© 2010 Beckman Coulter, Inc. P/N A87325A Page 1 of 16 Input DNA and Sample Quality Assessment...

© 2010 Beckman Coulter, Inc. P/N A87325A Page 1 of 16

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Intended Use For Research Use Only. Not for use in diagnostic procedures.

Intended for the preparation of 10 or 50 fragment libraries using the SPRI-TE Nucleic Acid Extractor. Fragment size is ideal for sequencing on the Illumina Genome Analyzer.

Summary and Explanation The SPRIworks Fragment Library System I uses the patented Agencourt SPRI paramagnetic particle-based technology to generate fragment libraries from fragmented or sheared input DNA.

The SPRIworks Fragment Library System I automated method is performed on the SPRI-TE Nucleic Acid Extractor, using pre-measured reagents in a sealed reagent cartridge, and enables the construction of three library types: no size selection; 200-400 bp size selection; and 300-600 bp size selection.

of 16 P/N A87325A © 2010 Beckman Coulter, Inc.

Principles of the Procedure The SPRIworks Fragment Library System I method begins with the off-line shearing of input DNA by nebulization or other means. Fragmented DNA is then loaded onto the SPRI-TE instrument which performs the subsequent steps in the library construction process.

The SPRIworks Fragment Library System I employs enzymatic reactions, SPRI-based reaction purifications, and optional SPRI-based size selection to automatically generate fragment libraries. All reagents required to construct a single library are contained within the reagent cartridge. Additional reagents and primers are required for subsequent enrichment of the library. The method can generate from one to ten libraries simultaneously per 5-hour run.

After processing on the SPRI-TE instrument, libraries are further amplified and used as templates for sequencing on the Illumina Genome Analyzer.

Product Information SPRIworks Fragment Library System I Kit

1. SPRIworks Fragment Library Kit I - 10 (REF A84801)•1 x 10 SPRIworks Fragment Library I cartridges, 1 library/reagent cartridge•1 x 50 Pipette Tips & Sheaths, 1 mL•1 x 50 Piercing Tips & Sheaths•1 x 50 Screw Cap Tubes, 2 mL•1 x 6mL SPRIworks Nebulization Buffer•1 x 10 SPRI-TE Trays

2. SPRIworks Fragment Library Kit I - 50 (REF A84803)•5 x 10 SPRIworks Fragment Library I cartridges, 1 library/reagent cartridge•2 x 50 Pipette Tips & Sheaths, 1 mL•1 x 50 Piercing Tips & Sheaths•3 x 50 Screw Cap Tubes, 2 mL•5 x 6mL SPRIworks Nebulization Buffer•5 x 10 SPRI-TE Trays


Specifications:

• Provided ready to use.• Store upright as indicated.• Stable until the expiration date stated on the label when stored properly.• If the cartridge is damaged or leaking, or if the foil is punctured, discard the cartridge.• Allow frozen components to thaw completely at 8 to 30°C prior to use.• Do not exceed three freeze-thaw cycles.

Contents: buffers, enzymes, ligase, nuclease-free water, reagents, ethanol, and sodium azide.

Warnings and Precautions • For Research Use Only.• Patient samples and blood-derived products may be routinely processed with minimum risk using the procedure

described. However, handle these products as potentially infectious according to universal precautions and good clinical laboratory practices, regardless of their origin, treatment, or prior certification. Use an appropriate disinfectant for decontamination. Store and dispose of these materials and their containers in accordance with local regulations and guidelines

• Sodium azide may react with lead and copper plumbing to form highly explosive metal azides. On disposal of liquids, flush with a large volume of water to prevent azide build-up1.

• R10: Flammable.• S7-16 Keep container tightly closed. Keep away from sources of ignition - No smoking.• The Material Safety Data Sheet (MSDS) is available upon request.

