Fluidigm Access Array User Manual

PN 100-5024 E2

Access Array System for the Ion Torrent
PGM Sequencing System

USER GUIDE

For Use Only with Access Array Reagents

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. Fo r licensing information,
contact [e-mail].

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. Fo r licensing information,
contact [e-mail].

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

For technical support visit fluidigm.com/support.

North America [phone] | Toll-free (US/CAN): 866 358 4354 | [e-mail]
Latin America [phone] | [e-mail]
Europe/Middle East/Africa/Russia [phone] | [e-mail]
Japan [phone] | [e-mail]
China (excluding Hong Kong) [phone] | [e-mail]
All other Asian countries/India/Australia [phone] | [e-mail]

2

Access Array System for the Ion Torrent PGM Sequencing System: User Guide

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

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

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

Access Array System for the Ion Torrent PGM Sequencing System: User Guide

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

3

Contents

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

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Access Array System for the Ion Torrent PGM Sequencing System: User Guide

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:

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.

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 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

5

About This Guide

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.

6

Access Array System for the Ion Torrent PGM Sequencing System: User Guide

Chapter 1: Access Array System Overview

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

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.

Access Array System for the Ion Torrent PGM Sequencing System: User Guide

7

Chapter 1: Access Array System Overview

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.

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Access Array System for the Ion Torrent PGM Sequencing System: User Guide

Chapter 1: Access Array System Overview

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

9

Chapter 1: Access Array System Overview

IFC Controller AX

Pre-PCR

FC1 cycler

IFC Controller AX

Post-PCR

Access Array System Components

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.

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Access Array System for the Ion Torrent PGM Sequencing System: User Guide

Access Array IFCs

Primer inlets

Sample inlets

Control Line Fluid

H1

H2

H3

H4

well

well

well

well

A1

Interface
accumulator

Containment
accumulator

Access Array reagents can be run on the:

• LP 48.48 IFC (PN 101-1926)

• Access Array 48.48 IFC (PN AA-M-48.48)

Chapter 1: Access Array System Overview

Access Array IFCs

The high-throughput LP 48.48 IFC (or Access Array 48.48 IFC) enables target enrichment of
48 unique samples at the same time.

Specifications

Description Parameter

Footprint dimensions 128 x 85 x 14 mm

Inlet spacing on input frame 4.5 mm pitch

Primer inlets 48

Sample inlets 48

Reaction chambers 2,304

Reaction volume 35 nL

Instrument compatibility IFC Controller AX, FC1 cycler, and stand-alone thermal cycler

Access Array System for the Ion Torrent PGM Sequencing System: User Guide

11

Chapter 1: Access Array System Overview

Access Array System Workflow

Access Array System Workflow

The simplicity of running experiments is illustrated in the process below.

The Access Array system workflow consists of five major steps:

Workflow Step

1 Dispense

Transfer samples and primers to the inlets on the LP 48.48 IFC (or Access Array 48.48 IFC)
from a standard 96-well plate, using an 8-channel pipette.

2 Load

Reaction mixtures are automatically loaded and assembled on the IFC using the pre-PCR
IFC Controller AX.

3 Thermal-cycle

Perform PCR amplification using the FC1 cycler.

4 Harvest

PCR products from each sample are automatically pooled and harvested using the

post-PCR IFC Controller AX.

5 Recover

Recover the PCR products from each of the 48 sample inlets, using an 8-channel pipette.

Quantify the sequencing library.

Materials

This section describes the materials required to perform an Access Array experiment
including the supported reagents and consumables.

Required Reagents

IMPORTANT

• 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).

• Store reagents according to manufacturer's storage recommendations as soon as they are
received.

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Access Array System for the Ion Torrent PGM Sequencing System: User Guide

Chapter 1: Access Array System Overview

Reagents Supplied by Fluidigm

Product Name Manufacturer Part Number Storage

20X Access Array Loading Reagent Fluidigm 100-0883

Materials

1X Access Array Harvest Solution

1X Access Array Harvest Solution is not packaged for
individual sale. It can be purchased in units of 10, under
the name Access Array Harvest Pack, PN 100-3155, or as
a component in the 48.48 Access Array Loading Reagent
Kit, PN 100-1032.

Access Array Hydration Reagent v2

Fluidigm 100-1031

–20 °C

Fluidigm 100-7966

IMPORTANT This reagent is only required when

using the Access Array 48.48 IFC, and it is not for
use with the LP 48.48 IFC.

