Access Array for Illumina Sequencing Systems - Fluidigm [PDF]

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PN 100-3770 L1

Access Array System for Illumina Sequencing Systems 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. For licensing information, contact [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, contact [email protected]. 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. 09/2018

For technical support visit fluidigm.com/support. North America +1 650 266 6100 | Toll-free (US/CAN): 866 358 4354 | [email protected] Latin America +1 650 266 6100 | [email protected] Europe/Middle East/Africa/Russia +44 1223 598100 | [email protected] Japan +81 3 3662 2150 | [email protected] China (excluding Hong Kong) +86 21 3255 8368 | [email protected] All other Asian countries/India/Australia +1 650 266 6100 | [email protected]

2

Access Array System for Illumina Sequencing Systems: User Guide

Contents About This Guide

5

Sample Quantitation

23

Safety Alert Conventions

5

Sample Normalization

23

Safety Data Sheets

6

Chapter 5: Multiplex Amplicon Tagging on the LP 48.48 IFC

24

Multiplex PCR on the LP 48.48 IFC

25

Target-Specific Primer Pooling

25

Chapter 1: Access Array System Overview

7

4-Primer Amplicon Tagging Overview

8

Access Array System Components

10

Reference Documents

25

Access Array IFCs

10

Materials

25

Required Reagents

25

Specifications

11

Access Array System Workflow

12

Prime the IFC

26

Materials

12

Prepare the 20X Primer Solution

28

Required Reagents

12

Required Consumables

13

Prepare Sample Pre-Mix and Samples

28

Required Equipment

14

Example Sample and Primer Layout in 96-Well Plates

15

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

Prepare the Sample Pre-Mix Solution

28

Prepare the Sample Mix Solution

29

Load the IFC

29

Thermal-Cycle the IFC

30

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 Solution

33

17

Thermal-Cycle the 96-Well PCR Plate

34

Reference Documents

17

Materials

17

Check PCR Products on the Agilent 2100 Bioanalyzer

34

Required Reagents

17

Next Step

35

Prepare Primer Validation Reactions

18

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

20

Chapter 6: 4-Primer Amplicon Tagging on the LP 48.48 IFC

36

Run the PCR Reactions

20

Check PCR Products on the Agilent 2100 Bioanalyzer

21

Reference Documents

37

Materials

38

Required Supplies

38

Required Reagents

38

Required Consumables

39

Required Equipment

40

Prime the IFC

41

Prepare the 20X Primer Solutions

42

Chapter 4: Sample Quantitation and Normalization

22

Reference Documents

22

Materials

22

Required Reagents

22

Prepare Sample Master Mix Solutions

43

22

Prepare the Sample Pre-Mix Solution

43

Required Equipment

Access Array System for Illumina Sequencing Systems: User Guide

3

Contents

44

Appendix C: Ordering Instructions for FL1 and FL2 Sequencing Primers

66

Thermal-Cycle the IFC

45

Order the Oligos for FL1 and FL2 Primers

66

Harvest the IFC

45

Prepare FL1 and FL2 Primer Mixes

67

Chapter 7: Post-PCR Amplicon Purification and Quantitation

47

Reference Documents

47

Appendix D: Sequencing Workflow Using Fluidigm FL1 and FL2 Sequencing Primers

69

Materials

47

Reference Documents

69

Required Equipment

47

Required Reagents

47

Prepare Reagents for Sequencing on the Illumina HiSeq Sequencing System

69

Quantify PCR Products

48

Prepare Read 1 Sequencing Primer HT1/FL1 for the cBot

69

Pool Products from Multiple Access Array IFCs

48

Add Read 2 Indexing Primer FL2 to HP8

70

Purify Harvested PCR Products

48

Agilent 2100 Bioanalyzer Quantification

49

Add Read 3 Sequencing Primer FL1 to HP7 (for Paired-End Sequencing)