Product Storage Conditions

SPRIworks Fragment Library I Cartridges -25 to -15°C

SPRI-TE Piercing Tips & Sheaths 8 to 30°C

SPRI-TE Pipette Tips & Sheaths, 1 mL 8 to 30°C

SPRI-TE 2 mL Screw Cap Tubes 8 to 30°C

SPRIworks Nebulization Buffer -25 to -15°C


Input DNA and SampleQuality Assessment

Beckman Coulter recommends 3 to 5 µg of genomic DNA for library construction. Observe local laboratory standard operating procedures for handling, processing, and storing samples. Input DNA should have the following characteristics:

• DNA is from the target organism and contains no contaminating DNA.• DNA is double-stranded, and fragments are larger than 1.5 kb.

NOTE: This does not apply if pooled PCR products are used as input DNA.

• DNA is not a product of whole-genome amplification. Whole genome amplification may result in unequal representation of different regions in the genome.

• DNA is free of particulate matter.• DNA has an OD260/280 ratio of approximately 1.8.• DNA has a minimum concentration of 50 ng/µL in water, or 10 mM TRIS (pH 7.4 to 8.0)• DNA is not otherwise contaminated. Any contamination in the starting material will carry contamination into input

DNA and into the fragment libraries constructed from the input DNA. Contaminated DNA may inhibit the library construction process.

Materials Provided SPRIworks Fragment Library Kit I - 10 (REF A84801) orSPRIworks Fragment Library Kit I - 50 ( REF A84803)

Materials Required But NotProvided

• DNA Fragmentation EquipmentSPRIworks Nebulizer Kit (REF A84871) or other commercial fragmentation equipment.

• AdaptersSPRIworks Adapter Kit I (REF A84879) or other commercial annealed adapter oligo mix.

• Library Enrichment ReagentsCommercial master mix; SPRIworks Adapter Kit I (REF A84879), which includes adapters and primers for single read sequencing on the Illumina Genome Analyzer; AMPure XP (REF A63880) for purification of PCR products; Agencourt SPRIPlate 96 Ring Super Magnet Plate (Agencourt P/N A32782), or other commercial low- or medium-throughput magnet stand; 70% ethanol.

• Thermal CyclerCommercial thermal cycler.


Procedure Prepare Reagent CartridgesBefore shearing DNA, Beckman Coulter recommends that you remove SPRIworks Fragment Library I cartridges from -20°C storage and allow the cartridges to thaw during shearing. Remove one cartridge for each library to be constructed. Thaw cartridges at room temperature (8 to 30°C) for approximately one hour, or until all contents are completely thawed.

Shear DNAPrior to library construction, sample DNA is sheared into fragments by nebulization or other means. Beckman Coulter recommends the use of the SPRIworks Nebulizer (REF A84871). Refer to the Instructions for Use or system documentation for information specific to the fragmentation equipment used by your laboratory.

Set up the SPRI-TE Nucleic Acid ExtractorThe instrument must be set up as follows for successful library construction.

NOTE: The setup procedure described below is not required if the SPRIworks Method Card for Fragment Library System I (REF A84868) is already in the instrument. If the SPRIworks Method Card for Fragment Library System I is already in the instrument, proceed to the Remove Racks procedure.

1. Turn Off the SPRI-TE Nucleic Acid Extractor.

2. Press the thin black eject button at the bottom of the method card reader to remove any method card that may be in the instrument.


3. Insert the SPRIworks Method Card for Fragment Library System I (REF A84868)..

Insert the method card vertically, with the label facing to the left and with the arrow pointing towards the instrument. Push the card firmly until it locks into place, then close the small metal cover.

CAUTIONS:

•Do not force the method card into the instrument.•Do not remove or insert a method card while the instrument is powered on.•Do not open the instrument door while the instrument is initializing.

4. Close the instrument door.

5. Turn the power on.


6. Open the door only when the LED Screen displays the following message:

Remove RacksRemove the Tip/Tube Rack and the Reagent Rack from the instrument.

