Agilent Technologies E5500 Series User guide

USER GUIDE
Fragment Library Preparation Using the
AB Library Builder™ System
5500 Series SOLiD™ Systems
Publication Part Number 4460965 Rev. A
Revision Date March 2011
design experiment
X prepare libraries
prepare beads
run sequencer
analyze data
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
This user guide is the proprietary material of Applied Biosystems, LLC or its affiliates and is protected by laws of copyright. The customer of the 5500 Series
SOLiD™ Sequencers is hereby granted limited, non-exclusive rights to use this user guide solely for the purpose of operating the 5500 Series SOLiD™
Sequencers. Copying, renting, modifying, or creating derivatives of this user guide not authorized by Applied Biosystems or its affiliates is prohibited.
Information in this document is subject to change without notice.
APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO
THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. TO THE FULLEST EXTENT ALLOWED BY LAW, IN NO EVENT SHALL APPLIED
BIOSYSTEMS 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 DOCUMENT, INCLUDING BUT NOT
LIMITED TO THE USE THEREOF, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT APPLIED BIOSYSTEMS IS ADVISED OF THE POSSIBILITY OF
SUCH DAMAGES.
NOTICE TO PURCHASER: DISCLAIMER OF LICENSE
The products in this User Guide may be covered by one or more Limited Use Label License(s). Please refer to the respective product documentation or the
Applied Biosystems website under www.appliedbiosystems.com for the comprehensive license information. By use of these products, the purchaser accepts
the terms and conditions of all applicable Limited Use Label Licenses. These products are sold for research use only, and are not intended for human or
animal diagnostic or therapeutic uses unless otherwise specifically indicated in the applicable product documentation or the respective Limited Use Label
License(s). For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way,
Carlsbad, California 92008.
TRADEMARKS
The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.
Bioanalyzer is a trademark of Agilent Technologies, Inc.
AMPure is a registered trademark of Beckman Coulter, Inc.
Biomek is a registered trademark of Beckman Coulter, Inc.
Covaris is a registered trademark of Covaris, Inc.
Freedom EVO is a registered trademark of Tecan Group Ltd.
Kimwipes is a registered trademark of Kimberly-Clark Corporation.
Latitude is a registered trademark of Dell, Inc.
NanoDrop is a registered trademark of NanoDrop Technologies.
TaqMan is a registered trademark of Roche Molecular Systems, Inc.
© Copyright 2011, Life Technologies Corporation. All rights reserved.
Part Number 4460965 Rev. A
03/2011
Contents
About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
CHAPTER 1
About the Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Library preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Product information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Kit contents and storage conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
CHAPTER 2
Prepare to Build the Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Quantitate the DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Shear the DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Set up the AB Library Builder™ System for size-selected or express fragment library
preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
CHAPTER 3
Build the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Start the run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Set up for a new run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
CHAPTER 4
Nick-Translate the Library with Optional Amplification . . . . . . . . 29
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Nick-translate the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
(Optional) Nick-translate and amplify the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Quantitate the DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Check the size distribution of the library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
(Optional) Pool equal molar barcoded libraries of similar size . . . . . . . . . . . . . . . . . . . . . . . . . 34
CHAPTER 5
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Instrument error codes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
3
Contents
APPENDIX A
Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Required Applied Biosystems reagent kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Required Applied Biosystems reagent kits for automated
liquid-handling systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Required equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Optional equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Replacement parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Required consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Optional consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
APPENDIX B
Supplemental Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Load and unload Covaris® microTUBE vials from the Covaris® microTUBE holder . . . . . . 47
AB Library Builder™ System operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
(Optional) Size-select and pool libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Quantitate the DNA with the NanoDrop® ND-1000 Spectrophotometer . . . . . . . . . . . . . . . . 55
APPENDIX C
Supplemental Background Information . . . . . . . . . . . . . . . . . . . . . 59
Why prepare fragment libraries? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Preparing fragment libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Sequence orientation from source DNA to sequence map . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
APPENDIX D
Library Construction Oligonucleotide Sequences . . . . . . . . . . . . . 63
PCR Primer and adaptor sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Barcoded adaptor sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
APPENDIX E
Checklist and workflow tracking form . . . . . . . . . . . . . . . . . . . . . . . 73
Workflow checklists: prepare a standard or express fragment library with the
AB Library Builder™ System Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Workflow tracking: prepare a standard or express fragment library with the
AB Library Builder™ System Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
APPENDIX F
Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
General chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Chemical waste safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
4
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Contents
Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
5
Contents
6
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
About This Guide
Safety information
Note: For important instrument safety information, refer to the AB Library Builder™
System User Guide (Part no. 4463421). For general safety information, see this section
and Appendix F, “Safety” on page 77. When a hazard symbol and hazard type appear
by a chemical name or instrument hazard, see the “Safety” Appendix for the complete
alert on the chemical or instrument.
Safety alert words
Four safety alert words appear in Applied Biosystems user documentation at points in
the document where you need to be aware of relevant hazards. Each alert word—
IMPORTANT, CAUTION, WARNING, DANGER—implies a particular level of
observation or action, as defined below:
IMPORTANT! – Indicates information that is necessary for proper instrument
operation, accurate chemistry kit use, or safe use of a chemical.
CAUTION! – Indicates a potentially hazardous situation that, if not avoided,
may result in minor or moderate injury. It may also be used to alert against
unsafe practices.
WARNING! – Indicates a potentially hazardous situation that, if not avoided,
could result in death or serious injury.
DANGER! – Indicates an imminently hazardous situation that, if not avoided,
will result in death or serious injury. This signal word is to be limited to the most
extreme situations.
Except for IMPORTANTs, each safety alert word in an Applied Biosystems document
appears with an open triangle figure that contains a hazard symbol. These hazard
symbols are identical to the hazard symbols that are affixed to Applied Biosystems instruments.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
7
About This Guide
Safety information
SDSs
The SDSs for any chemicals supplied by Applied Biosystems or Ambion are available
to you free 24 hours a day. For instructions on obtaining SDSs, see “SDSs” on page 78.
IMPORTANT! For the SDSs of chemicals not distributed by Applied Biosystems or
Ambion contact the chemical manufacturer.
8
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
CHAPTER 1
About the Products
Note: For complete site preparation and operating instructions of the AB Library
Builder™ System, refer to the AB Library Builder™ System Site Preparation Guide (Part
no. 4465106) and the AB Library Builder™ System User Guide (Part no. 4463421) at:
http://www.appliedbiosystems.com/librarybuilderguides.
For a more detailed overview of library types and the library preparation workflows,
see “Supplemental Background Information” on page 59.
Library preparation
Library preparation is the first step in which samples are adapted for sequencing on
the 5500 Series SOLiD™ Sequencers. During library preparation, forward and reverse
adaptors are added to the ends of DNA inserts (The bead is for illustration purposes
only and is not added until the bead preparation step):
Product information
Purpose of the
products
To prepare fragment and barcoded fragment libraries for sequencing on the
5500 Series SOLiD™ Sequencers, Life Technologies offers a system of kits and adaptors
to customize preparation of single to multiplexed, barcoded libraries (Life
Technologies part numbers are in parentheses. For comparison, the SOLiD™ 4 System
kits and adaptors are shown):
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
9
Chapter 1 About the Products
Product information
5500 Series SOLiD™ System
Library Core Kits
Adaptors
5500 SOLiD™ Fragment Library Core Kit
(4464412)
5500 SOLiD™ Fragment Library Standard Adaptors
(4464411)
5500 SOLiD™ 48 Fragment Library Core Kit
(4464415)
5500 SOLiD™ Fragment Library Barcode Adaptors
(4464404)
Library Builder™ Fragment Core Kit for 5500 SOLiD™
(4463763)
SOLiD™ 4 System
How to use the
core kits and
adaptors
Library Kits
Adaptors
SOLiD™ Fragment Library Construction Kit
(4443473)
SOLiD™ Fragment Library Oligos Kit
(4401151)
Library Builder™ Fragment Core Kit for SOLiD™ 4.0
(4463762)
SOLiD™ Fragment Library Barcoding Kit 1-96
(4449637)
This user guide describes how to use the Library Builder™ Fragment Core Kit for 5500
SOLiD™ with the 5500 SOLiD™ Fragment Library Standard Adaptors or the 5500
SOLiD™ Fragment Library Barcoding Adaptors. Use the 5500 SOLiD™ 48 Fragment
Library Core Kit with the adaptors for automated liquid-handling systems such as the
Beckman Coulter Biomek® FXp and Tecan Freedom EVO® instruments. To use the
5500 SOLiD™ Fragment Library Core Kit with the adaptors, refer to Fragment Library
Preparation: 5500 Series SOLiD™ Systems User Guide (Part no. 4460960).
Used with a wide range of barcoded adaptors, the Library Builder™ Fragment Core Kit
for 5500 SOLiD™ contains reagents and a protocol card to prepare fragment libraries
(100–250 bp, before adaptor ligation) or express libraries (100–550 bp, before adaptor
ligation). The protocol card directs the instrument to end-repair, ligate, and size-select
libraries.
After automated library preparation, you nick-translate and quantitate the library for
templated bead preparation on the Applied Biosystems SOLiD™ EZ Bead™ System
[refer to SOLiD™ EZ Bead™ Emulsifier Getting Started Guide (Part no. 4441486)].
10
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Chapter 1 About the Products
Kit contents and storage conditions
Kit contents and storage conditions
Kit contents
The Library Builder™ Fragment Core Kit for 5500 SOLiD™ (Part no. 4463763) contains
materials sufficient to prepare up to 13 standard or express fragment libraries:
Part
AB Library Builder™ Fragment
Reagents Module for 5500 SOLiD™
Description
Storage temperature
• 13 cartridges. Each
cartridge contains
ready-to-use
reagents.
–20°C
• 13 tubes of 5✕
Reaction Buffer
tubes.
• 1 tube of Shear
Buffer.
• 5 tubes of
Platinum® PCR
Amplification mix
and 1 tube each of
Library PCR
Primer 1 and Library
PCR Primer 2
AB Library Builder™ Plastics Module
Sample and elution
tubes, tips, and tip
holders.
Room temperature
The adaptors that are required to prepare fragment libraries with the AB Library
Builder™ System are sold separately:
Part
Description
Storage temperature
5500 SOLiD™ Fragment Library
Standard Adaptors (4464411)
One each
–20°C
5500 SOLiD™ Fragment Library
Barcode Adaptors (4464404)
One each
–20°C
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
11
Chapter 1 About the Products
Kit contents and storage conditions
12
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
CHAPTER 2
Prepare to Build the Library
Workflow
Quantitate the DNA (page 14)
Shear the DNA (page 14)
Set up the AB Library Builder™ System for size-selected or express fragment
library preparation (page 17)
Inspect the AB Library Builder™ Cartridges (page 17)
Insert or change the protocol card and power ON the instrument (page 18)
Load the racks and tubes (page 19)
Insert the racks into the AB Library Builder™ Device (page 23)
Procedural guidelines
• The protocol is designed for 10 ng–5 µg of genomic DNA or ligated PCR product.
• Use good laboratory practices to minimize cross-contamination of products.
• Adjust microcentrifuge speeds and times according to the g-forces specified in the
protocols. Applied Biosystems recommends the Eppendorf 5417R tabletop
microcentrifuge.
• Perform all steps requiring 0.5-mL and 1.5-mL tubes with 0.5-mL Eppendorf
LoBind Tubes (Eppendorf Part no. 022431005) and 1.5-mL Eppendorf LoBind
Tubes (Eppendorf Part no. 022431021).
• Thaw reagents on ice or at room temperature before use, but thaw Shear Buffer at
room temperature.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
13
Chapter 2 Prepare to Build the Library
Quantitate the DNA
Quantitate the DNA
For accuracy, determine sample DNA concentration using a double-stranded DNAspecific fluorescence assay. Use the HS Assay Kit to measure dsDNA concentrations
from 10 pg/µL to 100 ng/µL. For samples outside this range, use the dsDNA BR for
higher concentrations of DNA or PicoGreen® dsDNA Assay Kit for lower
concentrations:
• Invitrogen Qubit™ dsDNA HS Assay Kit (Invitrogen Part no. Q32851 or Q32854)
or
• Invitrogen Qubit™ dsDNA BR Assay Kit (Invitrogen Part no. Q32850 or Q32853).
or
• Invitrogen Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen Part no. P7589)
Shear the DNA
This step involves fragmenting the input DNA into small fragments with a mean
fragment size of 165 bp by using the Covaris® System. The conditions have been tested
for shearing 10 ng–5 µg DNA in a total volume of 120 µL. For certain DNA samples,
optimizing the shearing protocol may be necessary.
