adp 23 long-range pcr rev b

3
Advanced Development Protocol
Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Reference Documents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Required Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Required Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Long-Range PCR Primer Design Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Primer Validation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Priming the 48.48 Access Array IFC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Long-Range PCR Amplification on the 48.48 Access Array IFC . . . . . . . . . . . . . . . . . . . 7

Preparing the Sample Pre-Mix and Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Loading the 48.48 Access Array IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Thermal Cycling the 48.48 Access Array IFC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Harvesting PCR Products from the 48.48 Access Array IFC . . . . . . . . . . . . . . . . . . . . .13

Checking PCR Products on the Agilent 2100 BioAnalyzer . . . . . . . . . . . . . . . . . . . . . . . 15

For More Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Long-Range PCR on the 48.48 Access Array IFC

PN 100-2045 B 2

Fluidigm Advanced Development Protocol 23

2

Introduction

This document explains how to use the Fluidigm 48.48 Access Array™ IFC to generate Long-Range PCR products (1-10 kb). Long-Range PCR can be used to enrich specific target regions for high-throughput sequencing experiments carried out on the 454 GS FLX Sequencer, the Illumina Genome Analyzer or the Life Technologies SOLiD sequencing platform.

Figure 1: Long-Range PCR workflow on the Access Array system

Reference Documents

Fluidigm Access Array User Guide (PN 68000158)

Fluidigm IFC Controller AX User Guide (PN 68000157)

Agilent DNA 12000 Kit User Manual

Qualify Primer Sets

Select Best Primer Sets

Design PCR Primers

Qualify Samples

Clean up PCR product pool

Normalize Sample Concentrations

Load Samples and Primers on Access Array IFC

PCR amplify for 35 cycles

Recover PCR products from Access Array IFC

Qualify/Quantify PCR products

Select and Pool Samples

Library Preparation

Advanced Development Protocol 23 Fluidigm

Required Reagents

Stored at -20ºC

Stored at 4ºC

Stored at Room Temperature

Required Equipment

Phusion™ Flash High Fidelity PCR Master Mix (New England BioLabs, PN F-548)

Phusion™ HotStart High-Fidelity DNA Polymerase (New England BioLabs, PN F-540)

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

100 µM target-specific (TS) Forward Primer Plate

100 µM target-specific (TS) Reverse Primer Plate

50 ng/µL genomic DNA (Coriell, PN NA17317)

Agilent DNA 12000 Kit Reagents (Agilent, PN 5067-1506)

1X Access Array Harvesting Reagent (Fluidigm, PN 100-1031) cap

1X Access Array Long-Range PCR Hydration Reagent (Fluidigm, PN 100-1721)

1M Magnesium Chloride (TEKnova, PN M0300)

DNA Suspension Buffer (10 mM Tris, pH 8.0, 0.1 mM EDTA) (TEKnova, PN T0221)

PCR Certified Water (TEKnova, PN W3330)

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

48.48 Access Array IFC(s)

Fluidigm Stand-Alone Thermal Cycler (SATC) or Fluidigm FC1 Thermal Cycler

MicroAmp Optical Adhesive Film (Life Technologies, PN 431197)

Universal Black Microplate Lid (VWR, PN 77776-852)

TempPlate Sealing Foil (USA Scientific, PN 2923-0110)

3


4

Long-Range PCR Primer Design Guidelines

The Long-Range PCR protocol for the Access Array system supports production of amplicons between 1 and 10 kb in size. However, within this range of product size, PCR yields will vary with the size of the amplicon (shorter fragments will produce more DNA). We strongly recommend primer sets for Long-Range PCR experiments to be designed to generate PCR products within 20% of the average PCR product size. For example, for PCR products with an average size of 5 kb, we recommend using a size range between 4500-5500 bases for the primer sets. If PCR product sizes fall outside of this range, relative coverage of each of the products in the sequencing experiment will vary considerably.

1 We recommend designing primer sets so that adjacent amplicons have an overlap of 250-500 bases.

2 Primers can be designed using the free web tool at http://flypush.imgen.bcm.tmc.edu/primer/ and the following parameters:

a Primer Tm: 58ºC ≤ Tm ≤ 61ºC

b Primer Size: 20 nucleotides (nt) ≤ Length ≤ 32 nt

c Primer GC%: 40%<GC%<70%

d Max Poly-X: Avoid nucleotide runs ≥ 4 nt.

3 Refer to the Fluidigm Access Array User Guide for additional Primer Design guidelines.

Primer Validation

The target-specific (TS) Primer pairs should be validated using the protocol outlined below before running a 48.48 Access Array IFC. This protocol prepares enough reagents to perform 48 primer validation reactions in a 96-well plate.

