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CMS_093440 - Applied Biosystems

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1. 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTCATCAAGTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 054 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAAAAGGCGGACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 055 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAGCTTAAGCGCTGCTGTAC GGCCAAGGCGT 3 53 Barcode T 056 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGCATGTCACCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 057 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTCTAGTAAGAACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 058 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTTAAAGTGGCGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 059 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAAGTAATGTCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 060 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTGCCTCGGTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 061 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAAGATTATCGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 062 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAGGTGAGGGTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 063 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGCGGGTTCGACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 064 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTGCTACACCCTGCTGTACGGCCAAGGCGT 3 53 78 Fragment Library Preparati
2. fragment on a bead The sequencing direction for F3 reads of paired end libraries is 5 to 3 The sequencing direction for F5 reads of paired end libraries is 3 to 5 To support traditional 5 to 3 representation the complement of the reads are written For more information on sequencing tags refer to 5500 Series SOLiD Sequencers User Guide Part no 4456991 72 Fragment Library Preparation 5500 Series SOLID Systems User Guide APPENDIX D Oligonucleotide Sequences Library construction oligonucleotides PCR Primer and Note The internal adaptor used for DNA fragment libraries is different from the ada ptor sequences 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 uM 5 CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGA T 3 41 5 TCACCGACTGC CCATAGAGAGGAAAGCGGAGGCGTAGTGG C C 3 42 Standard Adaptor 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTGTAAGAGGCTGCTGTACGGCCAAGGCGT 3 53 Library PCR Primer 1 50 uM 5 CCACTACGCCTCCGCTTTCCTCTCTATG 3 28 Library PCR Primer 2 50 uM 5 CTGCCCCGGGTTCCTCATTCT 3 21 Barcoded adaptor sequences Barcoded adaptor sequence Barcode T 001 50 uM Length nt Fragment Library Preparation 5500 Series SOLiD Systems User Guide
3. Chemical waste safety Chemical waste CAUTION HAZARDOUS WASTE Refer to Safety Data Sheets and local hazards regulations for handling and disposal WARNING CHEMICAL WASTE HAZARD Wastes produced by Applied Biosystems instruments are potentially hazardous and can cause injury illness or death 88 Fragment Library Preparation 5500 Series SOLID Systems User Guide Appendix F Safety Chemical waste safety 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 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 Chemical waste To minimize the hazards of chemical waste safety guidelines 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 appro
4. 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 Figure 1 Fragment library preparation workflow overview EC EA a ci gt w Cs Ee Qn E fs Da Library molecule ligated Genomic DNA Sheared DNA with P1 T and barcoded adaptors The barcoded adaptor consists of 3 segments of sequence 1 Internal adaptor sequence which is necessary for sequencing the barcode 2 Barcode sequence 3 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 on page 70 Fragment Library Preparation 5500 Series SOLID Systems User Guide 69 au Appendix C Overview Preparing fragment libraries Figure 2 Fragment library design P1 T Adaptor ds 41 42 bp P1 T Adaptor P1 T Adaptor vy Phosphorothioate bond 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 on page 71 These primers can be used only for library amplification and not for alternative or modified library construction adaptor design
5. STOPPING POINT Store the purified DNA in Low TE Buffer at 4 C or proceed directly to Quantitate the size selected DNA Quantitate the size selected DNA Measure the DNA concentration of each library using e 2 uL of sample with the Qubit dsDNA HS Assay Kit Invitrogen Part no Q32851 and the Qubit 2 0 Fluorometer Invitrogen Part no Q32866 or e 2 uL of sample in the NanoDrop ND 1000 Spectrophotometer see Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer on page 62 or e 1 uL of sample in the Agilent Technologies 2100 Bioanalyzer IMPORTANT The average yield of size selected DNA is 30 of input quantity If the yield is substantially lt 20 troubleshoot the low yield then repeat the procedure from Shear the DNA on page 33 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 39 Chapter 3 Prepare Multiple Fragment Libraries Add a dA tail to the size selected DNA Add a dA tail to the size selected DNA STOPPING POINT Store the purified DNA in Low TE Buffer at 4 C or proceed directly to Add a dA tail to the size selected DNA A thermostable polymerase adds non templated dA to the 3 ends of the DNA The thermostable polymerase lacks 3 5 exonuclease activity at higher temperatures 1 5 In a new 1 5 mL LoBind Tube combine to prepare the master mix to add an A tail to each library Amount per Master mix for N Compo
6. 5 CGCCTTGGCCGTACAGCAG 3 19 CTGCCCCGGGTTCCTCATTCTCTGTGTAAGAGGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 002 50 uM 5 CGCCTTGGCCGTACAGCAG3 19 5 CTGCCCCGGGTTCCTCATTCTCTAGGGAGTGGTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 003 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTATAGGTTATACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 004 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGGATGCGGTCCTGCTGTACGGCCAAGGCGT 3 53 73 Appendix D Oligonucleotide Sequences Library construction oligonucleotides Barcoded adaptor sequence Length nt Barcode T 005 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTGGTGTAAGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 006 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGCGAGGGACACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 007 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGGGTTATGCCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 008 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGAGCGAGGATCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 009 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAGGTTGCGACCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 010 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCT GCGGTAAGCTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 011 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTGCGACACGCTGCTGTACGGCCAAGGCGT 3
7. Prepare 70 ethanol Component Volume Nuclease Free Water 300 pL Ethanol Absolute 700 uL Total 1000 pL 3 Transfer the PCR reaction from step 4 in Amplify the library to anew 1 5 mL LoBind Tube 4 Bind the DNA to the Agencourt AMPure XP Reagent a Prepare the bead suspension in the sample reaction Component Volume Amplified library 125 uL Agencourt AMPure XP Reagent 187 5 uLt t 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 the 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 remove and discard the supernatant 5 Wash the DNA 3 times For each wash a Add 200 uL of freshly prepared 70 ethanol to the tube mix by inverting the tube a few times then pulse spin b Place the tube in the DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant 6 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 uL pipettor 7 Open the tube then dry the beads at room temperature 20 25 C for 5 10 minutes 8 Elute the DNA a Remove the tube from the DynaMag 2 magnetic rack then add 30 uL Low TE Buffer directly to the p
8. 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 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 59 Appendix A Ordering Information Optional consumables 8 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 Vendor Agilent DNA 1000 Kit Agilent Technologies 5067 1504 t Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance 60 Fragment Library Preparation 5500 Series SOLID Systems User Guide APPENDIX B Supplemental Procedures This appendix covers Load and unload Covaris microTUBE vials from the Covaris microTUBE Load and unload Covaris microTUBE vials from the Covaris microTUBE holder Load Covaris 1 Use a thumb to push the stainless steel plunger up into the body of the microTUBE vials 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