Shear the DNA
You can shear the DNA with two supported shearing systems:
• The Covaris® S220 System (see “Shear the DNA with the Covaris® S220 System”).
or
• The Covaris® S2 System (see “Shear the DNA with the Covaris® S2 System” on
page 15.
Shear the DNA with the Covaris® S220 System
IMPORTANT! Ensure that the bath temperature during shearing is between
5–10°C. Higher shearing temperatures can be harmful to DNA.
1. For each library dilute the components below in a 1.5-mL LoBind Tube. Shear
Buffer reduces DNA damage from fragmentation.
Component
Amount
DNA
10 ng–5 µg
1✕ Low TE Buffer
Variable µL
Shear Buffer
1.2 µL
Total
120 µL
2. Prepare the Covaris® S220 Tank:
a. Ensure that the water in the Covaris® S220 tank is filled with fresh deionized
water to fill-line level 12 on the graduated fill-line label.
The water should cover the visible glass part of the tube.
14
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Chapter 2 Prepare to Build the Library
Shear the DNA
b. Set the chiller temperature to 2–5 °C to ensure that the temperature reading
in the water bath displays 5°C.
c. Supplement the circulated water chiller – not the actual water bath – with
20% ethylene glycol.
3. Load the DNA:
a. Place a Covaris® microTUBE into the loading station.
b. Keeping the snap-cap on the tube, use a tapered pipette tip to slowly transfer
the 120 µL of DNA sample through the pre-split septa.
Be careful not to introduce a bubble into the bottom of the tube.
Note: To load and unload the Covaris® microTUBE correctly from the
microTUBE holder, see “Load and unload Covaris® microTUBE vials from
the Covaris® microTUBE holder” on page 47.
4. Shear the DNA using the following Covaris® S220 System conditions:
IMPORTANT! Ensure that the bath temperature limit is set at 15°C, and keep the
bath temperature to ≤ 10°C.
Condition
Setting
Number of cycles
6
Bath temperature
5°C
Bath temperature limit
15°C
Mode
Frequency
sweeping
Water quality testing function
Off
Duty Factor
10%
Peak Incident Power
175 Watts
Cycles/burst
100
Time
60 seconds
5. Remove the sheared DNA:
a. Place the Covaris® microTUBE into the loading station.
b. While keeping the snap-cap on, insert a pipette tip through the pre-split
septa, then slowly remove the sheared DNA.
c. Transfer 110 µL of the sheared DNA into a new 1.5-mL sample tube
provided in the Library Builder™ Fragment Core Kit for SOLiD™ 5500.
Shear the DNA with the Covaris® S2 System
1. Prepare the Covaris® S2 Tank:
a. Ensure that the water in the Covaris® S2 tank is filled with fresh deionized
water to fill-line level 12 on the graduated fill-line label.
The water should cover the visible glass part of the tube.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
15
Chapter 2 Prepare to Build the Library
Shear the DNA
b. Set the chiller temperature to 2–5°C to ensure that the temperature reading in
the water bath displays 5°C.
c. Supplement the circulated water chiller with 20% ethylene glycol.
2. Dilute the desired amount of DNA to 120 µL in 1✕ Low TE Buffer in a LoBind
tube. Shear Buffer reduces DNA damage from fragmentation:
Component
Amount
DNA
10 ng to 5 µg
1✕ Low TE Buffer
Variable µL
Shear Buffer
1.2 µL
Total
120 µL
3. Load the DNA into the Covaris® S2 System:
a. Place a Covaris® microTUBE into the loading station.
b. Keeping the snap-cap on the tube, use a tapered pipette tip to slowly transfer
the 120 µL of DNA sample through the pre-split septa.
Be careful not to introduce a bubble into the bottom of the tube.
Note: To load and unload the Covaris® microTUBE correctly from the
microTUBE holder, see “Load and unload Covaris® microTUBE vials from
the Covaris® microTUBE holder” on page 47.
4. Shear the DNA using the following Covaris® S2 System conditions:
IMPORTANT! Ensure that the bath temperature limit is set at 15°C, and keep the
bath temperature to ≤ 10°C.
Condition
Setting
Number of cycles
6
Bath temperature
5°C
Bath temperature limit
15°C
Mode
Frequency
sweeping
Water quality testing function
Off
Duty cycle
10%
Intensity
5
Cycles/burst
100
Time
60 seconds
5. Remove the sheared DNA:
a. Place the Covaris® microTUBE into the loading station.
b. While keeping the snap-cap on, insert a pipette tip through the pre-split
septa, then slowly remove the sheared DNA.
c. Transfer 110 µL of the sheared DNA into a new 1.5-mL sample tube
provided in the Library Builder™ Fragment Core Kit for SOLiD™ 4.0.
16
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Chapter 2 Prepare to Build the Library
Set up the AB Library Builder™ System for size-selected or express fragment library preparation
Store the DNA in a supplied Sample Tube at 4°C for short-term
storage or at –20°C for long-term storage, or proceed directly to “Set up the
AB Library Builder™ System for size-selected or express fragment library
preparation”.
STOPPING POINT
Set up the AB Library Builder™ System for size-selected or
express fragment library preparation
About sizeselected and
express fragment
library preparation
AB Library Builder™ System prepares standard (size-selected) or express (not sizeselected) DNA fragment libraries. Use the AB Library Builder™ System with the
Library Builder™ Fragment Core Kit for SOLiD™ 5500 and Agencourt AMPure® XP
Reagent to end-repair, size-select (optional), ligate, and purify fragment libraries.
To install and set up the AB Library Builder™ System the AB Library Builder™ System
Site Preparation Guide (Part no. 4465106) and the AB Library Builder™ System User Guide
(Part no. 4463421).
IMPORTANT! To avoid data loss or run cancellation, always follow these practices:
• Before you insert or remove a protocol card, power the instrument OFF
• Before you power the instrument ON, insert the protocol card, then close the
instrument door.
• To pause the instrument during an extraction run, press Stop before you open the
instrument door
• When you are not performing a run or instrument test, you can open the instrument
door with the power OFF or ON
• Do not move instrument components such as the platform, magnets, and syringes
while the instrument is powered ON.
Before a run, follow these procedures to set up the instrument:
1. “Inspect the AB Library Builder™ Cartridges”.
2. “Insert or change the protocol card and power ON the instrument” on page 18.
3. “Load the racks and tubes” on page 19.
4. “Insert the racks into the AB Library Builder™ Device” on page 23.
Inspect the AB
Library Builder™
Cartridges
Each cartridge has 12 compartments for reagents:
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
17
Chapter 2 Prepare to Build the Library
Set up the AB Library Builder™ System for size-selected or express fragment library preparation
Cartridge compartment numbers
1
1300 µL
2–3
1200 µL
4
Empty
5–6
20 µL
7
85 µL
8
90 µL
9–10
20 µL
11: Tube to be added after 5✕ Reaction
Buffer tube prepared
12: Unsealed compartment for beads
Insert or change
the protocol card
and power ON the
instrument
Volumes
42.5 µL
(final volume
is 85 µL)
—
IMPORTANT! Do not remove the protocol card while the instrument is on. Removing
the card stops the run, and it may cause instrument data file loss. To remove the card,
see step 3.
If you accidentally remove the protocol card during a run, power off the instrument
immediately to minimize potential for instrument data loss.
For guidelines on handling protocol cards, see the AB Library Builder™ System User
Guide (Part no. 4463421).
1. Confirm that the power switch is in the OFF position.
Note: If you insert the card while the instrument is on, the instrument does not
recognize the card.
2. Open the card slot.
18
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Chapter 2 Prepare to Build the Library
Set up the AB Library Builder™ System for size-selected or express fragment library preparation
3. To remove a card that is already in the slot, push the button located below the
card slot (see the photo below), then pull the card out of the slot. Place the card in
the plastic cover in the box.
IMPORTANT! Do not remove the protocol card while the instrument is on.
4. Insert the appropriate protocol card in the slot with the arrow on the protocol
card pointing toward the instrument and the label facing left.
5. Push the card completely into the card slot, then close the card slot.
6. Close the door to the AB Library Builder™ Device.
7. Power ON the instrument.
When the card is fully inserted in the correct orientation, the display briefly
shows information including the instrument version, then displays the Main
menu.
8. Press START.
Load the racks and
tubes
Note: To ensure the best pipetting performance, use the cartridge rack and tip and
tube rack shipped with the instrument; these racks are calibrated with the instrument
at the factory. Before using other racks on a specific instrument, run the installation test
to qualify the racks for use on that instrument. Refer to the AB Library Builder™ System
User Guide for details
Wear gloves when you handle samples or load the cartridges, tips, and tubes in the
rack.
Remove the racks from the instrument
Open the instrument door (push up the door), then remove the tip and tube rack and
the cartridge rack:
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
19
Chapter 2 Prepare to Build the Library
Set up the AB Library Builder™ System for size-selected or express fragment library preparation
Load the cartridge rack
1. Remove up to 13 cartridges from the kit box.
IMPORTANT!
One cartridge is required per sample. Use only AB Library Builder™ Cartridges.
IMPORTANT!
Do not switch the supplied pre-filled reagents with any other buffers, because the
protocols are specifically optimized with the reagents supplied with the kit.
2. Thaw the cartridges at room temperature or on ice for ≤ 2 hours or until
completely thawed.
IMPORTANT! Avoid leaving the cartridges at room temperature for longer than
necessary to completely thaw them. Avoid repeated freeze-thawings of unused
cartridges.
IMPORTANT! Before loading the cartridges into the cartridge rack, ensure that the
cartridges are completely thawed, particularly reagents in cartridge wells 2 and 3.
3. Gently tap each cartridge on the laboratory bench until any liquid droplets that
might be underneath the foil seal are deposited into the bottom of the well.
20
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Chapter 2 Prepare to Build the Library
Set up the AB Library Builder™ System for size-selected or express fragment library preparation
4. Load the reagent cartridges into the cartridge rack by sliding each reagent
cartridge along the groove in the direction of the arrow until the reagent cartridge
clicks into place. Make sure that the notches in the cartridge align with the
notches in the cartridge rack.
Note: An incorrectly loaded cartridge rack may cause the instrument to stop
during a run.
Load adaptors in the cartridges
IMPORTANT! If you are preparing barcoded libraries for multiplexed sequencing, for
each sequencing run, use at least one of the following full sets of four barcodes:
Barcodes-T-001–004, 005–008, 009–012, 013–016, 017–020, 021–024, 025–028, 029–032,
033–036, 037–040, 041–044, 045–048, 049–052, 053–056, 057–060, 061–064, 065–068, 069–
072, 073–076, 077–080, 081–084, 085–088, 089–092, or 093–096. Use only one of the
barcoded-T-0XX adaptors for each ligation reaction, unless < 4 libraries are being
barcoded.
Use the barcodes according to these conditions:
• If <4 libraries are prepared for sequencing, then use multiple barcodes per library in
equal ratios. For example, for 2 libraries, use 2 barcodes for each library. For
3 libraries, use 4 barcode adaptors for each library for a total of 12 barcodes.
• If ≥4 libraries are prepared for sequencing and libraries are split into sets of 4 to use
full sets of barcodes, then use one set of barcodes for the remaining libraries (1,2,or
3 libraries). There is no need to use multiple barcodes per library in equal ratios.
1. Prepare the 5✕ Reaction Buffer tubes:
a. Spin the supplied 5✕ Reaction Buffer tubes for each sample.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
21
Chapter 2 Prepare to Build the Library
Set up the AB Library Builder™ System for size-selected or express fragment library preparation
b. Calculate the amount of each P1-T and barcoded adaptor (Y) needed. Y=µL
of P1-T=µL of barcoded adaptor:
Y μL adaptor needed
=
# μg DNA ×
×
A
9.2 pmol
1 μg DNA
× 10 ×
1 μL adaptor needed
50 pmol
where:
A = (value below), if...
Library type
0.3
Fragment
0.66
Express
fragment
Y µL adaptor = (value below), if...
0.56 µL (1:10 dilution)
<100 ng
(fragment
library)
1.21 µL (1:10 dilution)
<100 ng
(express
fragment
library
c. Calculate the amount of 1✕ Low TE Buffer required, based on the volumes of
P1-T and barcoded adaptors needed
μL 1X Low TE Buffer needed =
42.5 – 2Y
d. Add 1✕ Low TE Buffer, P1 and barcoded adaptors to the appropriate 5✕
Reaction Buffer tube. Vortex, then pulse-spin the tube and place it in open
position 11 of the cartridge.
Load Agencourt AMPure® XP Reagent in the cartridges
Thoroughly resuspend Agencourt AMPure® XP Reagent, then carefully transfer
500 µL to unsealed position 12.