1 Prepare the primer validation reaction components in a 1 mL tube.

96-well plate thermal cycler

Agilent 2100 BioAnalyzer

Agilent DNA 12000 Chips (included in the Agilent DNA 12000 Kit) (Agilent, PN 5067-1506)

96-well plate

IMPORTANT: The Long-Range PCR protocol for the Access Array system is only supported for primer pairs validated using the following procedure.


Table 1: Primer Validation Reaction Mix

2 Vortex the Primer Validation Reaction Mix for a minimum of 20 seconds, and centrifuge for at least 30 seconds to spin down all components.

3 Set up the PCR Reactions in a 96-well PCR plate.

a Add 10.0 μL of Primer Validation Reaction Mix to each well.

b Add 0.5 μL of the TS Forward Primer for 10 μM final concentration.

c Add 0.5 μL of the TS Reverse Primer for 10 μM final concentration.

The total PCR reaction volume is 11 μL.

4 Tightly seal the plate with a TempPlate Sealing Foil (USA Scientific, PN 2923-0110) to minimize dehydration during thermal cycling.

5 Vortex the 96-well PCR reaction plate for a minimum of 20 seconds, and centrifuge for 30 seconds to spin down all components.

6 Run the PCR Reactions.

d Load the 96-well plate onto the thermal cycler.

e Run the PCR protocol described below:

ComponentVolume per

Reaction (µL)Volume for 60 Reactions (µL)

Phusion Flash High-Fidelity PCR Master Mix (New England BioLabs, PN F-548)

5.0 300.0

20X Access Array Loading Reagent

(Fluidigm, PN 100-0883) cap

0.485 29.1

1M Magnesium Chloride (TEKnova, PN M0300)

0.015 0.9

Phusion HotStart High-Fidelity DNA Polymerase (New England BioLabs, F-540)

0.50 30.0

50 ng/µL genomic DNA 4.0 240.0

Total 10 600

NOTE: The volumes provided in the table above are sufficient for 60 11 μL reactions in a 96-well plate. We recommend preparing this amount to provide sufficient reagent to minimize errors due to pipetting.

5


6

Table 2: Long-Range PCR protocol for 1-10 kb amplicons

7 Check PCR Products on the Agilent 2100 BioAnalyzer.

a Dilute 2 µL of each PCR product with 10 µL of PCR Certified Water (PN W3330).

b Run 1 µL of the diluted PCR product from each of the PCR reactions on an Agilent DNA 12000 chip on the Agilent 2100 BioAnalyzer. Follow the Agilent DNA 12000 Kit User Manual for details.

c Inspect the results from the BioAnalyzer chip run. Please refer to the Fluidigm Access Array User Guide for details on checking PCR products on the Agilent 2100 BioAnalyzer.

Successful reactions will produce a single band of the expected size. If the PCR reactions contain multiple bands, or bands of incorrect size, the primer pair should be removed from the test set. In these cases, check the amplicon sequence for any problems such as a high G/C content or long nucleotide repeats. If there are no apparent problems with the amplicon sequence, redesign the primers by moving the primer location upstream or downstream by 1000-2000 bp or by splitting the target region into two amplicons.

8 Select the TS Primer pairs that produce the correct PCR products and continue with the 48.48 Access Array IFC.

Priming the 48.48 Access Array IFC

1 Inject control line fluid into both IFC accumulators.

2 Add 500 μL of 1X Access Array Harvest Reagent (Fluidigm, PN 100-1031) into the H1-H3 wells on the IFC.

Step Temperature Time

1 50ºC 2 min

2 70ºC 12 min

3 98ºC 10 sec

4 98ºC 1 sec

5 61ºC 5 sec

6 72ºC 5 min

7 Go to Step 4, repeat 34 times

8 72ºC 10 min

9 4ºC

NOTE: Control Line Fluid on the IFC or in the inlets makes the IFC unusable. Use only 48.48 syringes with 300 μL of Control Line Fluid (PN 89000020).


3 Add 500 μL of 1X Access Array Long-Range PCR Hydration Reagent (Fluidigm, PN100-1721) into the H4 well on the IFC.

Figure 2: Loading positions of the Harvest Reagent and Hydration Reagent on the 48.48 Access Array IFC

4 Place the 48.48 Access Array IFC into the Pre-PCR IFC Controller AX located in the Pre-PCR Lab and run Prime (151x) script.

Long-Range PCR Amplification on the 48.48 Access Array IFC

Prepare the 20X Primer Solutions1 Take the 100 μM target-specific (TS) Forward and Reverse Primer plates out of the

freezer and warm the plates up to room temperature. Vortex the 20X Primer solutions for a minimum of twenty seconds, and centrifuge for 30 seconds to spin down all components.

2 In a DNA-free hood, prepare the 20X Primer solution for each primer pair in a 96-well plate as shown below in Table 3.

NOTE: Make sure the H1, H2 and H3 wells are loaded with Access Array Harvest Reagent (Clear) and the H4 well is loaded with Access Array Hydration Reagent.

