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Apollo 324™ SystemPrepX™ ILM DNA Library Protocol

User Guide

© Copyright 2013, IntegenX Inc. All rights reserved.

LIMITATIONS

For research use only. Not intended for any human or animal diagnostic or therapeutic use. The purchaser of the product may use it for its internal research use only, and not for any commercial purposes. Information about licenses for commercial use is available from IntegenX. Unless expressly stated, the purchase of this product conveys no license or grant of any intellectual property rights, whether express, implied, by estoppel or otherwise.

Information in this protocol is subject to change without notice. IntegenX assumes no responsibility for any errors or omissions in this document.

INTEGENX AND ITS AFFILIATES HEREBY DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS PROTOCOL, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL INTEGENX OR ITS AFFILIATES BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS PROTOCOL, INCLUDING BUT NOT LIMITED TO YOUR USE THEREOF.

WARRANTY AND LIABILITY

Warranty information for the accompanying consumable product is set forth in IntegenX's Standard Terms and Conditions of Sale, available at www.integenx.com/tcos/. Notwithstanding anything in this document to the contrary, the limited warranty is subject to all exclusions, conditions, exceptions, and limitations set forth therein.

SAFETY DATA SHEET

Safety Data Sheets are available at www.integenx.com/msds/.

TRADEMARKS

Apollo 324, BeadX and PrepX are trademarks of IntegenX Inc.

AMPure and Agencourt are registered trademarks of Beckman Coulter, Inc.

TruSeq is a trademark of Illumina, Inc.

All other trademarks are the sole property of their respective owners.

IntegenX Inc.5720 Stoneridge Drive, Suite 300Pleasanton, CA 94588

925.701.3400

Email: [email protected]

www.IntegenX.com

Part Number P037482, Rev. A1/2013

Revision History

Revision Date Description

A 1/2013 Initial release

mailto: [email protected]

http://www.integenX.com

www.integenx.com/tcos/

www.integenx.com/msds/

Contents

PrepX ILM DNA Library Protocol User Guide iii

Chapter 1 Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1About the Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Processing Schematic. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Chapter 2 Preparing Samples and Reagents . . . . . . . . . . . . . . . . . . 3Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Materials for Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Reagent Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Customer-Supplied Reagents and Consumables. . . . . . . . . . . . . . . 4

Decontaminating the Lab Work Space and Instrument . . . . . . . . . . . . . . 5Preparing the Library Kit Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5Using 1X AMPure XP Beads. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Preparing 70% v/v EtOH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Preparing the Molecular Biology Grade Water Aliquot . . . . . . . . . . . . . . 6Preparing Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Shearing the DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Adjusting the DNA Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . 7Preparing the Fragmented Sample. . . . . . . . . . . . . . . . . . . . . . . . . . 8

Preparing Adapter Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Using Indexing Adapters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Using Non-indexing Adapters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Preparing Ligation Master Mix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Chapter 3 Setting Up and Running the Protocol . . . . . . . . . . . . . . 13Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Setting Up a Run. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Work Surface Layout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Launching the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Placing Consumables on the Work Surface . . . . . . . . . . . . . . . . . . 16Loading Samples and Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . 17Setting Up Runs for Fewer Than Eight Samples . . . . . . . . . . . . . . 21

Running the Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Starting a Run. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Monitoring a Run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Finishing a Run. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Retrieving and Handling the Processed Libraries. . . . . . . . . . . . . . 25

Contents

iv PrepX ILM DNA Library Protocol User Guide

Library Size Verification Using the Bioanalyzer . . . . . . . . . . . . . . . . . . . 25PCR Enrichment Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Thermocycling Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Library Input Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Adapter Dimer and Primer Dimer Cleanup . . . . . . . . . . . . . . . . . . . . . . 27Reagent and Reaction Locations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

PrepX ILM DNA Library Protocol User Guide 1

Chapter 1

Overview

In this chapter:

About the Protocol 1

Workflow 2

Processing Schematic 2

This PrepX ILM DNA Library Protocol User Guide provides the basic information necessary to use the Apollo 324™ System to prepare genomic DNA and cDNA libraries for sequencing.

This document assumes that you know how to use the Apollo 324™ System and the touchscreen interface. For details on using the system, refer to the Apollo 324 System User Guide.

IMPORTANT: IntegenX recommends that first-time users take advantage of user training offered with the installation of the system. A training video is available at http://integenx.com/324-training-video/.

About the ProtocolThe PrepX™ ILM DNA Library reagent kit (Catalog No. 400044) provides researchers the flexibility to prepare one to eight DNA libraries in one run for sequencing on Illumina platforms.

The entire process is completed in approximately 71 minutes, with the library products ready for amplification. Up to 40 libraries can be prepared in one eight-hour working day.

http://integenx.com/324-training-video/

2 PrepX ILM DNA Library Protocol User Guide

Chapter 1 Overview Workflow

Workflow1. Press the DNA button, and then press the PrepX ILM button on the touchscreen to

activate the Peltier heating/cooling units.

