Fluidigm Access Array User Manual

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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 outlicensing@lifetech.com.

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 outlicensing@lifetech.com.

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.

For technical support visit fluidigm.com/support.

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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

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

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

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.

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

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.

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

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.

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

Access Array IFCs

Primer inlets

Sample inlets

Control Line Fluid

well

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

Chapter 1: Access Array System Overview

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.

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

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.

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

Chapter 1: Access Array System Overview

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

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).

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

Chapter 3: Target-Specific Primer Validation for 4-Primer Amplicon Tagging on the LP 48.48 IFC

This chapter describes the validation procedure for the tagged TS primers designed in Chapter 2.

IMPORTANT Access Array

be ordered prior to primer validation. See Table 1 for oligo sequences and order through an oligonucleotide vendor.

™ Barcode 1 primers for the Ion Torrent™ PGM™ Sequencer - 96 must

Reference Documents

Agilent® DNA 1000 Kit Guide

Materials

Required Reagents

IMPORTANT

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

Reagents Supplied by Fluidigm

Product Name Manufacturer Part Number

20X Access Array Loading Reagent Fluidigm 100-0883

Access Array Barcode 1 primers for Ion Torrent with CS1/CS2 tags Fluidigm —

Target-specific primer pairs tagged with universal tags (CS1 forward tag, CS2 reverse tag)

50 M CS1–tagged TS forward primer

50 M CS2–tagged TS reverse primer

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

Fluidigm —

Chapter 3: Target-Specific Primer Validation for 4-Primer Amplicon Tagging on the LP 48.48 IFC

Prepare Primer Validation Reactions

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

60 ng/L genomic DNA (optional) Coriell NA17317

Prepare Primer Validation Reactions

The primer validation protocol prepares enough reagents to perform 48 primer validation reactions.

IMPORTANT It is essential to have Access Array Barcode 1 primers before proceeding with

the primer validation. See Table 1 below for the oligo sequences.

Table 1. Access Array Barcode 1 primers for Ion Torrent

Name Sequence (5′-3′) Total Length

A_BC1_CS1 forward primer CCATCTCATCCCTGCGTGTCTCCGACTCAGACGAGTG

CGTACACTGACGACATGGTTCTACA

P1_CS2 reverse primer CCTCTCTATGGGCAGTCGGTGATTACGGTA

GCAGAGACTTGGTCT

62 bp

45 bp

1 Prepare the 5X target-specific primer solution for 48 individual primer pairs as shown in

the table below.

Table 2. 5X target-specific primer solution preparation

Component Volume

(μL)

Concentration* (nM)

50 M CS1-tagged TS forward primer 1 250

50 M CS2-tagged TS reverse primer 1 250

PCR Certified Water (Teknova) 198

Total 200

* The final concentration in this table refers to the amount of each listed component in 5 L of

the final primer solution.

Final

2 Vortex the 5X target-specific primer solution for a minimum of 20 seconds and

centrifuge for 30 seconds.

NOTE The final tagged TS forward and reverse primer concentrations are 250 nM in the

5X primer solution. The final TS forward and reverse primer concentrations in the PCR reaction are 50 nM.

3 Prepare 2 M Access Array Barcode 1 primer solution.

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

Chapter 3: Target-Specific Primer Validation for 4-Primer Amplicon Tagging on the LP 48.48 IFC

Table 3. The 2 M Access Array Barcode 1 primers for Ion Torrent

Prepare Primer Validation Reactions

Component Volume

100 M A_BC1_CS1 forward primer 2.0 2

100 M P1_CS2 reverse primer 2.0 2

PCR Certified Water (Teknova) 96.0

Total 100

Final Concentration

(μL)

4 Prepare the primer validation reaction components.

IMPORTANT Warm the 20X Access Array Loading Reagent to room temperature before

use.

Table 4. 4-primer amplicon tagging primer validation reaction preparation

Component Volume

(μL)

10X FastStart High Fidelity Reaction Buffer without MgCl2 (Roche®)

25 mM MgCl

DMSO (Roche) 0.25 15.0 5%

(Roche) 0.9 54.0 4.5 mM

0.5 30.0 1X

Volume

for 60 (μL)

Final Concentration*

(M)

10 mM PCR Grade Nucleotide Mix (Roche) 0.10 6.0 200 M each

5 U/L FastStart High Fidelity Enzyme Blend (Roche)

20X Access Array Loading Reagent (Fluidigm)

2 M Access Array Barcode 1 primers for Ion Torrent (from step 3)

60 ng/L genomic DNA (Coriell)

PCR Certified Water (Teknova) 0.12 7.2

Total 4.0 240.0

* The final concentration in this reaction mix table refers to the amount of each listed component in 5 L of the

final reaction mix.

0.05 3.0 0.05 U/L

0.25 15.0 1X

1.0 60.0 400 nM

0.83 49.8 10 ng/L

5 Vortex the primer validation reaction mix for a minimum of 20 seconds and centrifuge for

30 seconds.

NOTE Table 4 displays the volumes required to prepare 48 reactions with a 25%

overage to evaluate 48 target-specific primer pairs. Scale up appropriately if a higher number of primer pairs are to be evaluated.

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

Chapter 3: Target-Specific Primer Validation for 4-Primer Amplicon Tagging on the LP 48.48 IFC

Set Up the PCR Reactions in a 384-Well PCR Plate

1 Prepare the primer validation PCR reactions:

a Add 4 L of primer validation reaction mix to each of the 48 wells.

b Add 1 L of the 5X target-specific primer solution.

The total PCR reaction volume is 5 L.

2 Vortex the 384-well PCR reaction plate for a minimum of 20 seconds and centrifuge for

30 seconds.