Load the tip and tube rack
IMPORTANT! If you are processing fewer than 13 samples, make sure to load the tips
and tubes in the same positions as the reagent cartridges that are loaded in the
cartridge rack.
Load the tip and tube rack in the following order:
1. Row S (fourth row): Load with sample/elution tubes containing 110 µL of
sheared DNA. If the sample volume is <110 µL, then add 1✕ TE to the sample for
a total volume of 110 µL. Ensure that the cap is off.
2. Row T1 and T2 (second and third rows): Load with AB Library Builder™ Tips
inserted into tip holders.
Note: Two tip and tip holder sets are required per sample.
22
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Chapter 2 Prepare to Build the Library
Set up the AB Library Builder™ System for size-selected or express fragment library preparation
3. Row E (first row): Load with labeled sample/elution tubes, with the caps removed
and secured:
S-Sample tube
T2-Tip and tip holder
T1-Tip and tip holder
E-Elution
(Optional) Place elution tube caps here
Insert the racks
into the AB Library
Builder™ Device
IMPORTANT!
• Insert the cartridge rack before the tip and tube rack. Changing the order of
loading the racks may cause the instrument to stop during a run.
• Use only AB Library Builder™ Sample Tubes (sample/elution tubes). Other tubes
may be picked up by the nozzle tips due to differences in tube height and shape,
stopping the run.
Insert the cartridge rack
IMPORTANT! Before inserting the cartridge rack into AB Library Builder™ Device,
ensure that the cartridges are completely thawed, particularly reagents in cartridge
wells 2 and 3.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
23
Chapter 2 Prepare to Build the Library
Set up the AB Library Builder™ System for size-selected or express fragment library preparation
Insert the loaded cartridge rack into the instrument:
WARNING! Do not touch the surface of the heat block. The temperature of the
heat block can reach 95°C. Touching the block can cause burns.
24
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Chapter 2 Prepare to Build the Library
Set up the AB Library Builder™ System for size-selected or express fragment library preparation
Insert the tip and tube rack
Insert the loaded tip and tube rack into the instrument with row E in the front:
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
25
Chapter 2 Prepare to Build the Library
Set up the AB Library Builder™ System for size-selected or express fragment library preparation
26
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
CHAPTER 3
Build the library
Workflow
Start the run (page 27) (run time: ~2.5 h)
Set up for a new run (page 28)
For additional instructions on instrument operation, see “AB Library Builder™ System
operation” on page 48.
Start the run
1. Press START to select the AB Library Builder™ System Kit option.
2. Confirm that you have loaded and inserted the cartridge rack and tip and tube
rack correctly.
3. Select the script for the kit you are using, then follow the on-screen prompts.
4. (Optional) Scan the sample, elution tube, and sample lane barcodes [refer to the
AB Library Builder™ System User Guide (Part no. 4463421)].
5. Close the door to the AB Library Builder™ Device.
6. Press START.
The screen shows the current step and the approximate incubation time
remaining.
IMPORTANT! Do not open the door during a protocol run. To pause or cancel the
run, see “AB Library Builder™ System operation” on page 48.
Note: If you lose power or the power cord is unplugged, the run stops. When the
power resumes, the digital display shows the Main menu. You cannot resume the
run. If the tips are still on the syringe unit when the power resumes, return the
tips to the original positions as described in “AB Library Builder™ System
operation” on page 48.
7. At the end of the run (the instrument beeps briefly and the digital display shows
“Finished Protocol”). To unload the instrument:
a. Press
to return to the Main menu, then open the instrument door.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
27
Chapter 3 Build the library
Set up for a new run
b. Remove the elution tubes. Confirm that they are properly labeled, then cap
the elution tubes containing the library in 100 µL.
c. If the library has a brown tint, place each tube in a DynaMag™-2 Magnetic
Rack for at least 1 minute until the solution is clear of brown tint when
viewed at an angle; then transfer the supernatant to a new tube.
d. Remove the tip and tube rack and cartridge rack.
e. Properly dispose of the used reagent cartridges, tips, and tubes.
f. Close the instrument door.
g. Clean the tip and tube rack as needed.
Note: No cooling period is required between runs.
Store the DNA in a supplied Sample Tubes at 4°C for short-term
storage or at –20°C for long-term storage, or proceed directly to “Nick-Translate
the Library with Optional Amplification” on page 29.
STOPPING POINT
Set up for a new run
WARNING! Do not clean the instrument with acids, or bases (such as bleach).
Acids and bases can react with the guanidine thiocyanate in the lysis buffer and
generate toxic gas.
1. Follow the set-up procedures for a new run (see “Set up the AB Library Builder™
System for size-selected or express fragment library preparation” on page 17).
Note: To set up for a new run using the same protocol card, leave the instrument
on. To set up for a new run with a different protocol card, power off the
instrument, then change the protocol card (see “Insert or change the protocol card
and power ON the instrument” on page 18).
2. Start the run (see “Start the run” on page 27).
28
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
CHAPTER 4
Nick-Translate the Library with
Optional Amplification
Workflow
Nick-translate the libraries
Prepare the reaction, then nick-translate
the library (page 29)
(Optional) Nick-translate and amplify
the libraries
Prepare the reaction, then nick-translate
and amplify the library (page 31)
Purify the nick-translated library (page 30)
Purify the nick-translated, amplified
library (page 32)
Quantitate the DNA (page 33)
Quantitate the DNA (page 33)
Stopping point
Stopping point
Check the size distribution of the
library (page 34)
Check the size distribution of the
library (page 34)
Stopping point
Stopping point
(Optional) Pool equal molar barcoded
libraries of similar size (page 34)
(Optional) Pool equal molar barcoded
libraries of similar size (page 34)
Stopping point
Stopping point
Nick-translate the library
Prepare the
reaction, then nicktranslate the
library
Note: To nick-translate and amplify the library, proceed to “(Optional) Nick-translate
and amplify the library” on page 31.
1. In a new 1.5-mL LoBind Tube, combine for each library:
Component
Platinum®
PCR Amplification Mix
Volume
400 µL
Library
100 µL
Total
500 µL
2. Vortex the reaction for 5 seconds, then pulse-spin.
3. Distribute 125-µL aliquots of combined library and PCR master mix between 4,
0.2-mL PCR tubes.
4. Incubate the library at 72°C for 20 minutes.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
29
Chapter 4 Nick-Translate the Library with Optional Amplification
Nick-translate the library
Purify the nicktranslated library
1. Resuspend the Agencourt AMPure® XP Reagent and allow the mixture to come
to room temperature (~30 minutes).
2. Prepare 70% ethanol for N number of libraries:
Component
Volume
Nuclease-Free Water
600 µL × N
Ethanol, Absolute
1400 µL × N
Total
2000 µL × N
3. For every nick-translated library, label a new 1.5-ml LoBind Tube.
4. Combine each set of the identical 4 PCR reactions (125 µL) to the appropriately
labeled 1.5-mL LoBind Tube. The total combined volume of the amplified library
is 500 µL.
5. Bind the DNA to the resuspended, ambient Agencourt AMPure® XP Reagent:
a. For each library, prepare the bead suspension:
Component
Nick-translated library
Agencourt
AMPure®
XP Reagent
Volume
500 µL
750 µL†
† Equal to 1.5 volumes of sample reaction.
b. Vortex the beads for 10 seconds, then pulse-spin.
c. Incubate the mixture at room temperature (20–25°C) for 5 minutes.
d. Place each tube in a DynaMag™-2 Magnetic Rack for at least 3 minutes until
the solution is clear of brown tint when viewed at an angle; then, remove and
discard the supernatant.
6. Wash the DNA 2 times. For each wash:
a. Without removing the tube from the magnet, add 750 µL of freshly prepared
70% ethanol and incubate for 30 seconds. Do not disturb the pellet.
b. Aspirate and discard ethanol.
7. Remove the tube from the DynaMag™-2 Magnetic Rack, pulse-spin the tube,
return the tube to the magnetic rack; then remove and discard the supernatant
with a 20-µL pipettor.
8. Open each tube, then dry the beads at room temperature (20–25°C) for
≤ 5 minutes.
9. Elute the DNA:
a. Remove each tube from the DynaMag™-2 Magnetic Rack, then add 50–
100 µL Low TE Buffer directly to the pellet to disperse the beads.
b. Vortex the beads for 10 seconds, then pulse-spin.
c. Incubate the beads for 2 minutes at room temperature.
d. Place the tube in the DynaMag™-2 Magnetic Rack for at least 1 minute until
the solution clears.
30
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Chapter 4 Nick-Translate the Library with Optional Amplification
(Optional) Nick-translate and amplify the library
e. Transfer the supernatant containing the amplified library to a new 1.5-mL
LoBind Tube.
10. Proceed to “Quantitate the DNA” on page 33.
(Optional) Nick-translate and amplify the library
Library amplification is useful to increase the amount of rare or low-input samples and
to enrich targeted sequences. Library amplification can, however, bias the library and
introduce base incorporation errors.
Prepare the
reaction, then nicktranslate and
amplify the library
1. In a new 1.5-mL LoBind Tube, combine for a PCR master mix:
Component
Volume per
amplification
Master mix for N
libraries
Platinum® PCR Amplification Mix
380 µL
380 µL × (1.1 × N)
Library PCR Primer 1, 50 µM
10 µL
10 µL × (1.1 × N)
Library PCR Primer 2, 50 µM
10 µL
10 µL × (1.1 × N)
Total
400 µL
400 µL × (1.1 × N)
2. Transfer 400 µL of the PCR master mix to each library. Each library is 100 µL in an
elution tube so that the total volume of the mix is 500 µL.
3. Vortex the reaction for 5 seconds, then pulse-spin.
4. Distribute 125-µL aliquots of combined library and PCR master mix between
four, 0.2-mL PCR tubes.
IMPORTANT! The current protocol is optimized for maximum yield from input
DNA. In many cases, library amplification is not needed. Quantitate the library to
assess the need to amplify it. If library amplification is needed, minimize the
number of cycles, based on the amount of starting input DNA. Use minimal
cycling to avoid over-amplification and production of redundant molecules.
5. Determine the number of PCR cycles:
Starting amount of DNA
Number of
cycles
10–100 ng
10 cycles
100 ng–1 µg
6–8 cycles
1–2 µg
4–6 cycles
2–5 µg
3–6 cycles
IMPORTANT! Minimize the number of PCR cycles to avoid over-amplification and
redundant molecules. Base the number of cycles on the amount of starting input
DNA.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
31
Chapter 4 Nick-Translate the Library with Optional Amplification
(Optional) Nick-translate and amplify the library
6. Run the PCR for each 125-µL aliquot:
Stage
Purify the nicktranslated,
amplified library
Step
Temp
Time
Holding
Nick translation
72°C
20 min
Holding
Denature
95°C
5 min
Cycling
Denature
95°C
15 sec
Anneal
62°C
15 sec
Extend
70°C
1 min
Holding
Extend
70°C
5 min
Holding
—
4°C
∞
1. Resuspend the Agencourt AMPure® XP Reagent and allow the mixture to come
to room temperature (~30 minutes).
2. Prepare 70% ethanol for N number of libraries:
Component
Volume
Nuclease-Free Water
600 µL × N
Ethanol, Absolute
1400 µL × N
Total
2000 µL × N
3. For every amplified library, label a new 1.5-ml LoBind Tube.
4. Combine each set of the identical 4 PCR reactions (125 µL) to the appropriately
labeled 1.5-mL LoBind Tube. The total combined volume of the amplified library
is 500 µL.
5. Bind the DNA to the resuspended, ambient Agencourt AMPure® XP Reagent:
a. For each library, prepare the bead suspension:
Component
Nick-translated and amplified library
Agencourt
AMPure®
XP Reagent
Volume
500 µL
750 µL†
† Equal to 1.5 volumes of sample reaction.
b. Vortex the beads for 10 seconds, then pulse-spin.
c. Incubate the mixture at room temperature (20–25°C) for 5 minutes.
d. Place each tube in a DynaMag™-2 Magnetic Rack for at least 3 minutes until
the solution is clear of brown tint when viewed at an angle; then, remove and
discard the supernatant.
6. Wash the DNA 2 times. For each wash:
a. Without removing the tube from the magnet, add 750 µL of freshly prepared
70% ethanol and incubate for 30 seconds. Do not disturb the pellet.
b. Aspirate and discard ethanol.
32
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Chapter 4 Nick-Translate the Library with Optional Amplification
Quantitate the DNA
7. Remove the tube from the DynaMag™-2 Magnetic Rack, pulse-spin the tube,
return the tube to the magnetic rack; then remove and discard the supernatant
with a 20-µL pipettor.
8. Open each tube, then dry the beads at room temperature (20–25°C) for
≤ 5 minutes.