2. Place consumables, reagents, magnetic beads and samples in the racks on the work surface.

3. Start the protocol run, using the touchscreen interface.

4. The samples undergo BeadX processing:

a. End repair

b. Intermediary bead-based cleanup

c. A-Tailing

d. Intermediary bead-based cleanup

e. Adapter ligation

5. Size selection and final bead-based cleanup is performed.

6. DNA samples are now ready for amplification and can be placed into the Agilent Bioanalyzer or equivalent DNA analyzer to determine the concentration after being centrifuged briefly.

Processing Schematic

FragmentedDNA Samples

EndRepair End Repair Bead Cleanup

AdapterMaster MixLigation Ligation

BeadCleanup

Bead Cleanup Libraries

A-TailingA-Tailing Bead Cleanup

SizeSelection

High Cut Low Cut

Ready for PCR


Chapter 2

Preparing Samples and Reagents

In this chapter:

Overview 3

Materials for Operation 3

Decontaminating the Lab Work Space and Instrument 5

Preparing the Library Kit Reagents 5

Using 1X AMPure XP Beads 6

Preparing 70% v/v EtOH 6

Preparing the Molecular Biology Grade Water Aliquot 6

Preparing Samples 6

Preparing Adapter Solution 8

Preparing Ligation Master Mix 10

OverviewThis chapter describes how to prepare samples and reagents for the PrepX™ ILM DNA Library protocol.

IMPORTANT: We only guarantee the Apollo 324™ System to perform using the recommended supplies and materials listed in this document.

Materials for Operation

Reagent Kit

The following reagents are provided in the PrepX ILM DNA Library Reagent Kit (Catalog No. 400040). The kit provides sufficient reagents for three 8-sample runs (24 samples).

The kit includes the following items, sufficient to process 24 samples:

• PrepX Universal DNA Library Kit (Box Enz)– 24 Enzyme strips (4 tubes in each strip, yellow labeled seal)– Ligase enzyme (3 tubes, 10 µL each)– Ligase buffer (1 tube, 360 µL)

• NaCl, 2.5M solution (1 bottle, 5 mL)

• Molecular biology grade water (1 bottle, 250 mL)

For a complete list of the materials and equipment required for using the Apollo 324 system, see the Apollo 324 System User Guide.


Chapter 2 Preparing Samples and Reagents Materials for Operation

Instruments

The following items are required.

Customer-Supplied Reagents and Consumables

The following reagents and consumables are recommended to run the Apollo 324 System.

Item Part Number Supplier

96-well thermocycler various various

Agilent 2100 Bioanalyzer G2938C Agilent

Agilent High Sensitivity DNA Kit (to perform Bioanalyzer runs for your prepared samples)

5067-4626 Agilent

Centrifuge for 0.2 mL 8-tube strips various various

Vortex mixer for preparing reagents and samples various various

Customer-Supplied Consumables Part Number Supplier

1.1 mL 12-tube strips (Axygen) 89005-580 VWR

0.2 mL 8-tube strips (Axygen) 10011-764 VWR

0.2 mL 8-cap strips (Axygen) 10011-786 VWR

Eppendorf 96-well skirted microtiter plate, conical, 150 µL 47744-122 VWR

50 mL Falcon test tube 21008-940 VWR

15 mL Falcon test tube 21008-929 VWR

1.5 mL DNA tube various various

Piercing tips 300028 IntegenX

Dispensing filter tips 300027 IntegenX

Reservoirs 300031 IntegenX

Micropipette tips (2 µL, 20 µL, 200 µL and 1000 µL) various various

Micropipettes (2 µL, 20 µL, 200 µL and 1000 µL) various various

Customer-Supplied Reagents Part Number Supplier

100% EtOH, reagent grade E7023 Sigma-Aldrich

AMPure Beads (450 mL kit) A63882 Agencourt

Adapters various various


Decontaminating the Lab Work Space and Instrument Chapter 2 Preparing Samples and Reagents

The recommended initial purchase quantities and usage per run of the consumables are as follows:

Decontaminating the Lab Work Space and Instrument1. Wipe the lab work surfaces with a 10% bleach solution, then wipe with water.

2. Wipe the lab work surfaces with 70% EtOH and dry thoroughly.

3. Wipe the Apollo 324 instrument work surface with 70% EtOH and dry thoroughly.

Preparing the Library Kit ReagentsTo prepare the reagents:

1. Retrieve the box containing the enzymes from the freezer and thaw the enzymes.

Only thaw the enzymes that you will be using for the run. Keep all reagents, including ligase enzyme, ligase buffer and annealed adapters on ice.

2. Vortex and spin down reagents briefly.

For IntegenX reagents, use an 8-strip adapter for the micro-centrifuge.

3. Visually inspect to be sure that the entire volume is at the bottom of each vial with no air pockets.

Requirement Recommended Initial Purchase Usage per Run

1.1 mL 12-tube strips 1 box 2 strips/run

0.2 mL 8-tube strips 1 box 5 strips /run

0.2 mL 8-cap strips 1 box 5 cap strips /run

Eppendorf 96-well skirted microtiter plate, conical, 150 µL

1 box 1 plate/run

50 mL Falcon test tube n/a varies

15 mL Falcon test tube n/a varies

1.5 mL DNA tube n/a varies

Piercing tips 1 box of 1000 125 runs - 8 tips/run

Dispensing filter tips 1 box of 960 17 runs - 56 tips/run

Reservoirs 1 box of 100 25 runs - 4 reservoirs/run

Micropipette tips (2 µL, 20 µL, 200 µL and 1000 µL) n/a varies

Micropipettes (2 µL, 20 µL, 200 µL and 1000 µL) n/a varies

Safety glasses, gloves and lab coats as required in your lab

n/a varies


Chapter 2 Preparing Samples and Reagents Using 1X AMPure XP Beads

Using 1X AMPure XP Beads

IMPORTANT: This protocol uses 1X AMPure XP beads. Just before starting the protocol, you will aliquot 200 µL of the AMPure beads into each tube of an empty 8-tube strip. Do not place the beads on ice.

