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
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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
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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