9. Elute the DNA:
a. Remove each tube from the DynaMag™-2 Magnetic Rack, then add 50–
100 µL Low TE Buffer directly to the pellet to disperse the beads.
b. Vortex the beads for 10 seconds, then pulse-spin.
c. Incubate the beads for 2 minutes at room temperature.
d. Place the tube in the DynaMag™-2 Magnetic Rack for at least 1 minute until
the solution clears.
e. Transfer the supernatant containing the amplified library to a new 1.5-mL
LoBind Tube.
Quantitate the DNA
Measure the DNA concentration by using:
• 2 µL of sample with the Qubit™ dsDNA HS Assay Kit (Invitrogen Part
no. Q32851) and the Qubit® 2.0 Fluorometer (Invitrogen Part no. Q32866). Use the
Qubit™ dsDNA HS Assay Kit to measure dsDNA concentrations from 10 pg/µL
to 100 ng/µL. For samples outside this range, use the Qubit™ dsDNA BR Assay
Kit for higher concentrations of DNA or the Invitrogen Quant-iT™ PicoGreen®
dsDNA Assay Kit for lower concentrations
or
• 2 µL of sample in the NanoDrop® ND-1000 Spectrophotometer (see “Quantitate
the DNA with the NanoDrop® ND-1000 Spectrophotometer” on page 55)
or
• 1 µL of sample in the Agilent Technologies 2100 Bioanalyzer™. If you used the
bioanalyzer, see “Check the size distribution of the library” on page 34.
and/or
• The appropriate volume in qPCR [refer to the Applied Biosystems SOLiD™ Library
TaqMan® Quantitation Kit protocol (Invitrogen Part no. A12120)]
STOPPING POINT Store the DNA in Elution Buffer (E1) at 4°C for short-term storage or at
–20°C for long-term storage. Proceed directly to emulsion PCR [refer to the SOLiD™
EZ Bead™ Emulsifier Getting Started Guide (Part no. 4441486)] or “Check the size
distribution of the library” on page 34.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
33
Chapter 4 Nick-Translate the Library with Optional Amplification
Check the size distribution of the library
Check the size distribution of the library
Use 1 µL of sample in the Agilent Technologies 2100 Bioanalyzer™. If you see the
expected size distribution, proceed directly to emulsion PCR [refer to the SOLiD™
EZ Bead™ Emulsifier Getting Started Guide (Part no. 4441486)]. If you do not see the
expected size distribution, troubleshoot or contact your Life Technologies Applications
Specialist.
Store the DNA in Low TE Buffer at 4°C for short-term storage or at
–20°C for long-term storage; or proceed to “(Optional) Pool equal molar barcoded
libraries of similar size”.
STOPPING POINT
(Optional) Pool equal molar barcoded libraries of similar size
IMPORTANT! To avoid library bias, do not pool the libraries until after gel purification
if:
• the libraries are of dissimilar sizes
• it is unacceptable to pool libraries of unequal library representation
• you prefer not to pool libraries of similar sizes
1. Quantitate the libraries to be pooled by qPCR (see “Quantitate the DNA” on page
33.
2. Mix together equal molar amounts of each barcoded library of similar size in an
appropriately sized LoBind Tube. Vortex the tube.
3. (Optional) size-select the pooled libraries [see “(Optional) Size-select and pool
libraries” on page 51].
STOPPING POINT Store the library DNA in Elution Buffer (E1) at 4°C, or proceed directly
to templated bead preparation [refer to SOLiD™ EZ Bead™ Emulsifier Getting Started
Guide (Part no. 4441486)].
34
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
CHAPTER 5
Troubleshooting
For symptoms other than those listed in this section, contact Technical Support
(“Obtaining support” on page 81).
Observation
Possible Cause
Recommended action
Before loading the cartridges in the cartridge rack
Precipitate in AB
Library Builder™
5✕ Reaction Buffer
tubes
5✕ Reaction Buffer tubes
were exposed to low
temperatures during
shipping or storage.
To dissolve precipitate that may have formed during shipping or storage,
incubate the 5✕ Reaction Buffer tubes at 37°C for 5 minutes or until
precipitate is no longer visible.
During the automated run
No power (the
digital display is
blank and the fan
does not turn on
when you power
on)
The digital display
is blank, but the fan
turns on when you
power on.
AC power cord is not
connected
Check AC power cord connections at both ends. Use the correct cords.
Fuse has blown
Check the integrity of the fuse and replace it if necessary (refer to the
AB Library Builder™ System User Guide).
If the problem persists after connecting the correct power cord and
replacing the fuse, contact Technical Support (“Obtaining support” on
page 81).
Protocol card is not
inserted correctly
Power off the instrument and re-insert the protocol card in the proper
orientation into the card slot (see “Insert or change the protocol card
and power ON the instrument” on page 18). Insert it completely into the
slot by manually pushing the card.
Protocol card was
inserted when the
instrument was powered
on
Power off the instrument, then power on the instrument.
Error code
displayed
—
See “Instrument error codes” on page 38.
Reagent
cartridges, tips, or
tubes are not
inserted in the
correct positions.
—
Press STOP to pause the run. Open the door, add the missing items,
then press START to resume the run. Do not open the door without
pausing the run.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
35
Chapter 5 Troubleshooting
Observation
Possible Cause
Recommended action
Run stops after an
initial start (you
may also see an
error code).
• Instrument door
opened during the run
IMPORTANT!
If you open the instrument door while the instrument is running, the run
stops, and it cannot be restarted. If you need to open the instrument
door during a run, first press Stop to pause the run, then open the door.
• Reagent cartridges,
tips, or tubes
incorrectly loaded in
the rack
• Racks incorrectly
loaded on the
instrument
1. Follow the procedure in “Instrument error codes” on page 38.
2. Before starting a new run, make sure that the reagent cartridges,
tips, and tubes are correctly loaded:
• Slide the reagent cartridges into the cartridge rack as described
in “Load the racks and tubes” on page 19.
• Load the cartridge rack before the tip and tube rack for proper
positioning.
• Do not cap the tubes.
3. If the instrument continues to stop during the run, contact Applied
Biosystems Technical Support.
Reagent cartridges not
completely thawed
1. Stop the run.
2. Remove the tip and tube rack, then remove the cartridge rack.
3. Inspect cartridge wells 2 and 3 for ice.
4. If any well is frozen, close the door to the AB Library Builder™
Device, then thaw the cartridges completely.
5. Replace the tips in position T2.
6. Insert the cartridge rack then the tip and tube rack onto the
AB Library Builder™ Device.
7. Restart the run.
No DNA yield
No sample added to tube
Add samples to tubes, load new reagent cartridges, then perform the
run again.
No liquid in tip, or
liquid in tip not
moving
No sample added to
tube, leading to wet filter
barrier on the tip and
blockage of nozzles
Add samples to tubes, load new reagent cartridges, then perform the
run again.
Buffer in the
bottom tray
Motor movements are
not smooth
Schedule preventive maintenance annually to ensure proper motor
movements.
Reagent cartridges, tips,
or tubes incorrectly
loaded in the rack
If you are processing fewer than 13 samples, make sure to load the tips
and tubes in the same positions as the reagent cartridges that are
loaded in the cartridge rack.
See below for leakage from tips.
Leakage from tips
or uneven liquid
handling between
nozzles
D-Rings are not greased
regularly or they need
replacement
You can continue the run, but maintain the D-rings as scheduled. To
prevent leakage, maintain or replace the D-rings (refer to the (AB
Library Builder™ System User Guide).
Blockage of tips
Too much starting
material causing clumps
or aggregates
Contact Technical Support (“Obtaining support” on page 81).
36
In future runs, use the sample volume recommended in the user guide
for the kit you are using.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Chapter 5 Troubleshooting
Observation
Possible Cause
Recommended action
Sample volume is lower
than the recommended
volume, leading to wet
filter barrier on the tip
and blockage of nozzles.
In future runs, use the recommended sample volume for the protocol
you are using.
Insufficient or no
adaptors added to the 5✕
Reaction Buffer tube
Add sufficient adaptor according to the adaptor calculations, and insert
the tube in position 11 of the cartridge (see “Load adaptors in the
cartridges” on page 21).
Enzymes or buffer not at
bottom of wells
Tap the wells down against a hard surface to move enzymes and buffer
to bottom of wells, then inspect the wells.
Observed DNA
peak size is
significantly
different from the
expected DNA peak
size
Incorrect volume in
sheared DNA or
prepared 5✕ Reaction
Buffer tube
Add the correct volumes to the sheared DNA and 5✕ Reaction Buffer
tubes.
Enzymes or buffer not at
bottom of wells
Tap the wells down against a hard surface to move enzymes and buffer
to bottom of wells, then inspect the wells.
Final library is
brownish
Beads in final library
1. Place the tube with the final library in a DynaMag™-2 Magnetic Rack
for at least 1 minute until the solution is clear of brown tint when
viewed at an angle.
After the automated run
No elution volume
No amplifiable
library
Long-term operation with lower-than-recommended sample volumes
can lead to issues with liquid handling performance.
2. Without disturbing the pellet, carefully transfer the supernatant,
which contains the final library, to a new 1.5-mL LoBind Tube.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
37
Chapter 5 Troubleshooting
Instrument error codes
Instrument error codes
If an extraction run is interrupted by an error, you cannot resume the interrupted run.
Follow the procedure below to resolve the error before you start a new run.
If you observe an error code:
1. Make a note of the error code, including the line number. Common error codes
are listed in the following table:
Code
Problem
Code
Problem
10
Failed return to origins, protocol cannot run
22
M axis time out, protocol in run
11
Limit error, protocol can not run
23
Y axis time out, protocol in run
12
Failed to return to Z Axis, protocol in run
24
Open door in motion
13
Failed to return to P axis, protocol in run
25
Abnormal input from bottom sensor in motion
14
Failed to return to M axis, protocol in run
26
Failed to initialize heating block
15
Failed to return to Y axis, protocol in run
27
Failed to initialize motion control board
16
Z axis limit error, protocol in run
110
System error; (Assigned greater than 10)
19
Y axis end limit, protocol in run
20
Z axis time out, protocol in run
21
P axis time out, protocol in run
2. Press ESC to return to the Main menu.
3. If there are tips attached to the nozzles, press 1 to select the Manual screen, then
press 2 to return the tips to the original position.
4. Power OFF the instrument, remove the protocol card, wait 5 minutes, insert the
protocol card, then power on the instrument.
5. Run the axis test (refer to the AB Library Builder™ System User Guide).
6. If the axis test:
• Is successful, start a new extraction run. Use new samples and plastics where
required.
• Is not successful, contact Technical Support (“Obtaining support” on page
81).
38
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
APPENDIX A
Ordering Information
A
This appendix covers materials for barcoded fragment library preparation:
■
Required Applied Biosystems reagent kits. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
■
Required equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
■
Optional equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
■
Replacement parts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
■
Required consumables. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
■
Optional consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Sufficient reagents are supplied in the AB Library Builder™ System Kit to prepare 13
libraries for high-throughput sequencing with the 5500 Series SOLiD™ System.
Upon receipt of the AB Library Builder™ System Kit, immediately store each
components at the temperature specified on the label.
Required Applied Biosystems reagent kits
Item (part number)†
Library Builder™ Fragment Core Kit for 5500
SOLiD™ (4463763)
Components
• AB Library Builder™
Reagents Module for
5500 SOLiD™
• AB Library Builder™
Plastics Module
† Applied Biosystems has validated this protocol using this specific material. Substitution
may adversely affect system performance.