1. Remove the Tip/Tube Rack from the instrument.

The Tip/Tube rack is a chrome-plated assembly that has five rows, each containing 10 holes. Grasp the rack by the handles and remove it from the instrument. Place the rack on a bench top in the same orientation as it was in the instrument.

2. Remove the Reagent Rack.

The Reagent Rack is the metal rack with ten lanes. Grasp it by its two handles (located at lanes 3 and 7). Lift and remove the rack from the instrument. Place the Reagent Rack on a bench top in the same orientation as it was in the instrument.

Prepare the Reagent RackFor each library to be constructed, insert one reagent cartridge.

1. Examine the reagent cartridges you removed from -20°C storage in the Prepare Reagent Cartridges procedure. Ensure that the contents of the reagent cartridges have completely thawed.

2. Ensure that all fluids and magnetic particles are at the bottom of the wells by grasping each reagent cartridge by the sides and tapping (foil side up) gently two or three times on a hard surface such as a bench top. If contents remain on the sides or top of the wells, tap the reagent cartridge gently onto a hard surface until all contents are at the bottom of the wells.


3. Load one reagent cartridge into the Reagent Rack for each library to be constructed.

Load the reagent cartridge into the rack by grasping the labeled lip and sliding the reagent cartridge into the grooved channel. Slide the reagent cartridge all the way to the back of the channel, until it is retained at front and back.

4. When all reagent cartridges are in position, load the Reagent Rack by grasping the handles and placing it into the extraction chamber.

Prepare the Tip/Tube RackFor each library to be constructed, insert one Piercing Tip & Sheath, two 1 mL Pipette Tips & Sheaths, one uncapped 2 mL Screw Cap Tube with Adapters, and one uncapped 2 mL Screw Cap Tube for the final library. If fewer than ten libraries are to be constructed during a run, align tips and tubes with the lane positions of the reagent cartridges in the Reagent Rack.

NOTE: Only use tips and tubes provided by Beckman Coulter.

For each library to be constructed:

1. Insert a Piercing Tip & Sheath into row 1.

2. Insert a 1 mL Pipette Tip & Sheath into row 2.

3. Insert a 1 mL Pipette Tip & Sheath into row 3.

4. Aliquot 10 µL of adapters (50 µM) into a labeled, uncapped 2 mL Screw Cap Tube, and insert the tube into row 4.

Label the adapter tube in accordance with your institutional guidelines.

5. Insert a labeled, uncapped 2 mL Screw Cap Tube into row 5. This tube will contain the fragment libraries at the conclusion of the run.

Label the library tube in accordance with your institutional guidelines.


6. The outcome looks like this:

7. When all tips and tubes are in position, load the Tip/Tube Rack by grasping the handles and placing it into the extraction chamber.

NOTE: The back edge of the Tip/Tube rack will overlap the Reagent Rack when loaded correctly.

8. Verify that tips and tubes are aligned with reagent cartridges in the Reagent Rack.

9. Seat tips firmly in the Tip/Tube rack by gently pressing down with the palm of your hand.

NOTE: Do not force the sheaths down into the rack. The underside of the sheath has a set of small ribs that are pressed into the rack by the pipettor module when the tip is loaded. These ribs are designed to secure the sheath in the rack to prevent it from being lifted out of the rack when the tip is removed.

Insert Input DNA for Library ConstructionFor each library to be constructed, insert an uncapped 2 mL Screw Cap Tube containing 3 to 5 µg (100 to 400 µL) of input DNA, prepared as described in the Shear DNA procedure, into the the circular hole in the next-to-last position in each reagent cartridge (position 13).

NOTE: Only use tubes provided by Beckman Coulter.

12

34

5

Piercing Tip1 mL Tip

1 mL TipAdapter Tube

Library Tube


Start the RunAfter loading the Reagent Rack and the Tip/Tube Racks onto the instrument and inserting the samples, start the run.

NOTE: Press the ESC key to return to the previous command at any time during method set up.