Run the PCR Reactions

1 Load the 384-well plate onto the PCR thermal cycler.

2 Run 35 cycles of PCR using the protocol described below:

Table 5. PCR protocol

PCR Stages Cycle

50 ºC 2 min 1

70 ºC 20 min 1

95 ºC 10 min 1

95 ºC 15 sec 60 ºC 30 sec 72 ºC 1 min

95 ºC 15 sec 80 ºC 30 sec 60 ºC 30 sec 72 ºC 1 min

95 ºC 15 sec 60 ºC 30 sec 72º 1 min

95 ºC 15 sec 80 ºC 30 sec 60 ºC 30 sec 72 ºC 1 min

95 ºC 15 sec 60 ºC 30 sec 72 ºC 1 min

95 ºC 15 sec 80 ºC 30 sec 60 ºC 30 sec 72 ºC 1 min

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

Chapter 3: Target-Specific Primer Validation for 4-Primer Amplicon Tagging on the LP 48.48 IFC

Check PCR Products on the Agilent 2100 Bioanalyzer

Use the Agilent DNA 1000 chips from the Agilent DNA 1000 Kit to check 1 L of PCR product from each of the PCR reactions described above. Follow the Agilent DNA 1000 Kit Guide for details.

On-target products should account for a minimum of 50% of the total yield (by mass) produced for a particular primer pair. If on-target products comprise <50% of total yield, primers may have to be redesigned.

NOTE The product size should be the sum of the target region and the length of the

Access Array Barcode 1 primers for Ion Torrent (103 bp).

Figure 1. Example of 48 primer pairs checked on an Agilent chip. The red-labeled PCR reactions generated multiple or incorrectly sized primers, which need to be redesigned and validated before continuing.

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

Chapter 4: Sample Quantitation and Normalization

This chapter provides a standard procedure to determine sample concentration using fluorimetry. Sample concentrations need to be normalized before proceeding with the LP 48.48 IFC protocol for Access Array™.

The Quant-iT sample concentration.

™

PicoGreen® fluorescent assay requires 1 L of sample DNA to determine

Reference Documents

Quant-iT™ PicoGreen® User Guide

Materials

Required Reagents

IMPORTANT Store reagents according to manufacturer's storage recommendations as

soon as they are received.

Product Name Manufacturer Part Number

Quant-iT PicoGreen dsDNA Assay Kit Thermo Fisher Scientific P11496

Required Equipment

Product Name Manufacturer Part Number

Fluorimeter-compatible 96- or 384-well microtiter plates Major laboratory supplier

—

(MLS)

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

Chapter 4: Sample Quantitation and Normalization

Sample Quantitation

Quantitate the samples by fluorimetry, using the Quant-iT PicoGreen dsDNA Assay Kit. Follow the manufacturer’s instructions.

Sample Normalization

The following recommendations apply to human genomic DNA samples. For other samples, the recommendations on sample concentrations may be different. Contact Technical

Support for assistance in this case.

• If the sample concentration is 50 ng/L gDNA input (a total of 50 ng gDNA), the sample is

ready for amplification on the LP 48.48 IFC. For germline mutation, you might be able to use as little as 525ng/L.

• If the sample concentration is below 50 ng/L gDNA, we recommend concentrating the

sample before amplification takes place on the Access Array.

• If the sample concentration is above 50 ng/L, we recommend diluting the sample to

50 ng/L using DNA Suspension Buffer before proceeding.

Sample Quantitation

Use the following formula to determine the correct volume of DNA Suspension Buffer required to dilute each sample to 50 ng/L:

Y = X (B/50 – 1)

Where X is the volume of the original sample (L) to be used in the dilution

Y is the DNA Suspension Buffer volume (L) needed to dilute X L of the original sample to 50 ng/L

B is the sample concentration (ng/L) measured by fluorimetry 50 is the desired sample concentration (ng/L)

For example: If a 10 L sample (X = 10 L) has a concentration of 200 ng/L (B = 200 ng/L):

Y = 10 L * ((200 ng/L)/(50 ng/L) –1)

Y = 30 L

Therefore: Dilute 10 L of the 200 ng/L sample in 30 L of DNA Suspension Buffer to obtain a 50 ng/L sample concentration.

• Normalize all sample concentrations to 50 ng/L.

Samples are now ready for amplification using the LP 48.48 IFC.

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

Chapter 5: Bidirectional Amplicon Tagging on the LP 48.48 IFC

This protocol outlines the bidirectional amplicon tagging strategy for the Ion Torrent™ PGM™

Sequencer - 96

The goal of this protocol is to sequence both ends of an amplicon with a single-read sequencing run. In the bidirectional amplicon tagging approach, tagged target-specific primer pairs are combined with two sets of sample-specific barcode primer pairs. The sample-specific barcode primer pairs are comprised of common sequence tags CS1 or CS2, appended with the Ion Torrent adapter sequences (A and P1, Table 6). This approach requires only one set of target-specific primer pairs, while the sample-specific barcode primers are universal and can be used in multiple experiments.

In the figure that follows, you can see that bidirectional amplicon tagging generates two types of PCR products per target region: one PCR product that allows for sequencing of the 5' end of the target region (Set A) and one PCR product that allows for sequencing of the 3' end of the target region (Set B). Because both PCR products are clonally amplified onto the Ion Sphere™ Particle (ISP) at the same time, one single-read sequencing run will yield sequence information for both ends of the target region. Bidirectional sequencing will produce high quality reads from both ends and across the full length of the amplicons.

for amplicon libraries that have been generated on the Access Array™ system.

Table 6. PCR products generated from bidirectional amplicon tagging

Primer Sequence

A_BC_CS1 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-[BC]-ACACTGACGACATGGTTCTACA-3'

P1_CS2 5'-CCTCTCTATGGGCAGTCGGTGATTACGGTAGCAGAGACTTGGTCT-3'

A_BC_CS2 5'-CCATCTCATCCCTGCGTGTCTCCGACTCAG-[BC]-TACGGTAGCAGAGACTTGGTCT-3'

P1_CS1 5'-CCTCTCTATGGGCAGTCGGTGATACACTGACGACATGGTTCTACA-3'

This strategy uses a two-step PCR approach: (1) The first PCR is run on the IFC in the presence of tagged, target-specific primers. (2) The harvested PCR product pools are then split into two subsequent PCR reactions with two sets of sample-specific barcode primers, Set A and Set B. Each is run on an independent 96-well plate. In Set A, the PCR reaction products that are generated allow for sequencing of the 5' end of the target region and utilize the A_BC_CS1 and P1_CS2 barcode primer combination. In Set B, the PCR reaction products that are generated allow for sequencing of the 3' end of the target region and utilize the A_BC_CS2 and P1_CS1 barcode primer combination.