70

PicoGreen Fluorimetry Quantification

50

Prepare Reagents for Sequencing on the Illumina MiSeq Sequencer

70

Prepare Read 1 Sequencing Primer FL1

70

Prepare Read 2 Indexing Primer FL2

71 71

Prepare the Sample Mix Solution

43

Load the IFC

Appendix A: 2-Primer Target-Specific PCR Amplification on the LP 48.48 IFC

51

Reference Documents

54

Prepare Read 3 Sequencing Primer FL1 (for Paired-End Sequencing only)

Materials

54

Perform a Sequencing Run

71

Prime the IFC

55

Index Read

72

Prepare the 20X Primer Solutions

56

Prepare Sample Mix Solutions

56

Load the IFC

58

Thermal-Cycle the IFC

58

Harvest the IFC

59

53

Appendix E: Electropherogram Examples 74 Appendix F: Access Array Barcodes for the Illumina Sequencing Systems

76

Access Array Barcode Library

76

Barcode Sequences

76

Appendix G: Related Documents

87

Appendix H: Safety

88

General Safety

88

Instrument Safety

88

Appendix B: Custom Sequencing Primers for NextSeq on the LP 48.48 IFC

61

Reference Documents

62

Required Reagents

62

Procedure

62

1 Prepare the Custom Sequencing Primers

63

2 Prepare the NextSeq Reagent Kit

63

Symbols on the Instrument

89

3 Finish Preparing the Custom Primers

64

Electrical Safety

90

4 Load the Sequencing Primers and Library

64

5 Sequence on the NextSeq 500 or 550

65

Chemical Safety

90

4

Access Array System for Illumina Sequencing Systems: 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 CAUTION ABBREVIATED SAFETY ALERTS. Hazard symbols and hazard types specified in procedures may be abbreviated in this document. For complete safety information, see Appendix H. 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.

Access Array System for Illumina Sequencing Systems: User Guide

5

About This Guide

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.

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 Illumina Sequencing Systems: User Guide

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 four major phases: 1) designing and validating target-specific primers for targeted sequencing, 2) quantifying and normalizing samples, 3) running an integrated fluidic circuit (IFC) for Access Array, and 4) qualifying and quantifying harvested PCR products for sequencing. The chapters in this user guide detail the following workflow chronologically:

Access Array System for Illumina Sequencing Systems: User Guide

7

Chapter 1: Access Array System Overview 4-Primer Amplicon Tagging Overview

Depending on the type of experiment, there are several Access Array amplicon tagging approaches for generating amplicon libraries to sequence on next-generation sequencers, such as the Illumina® MiSeq™, or HiSeq®. The sequencing method in the table below for prepared amplicons shows examples for single- or paired-end sequencing on HiSeq. Table 1. Some amplicon tagging strategies for sequencing on HiSeq Amplicon Preparation

Barcoding

Multiplex amplicon tagging on LP 48.48 IFC or Access Array 48.48 IFC

Barcode PCR products in a 96-well plate

(up to 480 amplicons per sample) (See Chapter 5) 4-primer amplicon tagging on LP 48.48 IFC or Access Array 48.48 IFC

Barcode PCR products in an IFC

(See Chapter 6) 2-primer amplicon tagging on LP 48.48 IFC or Access Array 48.48 IFC (singleplex reactions)

Barcode PCR products in a 96-well plate

(See Appendix A)

4-Primer Amplicon Tagging Overview The Access Array 4-primer amplicon tagging scheme is based on a step-out 4-primer PCR strategy where tagged target-specific (TS) primer pairs are combined with sample-specific primer pairs that contain a barcoding sequence, plus the adapter sequences used by Illumina sequencing systems. By incorporating sample-specific barcodes, all 2,304 PCR products generated from an LP 48.48 IFC (or Access Array 48.48 IFC) are unique and can be pooled together to run in a single sequencing experiment. First, the samples and sample-specific primers are loaded into each row of reaction wells on the IFC:

Tagged target-specific primers Sample + Barcode primers

8

Access Array System for Illumina Sequencing Systems: User Guide

Chapter 1: Access Array System Overview 4-Primer Amplicon Tagging Overview

Tagged TS forward and reverse primers are then loaded into each column of reaction wells on the IFC:

Tagged target-specific primers Sample + Barcode primers

After loading the IFC, each reaction well contains a unique combination of TS primer pairs and samples. Each reaction-well row also contains a sample-specific barcode primer pair:

Access Array System for Illumina Sequencing Systems: User Guide

9

Chapter 1: Access Array System Overview Access Array System Components

The resulting PCR products are pooled amplicons with unique barcodes:

Access Array System Components The Access Array system consists of the following components: •

IFCs for Access Array (either of these): •

LP 48.48 IFC



Access Array 48.48 IFC



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



FC1™ cycler

IFC Controller AX Pre-PCR

FC1 cycler

IFC Controller AX Post-PCR

NOTE The stand-alone thermal cycler has been discontinued. It has been replaced by the FC1 cycler.

Access Array IFCs Access Array reagents can be run on the: •

10

LP 48.48 IFC (PN 101-1926)

Access Array System for Illumina Sequencing Systems: User Guide

Chapter 1: Access Array System Overview Access Array IFCs



Access Array 48.48 IFC (PN AA-M-48.48) Primer inlets

Sample inlets

H1 well

Interface accumulator

H2 well

A1

H4

H3 well well

Containment accumulator

Control Line Fluid

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 Illumina Sequencing Systems: 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 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.

12

Access Array System for Illumina Sequencing Systems: User Guide

Chapter 1: Access Array System Overview Materials

Reagents Supplied by Fluidigm Product Name

Manufacturer

Part Number

20X Access Array Loading Reagent

Fluidigm

100-0883

1X Access Array Harvest Solution

Fluidigm

100-1031

Fluidigm

100-7966

Fluidigm

100-4876

Storage

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

–20 °C

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 Illumina Sequencers—384

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, 0.1 mM EDTA)

Teknova

T0221

Agencourt® AMPure® XP Reagent Beads

Beckman Coulter

A63880

100% ethanol

Major laboratory supplier (MLS)



Required Consumables Consumables Supplied by Fluidigm Product Name

Manufacturer

Part Number

Storage

LP 48.48 IFC or Access Array 48.48 IFC

Fluidigm

101-1926 AA-M-48.48

Room temperature

Fluidigm

101-2345

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

Access Array System for Illumina Sequencing Systems: User Guide

89000020

13

Chapter 1: Access Array System Overview Materials

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

Quant-iT™ PicoGreen® fluorescent assay

Thermo Fisher Scientific

P11496

Product Name

Manufacturer

Part Number

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

Fluidigm

IFC-AX

FC1™ cycler

Fluidigm

CYC-FC1

Product Name

Manufacturer

Part Number

DynaMag™-2 Magnet

Thermo Fisher Scientific

123-21D

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

Required Equipment Equipment Supplied by Fluidigm

NOTE The stand-alone thermal cycler has been discontinued. It has been replaced by the FC1 cycler.

Equipment from Other Suppliers

14



Access Array System for Illumina Sequencing Systems: User Guide

Chapter 1: Access Array System Overview Example Sample and Primer Layout in 96-Well Plates

Example Sample and Primer Layout in 96-Well Plates For ease of pipetting, fill the 96-well plates by columns, not rows.

Before You Begin IMPORTANT Before using the Access Array system, read and understand the detailed instructions and safety guidelines in Appendix H. •

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 Illumina Sequencing Systems: 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 μL per well in a single 96-well plate with mixed forward and reverse primers at a final concentration of 50 μM. Multiplex primer sets are provided in nuclease-free water at a final volume of 100 μL per well in six 96-well plates per set at a final concentration of 60 μM. 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). 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).