NOTE: Load the IFC in the Pre-PCR IFC Controller AX in the Pre-PCR Lab within 60 minutes of priming.

H1 H2

Harvest Reagent

Hydration Reagent

H3 H4

7


8

Table 3: 20X Primer solutions

3 Vortex the 20X Primer solution plate for 20 seconds, and centrifuge for 30 seconds to spin down all components. Store the plate at 4°C.

Preparing the Sample Pre-Mix and Samples

Prepare the Sample Pre-Mix Solution1 Normalize the concentration of each genomic DNA sample to 50 ng/µL.

2 In a DNA-free hood, combine the components listed in Table 4 below. This protocol prepares enough Sample Pre-Mix solution for 60 reactions. This is enough reagent to load one 48.48 Access Array IFC with 16 additional reactions to compensate for dead volume and pipetting error.

Component Volume (µL)

TS Forward Primer (100 μM) 0.5

TS Reverse Primer (100 μM) 0.5

20X Access Array Loading Reagent (Fluidigm, PN 100-0883) cap0.25

DNA Suspension Buffer 3.75

Total 5

NOTE: To avoid multiple freeze-thaws of primers, a larger volume of the 20X Primer solution can be prepared and aliquoted into multiple 96-well PCR plates. Store the unused 20X Primer solution plates at -20°C.

NOTE: The final TS Forward and Reverse Primer concentrations are 10 μM in the 20X Primer solution. The final TS Forward and Reverse Primer concentrations in the 48.48 Access Array IFC reaction chambers are 500 nM.


Table 4: Sample Pre-Mix solution

3 Vortex the tube for a minimum of 20 seconds, and centrifuge for 30 seconds to spin down all components.

Prepare the Sample Mix Solutions1 Combine the components listed below in a 96-well plate to prepare 48 individual Sample

Mix solutions.

Table 5: Sample Mix solutions

2 Vortex the Sample Mix solutions for a minimum of 20 seconds, and centrifuge for 30 seconds to spin down all components.

Loading the 48.48 Access Array IFC

1 Pipette 4 μL of 20X Primer solution into each of the primer inlets.

2 Pipette 4 μL of Sample Mix solution into each of the sample inlets.

ComponentVolume per

Reaction (µL)Volume per 60 Reactions (µL)

Phusion Flash High-Fidelity PCR Master Mix (New England BioLabs, PN F-548)

2.5 150.0

20X Access Array Loading Reagent

(Fluidigm, PN 100-0883) cap

0.242 14.52

1M Magnesium Chloride (Teknova, PN M0300)

0.008 0.48

Phusion HotStart High-Fidelity DNA Poly-merase (New England BioLabs, F-540)

0.25 15.0

Total 3 180

Component Volume (µL)

Sample Pre-Mix 3.0

50 ng/µL genomic DNA 2.0

Total 5

NOTE: It is essential that you vortex all components to ensure complete mixing.

9


10

Figure 3: 48.48 Access Array IFC Sample and Assay inlet pipetting map

H1 H2

H3 H4

Sample Mix Primer Mix


Figure 4: 48.48 Access Array IFC pipetting scheme

3 Load the 48.48 Access Array IFC into the Pre-PCR IFC Controller AX in the Pre-PCR Lab and run Load Mix (151x) script.

4 When the script is finished, press Eject to remove the 48.48 Access Array IFC from the Pre-PCR IFC Controller AX.

Thermal Cycling the 48.48 Access Array IFC

1 Check the top surface of the 48.48 Access Array IFC chip for any large particles or debris. Remove the particles gently with scotch tape.

2 Cut one of the MicroAmp Optical Adhesive Films (Life Technologies, PN 431197) into a 1.7 inch square using the Template for cutting the PCR Film shown in Figure 4. This will be used as the pre-cut PCR film for thermal cycling.

NOTE: We recommend using an 8-channel pipette to load the Sample Mix solution and 20X Primer solution. The recommended pipetting order is shown below.

147 8

11101316 17 18

211922 23

262932

24273033

3639424548

252831

34 3537 3840 41

4447

4346

20

14 15

9

12

5 62 3

Step 1

147 8

11101316 17 18

211922 23

262932

24273033

3639424548

252831

34 3537 3840 41

4447

4346

20

14 15

9

12

5 62 3

Step 2147 8

11101316 17 18

211922 23

262932

24273033

3639424548

252831

34 3537 3840 41

4447

4346

20

14 15

9

12

5 62 3

Step 3147 8

11101316 17 18

211922 23

262932

24273033

3639424548

252831

34 3537 3840 41

4447

4346

20

14 15

9

12

5 62 3

Step 4

212427

33

3147 8

11101316 17 181922 23

262932

30

3639424548

252831

34 3537 3840 41

4447

4346

20

14 15

9

12

5 62

Step 5147 8

11101316 17 18

211922 23

262932

24273033

3639424548

252831

34 3537 3840 41

4447

4346

20

14 15

9

12

5 62 3

Step 6

11


12

3 Place the film on the surface of the chip starting at one corner, smoothing the film gradually to avoid capturing air bubbles. Make sure to remove any air bubbles between the PCR film and the chip.