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

Chapter 5: Bidirectional Amplicon Tagging on the LP 48.48 IFC

Reference Documents

• IFC Controller AX User Guide (PN 68000157)

• Control Line Fluid Loading Procedure Quick Reference (PN 68000132)

•

Agilent® DNA 1000 Kit Guide

Materials

Required Reagents

Stored at –20 ºC

• FastStart™ High Fidelity PCR System, dNTPack (Sigma-Aldrich, PN 04738292001)

• 20X Access Array Loading Reagent (Fluidigm, PN 100-0883)

• 1X Access Array Harvest Solution (Fluidigm, PN 100-1031)

NOTE 1X Access Array Harvest Solution (Fluidigm, PN 100-1031) 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 System for the Ion Torrent PGM Sequencing System: User Guide

Chapter5:Bidirectional Amplicon Tagging on the LP 48.48 IFC

Accumulators

Sample

Primer

inlets

Prime the IFC

• 1X Access Array Hydration Reagent v2 (Fluidigm, PN 100-7966)

IMPORTANT This reagent is not required, but it ensures uniform harvest volumes if

used

during the priming step when using the Access Array 48.48 IFC. (It is not for use with

the LP 48.48 IFC.)

• Access Array Barcode Library for Ion Torrent PGM Sequencer - 96 (Fluidigm,

PN 100-4911)

• Target-specific primer pair with universal tags (CS1 forward, CS2 reverse), supplied in

separate forward and reverse primer pools in 96-well plates at the following stock concentrations:

• 50 M CS1-tagged TS forward primer

• 50 M CS2-tagged TS reverse primer

• Template DNA at 50ng/L

Stored at 4 ºC

Agilent DNA 1000 Kit Reagents (Agilent, PN 5067-1504)

Stored at Room Temperature

PCR Certified Water (Teknova, PN W3330)

Prime the IFC

NOTE The location of the sample inlets is different from 48.48 Gene Expression or

Genotyping IFCs.

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PRIMER INLETS

SAMPLE INLETS

INTERFACE ACCUMULATOR

CONTAINMENT ACCUMULATOR

IMPORTANT

• Use the LP 48.48 IFC (or the Access Array 48.48 IFC) within 24 hours of opening the package.

• Due to different accumulator volumes, use only 48.48 syringes with 300 μL of control line fluid (PN 89000020).

• Control line fluid on the IFC or in the inlets makes the IFC unusable.

• Load the IFC within 60 minutes of priming.

• Be certain that the reagents 1X Access Array Harvest Solution and 1X Access Array

Hydration Reagent v2 are thawed completely to room temperature and mixed thoroughly prior to use.

1 Inject Control Line Fluid into each accumulator on the IFC.

2 Add 500 L of 1X Access Array Harvest Solution (Fluidigm, PN 100-1031) into the

H1–H4 wells on the IFC.

Prime the IFC

NOTE If you are working with the Access Array 48.48 IFC, use 500 L of 1X Access

Array Hydration Reagent v2 (blue cap, Fluidigm, PN 100-7966) instead of 1X Access Array Harvest Solution in the H4 well. Hydration Reagent v2 ensures uniform harvest volumes when working with the Access Array 48.48 IFC, but it is not needed when working with the LP 48.48 IFC.

3 Remove and discard the blue protective film from the bottom of the AA IFC.

4 Load the IFC into the pre-PCR IFC Controller AX located in the pre-PCR lab.

5 Press Eject to move the tray out of the IFC Controller AX.

6 Place the IFC onto the tray by aligning the notched corner of the IFC to the A1 mark.

7 Press Load Chip to register the barcode of the IFC and activate the script selection.

8 Prime the LP 48.48 IFC for Access Array. Select Prime (155x) and Run Script.

NOTE For Access Array 48.48 IFCs, select Prime (151x) and Run Script.

9 Once the script is complete, press Eject to remove the IFC.

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SAMPLE INLETS

INTERFACE ACCUMULATOR

CONTAINMENT ACCUMULATOR

Prepare the 20X Primer Solutions

NOTE If you are using primers provided by the Fluidigm Assay Design Group, refer to the

order informatics packet for correct primer concentrations.

1 Prepare the 20X primer solutions for 48 individual primer pairs as shown in the table

below. These will be loaded into the primer inlets of an IFC.

IMPORTANT Warm the 20X Access Array Loading Reagent to room temperature before

use.

Table 7. Example for ASY-AA product (Fluidigm): Dilution of target-specific primers

Component Volume

50 M CS1-tagged TS forward primer/50 M CS2-tagged TS reverse primer

20X Access Array Loading Reagent (Fluidigm) 5.0 1X

PCR Certified Water (Teknova) 87.0

Total 100

* The final concentration in this table refers to the amount of each listed component in 5 L of the final primer

mix.

2 Vortex the 20X primer solutions for a minimum of 20 seconds and centrifuge for

30 seconds.

NOTE The final tagged TS forward and reverse primer concentrations are 4 M in the

20X primer solution. The final TS forward and reverse primer concentrations in the Access Array reaction chamber are 200 nM.

Final

(μL)

Concentration*

8.0 4 M

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

Chapter 5: Bidirectional Amplicon Tagging on the LP 48.48 IFC

Prepare Sample Pre-Mix and Samples

All DNA samples need to be added into the sample pre-mix individually, prior to loading the sample mix solutions into the sample inlets of an IFC.