1. Vortex the 1X AMPure beads. Tap the tubes on benchtop to remove the beads from the tube lid.

2. Aliquot 200 µL of 1X AMPure beads into each tube of the 8-tube strip.

NOTE: Do not centrifuge the bead strip. Be sure that no air is trapped beneath the bead volume. If there is an air pocket, use a pipette to remove the air.

3. Cap the tubes and set aside at room temperature until ready to load onto the Apollo 324 work surface.

Preparing 70% v/v EtOHCombine 10.5 mL of 100% EtOH and 4.5 mL of molecular biology grade water in a sterile 50 mL Falcon tube and mix. You will use 15 mL for each run.

Preparing the Molecular Biology Grade Water AliquotPipette 15 mL of molecular biology grade water into a sterile 50 mL Falcon tube. 15 mL will be consumed in the run.

Preparing SamplesSuccessful library preparation is highly dependent on sample quality.

Be very careful when pipetting. Do not allow any air pockets to form under the sample. If there are air pockets, the robot arm will pick up air instead of fluid.

The size of the source for the DNA libraries should be centered at approximately 400 bp. In order to ensure this, you must shear the DNA sample. Before shearing the DNA, it is important to assess the DNA quality and accurately determine the concentration.

We recommend that you perform both of the following methods to indicate sample quality:

• An absorbance measurement ratio of 260/280 that is between 1.8 and 2.0

• Fluorometric-based method such as Qubit or PicoGreen to quantify dsDNA

CAUTION


Preparing Samples Chapter 2 Preparing Samples and Reagents

Shearing the DNA

We recommend using Covaris systems to shear the starting DNA sample. For instructions on shearing the DNA, refer to the Covaris documentation. For optimal results, use Low TE or 10mM Tris as the shearing buffer.

We recommend the following settings on the Covaris S2. These settings were tested for E. coli samples (129 µL of sample, plus 1 µL of Tris). Other genome types might require different settings.

Adjusting the DNA Concentration

1. Run an Agilent high sensitivity chip analysis of the sheared DNA (diluted if required) by following the guidelines of the protocol supplied with the high sensitivity kit. The analysis will indicate the proper concentration for your samples.

2. Confirm that the shearing was successful in generating a DNA sample with a main peak at approximately 400 bp, as shown in the following example.

3. Dilute the sheared DNA with nuclease-free water to the working input concentration range of 10–300 ng (0.7–20 ng/µL).

You can now load the fragmented sample into the strip tubes.

Setting Value

Duty Cycle 10%

Intensity 4

Cycles/burst 200

Time/cycle 9

Number of Cycles 8

Total Process Time 72 seconds

100 ng

50 ng


Chapter 2 Preparing Samples and Reagents Preparing Adapter Solution

Preparing the Fragmented Sample

NOTE: Use color-coded Axygen strip tubes to ensure correct setup.

1. Pipette 15 µL of fragmented DNA sample (0.7–20 ng/µL input concentration) into each tube of an Axygen 8-tube strip.

Be very careful when pipetting. Do not allow any air pockets to form under the sample. If there are air pockets, the robot arm will pick up air instead of fluid.

2. To avoid contamination, temporarily cap the sample tubes with 8-tube strip Axygen caps until they are seated in the work surface.

3. Place the sample on ice until ready to load onto the Apollo 324 work surface.

IMPORTANT: The Apollo 324 System is calibrated for Axygen tubes only. Using other types might result in run failure.

Preparing Adapter SolutionThis section describes how to prepare the adapter solution.

• If you are using indexing adapters, see “Using Indexing Adapters.”• If you are using non-indexing adapters, see “Using Non-indexing Adapters.”

IMPORTANT: It is important to maintain this ratio of adapter to sample for the use range described for this protocol. Lowering the adapter input might result in poor adapter ligation. When using a low DNA input, you might need to use a lower adapter volume.

The tube strip containing the prepared adapter solution is placed in row 1 in Block 4 on the instrument work surface, as shown in Figure 2-1.

Figure 2-1 Adapter solution placement in Block 4

CAUTION

Adapter MMLigation MM

A B C D E F G H

121110987654321

Block 4


Preparing Adapter Solution Chapter 2 Preparing Samples and Reagents

Using Indexing Adapters

If you are using indexing adapters, prepare the adapter solution in four sterile 8-tube strips, as follows:

1. Thaw, vortex, and briefly centrifuge the annealed adapter tubes.

2. Prepare adapter stock solution to 25µM, if necessary.

3. Pipette the proper volume of adapter and water into each tube of an 8-tube strip, according to the order shown in Figure 2-1. See Table 2-1 for the recommended adapter solution component volumes.