9. 10 Load 2 uL of DNA sample onto the lower measurement pedestal and lower the sampling arm Fragment Library Preparation 5500 Series SOLiD Systems User Guide 65 66 Appendix B Supplemental Procedures Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer Fragment Library Preparation 5500 Series SOLID Systems User Guide APPENDIX C Overview This appendix covers Choosing the appropriate library type 67 Preparing fragment libraries 69 Sequence orientation from source DNA to sequence map 72 Choosing the appropriate library type TM These are the types of libraries that can be sequenced on the 5500 Series SOLiD Sequencers Library type Features Applications Go to Fragment Appropriate for sequence lengths lt 300 bp Adaptors on each end of sheared DNA insert Multiplexed sequencing The protocol is designed for 10 ng 5 ug of genomic DNA or ligated PCR product Compared to mate paired libraries fragment libraries yield a higher recovery of unique molecules when normalized to the same input amount Targeted resequencing primary library Genomic resequencing Methylation analysis Chapter 2 Prepare a Single Fragment Library on page 13 or Chapter 3 Prepare Multiple Fragment Libraries on page 31 Fragment Library Preparation 550
10. 2 0 Fluorometer Invitrogen Part no Q32866 or e 2 uL of sample in the NanoDrop ND 1000 Spectrophotometer see Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer on page 62 or e 1 uL of sample in the Agilent Technologies 2100 Bioanalyzer and or e The appropriate volume in qPCR refer to the Applied Biosystems SOLiD Library TagMan Quantitation Kit protocol Invitrogen Part no A12120 STOPPING POINT Store the purified DNA in its current buffer at 4 C or proceed directly to Optional Amplify the libraries Optional Amplify the libraries 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 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 45 Chapter 3 Prepare Multiple Fragment Libraries Optional Amplify the libraries Amplify the libraries 46 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 1 Transfer 20 0 uL of each purified library from Purify the DNA on page 42
11. Library PCR Primer 2 50 uM t 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 t Components 5500 SOLID Fragment 48 Library Core Kit 4464415 5500 SOLID 48 Fragment Library Enzyme Module 5500 SOLID 48 Fragment Library Amplification Module Fragment Library Preparation 5500 Series SOLiD Systems User Guide 55 Appendix A Ordering Information Optional Applied Biosystems Reagent Kits Item part no Components 5500 SOLiD 48 Fragment Library Enzyme e 10mM dNTP Module e End Polishing E1 eer e End Polishing E2 e 5X Reaction Buffer e A tailing Enzyme e T4 DNA Ligase 5 U uL e 10mM dATP e Shear Buffer 5500 SOLID 48 Fragment Library Platinum PCR Amplification Module Amplification Mix 4464417 t Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance Optional Applied Biosystems Reagent Kits Item part no Components SOLID Library Micro Column e Binding Buffer B2 L Purification Kit e Binding Buffer B2 S 4443751 e Wash
12. 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 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 5500 Series SOLiD Systems User Guide 93 Glossary P1 T Adaptor Standard Adaptor tag templated bead preparation 94 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 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 There are two uses for this term e Sequencing data from a single bead with a single primer set sometimes used interchangeably with read e 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 mate paired library sequencing and SAGE analysis respe
13. 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 Amplify the library Purify the DNA amplification and production of redundant molecules 1 Ina0 2 mL PCR tube prepare the PCR mixture Component Volume Adaptor ligated purified DNA 20 uLt Platinum PCR Amplification Mix 100 uL Library PCR Primer 1 50 uM 2 5 uL Library PCR Primer 2 50 uM 2 5 uL Total 125 uL t lt 20 uL is acceptable Do not adjust the PCR volume 2 Vortex the reaction for 5 seconds then pulse spin 3 Determine the number of PCR cycles Starting amount of DNA nos of 10 100 ng 10 cycles 100 ng 1 ug 6 to 8 cycles eut 4 to 6 cycles CL 0 to 3 cycles 4 Runthe PCR Stage Step Temp Time 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 co 1 Resuspend the Agencourt AMPure XP Reagent beads and allow the mixture to come to room temperature Fragment Library Preparation 5500 Series SOLiD Systems User Guide 27 Chapter 2 Prepare a Single Fragment Library Optional Amplify the library
14. Thermo Scientific ND 1000 Pipettors 20 uL Major Laboratory Supplier MLS t Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance j 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 R equired consumables Itemt Source Nuclease free Water 1 L Applied Biosystems AM9932 CF 1 Calibration Fluid Kitt Thermo Scientific CF 1 PR Conditioning Kit Thermo Scientific PR 1 Filtered pipettor tips Major Laboratory Supplier MLS t 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 Fragment Library Preparation 5500 Series SOLID Systems User Guide Appendix B Supplemental Procedures Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer Procedure 1 Ensure that the NanoDrop ND 1000 Spectroph
15. before adaptor ligation using the Covaris System The conditions have been tested for shearing 10 ng 5 ug DNA in a total volume of 120 uL For certain DNA samples optimizing the shearing protocol may be necessary You can shear the DNA with two supported shearing systems e The Covaris 5220 System see Shear the DNA with the Covaris 220 System or e The Covaris 2 System see Shear the DNA with the Covaris 2 System on page 35 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 33 Chapter 3 Prepare Multiple Fragment Libraries Shear the DNA Shear the DNA with IMPORTANT Ensure that the bath temperature during shearing is 5 10 C Higher the Covaris 220 shearing temperatures can be harmful to DNA System 1 Dilute the components below ina 1 5 mL LoBind Tube Shear Buffer reduces DNA damage from sonication Component Amount DNA 10 ng 5 ug 1X Low TE Buffer Variable uL Shear Buffer 1 2 uL Total 120 uL 2 Prepare the Covaris 220 Tank a Ensure that the water in the Covaris 220 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 b Set the chiller temperature to 2 5 C to ensure that the temperature reading in the water bath displays 5 C The circulated water chiller should be supplemented with 20 ethylene glycol 3 Load the DNA a Place
16. glycol 3 Load the DNA a Place a Covaris microTUBE into the loading station b With the snap cap on the tube use a tapered pipette tip to slowly transfer the 120 uL of DNA sample through the pre split septa IMPORTANT Do not 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 61 4 Shear the DNA using the following Covaris 5220 System conditions IMPORTANT Ensure that the bath temperature limit is set at 15 C and keep the bath temperature to lt 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 With the snap cap on insert a pipette tip through the pre split septa then slowly remove the sheared DNA c Transfer the sheared DNA into a new 1 5 mL LoBind tube 1 Prepare the Covaris 2 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 b Set the chiller temperat
17. 20 25 C for a Remove each tube from the DynaMag 2 magnetic rack then add 30 uL Low TE Buffer directly to the pellet to disperse the beads b Pipette the suspension up and down to mix Fragment Library Preparation 5500 Series SOLiD Systems User Guide 47 Chapter 3 Prepare Multiple Fragment Libraries Quantitate the DNA c Vortex the beads for 10 seconds then pulse spin d Place the tube in a DynaMag 2 magnetic rack for at least 1 minute until the solution clears e Transfer the supernatant containing the size selected DNA to a new 1 5 mL LoBind Tube Quantitate the DNA Measure the DNA concentration by using 2 uL of sample with the Qubit dsDNA HS Assay Kit Invitrogen Part no Q32851 and the Qubit 2 0 Fluorometer Invitrogen Part no Q32866 or 2 uL of sample in the NanoDrop ND 1000 Spectrophotometer see Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer on page 62 or 1 uL of sample in the Agilent Technologies 2100 Bioanalyzer If you used the bioanalyzer see Check the size distribution of the libraries 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 Low TE Buffer 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 Gettin