Prepare the Sample Pre-Mix Solution

1 Working in a DNA-free hood, combine the components listed in the table below. This

protocol prepares enough sample pre-mix for 60 reactions. This is enough reagent to load one IFC with 12 additional reactions to compensate for dead volume and pipetting error.

Table 8. Sample pre-mix solution

Component Volume

(μL)

10X FastStart High Fidelity Reaction Buffer without MgCl2 (Roche)

25 mM MgCl

DMSO (Roche) 0.25 15.0 5%

10 mM PCR Grade Nucleotide Mix (Roche) 0.1 6.0 200 M each

5 U/L FastStart High Fidelity Enzyme Blend (Roche)

20X Access Array Loading Reagent (Fluidigm)

PCR Certified Water (Teknova) 1.95 117.0

Total 4.0 240.0

* The final concentration in this sample pre-mix table refers to the amount of each listed component in 5 L of

the final sample mix.

(Roche) 0.9 54.0 4.5 mM

0.5 30.0 1X

0.05 3.0 0.05 U/L

0.25 15.0 1X

Volume

for 60 (μL)

Final

Concentration*

2 Vortex the sample pre-mix for a minimum of 20 seconds and centrifuge for 30 seconds.

Prepare the Sample Mix Solutions

1 Combine the components listed below in a 96-well plate to prepare 48 individual sample

mix solutions.

Table 9. Sample mix solution

Component Volume (μL) Final Concentration

Sample pre-mix 4.0

genomic DNA (50 ng/L) 1.0 10 ng/L

Total 5.0

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Chapter5:Bidirectional Amplicon Tagging on the LP 48.48 IFC

17 18

34 35

40 41

14 15

Step 1

17 18

34 35

40 41

Step 2

17 18

34 35

40 41

Step 3

17 18

34 35

40 41

14 15

Step 4

17 18

14 15

Step 5

34 35

40 41

14 15

Step 6

Load the IFC

2 Vortex the sample mix solution for a minimum of 20 seconds and centrifuge for

30 seconds.

IMPORTANT It is essential to vortex all components to ensure complete mixing.

Load the IFC

1 Pipet 4 L of 20X primer solution into each of the primer inlets.

2 Pipet 4 L of sample mix solution into each of the sample inlets.

IMPORTANT While pipetting, do not go past the first stop on the pipette. Doing so may

introduce air bubbles into the inlets.

NOTE An 8-channel pipette is recommended to load the sample mix and 20X primer

solutions. The recommended pipetting order is shown below.

3 Load the IFC into the pre-PCR IFC Controller AX in the pre-PCR lab.

4 Press Eject to move the tray out of the IFC Controller AX.

5 Place the IFC onto the tray by aligning the notched corner of the IFC to the A1 mark.

6 Press Load Chip to register the barcode of the IFC and activate the script selection.

7 Load the LP 48.48 IFC for Access Array. Select Load Mix (155x) and Run Script.

NOTE For Access Array 48.48 IFCs, select Load Mix v7 (151x) and Run Script. Load Mix

v7 (151x) is a script update from Load Mix (151x) for Access Array 48.48 IFCs and can be downloaded from fluidigm.com/software. Contact Technical Support for assistance.

8 Once the script is complete, press Eject to remove the IFC.

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

Chapter 5: Bidirectional Amplicon Tagging on the LP 48.48 IFC

Thermal-Cycle the IFC

IMPORTANT If you are using the Access Array reagents with the LP 48.48 IFC, use only the

AA 48x48 Standard v1 or AA48v1 thermal protocols specified in step 8. Do not use the LP- 48x48 protocol with Access Array reagents.

Place the IFC onto the FC1 cycler and start PCR by selecting the protocol AA 48x48 Standard v1. Refer to the FC1™ Cycler User Guide (PN 100-1279).

For stand-alone thermal cycler, select protocol AA48v1.

NOTE The protocol as programmed into the Fluidigm stand-alone thermal cycler takes

undershooting and overshooting of target temperatures into consideration and is therefore not identical to the PCR protocol for the FC1 cycler. Contact Technical Support if you need assistance in programming your cycler.

Harvest the IFC

1 After the PCR has finished, move the IFC into the Post-PCR lab for harvesting.

2 Remove the remaining fluids from the H1–H4 wells.

3 Pipet 600 L of fresh 1X Access Array Harvest Solution into each of the H1–H4 wells. (Do

not use Hydration Reagent v2 here.)

NOTE If you are working with the LP 48.48 IFC or the Access Array 48.48 IFC, use 1X

Access Array Harvest Solution in all four wells (H1-H4).

4 Pipet 2 L of 1X Access Array Harvest Solution into each of the sample inlets on the IFC.

5 Load the IFC into the Post-PCR IFC Controller AX located in the Post-PCR lab.

6 Press Eject to move the tray out of the IFC Controller AX.

7 Place the IFC onto the tray by aligning the notched corner of the IFC to the A1 mark.

8 Press Load Chip to register the barcode of the IFC and activate the script selection.

9 Harvest the LP 48.48 IFC for Access Array. Select Harvest (155x) and Run Script.

NOTE For Access Array 48.48 IFCs, select Harvest v7 (151x) and Run Script. Harvest v7

(151x) is a script update from Harvest v5 (151x) for Access Array 48.48 IFCs and can be downloaded from fluidigm.com/software. Contact Technical Support for assistance.

10 Once the script is complete, press Eject to remove the IFC.

11 Label a 96-well plate with the IFC barcode.

12 Carefully transfer 10 L of harvested PCR products from each of the sample inlets into

columns 1

–6 of a 96-well PCR plate, using an 8-channel pipette.

IMPORTANT Transfer PCR products from the IFC to the 96-well plate in the same order

as the IFC was loaded.

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DO NOT USE

H1H3H2

Sample inlets with harvested samples

INTERFACE ACCUMULATOR

CONTAINMENT ACCUMULATOR

Harvest the IFC

The following image displays a map of the PCR products when transferred from the IFC to the 96-well plate:

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

Chapter 5: Bidirectional Amplicon Tagging on the LP 48.48 IFC

Attach Sequence Tags and Sample Barcodes

NOTE All subsequent steps need to be carried out in a post-PCR lab to avoid contamination.