NOTE: For information about DNA adapters, refer to the section “TruSeq® DNA v1/v2/LT and RNA v1/v2/LT and ChIP Sample Prep Kits” in the document “2012-09-18_IlluminaCustomerSequenceLetter.pdf” on the Illumina website (https://icom.illumina.com/Download/Summary/5kKzNLtAtUCIhbHYc3NkUw). You must log in with your username and password to access the file.

Table 2-1 Adapter volumes

4. Temporarily cap the tubes and keep on ice or in the IntegenX 8-well cold block.

Using Non-indexing Adapters

If you are using non-indexing adapters, prepare the adapter solution in a sterile 8-tube strip, as follows:

NOTE: This section applies only if you are preparing your own adapter mix.

1. Resuspend lyophilized adapter oligonucleotides to 100µM each in 10mM Tris-EDTA, pH 8.0.

2. Add equal volumes of adapters to a 1.5 mL microfuge tube to produce a 50µM mixture of both, then vortex and spin down briefly.

3. Aliquot 50 µL of the adapters into PCR tubes, cap the tubes and anneal using the following thermocycling parameters:

• Step 1: 95 °C for 2:00 minutes

• Step 2: 0.1 °C/second to 25 °C

• Step 3: 25 °C for 5:00 minutes

• Step 4: 4 °C hold

4. Pool the annealed adapters into a sterile 1.5 mL tube.

5. Vortex and spin down briefly.

6. Aliquot into single use portions in PCR tubes and cap the tubes. Store frozen at -20 °C.

NOTE: Adapters can be prepared in advance and stored at -20 °C prior to use, but avoid freeze-thaw cycles.

7. Prepare the use-concentration by diluting 10-fold (50µM to 5µM) in 10mM Tris or reagent grade water.

Adapter Type

Adapter stock concentration

(µM)

Volume of stock adapter per reaction

(µL)

Volume of water per reaction

(µL)

Total volume of adapter solution

(µL)

Ordered by user 25 2 13 15

Illumina TruSeq adapters n/a 2.5 12.5 15

https://icom.illumina.com/Download/Summary/5kKzNLtAtUCIhbHYc3NkUw


Chapter 2 Preparing Samples and Reagents Preparing Ligation Master Mix

Preparing Ligation Master MixThe tube strip containing the prepared ligation mix is placed in row 2 in Block 4 on the instrument work surface, as shown in Figure 2-2.

Figure 2-2 Ligation mix placement in Block 4

Preparing Ligation Master Mix for Eight Samples

IMPORTANT: If you are processing fewer than eight samples in a run, follow the procedure in the section “Preparing Ligase Enzyme and Buffer for Fewer Than Eight Samples” to prevent pipetting loss of ligase enzyme and buffer.

This recipe prepares ligation mix for one run of eight samples. The ligation mix is aliquotted into one 8-tube strip; each tube contains ligation mix for one reaction. See Table 2-2 for ligation mix component volumes.

1. Prepare the ligation buffer and enzyme mix in a sterile microfuge tube (for one run of eight samples, plus one sample for volume loss to surfaces) as follows:

a. Thaw, vortex, and briefly centrifuge the Ligase Buffer tubes.

b. Thaw and briefly centrifuge the Ligase Enzyme tubes.

c. Combine 108 µL of T4 Ligase Buffer, 9 µL of T4 Ligase Enzyme, and 18 µL of nuclease-free water in a sterile microfuge tube. Pipette mix and briefly centrifuge. Keep the mixture on ice.

Table 2-2 Reagent volumes for ligation master mix

d. Aliquot 15 µL of this ligation mix into each tube of one 8-tube strip.

2. Temporarily cap the tubes and keep the ligation mix strip on ice or in the IntegenX 8-well cold block until ready to place onto the Apollo 324 work surface.

Visually inspect the tubes to be sure that the entire volume is at the bottom of the tubes, without air pockets.

1 Sample 8 (+1) Samples

T4 DNA Ligase Buffer 12 µL 108 µL

T4 DNA Ligase Enzyme 1 µL 9 µL

Nuclease-free water 2 µL 18 µL

Volume per sample 15 µL 135 µL


A B C D E F G H

121110987654321

Block 4


Preparing Ligation Master Mix Chapter 2 Preparing Samples and Reagents

Preparing Ligase Enzyme and Buffer for Fewer Than Eight Samples

1. Thaw, vortex, and briefly centrifuge the T4 DNA Ligase Enzyme tubes and T4 DNA Ligase Buffer tubes.

2. Aliquot 12 µL of T4 DNA Ligase Buffer into each tube of an Axygen 8-tube strip.

3. Aliquot 1 µL of T4 DNA Ligase Enzyme into each tube of an Axygen 8-tube strip.

4. Cap the tubes and freeze for later use.

5. When you are ready to use the reagents:

a. Thaw, vortex, and briefly centrifuge the T4 DNA Ligase Enzyme tubes and T4 DNA Ligase Buffer tubes.

b. Pipette the 1 µL of T4 DNA Ligase Enzyme into the 12 µL of T4 DNA Ligase Buffer in each tube.

c. Pipette 2 µL of nuclease-free water into each tube.

d. Temporarily cap the tubes and keep on ice or in the IntegenX 8-well cold block.