18. 53 Barcode T 012 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAAGAGGAAAACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 013 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGCGGTAAGGCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 014 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTGCGGCAGACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 015 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGAGTTGAATGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 016 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGGGAGACGTTCTGCTGTACGGCCAAGGCGT 3 53 74 Fragment Library Preparation 5500 Series SOLID Systems User Guide Appendix D Oligonucleotide Sequences Library construction oligonucleotides Barcoded adaptor sequence Length nt Barcode T 017 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGGCTCACCGCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 018 50 uM F CGCCTTGGCCGTACAGCAG3 19 5 CTGCCCCGGGTTCCTCATTCTCTAGGCGGATGACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 019 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTATGGTAACTGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 020 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTCAAGCTTTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 021 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTGCGGTTCCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 022 50 uM 5 CGCCTTG
19. Applied Biosystems Reagent Kits 56 Required equipment o 57 Optional equipment ooo 58 Required consumables faune dete se etd A eae es eo eee Aid 58 Optional consumables 2 2 60 Supplemental Procedures 61 Load and unload Covaris microTUBE vials from the Covaris microTUBE holder 61 Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer 62 OVERVIEW see sonner re ic ca 67 Choosing the appropriate library type 67 Preparing fragment libraries 2 69 Sequence orientation from source DNA to sequence map 72 Oligonucleotide Sequences 73 Library construction oligonucleotides 73 Checklist and workflow tracking form 83 Workflow checklists prepare a fragment library 83 Workflow tracking prepare a fragment library 85 Fragment Library Preparation 5500 Series SOLiD Systems User Guide Contents APPENDIX F Safety erario id a anaes 87 General chemical safety 2222 87 SDSS esos wide He ee ee de Se ee 88 Chemical waste safety ovaciones 88 Biological h
20. 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 outlicensingfalifetech 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 NanoDrop is a registered trademark of NanoDrop Technologies TaqMan is a registered trademark of Roche Molecular Systems Inc Copyright 2011 Life Technologies
21. a Covaris microTUBE into the loading station b With the snap cap on the tube use a tapered pipette tip to slowly transfer the 120 uL of DNA sample through the pre split septa IMPORTANT Do not 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 61 34 Fragment Library Preparation 5500 Series SOLID Systems User Guide Shear the DNA with the Covaris S2 System Fragment Library Preparation 5500 Series SOLiD Systems User Guide Chapter 3 Prepare Multiple Fragment Libraries Shear the DNA 4 Shear the DNA using the following Covaris 5220 System conditions IMPORTANT Ensure that the bath temperature limit is set at 15 C and keep the bath temperature to lt 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 With the snap cap on insert a pipette tip through the pre split septa then slowly remove the sheared DNA c Transfer the sheared DNA into a new 1 5 mL LoBind tube 1 Prepare the Covaris 2 Tank a Ensure
22. 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 microTUBE loading and unloading 1 depress plunger thumb 2 to load grip glass and insert tube not cap 3 to unload press on side of tube not cap IMPORTANT Do not press against the cap to load or unload microTUBE vials because pressing against the cap may dislodge or damage the cap Fragment Library Preparation 5500 Series SOLID Systems User Guide 61 Appendix B Supplemental Procedures Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer Unload Covaris microTUBE vials Quantitate the DNA with the NanoDrop ND 1000 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 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 Spectrophotometer Materials and equipment required 62 The Thermo Scientific NanoDrop 1000 Spectrophotometer measures nucleic acid samples from 2 ng uL 3700 ng uL without dilution Required equipment Itemt Source NanoDrop ND 1000 Spectrophotometer computer required
23. b Pipette the suspension up and down to mix c Vortex the beads for 10 seconds then pulse spin d Place the tube in the magnetic rack for at least 1 minute until the solution clears e Transfer the supernatant containing the size selected DNA to a new 1 5 mL LoBind Tube STOPPING POINT Store the purified DNA in Low TE Buffer at 4 C 8 Proceed as follows If you want to Then Further remove residual adaptors the Repeat Purify the DNA using Agencourt DNA AMPure XP Reagent on page 23 Use 30 pL of Agencourt AMPure XP Reagent with 20 uL of bead purified sample equal to 1 5X of Agencourt AMPure XP Reagent per sample volume see figure below Quantitate the DNA without additional Proceed to Quantitate the ligated DNA purification on page 26 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 Prepare a Single Fragment Library Ligate adaptors to the DNA A second purification of the ligated DNA with the Agencourt AMPure XP Reagent substantially removes unligated adaptors L 2 o One round e Two rounds FU 10 Purify the DNA with the SOLiD Library Micro Column Purification Kit 1 Pre spin an empty PureLink Micro column in a collection tube at 10 000 x g for 1 minute before use 2 If not already prepared prepare the Binding and Wash Buffers a Add sufficient 100 isopropanol to Binding Buffer B2 L to prepare Binding Buffer B2 L with 4
24. eed ae oe ad Ana ire ner 31 Procedural guidelines a E 33 Quantitatethe DNA ooo a da dean a 33 Shearithe DNA a cessere lei de rad te nt athe e diab Banos pai td siete 33 End polish the DNA a doe ee eb es al dd 36 Size select the DNA by Agencourt AMPure XP Reagent 37 Quantitate the size selected DNA 39 Add a dA tail to the size selected DNA 40 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 3 Contents CHAPTER 4 APPENDIX A APPENDIX B APPENDIX C APPENDIX D APPENDIX E Ligate adaptors tothe DNA 40 Quantitate the ligated DNA 2 45 Optional Amplify the libraries 45 Quantitatethe DNA A a acest adieu 48 Check the size distribution of the libraries 48 Optional Pool equal molar barcoded libraries 49 Troubleshooting td ane Dacha Ginn Rennes den ee 49 TFOUDLESNOOLING O 51 Ordering Information 53 Required Applied Biosystems reagent kits for library preparation 53 Required Applied Biosystems reagent kits for automated liquid handling systems 0 0 cece eee rrara nnan ranana 55 Optional
25. protocol using this specific material Substitution may adversely affect system performance Required consumables Itemt Source 1X Low TE Buffer Applied Biosystems 4389764 Nuclease free Water 1 L Applied Biosystems AM9932 58 Fragment Library Preparation 5500 Series SOLID Systems User Guide AppendixA Ordering Information Required consumables Itemt Source MicroAmp Optical 8 Tube Strip 0 2 mL Applied Biosystems 4316567 Invitrogen Qubit dsDNA HS Assay Kit or Invitrogen Qubit dsDNA BR Assay Kit or Invitrogen Quant iT PicoGreen dsDNA Assay Kit Invitrogen Q32851 or Q32854 Invitrogen Q32850 or Q32853 Invitrogen P7589 Agencourt AMPure XP 5 mL Kit Beckman Coulter or Genomics A63880 or Agencourt AMPure XP 60 mL Kit A63881 Covaris microTUBEs Covaris 520045 2 Propanol Sigma Aldrich 19516 Ethylene glycol American Bioanalytical AB00455 01000 0 5 mL LoBind Tubes Eppendorf 022431005 1 5 mL LoBind Tubes Eppendorf 022431021 CF 1 Calibration Fluid Kit Thermo Scientific CF 1 PR 1 Conditioning Kitt Thermo Scientific PR 1 Ethanol absolute Sigma Aldrich E7023 Filtered pipettor tips Major Laboratory Supplier MLS 8 t Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance The NanoDrop Conditioning Kit is useful for