Prepare the Sample Pre-Mix Solution

1 Working in a DNA-free hood, combine the components listed in the table below. This

protocol prepares enough sample pre-mix for 120 reactions. This is enough reagent to amplify the 48 PCR product pools harvested from one IFC for Set A and Set B bidirectional barcoding with 24 additional reactions to compensate for dead volume and pipetting error.

Table 10. Sample pre-mix solution

Component Volume

(μL)

10X FastStart High Fidelity Reaction Buffer without MgCl2 (Roche)

25 mM MgCl2 (Roche) 3.6 432.0 4.5 mM

DMSO (Roche) 1.0 120.0 5%

10 mM PCR Grade Nucleotide Mix (Roche) 0.4 48.0 200 M each

5 U/L FastStart High Fidelity Enzyme Blend (Roche)

PCR Certified Water (Teknova) 7.8 936.0

Total 15.0 1,800.0

* The final concentration in this sample pre-mix table refers to the amount of each listed component in 5 L of

the final sample mix.

2.0 240.0 1X

0.2 24.0 0.05 U/L

Volume

for 120

(μL)

Final

Concentration*

2 Vortex the sample pre-mix for a minimum of 20 seconds and centrifuge for 30 seconds.

Prepare a 100-Fold Dilution of the Harvested PCR Products

1 In a 96-well plate pipet 99 L PCR Certified Water into 48 wells.

2 Add 1 L of PCR product from each sample harvested from the IFC to a separate well in

the 96-well plate as described in the section Harvest the IFC on page 31.

3 Vortex the PCR product dilutions for a minimum of 20 seconds and centrifuge for

30 seconds.

Prepare the Sample Mix Solutions

1 Combine the components listed below in two 96-well plates to prepare two sets of 48

individual sample mix solutions.

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

Chapter5:Bidirectional Amplicon Tagging on the LP 48.48 IFC

Attach Sequence Tags and Sample Barcodes

Table 11. Sample mix solution - SET A

Component Volume

Sample pre-mix 15.0

(μL)

Access Array Barcode Library for Ion Torrent PGM Sequencer Plate A1

Diluted Harvested PCR Product Pool 1.0

Total 20.0

Table 12. Sample mix solution - SET B

Component Volume

Sample pre-mix 15.0

Access Array Barcode Library for Ion Torrent PGM Sequencer Plate B1

Diluted Harvested PCR Product Pool 1.0

Total 20.0

4.0

(μL)

4.0

IMPORTANT It is essential to vortex all components to ensure complete mixing.

NOTE

• The final concentrations of the forward and reverse barcode primers are 400 nM per well.

• Each well should receive a unique barcode primer pair.

2 Vortex the sample mix solutions for a minimum of 20 seconds and centrifuge for

30 seconds.

Thermal-Cycle the 96-Well PCR Plate

Place the PCR plates on two PCR thermal cyclers and run the following PCR protocol:

Table 13. PCR protocol for attaching sequence tags and sample barcodes

PCR Stages Cycle

95°C 10 min 1

95°C 15 sec

60°C 30 sec

72°C 1 min

72°C 3 min 1

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

Chapter 5: Bidirectional Amplicon Tagging on the LP 48.48 IFC

Check PCR Products on the Agilent 2100 Bioanalyzer

1 Use the Agilent DNA 1000 chips from the Agilent DNA 1000 Kit to check 1 L of PCR

product from each of the PCR reactions described above. Follow the Agilent DNA 1000

Kit Guide for details.

2 Check the results of the chip to determine if the PCR product pool has the expected size.

Depending on the expected sizes of the PCR products, a smear may be visible. Comparison of the electropherogram to a histogram of the expected sizes indicates correctness of the product size range. The PCR products of the barcoding step should exhibit a band shift of +63 bp when compared to Harvest Pool Products.

3 Store the PCR products at –20 ºC.

Next Step

Continue with your library preparation as described in Post-PCR Amplicon Purification and

Quantitation on page 36.

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

Chapter 6: Post-PCR Amplicon Purification and Quantitation

This chapter describes a standard Access Array™ procedure for the analysis of PCR products harvested from an LP 48.48 IFC.

The quality of the PCR products prepared on an LP 48.48 IFC (or a Access Array 48.48 IFC) is critical for successful amplicon sequencing. Any contamination of primers or primer dimers in the PCR products will be directly reflected in the quality of sequencing reads. Therefore, the PCR products generated on an IFC should to be qualified and purified before sequencing.

The PCR products generated on the IFC are first analyzed using an Agilent® 2100 Bioanalyzer to check for quality. Next, the PCR products are pooled together in equal volume to create one PCR product library. The PCR product library is then purified using AMPure XP beads, and quantified before proceeding to sequencing. It is recommended to use the Quant-iT PicoGreen dsDNA assay kit for quantification of the final (cleaned-up) library prior to sequencing.

Reference Documents

• Agilent® DNA 1000 Kit Guide

• Quant-iT™ PicoGreen® User Guide

Materials

Required Equipment

• SPRIPlate® 96R Magnet Plate (Agencourt, PN 000219)

or DynaMag™-2 magnet (Invitrogen, PN 123-21D)

• Agilent 2100 Bioanalyzer and DNA 1000 Kit (Agilent, PN 5067-1504)

• Fluorimeter-compatible 96- or 384-well microtiter plates

Required Reagents

Stored at 4 °C

• Agencourt AMPure XP Reagent beads (Beckman Coulter Genomics, PN A63880)

• Quant-iT

PicoGreen dsDNA Assay Kit (Invitrogen, P11496)

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

Chapter 6: Post-PCR Amplicon Purification and Quantitation

Quantitate PCR Products

Stored at Room Temperature

• DNA suspension buffer (10 mM TRIS, pH 8.0, 0.1 mM EDTA) (Teknova, PN T0221)

• 100% Ethanol

• PCR Certified Water (Teknova, PN W3330)

Quantitate PCR Products

The PCR product library can be quantified using an Agilent 2100 Bioanalyzer with DNA 1000 Chips.

