USER GUIDE Fragment Library Preparation Using the AB Library Builder™ System 5500 Series SOLiD™ Systems Publication Part Number 4460965 Rev.
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.
Contents About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Safety information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 CHAPTER 1 About the Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Library preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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 . . . . . . . . . . . . . . . . . . . . . . . . .
Contents Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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.
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.
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.
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) S
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.
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 D
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.
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.
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.
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”.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 dist
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.
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.
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.
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.
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.
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.
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.
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).
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.
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 . . . . . . .
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™ F
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.
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 Bio
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.
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.
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.
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.
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 sequencin
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.
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.
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.
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′-CGCCTTGG
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′-CGCCTTGGC
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′-CGCCTTGG
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′-CGCCTTGG
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′-CGCCTTGG
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′-CGCCTTGG
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′-CGCCTTGG
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′-CGCCTTGG
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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
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 Adapt
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. . . . . . . . . . . . . . . . . . . . . . . .
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.
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.
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.
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).
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.
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.
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.
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.
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.