26. take replicate measurements of the different quantitation methods according to method same sample for an average e Use more than one quantitation method Note The NanoDrop ND 1000 Spectrophotometer measures UV absorption of ssDNA dsDNA and free nucleotides Free nucleotides and ssDNA have higher extinction coefficients than dsDNA The Qubit 2 0 Fluorometer measures the fluorescence from probes bound to dsDNA The NanoDrop ND 1000 Spectrophotometer tends to overestimate DNA and the Qubit 2 0 Fluorometer tends to underestimate DNA The Agilent Technologies 2100 Bioanalyzer measures sample peak area against to a reference peak which could be subject to some error Fragment Library Preparation 5500 Series SOLiD Systems User Guide 51 Chapter 4 Troubleshooting Observation Possible cause Recommended action Library yields lower than expected DNA loss during purification Purification with the Agencourt AMPure XP Reagent e Ensure that the Agencourt AMPure XP Reagent beads are fresh and used before the expiration date e Prepare fresh 70 ethanol e Elute the DNA with a solution of low ionic strength such as Low TE Buffer Purification with the SOLID Library Micro Column Purification Kit e Pre spin the columns e Add 2 propanol to buffers B2 S and B2 L and ethanol to Wash Buffer W1 as instructed Adaptor ligation suboptimal If the DNA yield after end re
27. 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 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 100 uL in 1X Low TE Buffer in a LoBind tube Component Amount DNA 10 ng to 5 ug 1X Low TE Buffer Variable uL Shear Buffer 1 2 uL Total 120 uL 3 Load the DNA into the Covaris S2 System a Place a Covaris microTUBE into the loading station 35 Chapter 3 Prepare Multiple Fragment Libraries End polish the DNA b Keeping the snap cap on the tube use a tapered pipette tip to slowly transfer the 100 uL of DNA sample through the pre split septa Be careful not to introduce a bubble into the bottom of the tube 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 61 4 Shear the DNA using the following Covaris 2 System conditions IMPORTANT Ensure that the bath temperature limit is set at 15 C and keep the bath temperature to lt 10 C Condition Setting Number of cycles 6 Bath temperature 5 C Bath temperature limit 15 C
28. the last purification step If DNA fragments were sheared using the standard protocol for fragment library preparation the average insert size should be approximately 165 bp before adaptor ligation as shown in the calculation and example below ug to pmol _10pg 1tpmol 1 conversion factor 1 ug 660 pg Average insert size ug to pmol 1 uL adaptor needed Y pL adaptor needed ugDNA conversion factor we 50 pmol Example For 1 ug of purified end repaired DNA with an average insert size of 165 bp and 30 yield after size selection 0 3 ug of size selected DNA ug to pmol 10 pg 1 pmol 1 _ conversion factor Tug 660 pg A 65 T 9 2 pmol ug DNA 1 HL adaptor needed Y uL adaptor needed 0 3pg DNA x 9 2 pmol ug DNA x 10 x 50 pmol 0 55 uL adaptor needed Ligate adaptors to IMPORTANT Do not use P1 and P2 Adaptors that are designed for fragment library the DNA preparation and sequencing on the SOLiD 4 System These adaptors are not compatible with reverse read sequencing on the 5500 Series SOLiD Sequencers Use only P1 T and Barcode T 0XX Adaptors that are designed for the 5500 Series SOLiD Sequencers 1 Ina new 1 5 mL LoBind Tube combine for a ligation master mix Component Volume 5X Reaction Buffer 3 0 uL P1 T Adaptor 50 uM Yul Barcode T 0XX 50 uMt Yul T4 DNA Ligase 5 U uL 6 5 uL 10 mM dNTP 1 2 uL Nuclease free Water Variable uL Total 15 uL t If 5500 SOLID Fragmen
29. 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 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 88 IMPORTANT For the SDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer 8 Fragment Library Preparation 5500 Series SOLID Systems User Guide CHAPTER 1 About the Products IMPORTANT If you have purchased the AB Library Builder System and you want to prepare a fragment library with an automated system refer to the Fragment Library Preparation Using the AB Library Builder System 5500 Series SOLiD Systems User Guide Part no 4460965 For a more detailed overview of library types and the librar
30. 0 isopropanol b Add sufficient 100 ethanol to the Wash Buffer W1 to prepare Wash Buffer W1 with 80 ethanol 3 To1 volume 65 uL of ligation reaction add 4 volumes 260 uL of Binding Buffer B2 L with isopropanol 40 Mix well 4 Load the DNA onto the PureLink Micro column a Apply all of the sample from step 3 to the PureLink Micro column in a collection tube b Spin the column at 10 000 x g for 1 minute at room temperature then discard the flow through dsDNA is bound to the column c Ensure that the entire sample has been loaded onto the column 5 Wash the column a Return the PureLink Micro column to the same collection tube b Add 650 uL of Wash Buffer W1 with ethanol to wash the column c Spin the column at 10 000 x g for 1 minute at room temperature then discard the flow through Fragment Library Preparation 5500 Series SOLiD Systems User Guide 25 Chapter 2 Prepare a Single Fragment Library Quantitate the ligated DNA d Spin the column at 14 000 x g for 1 minute at room temperature to remove residual wash buffer and dry the silica membrane then discard the flow through and collection tube 6 Elute the DNA a Transfer the column to a clean Elution Tube b Add 22 uL of Elution Buffer E1 to the center of the column to elute the DNA then let the column stand for 1 minute at room temperature c Spin the column at 14 000 x g for 1 minute at room temperature 7 Optional To
31. 0 Series SOLiD Systems User Guide 67 68 Appendix C Overview Choosing the appropriate library type Library type Features Applications Go to Mate paired Two DNA insert tags 600 bp 6 kb apart Separated by an internal adaptor More input DNA required 1 5 pg Paired reads enable unique mapping in regions not accessible to single read sequencing Information on tag orientation and apparent distance between tags Increase mapping specificity over standard fragment library sequencing Detect large structural variations in the genome Bridge sequencing gaps De novo sequencing primary library Genomic resequencing primary library Methylation analysis Mate Paired Library Preparation 5500 Series SOLID Systems User Guide Part no 4460958 The type 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 and shorter read lengths are adequate For information about specific applications go to the 5500 Series SOLiD Sequencers website www appliedbiosystems com solid5500 Or contact your field applications specialist Fragment Library Preparation 5500 Series SOLID Systems User Guide Appendix C Overview Preparing fragment libraries
32. 4 50 uM each 5500 SOLID Fragment Library Barcode Adaptors 65 80 50 uM each 5500 SOLID Fragment Library Barcode Adaptors 81 96 50 uM each 5500 SOLID Fragment Library Barcode Adaptors 1 16 4464405 Barcode adaptors T 001 T 016 P1 T Adaptor 50 uM Library PCR Primer 1 50 uM Library PCR Primer 2 50 uM 5500 SOLiD Fragment Library Barcode Adaptors 17 32 4464406 Barcode adaptors T 017 T 032 P1 T Adaptor 50 uM Library PCR Primer 1 50 uM Library PCR Primer 2 50 uM Fragment Library Preparation 5500 Series SOLID Systems User Guide AppendixA Ordering Information Required Applied Biosystems reagent kits for automated liquid handling systems Item part no t Components 5500 SOLID Fragment Library Barcode Adaptors 33 48 4464407 Barcode adaptors T 033 T 048 P1 T Adaptor 50 uM Library PCR Primer 1 50 uM e Library PCR Primer 2 50 uM 5500 SOLID Fragment Library Barcode e Barcode adaptors Adaptors 49 64 T 049 T 064 4464408 P1 T Adaptor 50 uM Library PCR Primer 1 50 uM e Library PCR Primer 2 50 uM 5500 SOLID Fragment Library Barcode e Barcode adaptors Adaptors 65 80 T 065 T 080 4464409 P1 T Adaptor 50 uM Library PCR Primer 1 50 uM e Library PCR Primer 2 50 uM 5500 SOLiD Fragment Library Barcode e Barcode adaptors Adaptors 81 96 T 081 T 096 4464410 P1 T Adaptor 50 uM Library PCR Primer 1 50 uM
33. 8 description 88 obtaining 88 91 single fragment library optional Amplify the library 26 end polish the DNA 18 quantitate the library 29 Quantitate the sheared DNA 21 26 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 95 Index supplemental procedures 61 quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer 62 T training information on 91 W WARNING description 7 waste disposal guidelines 89 waste profiles description 89 96 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 0960977 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 technologies www lifetechnologies com
