emPCR Amplification Method Manual – Lib L LV...GS FLX+ Series— XL+, emPCR Amplification Method...

For life science research only. Not for use in diagnostic procedures.

GS FLX / X LR 70

GS FLX+ / XLR70

G S F L X + / X L +

GS Junior / Junior

Inst rument / K i t

emPCR Amplification Method Manual – Lib L LV

GS FLX+ Series — XL+

May 2011

emPCR Method Manual – Lib-L LV

GS FLX+ Series — XL+ 2 / 12 May 2011

1. WORKFLOW

The emulsion-based clonal amplification (emPCR amplification) of a DNA library sample involves 7 major steps, described in Figure 1. Variants of this procedure exist to accommodate different types of DNA libraries and different number of beads requirements for the experiment. The method described in this manual is for Rapid libraries (Lib-L) from the GS FLX+ Series and for Large Volume emulsions (LV).

This method is to amplify libraries that will be sequenced on the GS FLX+ Instrument, using a GS FLX Titanium Sequencing Kit XL+.

Preparation of the Emulsions and Amplification

Kit Part A

Amplification Reaction; Bead Recovery and Enrichment

Kit Part B

Approx. Time

1 - 2 h

4 – 5 h

1

Preparation of the Reagents and of the Emulsion Oil (section 3.1)

Preparation of the Reagents

Preparation of the Emulsion Oil

Preparation of the Mock Amplification Mix and Pre-Emulsions

Preparation of the Live Amplification Mix

DNA Library Capture (section 3.2)2

Emulsification (section 3.3)3

8 h

Amplification (section 3.4)

Dispensing the Emulsions

Amplification Reaction

4

6

DNA Library Bead Enrichment (section 3.6)

Preparation for Enrichment

Preparation of the Enrichment Beads

Enrichment of the DNA-Carrying Beads

Collection of the Enriched DNA Beads

Sequencing Primer Annealing (section 3.7)7

5

Bead Recovery (section 3.5)

Vacuum-Assisted Emulsion Breaking Set Up

Emulsion Collection and Initial Washes

Bead Washes and Recovery

Figure 1: Workflow of the emPCR Amplification method



2. BEFORE YOU BEGIN

Room temperature is + 15 to + 25°C.

2.1 Sample Handling Strategy for Large Volume Emulsions

Figure 2 shows sample handling in the large volume emulsion.

1 GS FLX Titanium LV emPCR Kit (Lib-L)1 GS FLX Titanium emPCR Breaking Kit. LV/MV, 12 pcs

Total of 2 emulsion cups

2 samples(1 Emulsion Cup each)

1 x Emulsion Cup

2 regions gasket(Large)

PCR Plate 1~90 wells

PCR Plate 2~90 wells

2 x 50 ml Collection Tubes

2 x 1.7 ml Collection Tubes

1 Seq Primer Tube

FOR EACH SAMPLE (x 2)

Figure 2: Sample handling for Large volume emulsions

2.2 Determining the Amount of Library to Use in emPCR Amplification

To clonally amplify the library, the optimal amount of DNA to add to the Capture Beads must be determined using one of two titration options. The simplest and less time consuming option is the emulsion titration, which consists of using the % of bead enrichment as an indicator of the sequencing quality. This is achieved by performing emPCR amplifications on various amounts of library DNA and determining the % of enriched beads. The more involved option is the Sequencing titration, which consists of sequencing the test samples obtained after an emulsion titration. Both options are described in detail in the GS FLX+ Series — XL+. emPCR Amplification Method Manual Lib-L SV Refer to this manual for the procedures.

However, if the library yield can be predicted from past experience, one can use a pre-determined number of DNA molecules per bead.

2.3 Library Requirements

The library processed through emPCR Amplification should be:

1. free of small DNA species

2. with appropriate 454 Adaptors on both ends for the emulsion format used

3. properly quantified, and diluted such that at least 1 µl will be pipetted

4. composed of fragments whose lengths are compatible with the emPCR Amplification method used



3. PROCEDURE

3.1 Preparation of the Reagents and of the Emulsion Oil

3.1.1 Preparation of the Reagents

1. Open the emPCR Reagents box and thaw the kit components at room temperature, except the enzymes which should be kept at -15 to -25°C. Once thawed, vortex for 5 seconds.