1 Run 1 L of the 48 pooled PCR products from each sample on a Bioanalyzer DNA 1000

Chip following the manufacturer’s instructions.

a Ensure that amplicon sizes and distribution are within the expected range (±5% for

amplicons in the range of 200-400 bp including tags).

b Ensure that primer-dimer contamination in the PCR product pool (in the range of

–130 bp) is less than 25%, based on the Bioanalyzer quantification shown in

Appendix A: Electropherogram Examples.

2 Continue with the purification procedure below for all of the pooled PCR products that

contain less than 25% primer dimers.

Purify Harvested PCR Products

1 Remove AMPure XP beads from refrigerator and warm to room temperature for

30 minutes.

2 Prepare 70% ethanol solution:

a To a 15 mL tube, add 3 mL of PCR-Certified Water and 7 mL of 100% ethanol.

b Vortex for 5 seconds.

3 Pool 1 L of each barcoded sample PCR product (see Attach Sequence Tags and Sample

Barcodes on page 33) into a new microcentrifuge tube.

4 Vortex AMPure XP beads for 10 seconds to resuspend. Bead solution should appear

homogeneous and consistent in color.

5 Pipet the barcoded sample pool, DNA suspension buffer, and AMPure XP beads into a

1.5 mL microtube according to the table below:

Component Volume (μL)

Barcoded sample pool 12.0

DNA suspension buffer 24.0

AMPure XP Beads 36.0

Total 72.0

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

Chapter 6: Post-PCR Amplicon Purification and Quantitation

PCR Product Library Quantitation Procedure

6 Vortex the tube and incubate at room temperature for 10 minutes.

7 Place the microtube onto a magnetic separator and allow it to sit for 1 minute.

8 Carefully pipet out the supernatant without disturbing the beads (remove as much liquid

as possible).

9 Add 180 L of 70% ethanol and vortex for 10 seconds.

10 Place the microtube onto a magnetic separator and allow it to set for 1 minute.

11 Carefully pipet the supernatant without disturbing the beads.

12 Add 180 L of 70% ethanol and vortex for 10 seconds.

13 Place the microtube onto a magnetic separator and allow it to set for 1 minute.

14 Carefully pipet out the supernatant without disturbing the beads.

15 Allow the beads to air dry for approximately 10 minutes by leaving the tube on the bench.

Make sure the tube is completely dry before proceeding.

16 Add 40 L of DNA suspension buffer to the microtube and vortex for 5 seconds.

17 Place the microtube onto a magnetic separator and allow it to set for one minute.

18 Carefully transfer the supernatant to a new 1.5 mL microtube.

PCR Product Library Quantitation Procedure

Use one of the following quantitation methods to quantify the PCR product library.

IMPORTANT The Ion Library Quantification Kit (Ion Torrent, PN.4468802) is not compatible

with the library generated with Access Array Barcode Library for Ion

- 96

(Fluidigm, PN 100-4911).

Agilent 2100 Bioanalyzer Quantification

1 Run 1 L of the PCR product library on a Bioanalyzer DNA 1000 Chip following the

manufacturer’s instructions.

2 Define a region of interest in the electropherogram to determine the PCR product library

concentration.

a Select the Region Table subtab on the bottom panel of the Electropherogram tab.

b Right-click the electropherogram and select Add region. Define the region to cover

all of the PCR product library peaks.

Torrent™ PGM™ Sequencer

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

Chapter 6: Post-PCR Amplicon Purification and Quantitation

PCR Product Library Quantitation Procedure

c The Region Table listed below the electropherogram will show the concentration of

the Region containing the PCR product library. Refer to the Agilent 2100 Bioanalyzer User Guide for additional information on Regions.

PicoGreen Fluorimetry Quantification

Quantitate the PCR product library by fluorimetry, using the Quant-iT PicoGreen dsDNA Assay Kit, following the manufacturer’s instructions.

Independent IFC libraries should be quantitated separately. After quantitation, libraries must be normalized and volumetrically combined into a single final library for sequencing. The final library concentration based on Quant-iT is then used for the downstream sequencing preparation.

Pool the Products from Multiple Access Array IFCs

To pool amplicons from more than one Access Array IFC, pool and purify each Access Array IFC separately.

1 Follow the steps outlined in this chapter under Purify Harvested PCR Products on

page 37 for each Access Array IFC.

2 For bidirectional amplicon tagged libraries (generated in Chapter 5), pool products A and

B from the same Access Array IFC (up to 48 samples) together for purification.

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

Chapter 6: Post-PCR Amplicon Purification and Quantitation

PCR Product Library Quantitation Procedure

3 Continue to quantify the pooled, purified library by calculating the PCR Product Library

Concentration outlined later in this chapter.

4 Once each IFC Library has been purified and quantified, normalize the concentration of

each IFC Library and pool libraries volumetrically by adding an equal volume of each library to a new microcentrifuge tube.

The pooled Access Array IFC library is now ready for sequencing.

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

Appendix A: Electropherogram Examples

The figures in this section are examples from an Agilent® 2100 Bioanalyzer electropherogram of a harvested PCR product pool. The first two figures show a DNA 1000 Chip electropherogram of a pooled PCR product with 48 amplicons ranging between 180– 350 bp.

Figure A shows the harvested PCR product pool from a Access Array 48.48 IFC, and Figure B shows the same harvested PCR product pool after SPRI cleanup:

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

Appendix A: Electropherogram Examples

The following figure shows a DNA 1000 Chip electropherogram overlay of an unpurified pooled PCR product library and the same purified pooled PCR product library after AMPure XP cleanup:

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

Appendix B: Access Array Barcode Library for the Ion Torrent PGM Sequencer - 96

The Access Array™ Barcode Library for the Ion Torrent™ PGM™ Sequencer - 96 (Fluidigm, PN 100-4911) is designed to be used for bidirectional sequencing strategy on the Ion Torrent sequencer for amplicon libraries that have been generated on the Access Array™ system.