Chapter 2 Preparing Samples and Reagents Preparing Ligation Master Mix


Chapter 3

Setting Up and Running the Protocol

In this chapter:

Overview 13

Setting Up a Run 13

Running the Protocol 23

Library Size Verification Using the Bioanalyzer 25

PCR Enrichment Recommendations 25

Adapter Dimer and Primer Dimer Cleanup 27

Reagent and Reaction Locations 28

OverviewThis chapter describes how to set up and run the protocol, providing guidance for placing the reagents, samples and consumables on the work surface, and then running the protocol.

There are two types of runs, each requiring a slightly different setup.

• Eight samples – The default setup of eight samples fills the available blocks on the work surface. For details, see “Setting Up a Run.”

• Fewer than eight samples – Tubes must be loaded specifically in the middle of the block to avoid side-to-side torque. For details, see “Setting Up Runs for Fewer Than Eight Samples.”

The work surface setup window in the touchscreen interface is mapped to the work surface blocks on the instrument. The window provides guidance for placing reagents, samples and consumables, and then running the protocol.

For details on placing:

• Consumables, see “Placing Consumables on the Work Surface.”

• Enzymes, samples and AMPure beads, see “Loading Samples and Reagents.”

For details on preparing the samples and reagents before loading them in the instrument, see Chapter 2, “Preparing Samples and Reagents.”

An accumulation of discarded pipettes and tips in the waste tip box can cause a run to fail. Be sure to open the waste tip box access door and check that the box has been emptied.

Setting Up a RunThis section describes how to load items on the instrument, using the setup window to verify placement.

CAUTION


Chapter 3 Setting Up and Running the Protocol Setting Up a Run

Work Surface Layout

The following illustration shows the layout of the work surface and placement of the consumables, reagents and samples for the protocol run. The default setup for eight samples fills the available blocks on the work surface. The setup windows provide guidance for setting up runs.

Setup Window

The setup window in the touchscreen interface provides guidance for setting up runs. In the setup window, the NEXT and BACK buttons enable you to navigate so you can easily view the setup for any block.

• NEXT magnifies the next sequential block.

• BACK returns to the last magnified block or the Start window.

For details on using the touchscreen interface, refer to the Apollo 324 System User Guide.

Launching the Software

1. Power on the instrument.

The software start-up window is displayed for a few seconds, and then the IntegenX splash screen appears. When you power on the instrument, the temperature in the heating/cooling units (Blocks 3 and 4) adjusts to 18 °C.

After the splash screen disappears, the initial Start window is displayed, showing the available protocols.

After the software is launched, the pipette head of the Apollo 324 System will initialize.

IMPORTANT: Pressing in the center of the window or pressing and holding any of the buttons for several seconds will initiate calibration of the touchscreen. If you accidentally do so, refer to “Calibrating the Touchscreen” in Chapter 3 of the Apollo 324 System User Guide.

Block 7 Block 8

NotUsed

NotUsed


NotUsed

2.5 M NaCl

A1 MICROPLATE H1A B C D E F G H

A B C D E F G H

A B C D E F G H

121110987654321

121110987654321

121110987654321

Block 1 Block 2 Block 4

Block 5 Block 6

PiercingTips

FilterTips

1.1 mlStrip

Tubes

Reservoir 4Empty

Reservoir 370% EtOHReservoir 2

Empty

Reservoir 1H2O

SamplesNot Used

Beads

NotUsed

Products

A B C D E F G H

Block 3

EnzymeStrips


Setting Up a Run Chapter 3 Setting Up and Running the Protocol

2. To select the protocol, in the Start window, press DNA, and then press PrepX ILM.

The work surface setup window is displayed.

On the instrument work surface, the left-hand heating/cooling unit (Block 3) remains at 18 °C, while the right-hand unit (Block 4) cools to 4 °C.

While Block 4 is cooling, the COOLING button is displayed.

When Block 4 reaches 4 °C, the RUN button appears.

3. While the Block 4 is cooling, pre-load consumables, samples and reagents on the work surface.

NOTE: You can place the consumables (tubes, plates, reservoirs and tips) on the work surface before starting the software and loading the samples and reagents. For details, see “Placing Consumables on the Work Surface.”

COOLING button displayed during cooling of Block 4

RUN button displayed when Block 4 reaches4 °C



Placing Consumables on the Work Surface

Placing the consumables on the work surface can be done in advance.

NOTE: For instructions on setting up runs for fewer than eight samples, see “Setting Up Runs for Fewer Than Eight Samples.”

1. Place 24 1.1 mL tubes (three 8-tube strips) in rows 1–3 of Block 1.

Verify that they are properly seated and level.

2. Place a clean, empty 96-well microtiter plate in Block 2, with the A1 well in the lower left corner.

Dispense 100 µL of the 2.5M NaCl solution into each well of row 1.



3. Place the piercing tips and dispensing pipette tips into Block 5 as follows:

a. Place 8 grey piercing tips into row 1.

b. Place 56 dispensing pipette tips into rows 2–8.

NOTE: Do not use the plastic carrier tray in Block 5, as it might cause tips to stick during automatic operation.

4. Place four empty reservoirs in Block 6. You will fill these later.

Make sure that the reservoirs are seated properly.