Item†
5500 SOLiD™ Fragment Library Standard
Adaptors (4464411)
Source
• Barcode-T-001, 50 µM
• P1-T Adaptor, 50 µM
• Library PCR Primer 1,
50 µM
• Library PCR Primer 2,
50 µM
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
39
A
Appendix A Ordering Information
Required Applied Biosystems reagent kits
Item†
5500 SOLiD™ Fragment Library Barcode
Adaptors 1–96
(4464404)
Source
• P1-T Adaptor, 50 µM
• Library PCR Primer 1,
50 µM
• Library PCR Primer 2,
50 µM
• 5500 SOLiD™ Fragment
Library Barcode
Adaptors 1–16, 50 µM
each
• 5500 SOLiD™ Fragment
Library Barcode
Adaptors 17–32, 50 µM
each
• 5500 SOLiD™ Fragment
Library Barcode
Adaptors 33–48, 50 µM
each
• 5500 SOLiD™ Fragment
Library Barcode
Adaptors 49–64, 50 µM
each
• 5500 SOLiD™ Fragment
Library Barcode
Adaptors 65–80, 50 µM
each
• 5500 SOLiD™ Fragment
Library Barcode
Adaptors 81–96, 50 µM
each
5500 SOLiD™ Fragment Library Barcode
Adaptors 1–16
(4464405)
• Barcode adaptors
T-001–T-016
• P1-T Adaptor, 50 µM
• Library PCR Primer 1,
50 µM
• Library PCR Primer 2,
50 µM
40
5500 SOLiD™ Fragment Library Barcode
Adaptors 17–32
(44644106‡
Barcode adaptors
T-017–T-032
5500 SOLiD™ Fragment Library Barcode
Adaptors 33–48
(44644107‡
Barcode adaptors
T-033–T-048
5500 SOLiD™ Fragment Library Barcode
Adaptors 49–64
(4464408)‡
Barcode adaptors
T-049–T-064
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix A Ordering Information
Required Applied Biosystems reagent kits for automated liquid-handling systems
Item†
A
Source
5500 SOLiD™ Fragment Library Barcode
Adaptors 65–80
(4464409)‡
Barcode adaptors
T-065–T-080
5500 SOLiD™ Fragment Library Barcode
Adaptors 81–96
(44644140)‡
Barcode adaptors
T-081–T-096
† Applied Biosystems has validated this protocol using this specific material. Substitution
may adversely affect system performance.
Required Applied Biosystems reagent kits for automated
liquid-handling systems
Note: Customers who have access to an automated liquid-handling system such as the
Beckman Coulter Biomek® FXp and Tecan Freedom EVO® instruments, can choose
from the kits below:
Item (part no.)†
5500 SOLiD™ Fragment 48 Library Core Kit
(4464415)
Components
• 5500 SOLiD™ 48
Fragment Library
Enzyme Module
• 5500 SOLiD™ 48
Fragment Library
Amplification Module
5500 SOLiD™ 48 Fragment Library Enzyme
Module
(4464416)
• 10 mM dNTP
• End Polishing E1
• End Polishing E2
• 5✕ Reaction Buffer
• A-tailing Enzyme I
• T4 DNA Ligase, 5 U/µL
• 10 mM dATP
• Shear Buffer
SOLiD™
5500
48 Fragment Library
Amplification Module
(4464417)
Platinum® PCR
Amplification Mix
† Applied Biosystems has validated this protocol using this specific material. Substitution
may adversely affect system performance.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
41
A
Appendix A Ordering Information
Required equipment
Required equipment
Item†
Source
AB Library Builder™ System
Applied Biosystems
The system includes:
4463592
• Library Builder Fragment Core Kits for
SOLiD™ 4 and 5500 series Protocol Card
• AB Library Builder™ Device
• Tip and Tube Tray
• Reagent Cartridge Rack
• Barcode Reader
• RS232C Cable
• CommViewer Barcode Software CD-ROM
• 13 empty reagent cartridges
• 52 sample/elution tubes
AB Library Builder™ System with Service
Installation
Applied Biosystems
4463794
The system includes:
• Library Builder Fragment Core Kits for
SOLiD™ 4 and 5500 series Protocol Card
• AB Library Builder™ Device
• Tip and Tube Tray
• Reagent Cartridge Rack
• Barcode Reader
• RS232C Cable
• CommViewer Barcode Software CD-ROM
• 13 empty reagent cartridges
• 52 sample/elution tubes
42
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix A Ordering Information
Required equipment
Item†
Covaris® S220 System‡
A
Source
Applied Biosystems
4465653
(110 V for U.S. customers)
(220 V for international customers)
The Covaris® S220 System includes:
• Covaris® S220 sonicator
• Universal Voltage Kit
• Latitude® laptop from Dell® Inc.
• MultiTemp III Thermostatic Circulator
• Covaris®-2 series Machine Holder for
(one) 1.5-mL microcentrifuge tube
• Covaris®-2 series Machine Holder for
(one) 0.65-mL microcentrifuge tube
• Covaris®-2 series Machine Holder for
(one)
13 mm × 65 mm tube
• Covaris®-2 Series Machine Holder for
(one) microTUBE
• Covaris® microTUBE Prep Station
• Covaris® Water Tank Label Kit
• Covaris® microTUBEs (1 pack of 25)
Covaris® S2 System§
(110 V for U.S. customers)
(220 V for international customers)
Microcentrifuge 5417R, refrigerated, without
rotor
Note: Fragment libraries
can be prepared with the
Covaris® S2 System. New
users should purchase the
Covaris® S220 System.
• Eppendorf††
022621807 (120 V/60
Hz)
• Eppendorf‡
022621840 (230 V/50
Hz)
FA-45-24-11, fixed-angle rotor,
24 × 1.5/2 mL, including aluminum lid,
aerosol-tight
96-well GeneAmp® PCR System 9700
(thermal cycler)
Eppendorf‡
022636006
• Applied Biosystems
N8050200 (Base)
• Applied Biosystems
4314443 (Block)‡
NanoDrop® ND-1000 Spectrophotometer
(computer required)
Thermo Scientific
E-Gel® iBase™ and E-Gel® Safe Imager™
Combo Kit
Invitrogen
ND-1000
G6465
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
43
A
Appendix A Ordering Information
Optional equipment
Item†
DynaMag™- 2 Magnet (magnetic rack)
Source
Invitrogen
123-21D
Vortexer
Major Laboratory Supplier
(MLS)
Picofuge
MLS
Pipettors, 2 µL
MLS
Pipettors, 20 µL
MLS
Pipettors, 200 µL
MLS
Pipettors, 1000 µL
MLS
† Applied Biosystems has validated this protocol using this specific material. Substitution
may adversely affect system performance.
‡ Or the Covaris® S2 System.
§ Or the Covaris® S220 System.
††Or equivalent but validation of the equipment for library preparation is required.
Optional equipment
Item†
Source
E-Gel® iBase™ and E-Gel® Safe Imager™
Combo Kit
Invitrogen
2100 Bioanalyzer™
Agilent Technologies
G6465
G2938C
Qubit™ Quantitation Starter Kit
Invitrogen
Q32860
Qubit®
2.0 Fluorometer
Invitrogen
Q32866
† Applied Biosystems has validated this protocol using this specific material.
Substitution may adversely affect system performance.
Replacement parts
Product name†
AB Library Builder™ Tips and Tip Holders
44
Vendor
4463781
AB Library
Builder™
and Tube Rack
4463776
AB Library
Builder™
Cartridge Rack
4463782
AB Library Builder™ D-Ring Tool
4465603
AB Library Builder™ Barcode Reader
4465657
AB Library Builder™ Sample Tubes
4463779
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix A Ordering Information
Required consumables
Product name†
AB Library Builder™ D-Rings
AB Library
Builder™
Plastics Module
Agilent DNA 1000 Kit
A
Vendor
4465602
4465605
Agilent Technologies
5067-1504
† Applied Biosystems has validated this protocol using this specific material.
Substitution may adversely affect system performance.
Required consumables
Item†
1✕ Low TE Buffer
Source
Applied Biosystems
4389764
Nuclease-free Water, 1 L
Applied Biosystems
AM9932
MicroAmp®
Optical 8-Tube Strip, 0.2 mL
Applied Biosystems
4316567
Invitrogen
Qubit™
dsDNA HS Assay Kit
Invitrogen
Q32851 or Q32854
or
Invitrogen Qubit™ dsDNA BR Assay Kit
Invitrogen
Q32850 or Q32853
or
Invitrogen Quant-iT™ PicoGreen® dsDNA
Assay Kit
Agencourt
AMPure®
XP:
Invitrogen
P7589
Beckman Coulter
Genomics
5 mL Kit
A63880
or
or
60 mL Kit
A63881
or
450 mL Kit
or
A63882
Covaris®
microTUBEs
Covaris
520045
Ethanol, absolute
Sigma-Aldrich
E7023
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
45
A
Appendix A Ordering Information
Optional consumables
Item†
Ethylene glycol
Source
American Bioanalytical
AB00455-01000
0.5-mL LoBind Tubes
Eppendorf
022431005
1.5-mL LoBind Tubes
Eppendorf
022431021
Filtered pipettor tips
Major Laboratory
Supplier (MLS)‡
† Applied Biosystems has validated this protocol using this specific material.
Substitution may adversely affect system performance.
‡ For the SDS of any chemical not distributed by Applied Biosystems, contact the
chemical manufacturer. Before handling any chemicals, refer to the SDS provided by
the manufacturer, and observe all relevant precautions.
Optional consumables
Product name†
50-bp ladder
Vendor
Invitrogen
10416-014
SOLiD™
Library Size Selection Gel
Applied Biosystems
4443733
CF-1 Calibration Fluid Kit
Thermo Scientific
CF-1
PR-1 Conditioning
Kit‡
Thermo Scientific
PR-1
Agilent DNA 1000 Kit
Agilent Technologies
5067-1504
† Applied Biosystems has validated this protocol using this specific material.
Substitution may adversely affect system performance.
‡ The NanoDrop® Conditioning Kit is useful for “reconditioning” the sample
measurement pedestals to a hydrophobic state if they become “unconditioned” (refer
to the Nanodrop® Conditioning Kit user’s manual for more information). The PR-1 kit
consists of a container of specially formulated polishing compound and a supply of
convenient applicators.
46
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
APPENDIX B
Supplemental Procedures
B
This appendix covers:
■
Load and unload Covaris® microTUBE vials from the Covaris® microTUBE
holder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
■
AB Library Builder™ System operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
■
(Optional) Size-select and pool libraries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
■
Quantitate the DNA with the NanoDrop® ND-1000 Spectrophotometer . . . . . . 55
Load and unload Covaris® microTUBE vials from the Covaris®
microTUBE holder
Load Covaris®
microTUBE vials
1. Use a thumb to push the stainless steel plunger up into the body of the
microTUBE holder.
2. Place the body of the microTUBE against the two amber plastic prongs with the
cap of the microTUBE positioned above the prongs.
3. Use a finger to press against the middle of the glass tube (not against the cap).
With a single motion, push the tube between the prongs to position the tube:
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
47
B
Appendix B Supplemental Procedures
AB Library Builder™ System operation
IMPORTANT! Do not press against the cap to load or unload microTUBE vials,
because pressing against the cap may dislodge or damage the cap.
4. Release the plunger. The plunger pushes the tube until the base of the cap rests
against the prongs. The tube and holder are now ready to be inserted into the S
Series instrument.
Unload Covaris®
microTUBE vials
1. Use a thumb to push the stainless steel plunger up into the body of the
microTUBE holder to relieve pressure on the cap.
2. Press against the side of the glass tube (not against the cap) to free the microTUBE
from the grip of the holder.
AB Library Builder™ System operation
Use the front panel
Parts of the front panel
The front panel provides tools for operating the instrument and tools for the service
engineer to maintain the instrument:
48
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix B Supplemental Procedures
AB Library Builder™ System operation
B
The front panel contains:
• A digital display that shows the steps of the protocol that is in use.
The digital display consists of 4 lines of information and menu choices.
For the Main menu, Tests menu, and Manual menu:
– The first line shows the current menu name
– The second and third line show the executable commands for the current
menu
– The fourth line describes the keys to use for executing the commands
For the protocols screen, the display provides current information on the protocol
step and allows you to choose options.
• Two LEDs: Green indicates the power is ON, and blinking red indicates an error
code
• The Keypad to enter parameters and operate the instrument:
Key
Description
0–9
To choose menu
ESC
To previous menu
START
To run or resume protocol
STOP
To stop or pause protocol
Enter (to confirm or enter the next menu)
BS
Backspace key to delete the last digit/character
SHIFT
Shift + Up/Down arrow keys to move the cursor right or left
during time/date setup
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
49
B
Appendix B Supplemental Procedures
AB Library Builder™ System operation
Manage the run
Pause a run
1. Press Stop to pause the run.
The display shows the following:
2. To resume the run after a pause, press Start.
The run continues from the last step before the pause.
Cancel a run
1. Press Stop to pause the run.
The display shows the following:
2. Press Stop again.
The instrument stops after the current step is completed. The screen returns to the
Main menu:
3. Press 1 to go to the Manual screen:
50
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix B Supplemental Procedures
(Optional) Size-select and pool libraries
B
4. Move the axes to the original positions and/or return the tip to the origin as
follows:
Note: When the run is interrupted, the axes and tip do not automatically return to
the original positions.
• If the tips need to be returned to the holders – Press 2 (Return Tip) to return
the tips to the tip holders and move all axes to the original position:
• If the tips do not need to be returned to the holders –
– Press 1 (ORG) to go to the ORG screen:
– Move each individual axis to the origin by pressing 1, 2, 3, 4,
respectively, or press 0 to return all axes to the origin.
5. Press ESC to return to Main menu:
You are now ready to set up for a new run.