2. Vortex and heat the tube of emPCR Additive at 55ºC for 15 minutes to aid dissolving. If a precipitate persists, centrifuge the tube and use the supernatant.

3. Spin all the kit components (including enzymes) in a bench top mini centrifuge for 10 seconds.

4. Return the enzymes to -15 to -25°C. The other kit reagents can be left at room temperature.

3.1.2 Preparation of the Emulsion Oil

Rattling sound: If the TissueLyser makes a loud rattling sound, turn it off immediately and make sure that both cups and adapters are screwed tightly in place.

1. Install the cups of Emulsion Oil securely in the TissueLyser, using the GS FLX Titanium emPCR Shaker Adapters, LV, provided as accessories to your GS FLX+ Instrument.

2. Shake the Emulsion Oil at 28 Hz for 2 minutes.

3. Remove the shaken Emulsion Oil from the TissueLyser.

3.1.3 Preparation of the Mock Amplification Mix and Pre-Emulsions

1. Dilute 2 ml of 5x Mock Amplification Mix with 8 ml of Molecular Biology Grade Water to make a working solution. Vortex.

2. Add 5 ml of the Mock Amplification Mix working solution to each cup of Emulsion Oil.

3. Invert the cups 2-3 times to mix, place on the TissueLyser, and secure firmly.

4. Shake in the TissueLyser at 28 Hz for 5 minutes.

5. Remove the emulsions cups from the TissueLyser.

3.1.4 Preparation of the Live Amplification Mix

emPCR amplification of Paired End libraries: Paired End libraries (20 kb, 8 kb and 3 kb Span) are amplified following the GS FLX Titanium Series methods and the corresponding kits. Refer to the emPCR Method Manuals – Lib-L LV, MV, and SV.

1. Prepare the Live Amplification Mix for the number of emulsion reactions being made, according to Table 1 for Rapid libraries. Add the reagents in the order they are listed in the table.

2. Vortex the Live Amplification Mix for 5 seconds, and store on ice.

Reagent Volume (μl)

1 LV Emulsion 2 LV Emulsions

Mol. Bio. Grade Water 1050 2100

emPCR Additive 1500 3000

5x Amplification Mix 860 1720

Amplification Primer 300 600

emPCR Enzyme Mix 200 400

PPiase 5 10

Total: 3915 7830

Table 1: Preparation of the Live Amplification Mix for Rapid libraries from the GS FLX+ Series — XL+

3.2 DNA Library Capture

1. Prepare 1x Capture Bead Wash Buffer TW by mixing 1 ml of 10x Capture Bead Wash Buffer TW with 9 ml of Molecular Biology Grade Water.



2. Vortex the tubes of DNA Capture Beads.

3. Pellet the beads in a bench top minifuge, by spinning for 10 seconds, rotating the tube 180°, and spinning again for 10 seconds.

4. Carefully remove and discard the supernatant without disturbing the bead pellet.

5. Wash each tube of beads twice with 1 ml of 1x Capture Bead Wash Buffer TW. Vortex to resuspend the beads, spin, and discard the supernatant after each wash.

6. Thaw an aliquot of the diluted DNA library to be amplified.

• If necessary, prepare a dilution of the library such that the volume to be added will be between 1 µl and 100 µl.

• Heat denature DNA prepared with the Rapid library method, by running the following program on a thermocycler, with the heated lid on:

95°C for 2 minutes 4°C on hold

7. To each tube of washed DNA Capture Beads, add the correct volume of the DNA library that will provide optimal amplification (e.g. per titration) to the bead pellet.

• Use the following equation:

μl) / molecules (in ionconcentrat librarytubeper beads 10 x 35 x beadper molecule desired tubeper library ofμl

6 =

8. Vortex the tubes for 5 seconds to mix their contents.

9. Prepare the DNA Capture Beads with library DNA mixes for individual emulsion reactions.

a. Transfer the captured library mixes from their DNA Capture Beads tubes to individual 15 ml tubes. Do not discard the original DNA Capture Beads tubes.

b. Add 3.75 ml of the Live Amplification Mix to each 15 ml tube.

c. From there, pipet 1 ml of the mix to the original DNA Capture Beads tubes that contained the captured library, vortex and transfer back the solution into the 15 ml tubes.

d. Repeat two times.

e. Vortex.