34. AG3 5 CTGCCCCGGGTTCCTCATTCTCTGGAAAGCGTTCTGCTGTACGGCCAAGGCGT 3 19 53 19 53 Barcode T 086 50 uM 5 CGCCTTGGCCGTACAGCAG 3 5 CTGCCCCGGGTTCCTCATTCTCTAGTACCAGGACTGCTGTACGGCCAAGGCGT 3 19 53 Barcode T 087 50 uM 5 CGCCTTGGCCGTACAGCAG 3 5 CTGCCCCGGGTTCCTCATTCTCTATAGCAAAGCCTGCTGTACGGCCAAGGCGT 3 19 53 Barcode T 088 50 uM 5 CGCCTTGGCCGTACAGCAG 3 5 CTGCCCCGGGTTCCTCATTCTCTGTTGATCATGCTGCTGTACGGCCAAGGCGT 3 80 Fragment Library Preparation 5500 Series SOLID Systems User Guide 19 53 Appendix D Oligonucleotide Sequences Library construction oligonucleotides Barcoded adaptor sequence Length nt Barcode T 089 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAGGCTGTCTACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 090 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTGACCTACTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 091 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGCGTAT TGGGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 092 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAAGGGATTACCTGCTGTAC GGCCAAGGCGT 3 53 Barcode T 093 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTTACGATGCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 094 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTATGGGTGTTTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 095 50 uM 5 CGCC
35. Buffer e Elution Buffer e Micro Spin Columns e Elution Tubes t Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance 56 Fragment Library Preparation 5500 Series SOLID Systems User Guide Required equipment AppendixA Ordering Information Required equipment Item Source Covaris 220 Systemt 110 V for U S customers 220 V for international customers The Covaris 220 System includes e Covaris 220 sonicator e Universal Voltage Kit e Latitude laptop from Dell Inc e MultiTemp III Thermostatic Circulator e Covaris 2 series Machine Holder for one 1 5 mL microcentrifuge tube e Covaris 2 series Machine Holder for one 0 65 mL microcentrifuge tube e Covaris 2 series Machine Holder for one 13 mm x 65 mm tube e Covaris 2 Series Machine Holder for one microTUBE e Covaris microTUBE Prep Station e Covaris Water Tank Label Kit e Covaris microTUBEs 1 pack of 25 Applied Biosystems 4465653 Covaris S2 System8 110 V for U S customers 220 V for international customers Note Fragment libraries can be prepared with the Covaris S2 System New users should purchase the Covaris S220 System Microcentrifuge 5417R refrigerated without rotor e Eppendorftt 022621807 120 V 60 Hz e Eppendorft 022621840 230 V 50 Hz FA 45 24 11 fixed angle rotor Epp
36. CGGCCAAGGCGT 3 53 Barcode T 043 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTCGCTAGGGTTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 044 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAAGGATGATCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 045 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTACTTGGCTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 046 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGGTCGTCGAACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 047 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGAGGGATGGCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 048 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGCCGTAAGTGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 049 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTATGTCATAAGCTGCTGTAC GGCCAAGGCGT 3 53 Barcode T 050 50 uM 5 CGCCTTGGCCGTACAGCAG3 19 5 CTGCCCCGGGTTCCTCATTCTCTGAAGGCTTGCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 051 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAAGCAGGAGTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 052 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTAATTGTAACTGCTGTACGGCCAAGGCGT 3 53 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 77 Appendix D Oligonucleotide Sequences Library construction oligonucleotides Barcoded adaptor sequence Length nt Barcode T 053 50 uM
37. CTCATTCTCTGTATCTGGGCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 076 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAAGTTTTAGGCTGCTGTACGGCCAAGGCGT 3 53 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 79 Appendix D Oligonucleotide Sequences Library construction oligonucleotides Barcoded adaptor sequence Length nt Barcode T 077 50 uM 5 CGCCTTGGCCGTACAGCAG 3 5 CTGCCCCGGGTTCCTCATTCTCTATCTGGTCTTCTGCTGTACGGCCAAGGCGT 3 19 53 Barcode T 078 50 uM 5 CGCCTTGGCCGTACAGCAG 3 5 CTGCCCCGGGTTCCTCATTCTCTGGCAATCATCCTGCTGTACGGCCAAGGCGT 3 19 53 Barcode T 079 50 uM 5 CGCCTTGGCCGTACAGCAG 3 5 CTGCCCCGGGTTCCTCATTCTCTAGTAGAATTACTGCTGTACGGCCAAGGCGT 3 19 53 Barcode T 080 50 uM 5 CGCCTTGGCCGTACAGCAG 3 5 CTGCCCCGGGTTCCTCATTCTCTGTTTACGGTGCTGCTGTACGGCCAAGGCGT 3 19 53 Barcode T 081 50 uM 5 CGCCTTGGCCGTACAGCAG 3 5 CTGCCCCGGGTTCCTCATTCTCTGAACGTCATTCTGCTGTACGGCCAAGGCGT 3 19 53 Barcode T 082 50 uM 5 CGCCTTGGCCGTACAGCAG 3 5 CTGCCCCGGGTTCCTCATTCTCTGTGAAGGGAGCTGCTGTACGGCCAAGGCGT 3 19 53 Barcode T 083 50 uM 5 CGCCTTGGCCGTACAGCAG 3 5 CTGCCCCGGGTTCCTCATTCTCTGGATGGCGTACTGCTGTACGGCCAAGGCGT 3 19 53 Barcode T 084 50 uM 5 CGCCTTGGCCGTACAGCAG 3 5 CTGCCCCGGGTTCCTCATTCTCTGCGGATGAACCTGCTGTACGGCCAAGGCGT 3 Barcode T 085 50 uM 5 CGCCTTGGCCGTACAGC
38. Corporation All rights reserved Part Number 4460960 Rev A 03 2011 Contents About This Guide lt oowoioronisita a eee eee 7 Safety information rota RA Bead ahs ed gles nt aan ta 7 CHAPTER 1 About the Products sense cir a 9 Library preparate os das atlas Ade eee 9 Productintormation ea dee ice ideada tdt do 9 Kit contents and storage conditions 11 CHAPTER 2 Prepare a Single Fragment Library 13 WOPKIIOW LE ts a A NE 13 Procedural quidelin s cita aa er did 14 Quantitatethe DNA cuco A a 15 Shear MODNA A E A AAA a 15 End polishthe DNA e te EA o ae ae e Ne 18 Size select the DNA by Agencourt AMPure XP Reagent 18 Quantitate the size selected DNA 21 Add a dA tail to the size selected DNA 21 Ligate adaptors to the DNA 22 Quantitate the ligated DNA 22 26 Optional Amplify the library 2 0 00 0c ccc ccc rr 26 Quantitatethe DNA cuca vest tee eek ied denim ed eee 29 Check the size distribution of the library 2 29 Troubleshooting 2 0 c v eeeer eh eee dn ree tr Hares de beats eI NA a 29 CHAPTER 3 Prepare Multiple Fragment Libraries 31 Work OW nevea erinra Soave dda Peed ads Pine
39. Ethanol Absolute 700 pL x N Total 1000 pL x N For every amplified library label a new 1 5 ml LoBind Tube Transfer each PCR reaction 125 uL from Optional Amplify the libraries on page 45 to the appropriately labeled 1 5 mL LoBind Tube Bind the DNA to the resuspended ambient Agencourt AMPure XP Reagent a For each library prepare the bead suspension Component Volume Amplified library 125 uL Agencourt AMPure XP Reagent 187 5 pL t 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 1 minute until the solution is clear of brown tint when viewed at an angle then remove and discard the supernatant Wash the DNA 3 times For each wash a Add 200 uL of freshly prepared 70 ethanol to each tube mix by inverting the tube a few times then pulse spin b Place each tube in the DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant Remove the tube from the DynaMag 2 magnetic rack pulse spin the tube return the tube to the DynaMag 2 magnetic rack then remove and discard the supernatant with a 20 uL pipettor 5 10 minutes Elute the DNA Open each tube then dry the beads at room temperature
40. GCCCCGGGTTCCTCATTCTCTGGTTGGGTGCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 033 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTCGTTGGATACCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 034 50 uM 5 CGCCTTGGCCGTACAGCAG3 19 5 CTGCCCCGGGTTCCTCATTCTCTTCGTTAAAGGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 035 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAAGCGTAGGACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 036 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTTCTCACATCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 037 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTCTGTTATACCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 038 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTCGTCTTAGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 039 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTTATCGTGAGT CTGCTGTACGGCCAAGGCGT 3 53 Barcode T 040 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAAAAGGGTTACTGCTGTACGGCCAAGGCGT 3 53 76 Fragment Library Preparation 5500 Series SOLID Systems User Guide Appendix D Oligonucleotide Sequences Library construction oligonucleotides Barcoded adaptor sequence Length nt Barcode T 041 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTTGTGGGATTGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 042 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGAATGTACTACTGCTGTA
41. GCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGAGAAGATGACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 023 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGCGGTGCTTGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 024 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGGGTCGGTATCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 025 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAACATGATGACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 026 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTCGGGAGCCCGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 027 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTCAGCAAACTTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 028 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAGCTTACTACCTGCTGTACGGCCAAGGCGT 3 53 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 75 Appendix D Oligonucleotide Sequences Library construction oligonucleotides Barcoded adaptor sequence Length nt Barcode T 029 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGAATCTAGGGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 030 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTAGCGAAGACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 031 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGCTGGTGCGTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 032 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CT
42. 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 uL of the sheared DNA into a new 1 5 mL sample tube provided in the Library Builder Fragment Core Kit for SOLiD 4 0 End polish the DNA 36 End Polishing E1 and E2 enzymes convert DNA with incompatible 5 protruding and or 3 protruding ends to blunt ended 5 phosphorylated blunt ended DNA The end polishing process converts DNA with overhangs to blunt ended DNA by exploiting the 5 to 3 polymerase and the 3 to 5 exonuclease activities of the enzymes used in the procedure below A kinase phosphorylates the 5 ends of the DNA Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 3 Prepare Multiple Fragment Libraries Size select the DNA by Agencourt AMPure XP Reagent 1 Ina new 1 5 mL LoBind Tube combine to prepare the end polishing master mix Component Amount per Master mix for N library libraries 5X Reaction Buffer 40 uL 40 uL x 1 1 x N 10 mM dNTP 8 0 uL 8 0 uL x 1 1 x N End Polishing E1 8 0 uL 8 0 uL x 1 1 x N End Polishing E2 10 uL 10 pL x 1 1 x N Nuclease Free Water 14 uL 14 pL x 1 1 x
43. N Total 80 pL 80 pL x 1 1 x N 2 Label 1 5 mL LoBind Tubes one tube for each library 3 Transfer 120 uL of each sheared DNA to the appropriately labeled tube 4 Pipette 80 uL of the master mix from step 1 into each labeled tube While pipetting the master mix take care not to cross contaminate libraries 5 Vortex each reaction for 5 seconds then pulse spin 6 Incubate each reaction at room temperature 20 25 C for 30 minutes Size select the DNA by Agencourt AMPure XP Reagent Use Agencourt AMPure XP Reagent purchased separately to size select the library with magnetic beads The first incubation with the AMPure XP beads selectively captures DNA gt 250 bp on the beads and DNA 250 bp is retained in the supernatant The second incubation with the retained supernatant and new beads selectively captures DNA gt 100 bp in the beads Therefore the retained beads contain DNA between 100 250 bp Bead based size selection provides on average higher yields over gel based size selection The first size selection with Agencourt AMPure XP reagent removes the longest DNA in the pellet The second size selection with Agencourt AMPure XP reagent removes the shortest DNA in the supernatant Follow the protocol for each library to size select lt 12 libraries Follow the manufacturer s instructions for handling the beads 1 Resuspend the Agencourt AMPure XP Reagent beads and allow the mixture to come to room te
44. TTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGAGTCCGGCACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 096 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAATCGAAGAGCTGCTGTACGGCCAAGGCGT 3 53 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 81 82 Appendix D Oligonucleotide Sequences Library construction oligonucleotides Fragment Library Preparation 5500 Series SOLID Systems User Guide APPENDIX E Checklist and workflow tracking form This appendix covers Workflow checklists prepare a fragment library 83 Workflow tracking prepare a fragment library 85 Workflow checklists prepare a fragment library Note The checklist includes only equipment and reagents needed to prepare libraries and excludes the usual and necessary standard laboratory equipment such as pipettes filtered pipette tips tubes vortexers microcentrifuges and nuclease free water Fragment Library Preparation 5500 Series SOLiD Systems User Guide 83 84 Appendix E Checklist and workflow tracking form Workflow checklists prepare a fragment library Equipment Reagents Preparation steps O Qubit 2 0 Fluorometer O Quant iT kit aa as O Covaris S220 System O 1x Low TE Buffer O Degas the water in the lt O Covaris microTube O Optional 100x Shear Buffer Covaris S2 or S220 z adaptor O Ethylene gl
45. USER GUIDE applied biosystems by Life technologies Fragment Library Preparation 5500 Series SOLiD Systems Publication Part Number 4460960 Rev A gt prepare libraries prepare beads run sequencer analyze data o technologies 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 M Sequencers is hereby granted limited non exclusive rights to use this user guide solely for the purpose of operating the 5500 Series SOLiD Sequencers Unauthorized copying renting modifying or creating derivatives of this user guide 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
46. aMag 2 magnetic rack for at least 1 minute until the solution clears The solution is clear of brown tint when viewed at an angle as shown below 4 Without disturbing the pellet carefully transfer the supernatant which contains the DNA of the desired size to a new 1 5 mL LoBind Tube Discard the pellet Fragment Library Preparation 5500 Series SOLID Systems User Guide 19 Chapter 2 Prepare a Single Fragment Library Size select the DNA by Agencourt AMPure XP Reagent 5 Bind the size selected DNA in the supernatant to the Agencourt AMPure XP Reagent a Combine in a 1 5 mL LoBind Tube Component Volume Supernatant 300 uL Agencourt AMPure XP Reagent 60 uL Total 360 uL t Equal to 0 3 volume of the end polish reaction volume of 200 pL 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 the tube in a DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant Save the pellet which contains the DNA 6 Wash the DNA bead complex 3 times For each wash a Add 200 uL of freshly prepared 70 ethanol to the tube mix by inverting the tube a few times then pulse spin b Place the tube in a DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant 7 Remove the tube from the DynaMag 2 magneti
47. azard safety 90 Documentation and Support 91 Related documentation 2220 91 Obtaining Support 522582 eS ede eee vied oe tweed ele eine Pee nm pu 91 A 93 Mir ie bee eee eee 95 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 5 Contents 6 Fragment Library Preparation 5500 Series SOLID Systems User Guide About This Guide Safety information Note For important instrument safety information refer to the 5500 Series SOLiD Sequencers User Guide Part no 4456991 For general safety information see this section and Safety on page 87 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 A CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used
48. beads selectively captures DNA gt 250 bp on the beads and DNA 250 bp is retained in the supernatant The second incubation with the retained supernatant and new beads selectively captures DNA gt 100 bp in the beads Therefore the retained beads contain DNA between 100 250 bp Bead based size selection provides on average higher yields over gel based size selection The first size selection with Agencourt AMPure XP reagent removes the longest DNA in the pellet The second size selection with Agencourt AMPure XP reagent removes the shortest DNA in the supernatant 1 Resuspend the Agencourt AMPure XP Reagent beads and allow the mixture to come to room temperature Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 Prepare a Single Fragment Library Size select the DNA by Agencourt AMPure XP Reagent 2 Prepare 70 ethanol Component Volume Nuclease Free Water 300 uL Ethanol Absolute 700 uL Total 1000 uL 3 Size select the DNA with Agencourt AMPure XP Reagent a Combine sheared DNA and resuspended ambient Agencourt AMPure XP Reagent in a 1 5 mL LoBind Tube Component Volume End polished DNA 200 uL Agencourt AMPure XP Reagent 100 pLt Total 300 pL t Equal to 0 5 volume of end polished DNA 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 the tube in a Dyn