1. Close the instrument door and select the green START key.

2. The method appears in the LCD screen as "SPRIworks Fragment Library System I." Specify size selection or no size selection as prompted.

Press 1 to perform size selection.Press 2 to skip size selection.

Select Enter (left arrow key) to confirm the size selection choice.

If performing size selection, select the size range:For 200-400 bp, select 1.For 300-600 bp, select 2.

Select Enter (left arrow key) to confirm the previous choice: 200-400 bp, 300-600 bp, or skip size selection. The run begins automatically.

IMPORTANT:

•Do not remove or insert a method card while the instrument is powered on.•If the door is opened while the method is running, the method will be canceled and cannot be

recovered.3. The method is finished when the instrument beeps three times.

At this point it is safe to open the door, retrieve and cap the tubes of libraries, and discard used tips, sheaths, and reagent cartridges.

NOTE: Remove all used tips, tubes, and reagent cartridges from the racks and dispose of materials according to local laboratory standard operating procedures for handling biohazardous waste.

4. After collection, libraries can be stored at 2 to 8° C for up to 6 hours. For long term storage temperatures of -20 C to -80° C are recommended.


Procedural Comments A. Refer to the system documentation for a specific description of installation, start-up, principles of operation, system performance characteristics, general operating instructions, operational limitations and precautions, hazards, main-tenance, and troubleshooting.

B. Ensure contents of new (un-punctured) reagent cartridges are at the bottom of the wells by tapping the bottom of the reagent cartridge on a hard surface a few times before loading on the instrument.

C. From the TOP MENU, limited error recovery operations can be performed, such as homing the tray and returning tips to their original sheaths.

Select 1 to see the following submenu:

•Select 1 to display another submenu to home any or all axes.•Select 2 to return the last-loaded tips to their sheaths. •Select ESC to return to the top-level menu.


Enrichment of Library DNA Perform enrichment of the library DNA using the protocol below. Other protocols suitable for use with the Illumina Genome Analyzer may also be used.

This protocol is based on 5 µg of input DNA for library construction.

1. Prepare the following PCR reaction mix:•DNA library (1 µL)•Finnzymes Phusion** High-Fidelity PCR Master Mix (25 µL)•SPRIworks PCR Primer F (1 µL)•SPRIworks PCR Primer R (1 µL)•Water (22 µL)The total volume of the PCR reaction mix is 50 µL.

2. Use a thermal cycler to perform the following PCR reaction. Follow the PCR protocol below:

30 seconds at 98°C10 cycles of:

10 seconds at 98°C30 seconds at 65°C30 seconds at 72°C

5 minutes at 72°C°Hold at 4°C

3. After PCR, spin down the tubes or plate in which the PCR was performed.4. Quantify the volume of PCR reaction.5. Add AMPure XP to the PCR reactions:

•For size-selected libraries, add 1.8x volumes of AMPure XP.•Nor non-size-selected libraries, add 1.2x volumes of AMPure XP.

NOTE: If you did not perform size selection during the library construction method, it is important that you perform purification using 1.2x volumes of AMPure XP to completely remove any adapter dimers.

6. Mix reagent and PCR reaction thoroughly by pipetting up and down 10 times.7. Incubate mixed samples for 5 minutes at room temperature (8 to 30°C).

NOTE: For maximum recovery, allow samples to incubate for the full incubation time.


8. Place the tubes or reaction plate onto a low- or medium-throughput magnet stand for 2 minutes to separate the paramagnetic particles from the solution.