Access Array Barcode Library for Ion Torrent PGM
Sequencer - 96

Fluidigm 100-4911

Reagents from Other Suppliers

Product Name Manufacturer Part Number

FastStart™ High Fidelity PCR System, dNTPack Sigma-Aldrich 04738292001

Agilent® DNA 1000 Kit Agilent 5067-1504

PCR Certified Water Teknova W3330

DNA Suspension Buffer (10 mM TRIS, pH 8.0, 0.1mM EDTA)

Agencourt

100% ethanol Major laboratory

®

AMPure® XP Reagent Beads Beckman

Teknova T0221

A63880

Coulter

—

supplier (MLS)

Required Consumables

Consumables Supplied by Fluidigm

Product Name Manufacturer Part Number Storage

LP 48.48 IFC or
Access Array 48.48 IFC

NOTE The Access Array 48.48 IFC has

been discontinued. It has been replaced by
the LP 48.48 IFC.

LP Control Line Fluid 48.48 or
Control Line Fluid for 48.48 IFCs

Fluidigm 101-1926

AA-M-48.48

Room temperature

Fluidigm 101-2345

89000020

Access Array System for the Ion Torrent PGM Sequencing System: User Guide

13

Chapter 1: Access Array System Overview

Example Sample and Primer Layout in 96-Well Plates

Consumables from Other Suppliers

Product Name Manufacturer Part Number

Microcentrifuge tubes, 1.5 mL MLS —

96- and 384-well PCR plates MLS —

Adhesive seals for PCR plates MLS —

P2-P1000 pipette tips Rainin —

Required Equipment

Equipment Supplied by Fluidigm

Product Name Manufacturer Part Number

IFC Controller AX (2 quantity, pre-PCR and post-PCR) Fluidigm IFC-AX

FC1 cycler

NOTE The stand-alone thermal cycler has been discontinued. It has

been replaced by the FC1 cycler.

Fluidigm CYC-FC1

Equipment from Other Suppliers

Product Name Manufacturer Part Number

DynaMag™-2 Magnet Thermo Fisher

Scientific

Agilent 2100 Bioanalyzer® Agilent G2939BA

Microcentrifuge MLS —

Vortex mixer MLS —

Plate centrifuge MLS —

96- and 384-well PCR thermal cycler MLS —

Single-channel P2-P1000 pipettes Rainin —

8-channel P20 pipette Rainin —

123-21D

Example Sample and Primer Layout in 96-Well Plates

For ease of pipetting, fill the 96-well plates by columns, not rows.

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Access Array System for the Ion Torrent PGM Sequencing System: User Guide

Chapter 1: Access Array System Overview

Before You Begin

Before You Begin

IMPORTANT Before using the Access Array system, read and understand the detailed

instructions and safety guidelines in Appendix D.

• Use good laboratory practices to minimize contamination of samples:

• Use a new pipette tip for every new sample.

• Whenever possible, separate pre- and post-PCR activities. Dedicate laboratory

materials to designated areas.

• Unless otherwise specified, thaw reagents at room temperature (15–30 °C), and then use

them at room temperature.

• Retrieve only the reagents required from each kit based on the number of IFCs you will

run.

• Only use the reagents provided in the required kit.

• Do not swap reagents between kit lots.

• Mix and centrifuge reagents as directed.

• Avoid creating bubbles when transferring reagents to the IFC.

•

This protocols in this document require the use of updated scripts for FC1 (v1.6 or later) and IFC

Controller AX (v2.8 or later) that can be downloaded from fluidigm.com/software.

Access Array System for the Ion Torrent PGM Sequencing System: User Guide

15

Chapter 2: Designing Targeted
Sequencing Primers

You can design your own Access Array target-specific primers using available online tools
(for example, UCSC website at genome.ucsc.edu/cgi-bin/hgGateway, Primer3 website at

bioinfo.ut.ee/primer3/, and in silico PCR at genome.ucsc.edu/cgi-bin/
hgPcr?hgsid=136961442) or by using the D3™ assay design service at Fluidigm.

Our D3™ assay design service provides Access Array target-specific primers for use in
Illumina MiniSeq™, MiSeq™, NextSeq™ and HiSeq® systems and in the Ion Torrent® PGM
system. They are designed to human genomic DNA. Uniplex primers are provided in
nuclease-free water at a final volume of 100 L per well in a single 96-well plate with mixed
forward and reverse primers at a final concentration of 50 M. Multiplex primer sets are
provided in nuclease-free water at a final volume of 100 L per well in six 96-well plates per
set at a final concentration of 60 M.

If you are using primers provided by the D3™ assay design service, refer to the order
informatics packet for primer sequences. If you are designing your own primers, see Access
Array–Generate Tagged Primers Workbook (Fluidigm, PN 100-3873).

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).

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Access Array System for the Ion Torrent PGM Sequencing System: User Guide