49. because they do not have 3 sequences necessary for the sequencing chemistry 70 Fragment Library Preparation 5500 Series SOLID Systems User Guide Appendix C Overview Preparing fragment libraries Figure 3 Fragment library amplification design Barcoded Adaptor 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 5500 Series SOLiD Systems User Guide 71 Appendix C Overview Sequence orientation from source DNA to sequence map Sequence orientation from source DNA to sequence map Mate pair Sheared size selected DNA DIE y Circularization with Internal Adaptor and Nick Translation Nick translation goes beyond the length of the read Sequencing of the F3 fragment on a bead PRE a Pr Reads are generated from F3 and R3 primers R3 maps upstream from F3 Note that R3 maps upstream of F3 in the source DNA fragment e The reads are expected to map at a distance equal to the average fragment length plus the average nick translation distance e The sequencing direction for mate pair libraries is 5 to 3 Paired end ae ae Sheared size selected DNA v Sequencing of the Be a5 Bm
50. 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 e 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 e Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html e 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 90 Fragment Library Preparation 5500 Series SOLID Systems User Guide Documentation and Support Related documentation For related documents refer to the 5500 Series SOLiD Systems User D
51. bsolute O Qubit 2 0 Fluorometer or O Quant iT dsDNA HS Assay Kit 2 O NanoDrop ND 1000 or A Spectrophotometer or O Applied Biosystems SOLID Eg Library TaqMan Quantitation ga O Agilent Technologies Kit g 2100 Bioanalyzer or ej Real time thermal cycler Fragment Library Preparation 5500 Series SOLID Systems User Guide Appendix E Checklist and workflow tracking form Workflow tracking prepare a fragment library Workflow tracking prepare a fragment library Sample Barcode Quantitation Lot number Step Quantity of DNA Step Lot number Starting Amount P1 Adaptor End Repair Library PCR Primer 1 Quantitative assay Library PCR Primer 2 Standard Adaptor Barcode 0XX Sample Barcode Quantitation Lot number Step Quantity of DNA Step Lot number Starting Amount P1 Adaptor End Repair Library PCR Primer 1 Quantitative assay Library PCR Primer 2 Standard Adaptor Barcode 0XX Sample Barcode Quantitation Lot number Step Quantity of DNA Step Lot number Starting Amount P1 Adaptor End Repair Library PCR Primer 1 Quantitative assay Library PCR Primer 2 Standard Adaptor Barcode 0XX Sample Barcode Quantitation Lot number Step Quantity of DNA Step Lot number Starting Amount P1 Adaptor End Repair Library PCR Primer 1 Quantitative as
52. c rack pulse spin the tube return the tube to the magnetic rack then remove and discard the supernatant with a 20 uL pipettor 8 Open the tube then dry the beads at room temperature 20 25 C for 5 10 minutes 9 Elute the DNA a Remove the tube from the DynaMag 2 magnetic rack then add 36 uL Low TE Buffer directly to the pellet to disperse the beads b Pipette the suspension up and down to mix c Vortex the beads for 10 seconds then pulse spin d Place the tube in a magnetic rack for at least 1 minute until the solution clears e Transfer the supernatant containing the size selected DNA to a new 1 5 mL LoBind Tube STOPPING POINT Store the purified DNA in Low TE Buffer at 4 C or proceed directly to Quantitate the size selected DNA on page 21 20 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 Prepare a Single Fragment Library Quantitate the size selected DNA Quantitate the size selected DNA Measure the DNA concentration using e 2 uL of sample with the Qubit dsDNA HS Assay Kit Invitrogen Part no Q32851 and the Qubit 2 0 Fluorometer Invitrogen Part no Q32866 or e 2 uL of sample in the NanoDrop ND 1000 Spectrophotometer see Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer on page 62 or e 1uL of sample in the Agilent Technologies 2100 Bioanalyzer IMPORTANT The average yield of size selected DNA is 30 of input quantity I
53. ce 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 uL of the sheared DNA into a new 1 5 mL sample tube provided in the Library Builder Fragment Core Kit for SOLiD 4 0 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 17 Chapter 2 Prepare a Single Fragment Library End polish the DNA End polish the DNA End Polishing E1 and E2 enzymes convert DNA with incompatible 5 protruding and or 3 protruding ends to blunt ended 5 phosphorylated DNA The end polishing process converts DNA with overhangs to blunt ended DNA by exploiting the 5 to 3 polymerase and the 3 to 5 exonuclease activities of the enzymes used in the procedure below A kinase phosphorylates the 5 ends of the DNA 1 Combine in a new 1 5 mL LoBind Tube Component Amount Sheared DNA 120 uL 5X Reaction Buffer 40 uL 10 mM dNTP 8 0 uL End Polishing E1 8 0 uL End Polishing E2 10 uL Nuclease Free Water 14 uL Total 200 uL 2 Vortex the reaction for 5 seconds then pulse spin 3 Incubate the mixture at room temperature 20 25 C for 30 minutes Size select the DNA by Agencourt AMPure XP Reagent 18 Use Agencourt AMPure XP Reagent purchased separately to size select the library with magnetic beads The first incubation with the AMPure XP
54. ction in a thermocycler with the lid heater on IMPORTANT Incubation nick translates the DNA Stage Temp Time Holding 20 C 30 min Holding 72 C 20 min Holding 4 C co Purify the DNA If you want to Then Purify the ligated DNA quickly with high Proceed to Purify the DNA using yield Agencourt AMPure XP Reagent Use 39 uL of Agencourt AMPure XP Reagent with 65 uL of ligated DNA equal to 0 6X of Agencourt AMPure XP Reagent per sample volume Purify the ligated DNA with a column for Proceed to Purify the DNA with the convenience SOLiD Library Micro Column Purification Kit on page 44 Purify the DNA using Agencourt AMPure XP Reagent 1 Resuspend the Agencourt AMPure XP Reagent beads and allow the mixture to come to room temperature 2 Prepare 70 ethanol Component Volume Nuclease Free Water 300 uL Ethanol Absolute 700 uL Total 1000 pL 3 Bind the DNA to the resuspended ambient Agencourt AMPure XP Reagent a Prepare the bead suspension in the sample reaction Component Volume Ligation reaction 65 uL AMPure XP Reagent 39 uLt t Equal to 0 6 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 42 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 3 Prepare Multiple Fragment Libraries Liga
55. ctively 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 5500 Series SOLID Systems User Guide barcoded fragment library preparation 9 13 31 51 biohazardous waste handling 90 C CAUTION description 7 checklists and workflow tracking forms 83 chemical hazard warning 87 chemical safety 87 chemical waste safety 88 89 D DANGER description 7 documentation related 91 G glossary 93 guidelines chemical safety 87 chemical waste disposal 88 chemical waste safety 89 H hazard warning chemical 87 hazards See safety l IMPORTANT description 7 L library preparation 13 31 M MSDS See SDS multiple fragment libraries 31 optional amplify the libraries 45 Index end polish the DNA 36 ligate adaptors to the DNA 40 quantitate the DNA 48 quantitate the ligated DNA 45 quantitate the sheared DNA 39 shear the DNA 15 33 troubleshooting 51 N NanoDrop ND 1000 Spectrophotometer 62 0 oligonucleotide sequences 73 P prepare a single fragment library 13 product information 9 product purpose of 9 R radioactive waste handling 89 required materials 53 S safety 87 biological hazards 90 chemical 87 chemical waste 88 guidelines 87 88 89 SDSs about
56. d 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 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 To minimize the hazards of chemicals e 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 88 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 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 87 Appendix F Safety SDSs e 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 e Check regularly for chemical leaks or spills If a leak or spill occurs f