3.3 Emulsification

1. Pour the content from each 15 ml tube of captured library from Section 3.2 into a prepared emulsion cup (from Section 3.1.3).

2. Invert the cups 3 times to mix.

3. Shake in the TissueLyser at 12 Hz for 5 minutes.

3.4 Amplification

3.4.1 Dispensing the Emulsions

1. After emulsification, dispense the emPCR amplification mixes into 96-well thermocycler plates, at 100 µl per well; each cup of emulsion will fill approximately 180 wells.

• Emulsion in cup lids: Some emulsion may have lodged in the cup lids. You can pipet it up from the lids but DO NOT spin the emulsion cups in an attempt to reclaim this material as this would risk breaking the emulsions.

• Air bubbles: dislodge any air bubbles that may be present at the bottom of the wells by gently tapping the plate.

2. Cap the wells and make sure that all the wells are properly sealed.

3. Clean up the area of any spilled reaction mix.

3.4.2 Amplification Reaction

1. Bring the plates containing the emulsified amplification reactions to the post-amplification area, place them in a thermocycler, and start the amplification program with the heated lid on. The program takes ~8 -9 h to complete.

1x 4 minutes at 94°C 50x 30 seconds at 94°C, 10 minutes at 60°C 10°C on hold

• Do not freeze the DNA beads: You can leave the amplified reactions at 10°C for up to 16 hours before further processing the samples.

• Emulsion breakage: Check all wells for emulsion breakage (i.e. faint yet distinct layers with a clearer middle or bottom layer). If the emulsion in any well appears broken, discard the entire well and do not recover the beads from it. See Section 4.2 for pictures of intact and broken emulsions.



3.5 Bead Recovery

3.5.1 Vacuum-Assisted Emulsion Breaking Set Up

1. Retrieve the Enhancing Fluid XT and the Annealing Buffer XT, and keep at room temperature.

2. Obtain a GS FLX Titanium emPCR Breaking Kit, LV/MV, 12 pcs. See Figure 3 for a general view of an assembled set up.

3. Attach a 50 ml conical tube to each of the two lids of the emPCR breaking Kit LV/MV. Save the caps.

4. Insert the blue connector into the top opening of the 8-pronged transpette.

5. Connect the other end of the tubing to a vacuum source (with liquid trap to capture the isopropanol waste).

If a vacuum source is not available, aspirate the emulsions from the microtiter plate wells with a syringe and blunt needle, as described for the SVE procedure, in the GS FLX+ Series— XL+, emPCR Amplification Method Manual Lib-L SV.

Figure 3: Schematic view of an assembled set up for vacuum-assisted emulsion breaking and bead recovery for Large Volume Emulsions

3.5.2 Emulsion Collection and Initial Washes

1. Aspirate the emulsions from each emulsion cup, 8 wells at a time and collect them in the two 50 ml collection tubes, using a slow circular motion of the transpette tips at the bottom of the wells.

• After aspirating all the emulsions, turn the transpette upside-down to help drain as much material as possible into the collection tubes.

2. Rinse the plates twice with 100 μl of isopropanol per well. Aspirate the rinse and turn the transpette upside-down to retrieve as much material as possible.



3. If you are processing more than one emulsion cup of the same library sample:

a. Turn off the vacuum.

b. Remove and cap the two 50 ml tubes containing the amplified DNA beads from the first emulsion cup (use the caps set aside).

c. Attach two new 50 ml collection tubes to the lids of the breaking set up. Save the caps.

d. Repeat Steps 1 and 2.

4. SLOWLY aspirate an additional (approximate) 5 ml of isopropanol to collect any beads that may remain in the tubing.

5. Turn off the vacuum, and remove and cap the two 50 ml collection tubes containing the amplified DNA beads.

6. If you are processing more than one library sample, repeat this section for each sample, using a new Emulsion Breaking assembly for each sample (see Figure 3).

3.5.3 Bead Washes and Recovery

• Tube requirement: All 1.7 ml tubes must be siliconized.

• Be careful not to disturb the pellet during the washes while discarding the supernatant.

1. Vortex each tube and mix the 50 ml collection tubes in pairs by transferring their contents back and forth, until the bead suspensions are of equivalent amount of emulsion oil.