5. Leave blocks 7 and 8 empty; they are not used.

6. Verify that the waste tip box is empty.

Loading Samples and Reagents

NOTE: For instructions on setting up runs for fewer than eight samples, see “Setting Up Runs for Fewer Than Eight Samples.”

1. If you have not already done so, press DNA in the Start window, and then PrepX ILM to initiate the protocol.


On the instrument work surface, Block 3 remains at 18 °C, Block 4 cools to 4 °C. While it is cooling, the COOLING button is displayed; when it reaches 4 °C, the RUN button appears.

While Block 4 is cooling, you can place samples and reagents on the work surface.

IMPORTANT: Do not load the adapter mix and ligation mix until Block 4 is at 4 °C and the RUN button appears (approximately three minutes).



2. Load the empty product tubes, prepared sample and reagents into Block 3 as follows:

a. Place the 8-tube strip with the samples (tube caps removed) into row 8.

b. Place the 8-tube strip with the AMPure beads into row 6.

NOTE: Do not centrifuge the bead strip before placing in row 8. Pipette out any air pockets in the tubes.

c. Place an empty 8-tube strip for product in row 5.

d. Place each of the eight 4-tube enzyme strips (yellow) vertically into rows 1–4 at the bottom of the block so that the arrows point toward the rear of the work surface.

NOTE: Verify that all tubes are oriented and seated correctly.

Visually inspect the tubes as you place them in the block to ensure that the entire volume is in the bottom of the tubes, without droplets on the side walls, bubbles or void volume. If necessary, centrifuge briefly before placing the tubes.

CAUTION



3. Place the metal retention plate over Block 3 to secure the tubes and keep them stable, aligning the guide pins at the top and bottom.

Rotate the side knobs to lock the retention plate in place.

4. After the RUN button appears, place the 8-tube strips with adapter master mix and ligation master mix in Block 4, as follows:

a. Place the strip with adapter master mix into row 1.

b. Place the strip with ligation master mix into row 2.





6. Fill the reagent reservoirs in Block 6 as follows:

a. Dispense 15 mL of freshly prepared 70% v/v EtOH into Reservoir 3.

b. Dispense 15 mL of molecular biology grade water into Reservoir 1.

c. Leave Reservoir 2 and Reservoir 4 empty; they are not used in this protocol.



7. Blocks 7 and 8 are not used and remain empty.

8. Verify placement of all reagents and consumables and check that all tubes, plates and reservoirs are seated properly.

Verify that all components are installed according to the setup window.

You are now ready to start the run. For instructions, see “Starting a Run.”

Setting Up Runs for Fewer Than Eight Samples

If you are running fewer than eight samples, several of the work surface blocks must be loaded from the center column, to avoid side-to-side torque. Use the procedure in this section to load the blocks, and consult the following table for the columns to load.

IMPORTANT: We recommend that you use complete empty strips rather than single tubes.

Where to Load Samples and AMPure Beads

For one to seven samples, load the sample and the AMPure beads in the appropriate columns as follows:

Number of Samples

Sample and Beads are Loaded in Columns

1 D

2 D E

3 C D E

4 C D E F

5 B C D E F

6 B C D E F G

7 B C D E F G H



As an example, the following sections describe the procedure to set up a run for one sample.

Placing Consumables on the Work Surface

1. Place one 12-tube strip of 1.1 mL tubes into column D of Block 1.

2. Place a clean, empty microtiter plate in Block 2, with the A1 well in the lower left corner.

Dispense 100 µL of the 2.5M NaCl solution into the column D well of row 1.

3. Place one grey piercing tip in column D, row 1 of Block 5.

4. Place seven dispensing pipette tips in column D, rows 2–8 of Block 5.

5. Place four empty reservoirs in the positions on Block 6.

Loading Samples and Reagents

1. If you have not already done so, press DNA in the Start window, and then PrepX ILM.


On the instrument work surface, Block 3 remains at 18 °C, while Block 4 cools to 4 °C. While it is cooling, the COOLING button is displayed; when it reaches 4 °C, the RUN button appears.

While Block 4 is cooling, you can load the samples and reagents.

IMPORTANT: Do not load the adapter mix and ligation mix until Block 4 is at 4 °C and the RUN button appears (approximately three minutes).

2. In Block 3:

a. Leave rows 9–12 empty.

b. Place the 8-tube strip with the samples into row 8. Use a complete strip rather than a single tube. The sample should be in column D.

c. Leave row 7 empty.

d. Place the 8-tube strip with the AMPure beads into row 6.

NOTE: Do not centrifuge the bead strip before placing in row 8. Pipette out any air pockets in the tubes.

e. Place the empty 8-tube strip for product in row 5.

f. Place one 4-tube enzyme strip into rows 1–4 of column D, with the arrow tab pointing up toward the rear of the work surface. Verify that orientation of the strip is correct.

NOTE: Verify that all tubes are oriented and seated correctly.



4. After the RUN button appears, place the 8-tube strips with adapter master mix and ligation master mix in Block 4, as follows:

a. Place the strip with adapter master mix into row 1.

b. Place the strip with ligation master mix into row 2.