Table 14. Access Array Barcode Library for Ion Torrent PGM Sequencer - 96

PN Plate

Number

100-4912 A1 Access Array Barcode Library for Ion Torrent PGM Sequencer P1_CS2 /

100-4913 B1 Access Array Barcode Library for Ion Torrent PGM Sequencer P1_CS1 /

Table 15. Barcode Sequences for Access Array Barcode Library for Ion Torrent PGM Sequencer - 96 (Fluidigm, PN100-4911)

Barcode Name Barcode Sequence Barcode Well Plate

MID-1 ACGAGTGCGT A1 A1 & B1

MID-2 ACGCTCGACA B1 A1 & B1

MID-3 AGACGCACTC C1 A1 & B1

MID-4 AGCACTGTAG D1 A1 & B1

MID-5 ATCAGACACG E1 A1 & B1

MID-6 ATATCGCGAG F1 A1 & B1

Reagent Description Volume

A_BC_CS1_1-96 at 2 M per oligo

A_BC_CS2_1-96 at 2 M per oligo

(L)

MID-7 CGTGTCTCTA G1 A1 & B1

MID-8 CTCGCGTGTC H1 A1 & B1

MID-10 TCTCTATGCG A2 A1 & B1

MID-11 TGATACGTCT B2 A1 & B1

MID-13 CATAGTAGTG C2 A1 & B1

MID-14 CGAGAGATAC D2 A1 & B1

MID-15 ATACGACGTA E2 A1 & B1

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

Appendix B: Access Array Barcode Library for the Ion Torrent PGM Sequencer - 96

Barcode Name Barcode Sequence Barcode Well Plate

MID-16 TCACGTACTA F2 A1 & B1

MID-17 CGTCTAGTAC G2 A1 & B 1

MID-18 TCTACGTAGC H2 A1 & B1

MID-19 TGTACTACTC A3 A1 & B1

MID-20 ACGACTACAG B3 A1 & B1

MID-21 CGTAGACTAG C3 A1 & B1

MID-22 TACGAGTATG D3 A1 & B1

MID-23 TACTCTCGTG E3 A1 & B1

MID-24 TAGAGACGAG F3 A1 & B1

MID-25 TCGTCGCTCG G3 A1 & B1

MID-26 ACATACGCGT H3 A1 & B1

MID-27 ACGCGAGTAT A4 A1 & B1

MID-28 ACTACTATGT B4 A1 & B1

MID-29 ACTGTACAGT C4 A1 & B1

MID-30 AGACTATACT D4 A1 & B1

MID-31 AGCGTCGTCT E4 A1 & B1

MID-32 AGTACGCTAT F4 A1 & B1

MID-33 ATAGAGTACT G4 A1 & B1

MID-34 CACGCTACGT H4 A1 & B1

MID-35 CAGTAGACGT A5 A1 & B1

MID-36 CGACGTGACT B5 A1 & B1

MID-37 TACACACACT C5 A1 & B1

MID-38 TACACGTGAT D5 A1 & B1

MID-39 TACAGATCGT E5 A1 & B1

MID-40 TACGCTGTCT F5 A1 & B1

MID-41 TAGTGTAGAT G5 A1 & B1

MID-42 TCGATCACGT H5 A1 & B1

MID-43 TCGCACTAGT A6 A1 & B1

MID-44 TCTAGCGACT B6 A1 & B1

MID-45 TCTATACTAT C6 A1 & B1

MID-46 TGACGTATGT D6 A1 & B1

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

Appendix B: Access Array Barcode Library for the Ion Torrent PGM Sequencer - 96

Barcode Name Barcode Sequence Barcode Well Plate

MID-47 TGTGAGTAGT E6 A1 & B1

MID-48 ACAGTATATA F6 A1 & B1

MID-49 ACGCGATCGA G6 A1 & B1

MID-50 ACTAGCAGTA H6 A1 & B1

MID-51 AGCTCACGTA A7 A1 & B1

MID-52 AGTATACATA B7 A1 & B1

MID-53 AGTCGAGAGA C7 A1 & B1

MID-54 AGTGCTACGA D7 A1 & B1

MID-55 CGATCGTATA E7 A1 & B1

MID-56 CGCAGTACGA F7 A1 & B1

MID-57 CGCGTATACA G7 A1 & B1

MID-58 CGTACAGTCA H7 A1 & B1

MID-59 CGTACTCAGA A8 A1 & B1

MID-60 CTACGCTCTA B8 A1 & B1

MID-61 CTATAGCGTA C8 A1 & B1

MID-62 TACGTCATCA D8 A1 & B1

MID-63 TAGTCGCATA E8 A1 & B1

MID-64 TATATATACA F8 A1 & B1

MID-65 TATGCTAGTA G8 A1 & B1

MID-66 TCACGCGAGA H8 A1 & B1

MID-67 TCGATAGTGA A9 A1 & B1

MID-68 TCGCTGCGTA B9 A1 & B1

MID-69 TCTGACGTCA C9 A1 & B1

MID-70 TGAGTCAGTA D9 A1 & B1

MID-71 TGTAGTGTGA E9 A1 & B1

MID-72 TGTCACACGA F9 A1 & B1

MID-73 TGTCGTCGCA G9 A1 & B1

MID-74 ACACATACGC H9 A1 & B1

MID-75 ACAGTCGTGC A10 A1 & B1

MID-76 ACATGACGAC B10 A1 & B1

MID-77 ACGACAGCTC C10 A1 & B1

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

Appendix B: Access Array Barcode Library for the Ion Torrent PGM Sequencer - 96

Barcode Name Barcode Sequence Barcode Well Plate

MID-78 ACGTCTCATC D10 A1 & B1

MID-79 ACTCATCTAC E10 A1 & B1

MID-80 ACTCGCGCAC F10 A1 & B1

MID-81 AGAGCGTCAC G10 A1 & B1

MID-82 AGCGACTAGC H10 A1 & B1

MID-83 AGTAGTGATC A11 A1 & B1

MID-84 AGTGACACAC B11 A1 & B1

MID-85 AGTGTATGTC C11 A1 & B1

MID-86 ATAGATAGAC D11 A1 & B1

MID-87 ATATAGTCGC E11 A1 & B1

MID-88 ATCTACTGAC F11 A1 & B1

MID-89 CACGTAGATC G11 A1 & B1

MID-90 CACGTGTCGC H11 A1 & B1

MID-91 CATACTCTAC A12 A1 & B1

MID-92 CGACACTATC B12 A1 & B1

MID-93 CGAGACGCGC C12 A1 & B1

MID-94 CGTATGCGAC D12 A1 & B1

MID-95 CGTCGATCTC E12 A1 & B1

MID-96 CTACGACTGC F12 A1 & B1

MID-97 CTAGTCACTC G12 A1 & B1

MID-98 CTCTACGCTC H12 A1 & B1

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

Appendix C: Related Documents

This document is intended to be used in conjunction with these related documents:

Document Title Part Number

Access Array™ Multiplex 20X Primer Solution Preparation 100-3895

4-Primer Amplicon Tagging with the LP 48.48 IFC 68000161

2-Primer Amplicon Tagging with the LP 48.48 IFC 68000148

Access Array

Advanta NGS Library Preparation with Access Array Protocol 101-7885

Advanta NGS Library Preparation on the LP 48.48 IFC with Access Array Quick Reference

IFC Controller AX User Guide 68000157

FC1™ Cycler User Guide 100-1279

Control Line Fluid Loading Procedure Quick Reference 68000132

–Generate Tagged Primers (an Excel Workbook) 100-3873

101-7886

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

Appendix D: Safety