(Optional) Size-select and pool libraries
Size-select the
barcoded libraries
Prepare the SOLiD Library Size Selection gel
The DNA is run on a SOLiD™ Library Size Selection gel. The correctly sized ligation
products (~240–270 bp) are electrophoresed to the collection wells of the SOLiD™ Size
Selection Gel. The eluates in each collection well are pooled.
1. Remove a SOLiD™ Library Size Selection gel from its package. Remove the combs
from top sample-loading wells and middle collection wells.
2. Set the SOLiD Library Size Selection Gel on the E-Gel® iBase™ system linked with
the E-Gel® Safe Imager™ Real-Time Transilluminator.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
51
B
Appendix B Supplemental Procedures
(Optional) Size-select and pool libraries
Load the gel
For exact fill volumes of the wells, refer to the Invitrogen E-Gel® SizeSelect™ Agarose Gels
Quick Reference Card.
1. Load 16 µL (≤ 1 µg/lane) of the (pooled) library DNA into wells 2, 3, 6 or 7 of the
top row of wells. If the sample volume is < 20 µL, add Nuclease-free Water to the
well for a total volume of 20 µL. Skip the center well (smaller well in the top
center of the gel for the ladder); and skip a single well to the right and left of the
center top well. Skip the two outermost wells (to avoid edge effects). Do not load
more than 1 µg of DNA per lane.
2. Load 10 µL of 50-bp ladder at 0.1 µg/µL to the center top well. Add 7 µL of water
to fill the well.
3. Fill the empty wells in the top row with 20 µL of Nuclease-free Water.
4. Fill each of the collection wells in the middle of the gel with 25 µL of Nuclease-free
Water. Add 20 µL of Nuclease-free Water to the middle center well.
52
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix B Supplemental Procedures
(Optional) Size-select and pool libraries
B
The following figure shows you where to load DNA, ladder, and Nuclease-free
Water on a SOLiD™ Library Size Selection gel to size-select the DNA (“M” is the
middle well for the ladder):
Nucleasefree
Water
DNA
NucleaseNucleasefree
free
Water Ladder Water
25 μL
Nucleasefree
Water
20 μL
Nucleasefree
Water
DNA
Nucleasefree
Water
25 μL
Nucleasefree
Water
Run the SOLiD™ Library Size Selection Gel
1. Run the gel:
• iBase system program: SizeSelect 2%
• Run time: 14:30 (14 minutes and 30 seconds)
Monitor the SOLiD™ Library Size Selection gel in real-time with the E-Gel® Safe
Imager Real-Time Transilluminator.
2. During the run, fill the middle collection wells with additional Nuclease-free
Water to ensure optimal migration of the DNA through the wells.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
53
B
Appendix B Supplemental Procedures
(Optional) Size-select and pool libraries
3. When the 250-bp band (~240–270-bp region) from the marker (ladder) lane is at
the top of the collection well, stop the run if the run has not already stopped:
Sample wells
250-bp marker
Collection wells
Note: After amplification, the total size of the product is ~240–270 bp, and the
estimated insert size after size selection is ~150–180 bp.
Collect the sample from the SOLiD™ Library Size Selection Gel
1. Collect the solution from the wells and pool according to samples.
2. Wash each collection well with 25 µL with Nuclease-free Water, then retrieve the
wash solution with the solution collected in Step 6.
3. (Optional) Concentrate the DNA with a SOLiD™ Library purification column.
(Optional) Pool
remaining libraries
that will be
combined into a
single emulsion
1. Quantitate the libraries to be pooled by qPCR (see “Quantitate the DNA” on page
33).
2. Mix together equal molar amounts of each barcoded library in an appropriately
sized LoBind Tube. Vortex the tube.
Store the purified DNA in Elution Buffer (E1) at – 20°C, or proceed
directly to emulsion PCR, as describe in the SOLiD™ EZ Bead™ Emulsifier Getting
Started Guide (Part no. 4441486).
STOPPING POINT
54
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix B Supplemental Procedures
Quantitate the DNA with the NanoDrop® ND-1000 Spectrophotometer
B
Quantitate the DNA with the NanoDrop® ND-1000
Spectrophotometer
The Thermo Scientific NanoDrop® 1000 Spectrophotometer measures nucleic acid
samples from 2 ng/µL–3700 ng/µL without dilution.
Materials and
equipment
required
Required equipment
Item†
NanoDrop® ND-1000 Spectrophotometer (computer required)
Source
Thermo Scientific
ND-1000
Pipettors (20 µL)
Major Laboratory
Supplier (MLS)‡
† Applied Biosystems has validated this protocol using this specific material. Substitution may adversely affect
system performance.
‡ For the SDS of any chemical not distributed by Applied Biosystems, contact the chemical manufacturer.
Before handling any chemicals, refer to the SDS provided by the manufacturer, and observe all relevant
precautions.
Required consumables
Item†
Nuclease-free Water (1 L)
Source
Applied Biosystems
AM9932
CF-1 Calibration Fluid Kit‡
Thermo Scientific
CF-1
PR Conditioning Kit
Thermo Scientific
PR-1
Filtered pipettor tips
Major Laboratory
Supplier (MLS)
† Applied Biosystems has validated this protocol using this specific material. Substitution may adversely affect
system performance.
‡ The NanoDrop® Conditioning Kit is useful for “reconditioning” the sample measurement pedestals to a
hydrophobic state if they become “unconditioned.” (Refer to the NanoDrop® Conditioning Kit user's manual
for more information.) The PR-1 kit consists of a container of specially formulated polishing compound and
a supply of convenient applicators.
Procedure
1. Ensure that the NanoDrop® ND-1000 Spectrophotometer is properly calibrated.
Use the CF-1 Calibration Fluid Kit if necessary.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
55
B
Appendix B Supplemental Procedures
Quantitate the DNA with the NanoDrop® ND-1000 Spectrophotometer
2. Open the NanoDrop® ND-1000 Spectrophotometer software to display a dialog
box:
3. Select the Nucleic Acid button.
56
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix B Supplemental Procedures
Quantitate the DNA with the NanoDrop® ND-1000 Spectrophotometer
B
4. Lift the sampling arm and load 2 µL of Nuclease-free Water onto the lower
measurement pedestal and lower the sampling arm:
5. In the dialog box, click OK and allow the instrument to initialize.
6. Lift the sampling arm and use Kimwipes® to remove water from the
measurement pedestal and the sampling arm.
7. Load 2 µL of the same buffer that was used to resuspend or elute the DNA onto
the measurement pedestal and lower the sampling arm.
8. Click Blank and allow the instrument to take a measurement:
9. Lift the sampling arm and wipe away the buffer from both the upper and lower
measurement pedestals with Kimwipes®. The instrument is now ready to take
readings.
10. Load 2 µL of DNA sample onto the lower measurement pedestal and lower the
sampling arm.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
57
B
58
Appendix B Supplemental Procedures
Quantitate the DNA with the NanoDrop® ND-1000 Spectrophotometer
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
APPENDIX C
Supplemental Background
Information
C
This appendix covers:
■
Why prepare fragment libraries? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
■
Preparing fragment libraries. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
■
Sequence orientation from source DNA to sequence map. . . . . . . . . . . . . . . . . . . 62
Why prepare fragment libraries?
Features
• Appropriate for sequence lengths ≤ 300 bp.
• Adaptors on each end of sheared DNA insert.
• Multiplexed sequencing.
• The protocol is designed for 10 ng–5 µg of genomic DNA.
• Compared to mate-paired libraries, fragment libraries yield a higher recovery of
unique molecules, when normalized to the same input amount.
Applications
• Targeted resequencing, primary library
• Genomic resequencing
• Methylation analysis
Complexity
The amount of library used depends on the application and information needed. For
deeper coverage of large and complex genomes (for example, human genomes), more
DNA is required to prepare libraries. For smaller and less complex genomes (for
example, microbial genomes), less DNA can be used. For information about specific
applications, go to the 5500 Series SOLiD™ Sequencers website:
www.appliedbiosystems.com/solid5500
Or, contact your field applications specialist.
Preparing fragment libraries
Fragment library preparation involves shearing DNA into small fragments and
ligating P1-T and barcoded adaptors specific for fragment library preparation (see
Figure 1 on page 60).
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
59
Appendix C Supplemental Background Information
C
Preparing fragment libraries
Figure 1 Fragment library preparation workflow overview
Genomic DNA
Library molecule ligated
with P1-T and barcoded
adaptors
Sheared DNA
The barcoded adaptor consists of 3 segments of sequence:
• Internal adaptor sequence, which is necessary for sequencing the barcode
• Barcode sequence
• P2 Adaptor sequence, which is used for library amplification and emulsion PCR
Different libraries to be multiplexed in the same sequencing run are ligated to
barcoded adaptors with different barcode sequences. Ninety-six barcode sequences
are available to tag different libraries (see Figure 2).
Figure 2 Fragment library design
P1-T Adaptor (ds)
41/42 bp
5'
C
C
C
C
A
C
T
A
C
G
C
C
T
C
C
G
C
T
T
T
C
C
T
C
T
C
T
A
T
G
G
G
C
A
G
T
C
G
G
T
G
A
G
G
T
G
A
T
G
C
G
G
A
G
G
C
G
A
A
A
G
G
A
G
A
G
A
T
A
C
C
C
G
T
C
A
G
C
C
A
C
T
T
3'
Barcoded Adaptor
P1-T Adaptor
Sheared DNA
Internal Adaptor
P1-T Adaptor
Sheared DNA
Internal Adaptor
Barcode0XX
P2 Adaptor
3'
T
C
G
C
C
T
T
G
G
C
C
G
T
A
C
A
G
C
A
G
G
C
G
G
A
A
C
C
G
G
C
A
T
G
T
C
G
T
C
T
C
T
C
T
T
A
C
T
C
C
T
T
G
G
G
C
C
C
C
G
T
C
5'
Internal Adaptor (ds)
Barcode-0XX (ss)
P2 Adaptor (ss)
20 bp
10 bp
23 bp
Barcoded Adaptor (ds)
19/53 bp
Phosphorothioate bond
60
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix C Supplemental Background Information
C
Preparing fragment libraries
After P1-T and barcoded adaptors are ligated to the sheared DNA, the library is
amplified using Library PCR Primers 1 and 2, specific to the P1 and barcoded adaptors
(see Figure 3). These primers can be used only for library amplification and not for
alternative or modified library construction adaptor design, because they do not have
3′sequences necessary for the sequencing chemistry.
Figure 3 Fragment library amplification design
Library PCR Primer 1 (ss)
28 bp
3'
5'
C
C
C
C
A
C
T
A
C
G
C
C
T
C
C
G
C
T
T
T
C
C
T
C
T
C
T
A
T
G
G
G
T
G
A
T
G
C
G
G
A
G
G
C
G
A
A
A
G
G
A
G
A
G
A
T
A
C
C
C
G
T
C
A
G
C
C
A
C
T
A
C
C
A
C
T
A
C
G
C
C
T
C
C
G
C
T
T
T
C
C
T
C
T
C
T
A
T
G
G
G
C
A
G
T
C
G
G
T
G
A
T
3'
5'
Barcoded Adaptor
Library PCR
Primer 1
Sheared DNA
P1-T Adaptor
Sheared DNA
P1-T Adaptor
Internal Adaptor
Barcode-0XX
P2 Adaptor
Internal Adaptor
Barcode-0XX
P2 Adaptor
Library PCR
Primer 2
T
C
T
C
T
T
A
C
T
C
C
T
T
G
G
G
C
C
C
C
G
T
C
5'
3'
A
G
A
G
A
A
T
G
A
G
G
A
A
C
C
C
G
G
G
G
C
A
G
T
C
T
T
A
C
T
C
C
T
T
G
G
G
C
C
C
C
G
T
C
3'
T
T
5'
Library PCR Primer 2 (ss)
21 bp
For RNA applications, an alternative method to generate barcoded libraries is
described in the protocols for the SOLiD™ RNA Barcode Module 1-16 (Part
no. 4427046), SOLiD™ RNA Barcode Module 17-32 (Part no. 4453189), and SOLiD™
RNA Barcode Module 33-48 (Part no. 4453191).
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
61
C
Appendix C Supplemental Background Information
Sequence orientation from source DNA to sequence map
Sequence orientation from source DNA to sequence map
Mate-pair
Sheared,
size-selected DNA
F3
R3
R3
F3
IA
F3
IA
Circularization with
Internal Adaptor and
Nick Translation
Nick-translation goes
beyond the length of the read.