16

Access Array System for Illumina Sequencing Systems: 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™ Barcode 1 primers for the Illumina® Sequencer must be ordered prior to primer validation. See the table in Prepare Primer Validation Reactions on page 18 for oligo sequences, and order through an oligonucleotide vendor.

Reference Documents Agilent® DNA 1000 Kit Guide

Materials 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.

Reagents Supplied by Fluidigm Product Name

Manufacturer

Part Number

20X Access Array Loading Reagent

Fluidigm

100-0883

Access Array Barcode 1 primers for Illumina with CS1/CS2 tags

Fluidigm



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

Fluidigm



50 μM CS1–tagged TS forward primer 50 μM CS2–tagged TS reverse primer

Access Array System for Illumina Sequencing Systems: User Guide

17

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/μL 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 the following table for oligo sequences, and order through an oligonucleotide vendor. Table 2. Access Array Barcode 1 primers for Illumina Name

Sequence (5′-3′)

PE1_CS1 forward primer

AATGATACGGCGACCACCGAGATCTACACTGACGACA TGGTTCTACA

47 bp

PE2_BC_CS2 reverse primer

CAAGCAGAAGACGGCATACGAGATGTATCGTCGTTAC GGTAGCAGAGACTTGGTCT

56 bp

1

Total Length

Prepare the 5X target-specific primer solutions for 48 individual primer pairs as follows: Table 3. 5X target-specific primer solution preparation Component

Volume (μL)

Final Concentration* (nM)

50 μM CS1-tagged TS forward primer

1

250

50 μM 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 μL of the final primer solution.

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

18

Prepare 2 μM Access Array Barcode 1 primer solution.

Access Array System for Illumina Sequencing Systems: User Guide

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

Table 4. The 2 μM Access Array Barcode 1 primers for Illumina Component

Volume (μL)

Final Concentration (μM)

100 μM PE1_CS1 forward primer

2.0

2

100 μM PE2_BC_CS2 reverse primer

2.0

2

PCR Certified Water (Teknova)

96.0

Total

4

100

Prepare the primer validation reaction mix for 4-primer amplicon tagging. IMPORTANT Allow the 20X Access Array Loading Reagent warm to room temperature before use. Table 5. The 4-primer amplicon tagging primer validation reaction mix Component

Volume (μL)

Volume for 60 (μL)

Final Concentration*

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

0.5

30.0

1X

25 mM MgCl2 (Roche)

0.9

54.0

4.5 mM

DMSO (Roche)

0.25

15.0

5%

10 mM PCR Grade Nucleotide Mix (Roche)

0.10

6.0

200 μM each

5 U/μL FastStart High Fidelity Enzyme Blend (Roche)

0.05

3.0

0.05 U/μL

20X Access Array Loading Reagent (Fluidigm)

0.25

15.0

1X

1.0

60.0

400 nM

60 ng/μL genomic DNA (Coriell)

0.83

49.8

10 ng/μL

PCR Certified Water (Teknova)

0.12

7.2

Total

4.0

240.0

2 μM Access Array Barcode 1 primers for Illumina (from step 3)

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

5

Vortex the primer validation reaction mix for a minimum of 20 seconds and centrifuge for 30 seconds. NOTE Table 5 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 Illumina Sequencing Systems: User Guide

19

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

Set Up the PCR Reactions in a 384-Well PCR Plate 1

Prepare the primer validation PCR reactions: a Add 4 μL of primer validation reaction mix to each of the 48 wells. b Add 1 μL of the 5X target-specific primer solution. The total PCR reaction volume is 5 μL.

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 6. 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

10

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

20

2

8

2

95 ºC 15 sec 60 ºC 30 sec 72 ºC 1 min

8

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

5

Access Array System for Illumina Sequencing Systems: 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

Check PCR Products on the Agilent 2100 Bioanalyzer Use the Agilent DNA 1000 chips from the Agilent DNA 1000 Kit to check 1 μL 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

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