2. Add isopropanol to a final volume of 40 ml in each collection tube and vortex.

3. Pellet the beads in a centrifuge at 930 x g for 5 min (2000 rpm in a Beckman Allegra centrifuge, rotor SX4750) and carefully remove the supernatant.

4. Add 35 ml of Enhancing Fluid XT and vortex well to resuspend (use glass rod, if necessary).

5. Centrifuge and discard the supernatant as in Step 3.

6. Add 35 ml of isopropanol and vortex well.


8. Add 35 ml of isopropanol and vortex well.


10. Add 35 ml of ethanol and vortex well.


12. Add 35 ml of Enhancing Fluid XT and vortex well.

13. Centrifuge and discard the supernatant as in Step 3, leaving approximately 2 ml of Enhancing Fluid XT.

14. Transfer the DNA bead suspension using a 1000 µl pipette in two 1.7 ml tubes for each emulsion cup processed (a total of 4 tubes per full LV kit).

15. Spin-rotate-spin and discard the supernatant.

16. Repeat Steps 14 and 15 until the entire DNA bead suspension has been transferred.

17. Rinse each of the 50 ml collection tubes with 600 μl of Enhancing Fluid XT, vortex, and add this rinse to the 1.7 ml tubes. Spin-rotate-spin and discard the supernatant.

18. Thoroughly rinse each bead pellet twice with 1 ml of Enhancing Fluid XT. Spin-rotate-spin and discard the supernatant

19. Add 1 ml of Enhancing Fluid XT to each bead pellet and vortex.

3.6 DNA Library Bead Enrichment

• At this point, you will have two 1.7 ml tubes for each emulsion cup processed.

• The reagent volumes listed in this section apply to each tube of beads.

3.6.1 Preparation for Enrichment

Hazardous Chemical – Sodium Hydroxide Solution: Sodium hydroxide (present in the Melt Solution) is a highly corrosive chemical that may cause burns if it contacts eyes or skin. Read the Material Safety Data Sheet for handling precautions.

1. Turn on the heating dry-block and set it to 65°C.

2. Prepare the Melt Solution by mixing 125 μl of NaOH (10 N) in 9.875 ml of Molecular Biology Grade Water.

3. Spin-rotate-spin the tubes of beads and discard the supernatant.

4. Add 1 ml of Melt Solution per tube of beads and vortex. Incubate for 2 minutes at room temperature. Spin-rotate-spin and discard the supernatant.

5. Repeat Step 4 once.

6. Add 1 ml of Annealing Buffer XT per tube of beads and vortex. Spin-rotate-spin and discard the supernatant.



7. Repeat Step 6 once.

8. Optional: To determine the % breaking efficiency, do the following:

a. Add 1 ml of Annealing Buffer XT and vortex.

b. Count a 3 µl aliquot of the bead suspension from each tube and add the two values to calculate the total % bead recovery. A recovery of over 85% would be typical at this point of the procedure. Calculate the % bead recovery using following equation:

x10010 x 35

recovered beads ofNumber Recovery Bead % 6 =

c. Spin-rotate-spin and discard the supernatant.

9. Add 45 μl of Annealing Buffer XT per tube and 25 μl of Enrichment Primer per tube, and vortex.

10. Place the tubes in a heat block at 65°C for 5 minutes, and then promptly cool on ice for 2 minutes.

11. Add 800 μl of Enhancing Fluid XT per tube and vortex. Spin-rotate-spin and discard the supernatant.

12. Add 1 ml of Enhancing Fluid XT per tube and vortex. Spin-rotate-spin and discard the supernatant.

13. Repeat Step 12.

14. Add 800 μl of Enhancing Fluid XT per tube and vortex.

3.6.2 Preparation of the Enrichment Beads

1. Vortex the tube of brown Enrichment Beads for 1 minute to resuspend its contents completely.

• If you are processing half an LV emPCR Kit (1 emulsion cup), aliquot 160 µl and store the rest at +2 to +8ºC.