R3
F3
Sequencing of the
fragment on a bead
P1
R3
IA
P2
ǩ5HDGVDUHJHQHUDWHGIURP)DQG5SULPHUV5PDSVXSVWUHDPIURP)1RWHWKDW
5PDSVXSVWUHDPRI)LQWKHVRXUFH'1$IUDJPHQW
ǩ 7KHUHDGVDUHH[SHFWHGWRPDSDWDGLVWDQFHHTXDOWRWKHDYHUDJHIUDJPHQWOHQJWK
SOXVWKHDYHUDJHQLFNWUDQVODWLRQGLVWDQFH
ǩ7KHVHTXHQFLQJGLUHFWLRQIRUmate-pair libraries is 5′ to 3′.
Paired-end
F3
F5
Sheared,
size-selected DNA
Sequencing of the
fragment on a bead
F3
P1
F5
BC
IA
P2
ǩ 7KHVHTXHQFLQJGLUHFWLRQIRU)UHDGVRIpaired-end libraries is 5′ to 3′.
ǩ 7KHVHTXHQFLQJGLUHFWLRQIRU)UHDGVRIpaired-end libraries is 3′ to 5′7RVXSSRUW
traditional 5′ to 3′UHSUHVHQWDWLRQWKHFRPSOHPHQWRIWKHUHDGVDUHZULWWHQ
For more information on sequencing tags, refer to 5500 Series SOLiD™ Sequencers User
Guide (Part no. 4456991).
62
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
APPENDIX D
Library Construction
Oligonucleotide Sequences
D
PCR Primer and adaptor sequences
Note: The internal adaptor used for DNA fragment libraries is different from the
internal adaptor used for RNA libraries.
Note: The “~” is a phosphorothioate bond, which protects a sequence from nucleases.
Adaptor and primer sequences
Length (nt)
P1-T Adaptor, 50 µM
5′-CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGA~T-3′
41
5′-TCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGG~C~C-3′
42
Standard Adaptor, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTGTAAGAGGCTGCTGTACGGCCAAGGCGT-3′
53
Library PCR Primer 1, 50 µM
5′-CCACTACGCCTCCGCTTTCCTCTCTATG-3′
28
Library PCR Primer 2, 50 µM
5′-CTGCCCCGGGTTCCTCATTCT-3′
21
Barcoded adaptor sequences
Barcoded adaptor sequence
Length (nt)
Barcode-T-001, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTGTAAGAGGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-002, 50 µM
5′-CGCCTTGGCCGTACAGCAG3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAGGGAGTGGTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-003, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTATAGGTTATACTGCTGTACGGCCAAGGCGT-3′
53
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
63
D
Appendix D Library Construction Oligonucleotide Sequences
Barcoded adaptor sequences
Barcoded adaptor sequence
Length (nt)
Barcode-T-004, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGGATGCGGTCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-005, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTGGTGTAAGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-006, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCGAGGGACACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-007, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGGGTTATGCCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-008, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGAGCGAGGATCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-009, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAGGTTGCGACCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-010, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCGGTAAGCTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-011, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTGCGACACGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-012, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAAGAGGAAAACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-013, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCGGTAAGGCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-014, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTGCGGCAGACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-015, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGAGTTGAATGCTGCTGTACGGCCAAGGCGT-3′
53
64
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix D Library Construction Oligonucleotide Sequences
Barcoded adaptor sequences
Barcoded adaptor sequence
D
Length (nt)
Barcode-T-016, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGGGAGACGTTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-017, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGGCTCACCGCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-018, 50 µM
5′-CGCCTTGGCCGTACAGCAG3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAGGCGGATGACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-019, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTATGGTAACTGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-020, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTCAAGCTTTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-021, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTGCGGTTCCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-022, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGAGAAGATGACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-023, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCGGTGCTTGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-024, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGGGTCGGTATCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-025, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAACATGATGACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-026, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTCGGGAGCCCGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-027, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTCAGCAAACTTCTGCTGTACGGCCAAGGCGT-3′
53
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
65
D
Appendix D Library Construction Oligonucleotide Sequences
Barcoded adaptor sequences
Barcoded adaptor sequence
Length (nt)
Barcode-T-028, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAGCTTACTACCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-029, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGAATCTAGGGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-030, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTAGCGAAGACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-031, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCTGGTGCGTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-032, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGGTTGGGTGCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-033, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTCGTTGGATACCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-034, 50 µM
5′-CGCCTTGGCCGTACAGCAG3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTTCGTTAAAGGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-035, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAAGCGTAGGACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-036, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTTCTCACATCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-037, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTCTGTTATACCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-038, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTCGTCTTAGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-039, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTTATCGTGAGTCTGCTGTACGGCCAAGGCGT-3′
53
66
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix D Library Construction Oligonucleotide Sequences
Barcoded adaptor sequences
Barcoded adaptor sequence
D
Length (nt)
Barcode-T-040, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAAAAGGGTTACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-041, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTTGTGGGATTGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-042, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGAATGTACTACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-043, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTCGCTAGGGTTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-044, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAAGGATGATCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-045, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTACTTGGCTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-046, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGGTCGTCGAACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-047, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGAGGGATGGCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-048, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCCGTAAGTGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-049, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTATGTCATAAGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-050, 50 µM
5′-CGCCTTGGCCGTACAGCAG3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGAAGGCTTGCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-051, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAAGCAGGAGTCTGCTGTACGGCCAAGGCGT-3′
53
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
67
D
Appendix D Library Construction Oligonucleotide Sequences
Barcoded adaptor sequences
Barcoded adaptor sequence
Length (nt)
Barcode-T-052, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTAATTGTAACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-053, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTCATCAAGTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-054, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAAAAGGCGGACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-055, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAGCTTAAGCGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-056, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCATGTCACCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-057, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTCTAGTAAGAACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-058, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTTAAAGTGGCGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-059, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAAGTAATGTCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-060, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTGCCTCGGTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-061, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAAGATTATCGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-062, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAGGTGAGGGTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-063, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCGGGTTCGACTGCTGTACGGCCAAGGCGT-3′
53
68
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix D Library Construction Oligonucleotide Sequences
Barcoded adaptor sequences
Barcoded adaptor sequence
D
Length (nt)
Barcode-T-064, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTGCTACACCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-065, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGGGATCAAGCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-066, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGATGTAATGTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-067, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTCCTTAGGGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-068, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCATTGACGACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-069, 50 µM
5′-CGCCTTGGCCGTACAGCAG3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGATATGCTTTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-070, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCCCTACAGACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-071, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTACAGGGAACGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-072, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAAGTGAATACCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-073, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCAATGACGTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-074, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAGGACGCTGACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-075, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTATCTGGGCCTGCTGTACGGCCAAGGCGT-3′
53
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
69
D
Appendix D Library Construction Oligonucleotide Sequences
Barcoded adaptor sequences
Barcoded adaptor sequence
Length (nt)
Barcode-T-076, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAAGTTTTAGGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-077, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTATCTGGTCTTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-078, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGGCAATCATCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-079, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAGTAGAATTACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-080, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTTTACGGTGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-081, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGAACGTCATTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-082, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTGAAGGGAGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-083, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGGATGGCGTACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-084, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCGGATGAACCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-085, 50 µM
5′-CGCCTTGGCCGTACAGCAG3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGGAAAGCGTTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-086, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAGTACCAGGACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-087, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTATAGCAAAGCCTGCTGTACGGCCAAGGCGT-3′
53
70
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix D Library Construction Oligonucleotide Sequences
Barcoded adaptor sequences
Barcoded adaptor sequence
D
Length (nt)
Barcode-T-088, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTTGATCATGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-089, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAGGCTGTCTACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-090, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTGACCTACTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-091, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGCGTATTGGGCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-092, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAAGGGATTACCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-093, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGTTACGATGCCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-094, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTATGGGTGTTTCTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-095, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTGAGTCCGGCACTGCTGTACGGCCAAGGCGT-3′
53
Barcode-T-096, 50 µM
5′-CGCCTTGGCCGTACAGCAG-3′
19
5′-CTGCCCCGGGTTCCTCATTCTCTAATCGAAGAGCTGCTGTACGGCCAAGGCGT-3′
53
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
71
D
72
Appendix D Library Construction Oligonucleotide Sequences
Barcoded adaptor sequences
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
APPENDIX E
Checklist and workflow tracking
form
E
This appendix covers:
■
Workflow checklists: prepare a standard or express fragment library with the
AB Library Builder™ System Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
■
Workflow tracking: prepare a standard or express fragment library with the
AB Library Builder™ System Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Workflow checklists: prepare a standard or express fragment
library with the AB Library Builder™ System Kit
Note: The checklist includes only equipment and reagents needed to prepare libraries
and excludes the usual and necessary standard laboratory equipment, such as pipets,
filtered pipet tips, tubes, vortexers, microcentrifuges, and nuclease-free water.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
73
E
Appendix E Checklist and workflow tracking form
Workflow checklists: prepare a standard or express fragment library with the AB Library Builder™ System Kit
Equipment
®
Covaris S220
System
† Covaris microTube
adaptor
† Covaris microTube
loading station
®
† Covaris microTube
Reagents
† 1× Low TE Buffer
† Shear Buffer
† Ethylene glycol
†
†
†
†
Protocol Card
Cartridge Rack
Tip and Tube Rack
AB Library Builder™
System
† Library Builder Fragment
Core Kit for 5500 SOLiD™
† AMPure™ XP Reagent Kit
† Library Builder™ Fragment
Core Kit for 5500 SOLiD™
™
† 5500 SOLiD Fragment
Library Standard Adaptors or
5500 SOLiD™ Fragment
Library Barcode Adaptors
†
†
Thermal cycler
PCR strip tubes
† AB Library Builder™
Amplification Reagents
†
Real-time PCR
system
† SOLiD Library TaqMan
Quantitation Kit
—
—
—
† E-Gel™ 2% SizeSelect™ gel
† 50-bp DNA Ladder
† Nuclease-free Water
† Thaw 50 bp DNA
Ladder on ice.
—
—
Quantitate
the library
Nicktranslate,
then
amplify the
library
Set up the AB Library
Builder™ System
Shear the DNA
†
(Optional)
Pool
equimolar
libraries of
similar size
—
74
—
iBase™ System
®
E-gel Safe
Imager™ instrument
™
®
emulsion
Pool
remaining
libraries to be
combined into
a single
(Optional) P
(Optional)
Gel-purify
the libraries
†
†
™
Preparation steps
† Degas the water in the
®
Covaris S220 System
30 minutes prior to use.
† Supplement the
circulated water chiller
with 20% ethylene
glycol.
† Thaw Shear Buffer at
room temperature.
† Thaw cartridges
completely.
† Add Agencourt
AMPure® XP Reagent to
cartridges.
† Add library adaptors to
Ligation Buffer Tubes.
† Load all tubes, tips, and
cartridges into the AB
Library Builder™ Device.
† Thaw Library PCR
Primers 1 and 2 on ice.
† Thaw Platinum™ PCR
Amplification Mix on
ice.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix E Checklist and workflow tracking form
Workflow tracking: prepare a standard or express fragment library with the AB Library Builder™ System Kit
E
Workflow tracking: prepare a standard or express fragment
library with the AB Library Builder™ System Kit
Sample:
Barcode:
Quantitation
Step
Quantity of DNA
Starting Amount
Quantitative PCR
Lot number
Step
Library Builder™ Fragment
Core Kit for 5500 SOLiD™
5500 SOLiD™ Fragment Library
Standard Adaptors or
5500 SOLiD™ Fragment Library
Barcode Adaptors
Sample:
Lot number
Barcode:
Quantitation
Step
Quantity of DNA
Starting Amount
Quantitative PCR
Lot number
Step
Library Builder™ Fragment
Core Kit for 5500 SOLiD™
5500 SOLiD™ Fragment Library
Standard Adaptors or
5500 SOLiD™ Fragment Library
Barcode Adaptors
Sample:
Lot number
Barcode:
Quantitation
Step
Starting Amount
Quantitative PCR
Quantity of DNA
Lot number
Step
Library Builder™ Fragment
Core Kit for 5500 SOLiD™
5500 SOLiD™ Fragment Library
Standard Adaptors or
5500 SOLiD™ Fragment Library
Barcode Adaptors
Lot number
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
75
E
76
Appendix E Checklist and workflow tracking form
Workflow tracking: prepare a standard or express fragment library with the AB Library Builder™ System Kit
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
APPENDIX F
Safety
F
This appendix covers:
■
General chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
■
SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
■
Chemical waste safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
■
Biological hazard safety. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Note: For instrument safety and biohazard guidelines, refer to the “Safety” section in
the AB Library Builder™ System User Guide (Part no. 4463421).
General chemical safety
WARNING! CHEMICAL HAZARD. Before handling any chemicals, refer to
the Safety Data Sheet (SDS) provided by the manufacturer, and observe all
relevant precautions.