9. Aspirate the supernatant from the tubes or reaction plate and discard.10.Add 200 µL of 70% ethanol to the tubes or reaction plate and incubate at room temperature (8 to 30°C) for a

minimum of 30 seconds.11.Aspirate the ethanol from the tubes or reaction plate and discard.12.Repeat steps 10 and 11 for a total of two washes.13.Remove the tubes or reaction plate from the magnet stand.14.Add 40 µL of elution buffer (reagent-grade water, TRIS acetate with a pH of 8.0, or TE).15.Mix the solution thoroughly by pipetting up and down 10 times.16.Incubate the solution at room temperature (8 to 30°C) for 2 minutes.17.Place the tubes or reaction plate onto the magnet stand for 1 minute to separate the paramagnetic particles from the

solution.18.Transfer the purified product to a new plate.

Troubleshooting GuideProblem Observation Reccomendations

No library is observed at the end of the SPRIworks library construction run or after analysis of library DNA with a quantitative trace method.

• Input DNA amount was insufficient.

• Input DNA was degraded.

• No DNA was added.

• Reagent cartridges were stored improperly.

• Evaluate input DNA using fluorometric methods to ensure starting quantity is sufficient.

• Evaluate the quality of input DNA using agarose gel or your preferred quantitative method for determining input DNA quality and fragment size.

Constructed library fails qPCR QC or does not yield a product during enrichment of library DNA.

• Adapters were not added to the adapter tube or adapters are incompatible with the SPRIworks Fragment Library System I.

• Initial shear was not within the size range selected during the method setup.

• Use adapters recommended for library construction.

• Evaluate the input DNA using a quantitative method for determining fragment size. Ensure there is DNA in the desired region that is size-selected.

Instrument stops during a run. The door of the SPRI-TE instrument was opened during the run.

Do not open the instrument door during a run. If the door is opened while the instrument is running, the method will be canceled and cannot be recovered.


Ordering Information The SPRIworks Fragment Library System I includes the components listed below. Use the following information to order components in the SPRIworks Fragment Library System I. To order replacement parts or for other instrument ordering information, refer to the SPRI-TE Operator's Manual.

1. SPRI-TE Nucleic Acid Extractor (REF A50100)2. SPRIworks Fragment Library Kit I - 10 (REF A84801)3. SPRIworks Fragment Library Kit I - 50 (REF A84803)4. SPRIworks Method Card Kit for Fragment Library System I (REF A84868)5. SPRIworks Accessories Kit (REF A88270)

NOTE: The SPRIworks Accessory Kit replaces the SPRI-TE Startup Kit and includes the documentation and consumables required for installation and maintenance of the SPRI-TE instrument.

Technical Support If you have any questions about this information, or for technical assistance regarding SPRIworks products or the SPRI-TE Nucleic Acid Extractor, contact Beckman Coulter.

•In the U.S.A. or Canada, contact Beckman Coulter Technical Support by phone at 800-854-3633, or online at www.beckman.com/customersupport. Before using online support the first time, you will need to register online.

•Outside the U.S.A. and Canada, contact your local technical support representative.


References 1 DHHS (NIOSH) Publication No. 78-127, August 1976. Current Intelligence Bulletin 13 - Explosive Azide Hazard. Available http://www.cdc.gov/niosh.

2 Illumina, Inc. Preparing Samples for Sequencing Genomic DNA Using the Genomic DNA Sample Prep Oligo Only Kit. 2007. Available http://www.illumina.com.

3 Beckman Coulter, Inc. Agencourt AMPure XP PCR Purification. 2009. Available http://www.beckmangenomics.com.

Beckman Coulter, SPRI, SPRI-TE, SPRIworks, SPRIPlate, and AMPure are trademarks of Beckman Coulter, Inc.; Beckman Coulter, SPRI, SPRI-TE, SPRIPlate, and AMPure are registered in the USPTO.

* Illumina is a trademark of Illumina, Inc.

** Phusion is a trademark of Finnzymes Oy.

Manufactured by Beckman Coulter, Inc.

Printed in U.S.A.

Made in U.S.A.

Issued February 2010

Copyright 2010 Beckman Coulter, Inc.

© 2010 Beckman Coulter, Inc. P/N A87325A Page 1 of 16 Input DNA and Sample Quality Assessment...

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