Running the Protocol Chapter 3 Setting Up and Running the Protocol



6. In Block 6, fill the reagent reservoirs as for eight samples.

7. Blocks 7 and 8 are not used and remain empty.

8. Verify placement of all reagents and consumables and check that all tubes, plates and reservoirs are seated properly.

Verify that all components are installed according to the setup window.

You are now ready to start the run. For instructions, see “Starting a Run.”

Running the Protocol

Starting a Run

Re-check placement of all consumables and reagents in each block.

When you are ready to start the protocol, close the instrument door and press RUN. If you are not ready, press NEXT, BACK or any block to review the setup and make changes to items on the work surface.

IMPORTANT: You must close the door in order to start the run.

After you press RUN, the front door of the instrument locks and the run starts. The status of each step of the run is displayed in a progress bar.


Chapter 3 Setting Up and Running the Protocol Running the Protocol

Monitoring a Run

After a run begins, the progress window shows the status of the process. A countdown timer shows how much time (hours and minutes) is left for the process. From this window, you can only stop a run completely.

The STOP button appears only after a run has started.

Only press the STOP button to abort a run in progress.

Finishing a Run

After the products have been eluted off the beads, the run is finished. A message informs you when the protocol is complete.

Press OK to display the Start window. At this point, the door unlocks.

Do NOT attempt to open the instrument door until the run is finished. Do NOT assume that a protocol is complete until the “PROTOCOL FINISHED” message is displayed.

CAUTION

DANGER


Library Size Verification Using the Bioanalyzer Chapter 3 Setting Up and Running the Protocol

Retrieving and Handling the Processed Libraries

1. Open the door and remove the 8-tube product strip (in row 5, Block 3).

2. Cap the processed samples immediately and store them on ice or at -20 °C.

3. Remove and discard used reagents and consumables.

The library products are now ready for amplification.

Library Size Verification Using the Bioanalyzer1. Run the diluted PrepX ILM DNA libraries on an Agilent high sensitivity chip by

following the guidelines of the Agilent protocol supplied with the high sensitivity kit.

Include the source sample (also diluted to ~10 ng/µL) for reference.

2. Verify that the library size distribution is similar to the following example, with a peak between 300–400 bp.

PCR Enrichment RecommendationsPCR is used to enrich the output library mixture into a uniform, sequenceable population, which is subsequently quantitated before depositing on the flow cell. Our experience has shown that higher quality DNA (as shown by A260/280 ratios) requires fewer or no cycles of PCR enrichment. The reason is that while the ligase reaction is extremely efficient, the quality of the input sample directly affects the efficiency of the ligation reaction. For example, input sample with a high ratio of ssDNA or contaminants will have a lower ligation efficiency and therefore will have a lower percentage of PCR competent fragments (i.e., libraries will have a full complement of ligated adapters). Such samples with a lower percentage of PCR competent fragments will need more PCR cycles.


Chapter 3 Setting Up and Running the Protocol PCR Enrichment Recommendations

Thermocycling Conditions

When enriching samples by PCR, it is important to use thermocycling conditions recommended by the manufacturers of your adapters and PCR primers. It is critical to pair your adapters with the correct PCR primers. For example, if you are using Illumina TruSeq adapters, follow Illumina’s recommended 10 cycles of PCR using their PCR primers and thermocycling conditions. You can also choose to empirically determine the minimal number of PCR cycles required for your sequencing needs if you do not want to perform the full 10-cycle PCR. In some cases, as few as 3 to 4 cycles of PCR will be sufficient; however, more challenging samples and very low input samples might require the full 10 cycles of PCR.

Table 3-1 provides general guidance for the number of PCR cycles based on DNA quality assessed using A260/280 ratios.

Table 3-1 Library quality (A260/280) and PCR cycle guidelines

NOTE: If you input more than 5 ng for PCR, reduce the number of cycles accordingly to prevent over-amplification You can input up to 10 ng for PCR.

Library Input Volume

Using the data from the Agilent high sensitivity run (or alternatively, from a Qubit or PicoGreen assay) calculate the volume of the sample required to input 5 ng of the library to the PCR reaction. For low input samples that yield less than 5 ng of library output, add the entire volume to the PCR reaction.

IMPORTANT: DO NOT use a spectrophotometer to determine input concentration to PCR. Quantitation will be inaccurate and can result in poor amplification.

Table 3-2 serves only as a guide to the volume of library that one may add to a PCR reaction based on the amount of input DNA sample used. The volume equivalent to 5 ng of library generated is only an approximation based on Good and Excellent DNA quality (Table 3-1), but the volume will vary depending on the starting quality of DNA sample. It is critical that you assess your library quality and quantity to determine the proper volume of library to use in PCR.

Table 3-2 PCR volume guidelines

DNA Quality (A260/280 Ratio)

Marginal (< 1.8) Good (1.8–1.9) Excellent (1.9–2.0)

5–10 cycles 3–5 cycles 0–3 cycles

DNA Input to Apollo 324 (ng)

Volume Equivalent to 5 ng (µL)

10 15 (entire sample)

11–50 4–15

51–100 2–4

101–300 1–2


Adapter Dimer and Primer Dimer Cleanup Chapter 3 Setting Up and Running the Protocol

Adapter Dimer and Primer Dimer CleanupClean up post-PCR libraries using equal volumes of bead and sample (1:1 ratio) to remove any excess adapter dimers and primer dimers. An example of an Agilent HS Bioanalyzer trace of a 10-cycle post-PCR library after AMPure bead clean-up is shown.