WARNING! CHEMICAL HAZARD. All chemicals in the instrument, including
liquid in the lines, are potentially hazardous. Always determine what chemicals
have been used in the instrument before changing reagents or instrument
components. Wear appropriate eyewear, protective clothing, and gloves when
working on the instrument.
WARNING! CHEMICAL HAZARD. Four-liter reagent and waste bottles can
crack and leak. Each 4-liter bottle should be secured in a low-density
polyethylene safety container with the cover fastened and the handles locked in
the upright position. Wear appropriate eyewear, clothing, and gloves when
handling reagent and waste bottles.
WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in
a glass container because of the risk of breaking or shattering. Reagent and
waste bottles can crack and leak. Each waste bottle should be secured in a lowdensity polyethylene safety container with the cover fastened and the handles
locked in the upright position. Wear appropriate eyewear, clothing, and gloves
when handling reagent and waste bottles.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
77
F
Appendix F Safety
SDSs
Chemical safety
guidelines
To minimize the hazards of chemicals:
• Read and understand the Safety Data Sheets (SDSs) provided by the chemical
manufacturer before you store, handle, or work with any chemicals or hazardous
materials. (See “About SDSs” on page 78.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the SDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use
only with adequate ventilation (for example, fume hood). For additional safety
guidelines, consult the SDS.
• Check regularly for chemical leaks or spills. If a leak or spill occurs, follow the
manufacturer’s cleanup procedures as recommended in the SDS.
• Comply with all local, state/provincial, or national laws and regulations related to
chemical storage, handling, and disposal.
SDSs
About SDSs
Chemical manufacturers supply current Safety Data Sheets (SDSs) with shipments of
hazardous chemicals to new customers. They also provide SDSs with the first
shipment of a hazardous chemical to a customer after an SDS has been updated. SDSs
provide the safety information you need to store, handle, transport, and dispose of the
chemicals safely.
Each time you receive a new SDS packaged with a hazardous chemical, be sure to
replace the appropriate SDS in your files.
Obtaining
SDSs
The SDS for any chemical supplied by Applied Biosystems is available to you free 24
hours a day. To obtain SDSs:
1. Go to www.appliedbiosystems.com, click Support, then select SDS.
2. In the Keyword Search field, enter the chemical name, product name, SDS part
number, or other information that appears in the SDS of interest. Select the
language of your choice, then click Search.
3. Find the document of interest, right-click the document title, then select any of the
following:
• Open – To view the document
• Print Target – To print the document
• Save Target As – To download a PDF version of the document to a
destination that you choose
Note: For the SDSs of chemicals not distributed by Applied Biosystems, contact the
chemical manufacturer.
Chemical waste safety
Chemical waste
hazards
78
CAUTION! HAZARDOUS WASTE. Refer to Safety Data Sheets and local
regulations for handling and disposal.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Appendix F Safety
Chemical waste safety
F
WARNING! CHEMICAL WASTE HAZARD. Wastes produced by Applied
Biosystems instruments are potentially hazardous and can cause injury, illness,
or death.
WARNING! CHEMICAL STORAGE HAZARD. Never collect or store waste in
a glass container because of the risk of breaking or shattering. Reagent and
waste bottles can crack and leak. Each waste bottle should be secured in a lowdensity polyethylene safety container with the cover fastened and the handles
locked in the upright position. Wear appropriate eyewear, clothing, and gloves
when handling reagent and waste bottles.
Chemical waste
safety guidelines
WARNING! Do not add acids, or bases (such as bleach), to any wastes
containing lysis buffer (present in reagent cartridges or tubes). Acids and bases
can react with guanidine thiocyanate in the lysis buffer and generate toxic gas.
To minimize the hazards of chemical waste:
• Read and understand the Safety Data Sheets (SDSs) provided by the
manufacturers of the chemicals in the waste container before you store, handle, or
dispose of chemical waste.
• Provide primary and secondary waste containers. (A primary waste container
holds the immediate waste. A secondary container contains spills or leaks from
the primary container. Both containers must be compatible with the waste
material and meet federal, state, and local requirements for container storage.)
• Minimize contact with chemicals. Wear appropriate personal protective
equipment when handling chemicals (for example, safety glasses, gloves, or
protective clothing). For additional safety guidelines, consult the SDS.
• Minimize the inhalation of chemicals. Do not leave chemical containers open. Use
only with adequate ventilation (for example, fume hood). For additional safety
guidelines, consult the SDS.
• Handle chemical wastes in a fume hood.
• After emptying a waste container, seal it with the cap provided.
• Dispose of the contents of the waste tray and waste bottle in accordance with
good laboratory practices and local, state/provincial, or national environmental
and health regulations.
Waste disposal
If potentially hazardous waste is generated when you operate the instrument, you
must:
• Characterize (by analysis if necessary) the waste generated by the particular
applications, reagents, and substrates used in your laboratory.
• Ensure the health and safety of all personnel in your laboratory.
• Ensure that the instrument waste is stored, transferred, transported, and disposed
of according to all local, state/provincial, and/or national regulations.
IMPORTANT! Radioactive or biohazardous materials may require special
handling, and disposal limitations may apply.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
79
F
Appendix F Safety
Biological hazard safety
Biological hazard safety
General biohazard
80
WARNING! BIOHAZARD. Biological samples such as tissues, body fluids,
infectious agents, and blood of humans and other animals have the potential to
transmit infectious diseases. Follow all applicable local, state/provincial, and/or
national regulations. Wear appropriate protective equipment, which includes
but is not limited to: protective eyewear, face shield, clothing/lab coat, and
gloves. All work should be conducted in properly equipped facilities using the
appropriate safety equipment (for example, physical containment devices).
Individuals should be trained according to applicable regulatory and company/
institution requirements before working with potentially infectious materials.
Read and follow the applicable guidelines and/or regulatory requirements in
the following:
• U.S. Department of Health and Human Services guidelines published in
Biosafety in Microbiological and Biomedical Laboratories; http://www.cdc.gov/
biosafety/publications/index.htm).
• Occupational Safety and Health Standards, Bloodborne Pathogens (29
CFR§1910.1030; www.access.gpo.gov/ nara/cfr/waisidx_01/
29cfr1910a_01.html).
• Your company’s/institution’s Biosafety Program protocols for working with/
handling potentially infectious materials.
Additional information about biohazard guidelines is available at:
www.cdc.gov
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Documentation and Support
Related documentation
For related documents, refer to the 5500 Series SOLiD™ Systems User Documentation
Quick Reference (Part no. 4465102).
Obtaining support
For the latest services and support information for all locations, go to:
www.appliedbiosystems.com
At the Applied Biosystems website, you can:
• Access worldwide telephone and fax numbers to contact Applied Biosystems
Technical Support and Sales facilities.
• Search through frequently asked questions (FAQs).
• Submit a question directly to Technical Support.
• Order Applied Biosystems user documents, SDSs, certificates of analysis, and
other related documents.
• Download PDF documents.
• Obtain information about customer training.
• Download software updates and patches.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
81
Documentation and Support
Obtaining support
82
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Glossary
barcode
A short, unique sequence that is incorporated into a library that enables identification
of the library during multiplex sequencing.
Barcoded Adaptor
During fragment library preparation, the double-stranded oligonucleotide that is
ligated to the genomic DNA fragment such that the internal adaptor, barcode
sequence, and the P2 Adaptor are at the 3' end of the sequencing template.
barcoded library
A library that has a unique barcode sequence incorporated that enables identification
of the library during multiplex sequencing.
fragment library
A library that has a single insert prepared from genomic DNA for sequencing on the
SOLiD™ System. Fragment libraries compatible with the 5500 Series SOLiD™
Sequencers can be sequenced with a forward-only run or with a paired-end run.
internal adaptor (IA)
The internal adaptor sequence is incorporated into the template during library
construction and provides a common hybridization target for SOLiD™ sequencing
primers. The IA sequence is different in DNA-source libraries and RNA-source
libraries, therefore sequencing primers specific for RNA and DNA libraries must be
used for reverse reads (F5 tag). The IA-containing adaptors used during mate-paired
library preparation are different from the adaptors used for fragment library
preparation, but the sequencing primers used for forward reads originating in the IA
sequence (R3 and BC tags) are the same. See the 5500 Series SOLiD™ Systems Sequencing
Products Ordering Guide for a schematic of sequencing primers compatible with each
type of SOLiD™ library.
library
A set of DNA or cDNA molecules prepared from the same biological specimen and
prepared for sequencing on the SOLiD™ System.
Library PCR
Primer 1
Single-stranded oligonucleotide used in library amplification and corresponding to the
P1-T Adaptor sequence.
Library PCR
Primer 2
Single-stranded oligonucleotide used in library amplification and corresponding to the
P2 Adaptor sequence.
mate-paired library
Library consisting of two DNA segments that reside a known distance apart in the
genome, linked by an internal adaptor, and with P1 and P2 Adaptors ligated to the 5'
and 3' ends of the template strand, respectively.
multiplex
sequencing
Sequencing runs in which multiple barcoded libraries are simultaneously sequenced
in a single flowchip lane. Each bead is assigned to the correct library after the
sequencing run according to the sequence of its barcode.
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
83
Glossary
P1-T Adaptor
A T-tailed double-stranded oligonucleotide containing the P1 sequence that is ligated
to A-tailed DNA segments during library construction; the result is that the P1
sequence is attached to the 5' end of the template strand.
Standard Adaptor
During fragment library preparation, the double-stranded oligonucleotide that is
ligated to the genomic DNA fragment such that the internal adaptor, barcode sequence
BC-001, and the P2 Adaptor are at the 3' end of the sequencing template.
tag
There are two uses for this term.
• A length of DNA or cDNA to be sequenced; especially, a relatively short stretch of
DNA or cDNA that is used to infer information about the longer native molecule
from which it is derived, such as in SAGE™ analysis and mate-pair library
sequencing.
• Sequencing data from a single bead with a single primer set; sometimes used
interchangeably with read.
templated bead
preparation
84
Process of covalently attaching and clonally amplifying template strands to beads by
emulsion PCR, enriching the beads to remove beads without template, then modifying
the 3' end of the template on the beads to prepare for bead deposition and sequencing
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
Index
list of 38
troubleshooting 35
A
AB Library Builder System
running 27
AB Library Builder™ Kits
contents 9, 10
about the kit 9
B
barcoded fragment library preparation 9, 13, 27, 29,
35
biohazardous waste, handling 80
build the library 27
G
glossary 83
guidelines
chemical safety 78
chemical waste disposal 78
chemical waste safety 79
H
hazard warning, chemical 77
hazards. See safety
C
cartridge rack
insert in instrument 24
load cartridges 19
CAUTION, description 7
change protocol card 18
checklists and workflow tracking forms 73
chemical hazard warning 77
chemical safety 77, 78
chemical waste safety 78, 79
cooling period, not required 28
Covaris microTUBE holder, vials, system,
programs 47
D
DANGER, description 7
digital display
description 49
troubleshooting 35
DNA yield, troubleshooting 36
documentation, related 81
E
elution volume, troubleshooting 36, 37
error codes
I
IMPORTANT, description 7
insert protocol card 18
instrument
cancel run 50
display 49
error codes 38
pause run 50
start run 27
troubleshooting 35
K
kit contents 10
L
leakage, troubleshooting 36
M
MSDS. See SDS
multiple fragment libraries
(optional) amplify the libraries 31
quantitate the DNA 33
shear the DNA 14, 33
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
85
Index
troubleshooting 35
N
NanoDrop® ND-1000 Spectrophotometer 55
U
O
using the front panel 48
oligonucleotide sequences 63
ordering information 39
W
P
pause instrument run 50
power, troubleshooting 35
prepare to build the library 13
protocol card
accidental removal during run 18
insert or change 18
WARNING, description 7
waste disposal, guidelines 79
waste profiles, description 79
workflow tracking forms and checklists 73
Q
quantitating the library 33
R
rack. See cartridge rack, tip and tube rack
radioactive waste, handling 79
remove protocol card 18
required materials 39
S
safety 77
biological hazards 80
chemical 77
chemical waste 78
guidelines 78, 79
script, select 27
SDSs
about 8
description 78
obtaining 78, 81
start instrument run 27
starting the run 27
stop instrument run 50
supplemental procedures 47
quantitate the DNA with the NanoDrop® ND1000 Spectrophotometer 55
T
training, information on 81
86
Fragment Library Preparation Using the AB Library Builder™ System: 5500 Series SOLiD™ Systems User Guide
4460965A
Headquarters
5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone +1 760 603 7200 | Toll Free in USA 800 955 6288
For support visit www.appliedbiosystems.com/support
www.lifetechnologies.com