IMPORTANT For translations of the instrument safety information, see Safety Information for

Genomics Instruments (PN 101-6810).

General Safety

In addition to your site-specific safety requirements, Fluidigm recommends the following general safety guidelines in all laboratory and manufacturing areas:

• Use the appropriate personal protective equipment (PPE): safety glasses, fully enclosed shoes, lab coats, and gloves, according to your laboratory safety practices.

• Know the locations of all safety equipment (fire extinguishers, spill kits, eyewashes/ showers, first-aid kits, safety data sheets, etc.), emergency exit locations, and emergency/injury reporting procedures.

• Do not eat, drink, or smoke in lab areas.

• Maintain clean work areas.

• Wash hands before leaving the lab.

Instrument Safety

The instrument should be serviced by authorized personnel only.

WARNING Do not modify this instrument. Unauthorized modifications may create a safety hazard.

WARNING BIOHAZARD. If you are putting biohazardous material on the instrument, use appropriate personal protective equipment and adhere to Biosafety in Microbiological and Biomedical Laboratories (BMBL), a publication from the Centers for Disease Control and Prevention, and to your lab's safety protocol to limit biohazard risks. If biohazardous materials are used, properly label the equipment as a biohazard. For more information, see the BMBL guidelines online at

cdc.gov/biosafety/publications/index.htm.

WARNING PHYSICAL INJURY HAZARD. Do not attempt to lift or move any boxed or crated items unless you use proper lifting techniques. The weight of the boxed IFC Controller AX is 27 kg (59 lb) and the FC1 cycler is 11 kg (25 lb). If you choose to lift or move the instrument after it has been installed, do not attempt to do so without the assistance of others. Use appropriate moving equipment and proper lifting techniques to minimize the chance of physical injury.

CAUTION PINCH HAZARD. The instrument door and tray can pinch your hand. Make sure your fingers, hands, and shirtsleeves are clear of the door and tray when loading or ejecting an integrated fluidic circuit (IFC).

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

Symbols on the Instrument

CAUTION HOT SURFACE HAZARD. Never press down on the integrated fluidic circuit (IFC) when it is on the thermal cycler chuck. If you encounter a vacuum problem, turn off the system, allow it to cool down, and remove the IFC. Clean the bottom of the IFC and/or chuck surface with a lint-free cloth and 70% isopropyl alcohol.

CAUTION HOT SURFACE HAZARD. Make sure the chuck has had time to cool. It can get very hot and cause burn injury.

Symbols on the Instrument

The following table describes the hazard symbols that may be used in this document or on labels on the instrument.

Symbol Description

Hazard. Consult the user guide for further information.

Hot surface hazard. Do not touch; potential for personal injury.

Appendix D: Safety

Biohazard.

Electricity hazard. Indicates high electricity levels and a threat of electric shock from machines and/or equipment in the vicinity. You may suffer severe injuries or death.

Pinch hazard. Indicates where pinch hazards exist. Exercise caution when operating around these areas.

Lifting hazard.

Power and standby symbol.

Power switch is in the Off position.

Power switch is in the On position.

Protective conductor terminal (main ground). It must be connected to earth ground before any other electrical connections are made to the instrument.

To minimize negative environmental impact from disposal of electronic waste, do not dispose of electronic waste in unsorted municipal waste.

Follow local municipal waste ordinances for proper disposal provision. Contact customer service for information about responsible disposal options.

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

Appendix D: Safety

Electrical Safety

NOTE The main power disconnect is on the rear panel of the instrument.

WARNING ELECTRICAL HAZARD. DO NOT REMOVE THE COVERS. Electrical shock can result if the instrument is operated without its protective covers. No internal components are serviceable by the user.

WARNING ELECTRICAL HAZARD. Plug the instrument into a properly grounded receptacle with adequate current capacity.

Chemical Safety

The responsible individuals must take the necessary precautions to ensure that the surrounding workplace is safe and that instrument operators are not exposed to hazardous levels of toxic substances. When working with any chemicals, refer to the applicable safety data sheets (SDSs) provided by the manufacturer or supplier.

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

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Fluidigm Access Array User Manual

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