NOTE: Contact your local Field Application Specialist if you have questions about PCR cleanup.

Chapter 3 Setting Up and Running the Protocol Reagent and Reaction Locations


Reagent and Reaction LocationsBlock 1

Axygen 1.1 mL TubesBlock 2

Microtiter PlateBlock 3

0.2 mL PCR Tubes (PCR 0)Block 4

0.2 mL PCR Tubes (PCR 1)

Row Description Row Description Row Material Description Row Description

12 12 12 Not used 12

11 11 11 Not used 11

10 10 10 Not used 10

9 9 9 Not used 9

8 8 8 Samples x8 (15 µL) Sample tube, user input 8

7 7 7 Not used 7

6 6 6 1X AMPure Bead (200 µL) User input 1X beads 6

5 5 5 Product collection (15 µL) Empty strip tube 5

4 4 4 ER Enzyme (5 µL) Enzyme strip (kit) 4

3 70% EtOH (800 µL) 3 3 ER Buffer (15 µL) Enzyme strip (kit) 3

2 Water wash (600 µL) 2 2 AT Enzyme (5 µL) Enzyme strip (kit) 2 Ligation Master Mix (15 µL)

1 Elution water (600 µL) 1 NaCl, 2.5M (100 µL) 1 AT Buffer (15 µL) Enzyme strip (kit) 1 Adapter Master Mix (15 µL)

Block 5Tip Rack

Block 6Reservoirs

Block 7 Empty, Not Used

Block 8 Empty, Not Used

Row Description Description

12 Not used

Empty11 Not used

10 Not used

9 Not used

70% EtOH (15 mL)

8 Pipette tip

7 Pipette tip

6 Pipette tip

Empty5 Pipette tip

4 Pipette tip

3 Pipette tip

H2O(15 mL)

2 Pipette tip

1 Piercing tip


Index

Aadapter solution

placing tubes 19, 22, 28preparing using indexing adapters 9preparing using non-indexing adapters 9tube location 28

AMPure beadsloading in tubes 6placing tubes 18, 22preparing 6

Bbeads

loading in tubes 6placing tubes 18, 22preparing 6

buffers, supplied 3

Cconsumables

placing on the work surface 16, 28supplied by customer 4

DDNA libraries

recommendations for PCR enrichment 25retrieving and handling 25size verification 25

DNA samplesadjusting concentration of sheared samples 7preparing fragmented samples 8shearing before running the protocol 6, 7

Eenzymes

placing tubes 18, 22, 28supplied 3tube location 28

EtOHplacing 20preparing 70% 6

Ffilter tips

locations 28placing 17, 28

Hheating/cooling units, placing retention plates 19, 20

Iinstruments required for runs 4

Llibraries

recommendations for PCR enrichment 25retrieving and handling 25size verification 25

ligation master mixplacing tubes 19, 22, 28preparing 10tube location 28

Mmaterials and equipment required 3materials for operation

customer-supplied reagents and consumables 4instruments 4reagent kit 3

microtiter plate, placing 16molecular biology grade water

placing 20preparing aliquot 6

PPCR enrichment recommendations 25piercing tips

location 28placing 17, 22

pipette tipslocation 28placing 17, 22, 28


Index

PrepX ILM DNA Library protocolabout 1reagent kit 1, 3

productplacing tubes 18, 22, 28retrieving and handling 25tube location 28

protocol overview 1protocol runs

ending 24for fewer than eight samples 21monitoring progress 24performing 13placing consumables 16placing reagents and samples 18setting up 14starting 23

Rreagent and reaction locations 28reagent kit contents 3reagent reservoirs

filling 20, 23location 28placing 17, 22

reagentslocations on work surface 28placing tubes for dispensed reagents 18, 22, 28preparing 3PrepX ILM DNA Library Reagent Kit 3, 5supplied by customer 4

retention plates, on heating/cooling units 19, 20, 22, 23

Ssamples

adjusting concentration of sheared DNA 7handling processed samples 25location 28placing tubes 18, 22, 28preparing 3preparing fragmented samples 8recommendations for PCR enrichment 25shearing before running the protocol 6, 7

setting up and running the protocol 13setup window, starting runs 23software

finishing the protocol run 24monitoring progress 24setup window 14start window 14starting runs 23touchscreen interface 14

starting runs 23

Ttechnical support, contacting iitips

locations 28placing 17, 28

touchscreen interface 14training, obtaining information about ii

Wwaste tip box 17work surface

reaction locations 28reagent and tip layout 28

workflowprocessing schematic 2steps 2

IntegenX Inc.5720 Stoneridge Drive, Suite 300Pleasanton, CA 94588

925.701.3400

[email protected]

www.IntegenX.com

Part Number P037482, Rev. A1/2013

mailto:[email protected]

http://www.integenX.com

Documents

Apollo 324 System - content.stockpr.comcontent.stockpr.com/wafergen/db/.../Apollo324_PrepX... · Overview In this chapter: About the Protocol 1 Workflow 2 Processing Schematic 2 This