2. Pellet the Enrichment Beads using a Magnetic Particle Concentrator (MPC).

3. Discard the supernatant, taking care not to draw off any Enrichment Beads.

4. Add 1 ml of Enhancing Fluid XT and vortex.

5. Pellet the Enrichment Beads using an MPC.

6. Discard the supernatant, taking care not to draw off any Enrichment Beads.

7. Repeat Steps 4 to 6.

8. After discarding the supernatant, remove the tube from the MPC.

9. Add 320 μl for 2 emulsion cups (or 160 μl for 1 emulsion cup) of Enhancing Fluid XT and vortex.

3.6.3 Enrichment of the DNA-Carrying Beads

1. Add 80 μl of washed Enrichment Beads to each tube of amplified DNA beads (from Section 3.6.1) and vortex.

2. Rotate the tubes on the rotator, at room temperature (+15 to +25ºC) for 5 minutes.

3. Place the tubes in the MPC, and wait 3-5 minutes to pellet the Enrichment Beads.

4. Invert the MPC several times.

5. Carefully discard the supernatant from each tube using a 1000 µl pipette, taking care not to draw off any brown Enrichment Beads.

• Optionally, collect the supernatants containing “anti-enriched” beads for troubleshooting.

6. Wash the beads with Enhancing Fluid XT until there are no visible beads remaining in the supernatants, as follows:

a. Add 1 ml of Enhancing Fluid XT per tube.

b. Remove the tubes from the MPC and vortex the tubes.

c. Place the tubes back into the MPC to pellet the beads on the wall of the tube with the magnet. Invert the MPC.

d. Carefully discard the supernatant from each tube with a 1000 µl pipette, taking care not to draw off any Enrichment Beads.

e. Repeat 6 to 10 times until white DNA beads are no longer being aspirated.

• Optionally, collect the supernatants of each wash, and spin to monitor when washes are complete.

3.6.4 Collection of the Enriched DNA Beads

1. Remove the tubes of enriched beads (enrichment tubes) from the MPC and resuspend each bead pellet in 700 μl of Melt Solution.

2. Vortex for 5 seconds, and place the enrichment tubes in the MPC until the Enrichment Beads have pelleted.

3. Transfer the supernatants containing enriched DNA beads from each sample (> 1.4 ml) into a corresponding 1.7 ml collection tube (one sample per 1.7 ml tube).

4. Spin-rotate-spin the single 1.7 ml collection tube, and discard the supernatants.



5. Add once again 700 μl of Melt Solution to the enrichment tubes.

6. Vortex for 5 seconds, and place the enrichment tubes in the MPC until the Enrichment Beads have pelleted.

7. Transfer the supernatants containing enriched DNA beads into the same 1.7 ml collection tube.

8. Discard the enrichment tubes.

9. Spin-rotate-spin the 1.7 ml collection tube and discard the supernatants.

10. Add 1 ml of Annealing Buffer XT per collection tube and vortex for 5 seconds.

11. Spin-rotate-spin and discard the supernatant.

12. Repeat Steps 10 and 11 two more times.

13. Resuspend each bead pellet in 200 μl of Annealing Buffer XT.

3.7 Sequencing Primer Annealing

1. Add 50 μl of Sequencing Primer to each collection tube and vortex.

2. Place the collection tubes in a heat block at 65°C for 5 min, and then promptly on ice for 2 min.

3. Add 800 μl of Annealing Buffer XT per collection tube and vortex for 5 seconds. Spin-rotate-spin, and discard the supernatant.

4. Repeat Step 3 two times with 1 ml of Annealing Buffer XT.

5. Add 1 ml of Annealing Buffer XT to each bead pellet and vortex.

6. To determine the % bead enrichment, do the following:

a. Count a 3 µl aliquot of the beads with a particle counter.

b. Calculate the % bead enrichment using the following equation:

x100beads/cup10 x 35

recovered beads enriched ofNumber Recovery Bead % 6 =

c. Percent enrichment values between 5 and 20% are indicative of libraries that usually yield good sequencing results.

7. Store the beads at +2 to +8°C and sequence them within two weeks.



4. APPENDIX

4.1 Bead Counter Settings

454 Life Sciences Corporation, a Roche company, recommends the use of the Innovatis CASY TTC, TT, and DT models, as well as the Coulter Counter Multisizer 3, Z2, and Z1 models from Beckman Coulter for the quantitation of amplified DNA library beads obtained from emPCR amplification reactions. Table 2 shows the settings of the CASY models for bead counting. Figure 4 shows the settings of the Coulter Counter Multisizer 3 model and Table 3, the settings of the Coulter Counter Multisizer Z2 or Z1 model.

