification with
Fluidigm IFCs
A license to use Thermo Fisher Scientific's patented
preamplification method workflows involving a Fluidigm
integrated fluidic circuit (IFC) can be obtained (i) with purchase
of a Fluidigm IFC from Fluidigm Corporation or (ii) by a separate
license from Thermo Fisher Scientific. For licensing information,
contact outlice[email protected].
Limited Digital PCR License
A license to use Thermo Fisher Scientific's patented digital PCR
method in all fields other than in the Sequencing Field, the
Mass Spectrometry Field, and the Prenatal Field in workflows
involving a Fluidigm IFC can be obtained (i) with purchase of a
Fluidigm IFC from Fluidigm Corporation or (ii) by a separate
license from Thermo Fisher Scientific. For licensing information,
Trademarks
Fluidigm, the Fluidigm logo, Access Array, Advanta, D3, and
FC1 are trademarks or registered trademarks of Fluidigm
Corporation in the United States and/or other countries. All
other trademarks are the sole property of their respective
owners.
For EU's WEEE directive information, go to fluidigm.com/
compliance.
© 2018 Fluidigm Corporation. All rights reserved. 06/2018
Access Array System for the Ion Torrent PGM Sequencing System: User Guide 3
Contents
About This Guide 5
Safety Alert Conventions 5
Safety Data Sheets 6
Chapter 1: Access Array System
Overview 7
Access Array System Components 10
Access Array IFCs 11
Specifications 11
Access Array System Workflow 12
Materials 12
Required Reagents 12
Required Consumables 13
Required Equipment 14
Example Sample and Primer Layout in 96-Well
Plates 14
Before You Begin 15
Chapter 2: Designing Targeted
Sequencing Primers 16
Chapter 3: Target-Specific Primer
Validation for 4-Primer Amplicon
Tagging on the LP 48.48 IFC 17
Reference Documents 17
Materials 17
Required Reagents 17
Prepare Primer Validation Reactions 18
Set Up the PCR Reactions in a 384-Well PCR
Plate 20
Run the PCR Reactions 20
Check PCR Products on the Agilent 2100
Bioanalyzer 21
Chapter 4: Sample Quantitation and
Normalization 22
Reference Documents 22
Materials 22
Required Reagents 22
Required Equipment 22
Sample Quantitation 23
Sample Normalization 23
Chapter 5: Bidirectional Amplicon
Tagging on the LP 48.48 IFC 24
Reference Documents 25
Materials 25
Required Reagents 25
Prime the IFC 26
Prepare the 20X Primer Solutions 28
Prepare Sample Pre-Mix and Samples 29
Prepare the Sample Pre-Mix Solution 29
Prepare the Sample Mix Solutions 29
Load the IFC 30
Thermal-Cycle the IFC 31
Harvest the IFC 31
Attach Sequence Tags and Sample Barcodes 33
Prepare the Sample Pre-Mix Solution 33
Prepare a 100-Fold Dilution of the Harvested PCR
Products 33
Prepare the Sample Mix Solutions 33
Thermal-Cycle the 96-Well PCR Plate 34
Check PCR Products on the Agilent 2100
Bioanalyzer 35
Next Step 35
Chapter 6: Post-PCR Amplicon
Purification and Quantitation 36
Reference Documents 36
Materials 36
Required Equipment 36
Required Reagents 36
Quantitate PCR Products 37
Purify Harvested PCR Products 37
PCR Product Library Quantitation Procedure 38
Agilent 2100 Bioanalyzer Quantification 38
PicoGreen Fluorimetry Quantification 39
Pool the Products from Multiple Access Array
IFCs 39
Access Array System for the Ion Torrent PGM Sequencing System: User Guide
Contents
4
Appendix A: Electropherogram Examples 41
Appendix B: Access Array Barcode
Library for the Ion Torrent PGM
Sequencer - 96 43
Appendix C: Related Documents 47
Appendix D: Safety 48
General Safety 48
Instrument Safety 48
Symbols on the Instrument 49
Electrical Safety 50
Chemical Safety 50
Access Array System for the Ion Torrent PGM Sequencing System: User Guide 5
About This Guide
IMPORTANT
•Before using the instrument, read and understand the safety guidelines in this document.
Failure to follow these guidelines may result in undesirable effects, injury to personnel,
and/or damage to the instrument or to property.
•This guide is for preparing sequencing libraries with Access Array™ reagents. To prepare
targeted DNA sequencing libraries with Advanta™ NGS Library Reagent Kits, see the
Advanta NGS Library Preparation with Access Array Protocol (PN 101-7885).
Safety Alert Conventions
Fluidigm documentation uses specific conventions for presenting information that may
require your attention. Refer to the following safety alert conventions.
Safety Alerts for Chemicals
For hazards associated with chemicals, this document follows the United Nations Globally
Harmonized System of Classification and Labelling of Chemicals (GHS) and uses indicators
that include a pictogram and a signal word that indicates the severity level:
Safety Alerts for Instruments
For hazards associated with instruments, this document uses indicators that include a
pictogram and signal words that indicate the severity level:
Indicator Description
Pictogram (see example) consisting of a symbol on a white background within a red
diamond-shaped frame. Refer to the individual safety data sheet (SDS) for the applicable
pictograms and hazards pertaining to the chemicals being used.