Figure 5A: Placement of the pre-cut PCR film onto the 48.48 Access Array IFC chipFigure 5B: Template for cutting the PCR film

4 Remove the blue IHS protector from the bottom of the 48.48 Access Array IFC.

5 Place the 48.48 Access Array IFC on the Thermal Cycler.

6 Place the Universal Microplate Lid (PN 77776-852) on top of the 48.48 Access Array IFC carrier as shown in Figure 5.

Figure 6: Placement of the Universal Microplate Lid onto the 48.48 Access Array IFC carrier

A B

1.7”

1.7”Template for

cutting the PCR film


7 Turn on the vacuum.

8 Select the AALR48V1 PCR Protocol listed in Table 6 (for the SATC) or AA 48x48 Long Range v1 (for the FC1), and follow the Thermal Cycler instructions in the Fluidigm Access Array User Guide.

9 Press START to begin PCR.

Table 6: AALR48V1 - Long-Range PCR protocol for 2-10 kb amplicons

Harvesting PCR Products from the 48.48 Access Array IFC

1 After the PCR has finished, press ENTER and turn off the vacuum on the Fluidigm Thermal Cycler.

2 Move the 48.48 Access Array IFC into the Post-PCR Lab for harvesting.

3 Remove the Universal Microplate Lid (PN 77776-852) from the 48.48 Access Array IFC.

4 Remove the remaining 1X Access Array Harvest Reagent from the H1-H3 wells.

5 Remove the remaining 1X Access Array Hydration Reagent from the H4 well.

6 Pipette 600 μL of fresh 1X Access Array Harvest Reagent into each of the H1-H4 wells.

7 Pipette 2 μL of 1X Access Array Harvest Reagent into each of the sample inlets on the IFC.

Step Temperature Time

1 50ºC 2 min

2 70ºC 12 min

3 98ºC 10 sec

4 98ºC 1 sec

5 61ºC 5 sec

6 72ºC 5 min

7 Go to Step 4, repeat 34 times

8 72ºC 10 min

9 4ºC Hold at 4ºC

13


14

Figure 7: Harvest Reagent loading positions on Post-PCR 48.48 Access Array IFC

8 Place the 48.48 Access Array IFC into the Post-PCR IFC Controller AX located in the Post-PCR Lab and run Harvest (151x) script.

9 When the Harvest (151x) script has finished, remove the 48.48 Access Array IFC from the Post- PCR IFC Controller.

10 Label a 96-well plate with the 48.48 Access Array IFC barcode. Carefully transfer the harvested PCR products from each of the sample inlets into columns 1-6 of the 96-well PCR plate.

11 Store the PCR products at -20°C.

NOTE: Remove PCR products from the 48.48 Access Array IFC using an 8-channel pipette in the same order as you loaded the IFC.

H1 H2

H3 H4

2 µL/inlet 600 µLHarvest Reagent Harvest Reagent

600 µLHarvest Reagent


Figure 8: PCR product transfer map from the 48.48 Access Array IFC to a 96-well PCR plate

Checking PCR Products on the Agilent 2100 BioAnalyzer

1 Use the Agilent DNA 1200 chips from the Agilent DNA 1200 Kit to check 1 μL of PCR product from each of the PCR reactions described above. Follow the Agilent DNA 1200 Kit User Manual for details.

2 Check the PCR product pool to confirm that products of the correct size have been generated. The product pool contains a combination of all 48 PCR products generated by the Access Array IFC, and will appear as a smear on the BioAnalyzer. A histogram of expected PCR product sizes can be used for comparison with the electropherogram trace from the PCR product pool.

3 Store the PCR products at -20ºC.

4 Refer to the Fluidigm Access Array User Guide for more information about this workflow.

15


16

For More Information

To find out more about the information in this or any other Fluidigm Advanced Development Protocol, contact [email protected] or call:

North America: 1 866 358 4354

Europe: +33 1 60 92 42 40

Japan: +81 3 3555 2351

All other countries: +1 650 266 6100

© Copyright, Fluidigm Corporation. All rights reserved. Fluidigm, the Fluidigm logo, BioMark, Access Array, Dynamic Array, Digital Array, FC1, Topaz, and NanoFlex are trademarks or registered trademarks of Fluidigm Corporation. All other trademarks are the property of their respective owners. Part Number 100-2045 B

adp 23 long-range pcr rev b

Documents

access array ifc

range pcr

primer sets

england biolabs

range

size

agilent

long