Parameter Settings

Dual Thresholds

Upper Size (Tu) 25 µm

Lower Size (Tl) 15 µm

Count Mode Between

Aperture 150 µm

Metered Volume 1.2 ml (400 µl x 3)

Result Type Concentration

Dilution Factor* 3333 (10 ml diluent)

Table 2: Innovatis CASY TTC, TT, and DT models settings for bead counting.

* for 3 µl of beads

Figure 4: Coulter Counter Multisizer 3 settings for bead counting



Parameter Settings

Dual Thresholds Single Threshold

Upper Size (Tu) 25 µm --

Lower Size (Tl) 15 µm 15 µm

Count Mode Between Above Tl

Aperture 100 µm 100 µm

Metered Volume 1.0 1.0

Result Type Concentration Concentration

Dilution Factor* 3333 (10 ml diluent) 3333

Table 3: Coulter Counter Z2 or Z1 models settings for bead counting. Dual thresholds are preferable, if available.

* for 3 μl of beads

4.2 Pictures of Emulsions

A B

C

Figure 5: A and B show intact emulsions appearing as an homogenous suspension (A), at times layered by a clearer phase (B). The multiple bands in C are a sign of a broken emulsion.



Table of Contents 1. WorkFlow ................................................................................................................... 2 2. Before You Begin ....................................................................................................... 3

2.1 Sample Handling Strategy for Large Volume Emulsions ......................................... 3 2.2 Determining the Amount of Library to Use in emPCR Amplification .................. 3 2.3 Library Requirements..................................................................................................... 3

3. Procedure ................................................................................................................... 4 3.1 Preparation of the Reagents and of the Emulsion Oil ............................................... 4

3.1.1 Preparation of the Reagents ....................................................................................... 4 3.1.2 Preparation of the Emulsion Oil ................................................................................ 4 3.1.3 Preparation of the Mock Amplification Mix and Pre-Emulsions ........................ 4 3.1.4 Preparation of the Live Amplification Mix .............................................................. 4

3.2 DNA Library Capture .................................................................................................... 4 3.3 Emulsification ................................................................................................................. 5 3.4 Amplification ................................................................................................................... 5

3.4.1 Dispensing the Emulsions .......................................................................................... 5 3.4.2 Amplification Reaction ............................................................................................... 5

3.5 Bead Recovery ................................................................................................................. 6 3.5.1 Vacuum-Assisted Emulsion Breaking Set Up ......................................................... 6 3.5.2 Emulsion Collection and Initial Washes .................................................................. 6 3.5.3 Bead Washes and Recovery ........................................................................................ 7

3.6 DNA Library Bead Enrichment .................................................................................... 7 3.6.1 Preparation for Enrichment ....................................................................................... 7 3.6.2 Preparation of the Enrichment Beads ....................................................................... 8 3.6.3 Enrichment of the DNA-Carrying Beads ................................................................. 8 3.6.4 Collection of the Enriched DNA Beads .................................................................... 8

3.7 Sequencing Primer Annealing ...................................................................................... 9 4. Appendix .................................................................................................................... 10

4.1 Bead Counter Settings .................................................................................................. 10 4.2 Pictures of Emulsions ................................................................................................... 11

Published by 454 Life Sciences Corp. A Roche Company Branford. CT 06405 USA © 2011 454 Life Sciences Corp. All rights reserved. For life science research only. Not for use in diagnostic procedures. 454, 454 LIFE SCIENCES, 454 SEQUENCING, GS FLX, GS FLX TITANIUM, GS JUNIOR, EMPCR, PICOTITERPLATE, PTP, NEWBLER, REM, GTYPE, AMPLITAQ, AMPLITAQ GOLD, FASTSTART, NIMBLEGEN, SEQCAP, CASY, and INNOVATIS, are trademarks of Roche. Other brands or product names are trademarks of their respective holders. License disclaimer information is subject to change or amendment. For current license information, please visit our web site at www.454.com. (1) 0511

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emPCR Amplification Method Manual – Lib L LV...GS FLX+ Series— XL+, emPCR Amplification Method...

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