DANGER Signal word that indicates more severe hazards.
WARNING Signal word that indicates less severe hazards.
Indicator Description
Pictogram (see example) consisting of a symbol on a white background within a black
triangle-shaped frame. Refer to the instrument user guide for the applicable
pictograms and hazards pertaining to instrument usage.
DANGER Signal word that indicates an imminent hazard that will result in severe injury or death
if not avoided.
WARNING Signal word that indicates a potentially hazardous situation that could result in serious
injury or death if not avoided.
CAUTION Signal word that indicates a potentially hazardous situation that could result in minor
or moderate personal injury if not avoided.
IMPORTANT Signal word that indicates information necessary for proper use of products or
successful outcome of experiments.
Access Array System for the Ion Torrent PGM Sequencing System: User Guide
About This Guide
6
Safety Data Sheets
Read and understand the SDSs before handling chemicals. To obtain SDSs for chemicals
ordered from Fluidigm, either alone or as part of this system, go to fluidigm.com/sds and
search for the SDS using either the product name or the part number.
Some chemicals referred to in this user guide may not have been provided with your system.
Obtain the SDSs for chemicals provided by other manufacturers from those manufacturers.
Access Array System for the Ion Torrent PGM Sequencing System: User Guide 7
Chapter 1: Access Array System Overview
Amplicon tagging on the Access Array™ system significantly reduces the time required for
enrichment of targeted sequences by combining amplicon generation with library
preparation. The Access Array system workflow consists of five major phases: 1) designing
and validating target-specific primers for targeted resequencing, 2) quantifying samples, 3)
running an integrated fluidic circuit (IFC, either the LP 48.48 IFC or the Access Array 48.48
IFC), 4) attaching sequence tags and sample barcodes, and 5) qualifying and quantifying
harvested PCR products for sequencing. The chapters in this user guide detail this workflow
chronologically:
This Access Array target-specific PCR amplification scheme combines 48 target-specific (TS)
forward and reverse primers with 48 samples to generate 2,304 PCR reactions.
Design PCR primers and add tags
Qualify tagged primer sets
Select best tagged primer sets
Quantify samples
Normalize sample concentrations
Load samples and primers on Access Array IFC
PCR amplify for 35 cycles
Recover PCR products from Access Array IFC
Attach sequence tags and sample barcodes
Qualify/quantify PCR products
Select and pool samples
Clean up PCR product pool
Quantify PCR product pool for emulsion PCR
Access Array System for the Ion Torrent PGM Sequencing System: User Guide
Chapter 1: Access Array System Overview
8
The following image shows a 3 x 3 grid of reaction wells containing the target-specific
primers and samples before reagents are loaded into the LP 48.48 IFC for library preparation
prior to sequencing on the Ion Torrent™ PGM™ Sequencer - 96:
First, TS forward and reverse primers are loaded into each column of the reaction wells on
the LP 48.48 IFC.
Access Array System for the Ion Torrent PGM Sequencing System: User Guide
Chapter 1: Access Array System Overview
9
Samples are then loaded into each row of reaction wells on the LP 48.48 IFC:
After loading the IFC, each reaction well contains a unique combination of TS primer pairs
and samples:
Access Array System for the Ion Torrent PGM Sequencing System: User Guide
Chapter 1: Access Array System Overview
Access Array System Components
10
The resulting PCR products of pooled amplicons are as follows:
Access Array System Components
The Access Array system consists of the following components:
•IFCs for Access Array:
•LP 48.48 IFC
•Access Array 48.48 IFC
•IFC Controller AX (2 quantity, for pre-PCR and post-PCR)
•FC1™ cycler
NOTE The stand-alone thermal cycler has been discontinued. It has been replaced by the
FC1 cycler.
IFC Controller AX
Pre-PCR FC1 cycler IFC Controller AX
Post-PCR
Inhaltsverzeichnis
Andere Fluidigm Laborausrüstung Handbücher
Beliebte Laborausrüstung Handbücher anderer Marken
Agilent Technologies
Agilent Technologies 5800 ICP-OES Bedienungsanleitung
Endress+Hauser
Endress+Hauser Cleanfit CPA875 Bedienungsanleitung
NI
NI PXI-5422 Handbuch
Collomix
Collomix Aqix Bedienungsanleitung
SPEX SamplePrep
SPEX SamplePrep 6875 Freezer/Mill Series Bedienungsanleitung
Ocean Insight
Ocean Insight FLAME-NIR+ Bedienungsanleitung
Parker
Parker ALIGN-MG-NA Bedienungsanleitung
BD
BD 644787 Bedienungsanleitung
DENTAURUM
DENTAURUM Compact Megaplus Bedienungsanleitung
Biuged Laboratory Instruments
Biuged Laboratory Instruments BGD 626 Bedienungsanleitung
VWR
VWR SAS Super IAQ Bedienungsanleitung
illumina
illumina MiSeqDx Bedienungsanleitung