57. des for each library For 3 libraries use 4 barcode adaptors for each library for a total of 12 barcodes If 24 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 Fragment Library Preparation 5500 Series SOLID Systems User Guide Calculate the amount of adaptor to use for ligation Ligate adaptors to the DNA Chapter 3 Prepare Multiple Fragment Libraries Ligate adaptors to the DNA If the input DNA before shearing is e lt 100 ng Use 0 06 uL of each adaptor or an equivalent amount of adaptor after dilution For example use 0 6 uL of a 10 fold dilution of an adaptor or 1 2 uL of a 20 fold dilution e 2100 ng Calculate the amount of adaptor needed Y for the reaction based on the amount of DNA from the last purification step If DNA fragments were sheared using the standard protocol for fragment library preparation the average insert size should be approximately 165 bp before adaptor ligation as shown in the calculation and example below ug to pmol _10pg tpmol 1 conversion factor 1 ug 660 pg Average insert size ug to pmol 1 uL adaptor needed Y uL adaptor needed pgDNA x conversion factor 10x 50 pmol Example For 1 ug of purified end repaired DNA with an average insert size of 165 bp and 30 yield after
58. duct e If you are trying to construct a targeted resequencing library with small sized PCR products lt 500 bp then you must first perform a PCR product ligation step to concatenate the DNA For a concatenation protocol contact your field application specialist e Use good laboratory practices change gloves frequently to minimize cross contamination of products e Adjust microcentrifuge speeds and times according to the g forces specified in the protocols Applied Biosystems recommends the Eppendorf 5417R tabletop microcentrifuge e Perform all steps requiring 0 5 mL and 1 5 mL tubes with Eppendorf LoBind Tubes e Thaw Shear Buffer at room temperature just before use e Thaw all other reagents on ice just before use DNA For accuracy determine sample DNA concentration using a double stranded DNA specific fluorescence assay Use the HS Assay Kit to measure dsDNA concentrations from 10 pg uL to 100 ng uL For samples outside this range use the dsDNA BR for higher concentrations of DNA or PicoGreen dsDNA Assay Kit for lower concentrations e Invitrogen Qubit dsDNA HS Assay Kit Invitrogen Part no Q32851 or Q32854 or e Invitrogen Qubit dsDNA BR Assay Kit Invitrogen Part no Q32850 or Q32853 or e Invitrogen Quant iT PicoGreen dsDNA Assay Kit Invitrogen Part no P7589 This step involves sonicating the input DNA into small fragments with a mean fragment size of 160 bp and a fragment size range of 100 250 bp
59. ellet to disperse the beads b Pipette the suspension up and down to mix c Vortex the beads for 10 seconds then pulse spin d Place the tube in a magnetic rack for at least 1 minute until the solution clears e Transfer the supernatant containing the size selected DNA to a new 1 5 mL LoBind Tube 28 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 Prepare a Single Fragment Library Quantitate the DNA Quantitate the DNA Measure the DNA concentration by using e 2 uL of sample with the Qubit dsDNA HS Assay Kit Invitrogen Part no Q32851 and the Qubit 2 0 Fluorometer Invitrogen Part no Q32866 or 2 uL of sample in the NanoDrop ND 1000 Spectrophotometer see Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer on page 62 or 1 uL of sample in the Agilent Technologies 2100 Bioanalyzer If you used the bioanalyzer see Check the size distribution of the library and or TM 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 Low TE Buffer at 4 C for short term storage or at 20 C for long term storage Check the size distribution of the library Use 1 uL 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 Em
60. endorft 24 x 1 5 2 mL including aluminum lid 022636006 aerosol tight 96 well GeneAmp PCR System 9700 thermal cycler e Applied Biosystems N8050200 Base e Applied Biosystems 4314443 Block Fragment Library Preparation 5500 Series SOLiD Systems User Guide 57 Appendix A Ordering Information Optional equipment Item Source DynaMag 2 Magnet magnetic rack Invitrogen 123 21D NanoDrop ND 1000 Spectrophotometer Thermo Scientific computer required ND 1000 Qubit 2 0 Fluorometer Invitrogen Q32866 Vortexer Major Laboratory Supplier MLS PicoFuge Microcentrifuge MLS Pipettors 2 uL MLS Pipettors 20 uL MLS Pipettors 200 uL MLS Pipettors 1000 uL MLS t Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance Or the Covaris S2 System Or the Covaris 220 System tt Or equivalent but validation of the equipment for library preparation is required tt 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 equipment Itemt Source 2100 Bioanalyzer Agilent Technologies G2938C Qubit Quantitation Starter Kit Invitrogen Q32860 t Applied Biosystems has validated this
61. ent Library Preparation 5500 Series SOLiD Systems User Guide Kit contents and storage conditions Chapter 1 About the Products Kit contents and storage conditions Kit contents The 5500 SOLiD Fragment Library Core Kit Part no 4464412 contains materials sufficient to prepare 12 fragment libraries Part part no Description Storage temperature 5500 SOLID Fragment Library One each 20 C Enzyme Module 4464413 5500 SOLID Fragment Library One each 20 C Amplification Module 44644 14 The adaptor kits contain materials sufficient to prepare 12 fragment libraries when using 5 ug of input DNA Part Description Storage temperature 5500 SOLiD Fragment Library One each 20 C Standard Adaptors 4464411 5500 SOLiD Fragment Library One each 20 C Barcode Adaptors 4464404 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 11 Chapter 1 About the Products Kit contents and storage conditions 12 Fragment Library Preparation 5500 Series SOLID Systems User Guide Workflow CHAPTER 2 Prepare a Single Fragment Library IMPORTANT Customers who have purchased the AB Library Builder System and who wish to prepare a fragment library with an automated system refer to the Fragment Library Preparation Using the AB Library Builder System 5500 Series SOLiD Systems User Guide Part no 4460965 For an overview of library types that can be
62. f the yield is substantially lt 20 troubleshoot the low yield then repeat the procedure from Shear the DNA on page 15 STOPPING POINT Store the purified DNA in Low TE Buffer at 4 C or proceed directly to Add a dA tail to the size selected DNA Add a dA tail to the size selected DNA A thermostable polymerase adds non templated dA to the 3 ends of the DNA The thermostable polymerase lacks 3 5 exonuclease activity at higher temperatures 1 Combine in a 1 5 mL LoBind Tube Component Amount Size selected DNA 34 uL 5X Reaction Buffer 10 uL 10 mM dATP 1 0 uL A Tailing Enzyme 5 0 uL Total 50 uL 2 Incubate the mixture at 68 C for 30 minutes then cool to room temperature Note While the reaction is incubating calculate the amount of adaptors needed for ligation see Calculate the amount of adaptor to use for ligation on page 22 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 21 Chapter 2 Prepare a Single Fragment Library Ligate adaptors to the DNA Ligate adaptors to the DNA Calculate the If the input DNA before shearing is amount of adaptor e lt 100 ng Use 0 06 uL of each adaptor or an equivalent amount of adaptor after to use for ligation dilution For example use 0 6 uL of a 10 fold dilution of an adaptor or 1 2 uL of a 20 fold dilution e 2100 ng Calculate the amount of adaptor needed Y for the reaction based on the amount of DNA from
63. g Started Guide Part no 4441486 Check the size distribution of the libraries Use 1 uL 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 STOPPING POINT 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 on page 49 48 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 3 Prepare Multiple Fragment Libraries Optional Pool equal molar barcoded libraries Optional Pool equal molar barcoded libraries 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 lt 4 libraries are being barcoded Use the barcodes according to these conditions e If lt 4 libraries are prepared for sequencing
64. gent on page 42 Use 30 uL of Agencourt AMPure XP Reagent with 20 uL of bead purified sample equal to 1 5X of Agencourt AMPure XP Reagent per sample volume see figure below Quantitate the DNA without additional purification Proceed to Quantitate the ligated DNA on page 45 Fragment Library Preparation 5500 Series SOLID Systems User Guide 43 Chapter 3 Prepare Multiple Fragment Libraries Ligate adaptors to the DNA A second purification of the ligated DNA with the Agencourt AMPure XP Reagent substantially removes unligated adaptors One round hd Two rounds Fu Purify the DNA with the SOLID Library Micro Column Purification Kit 1 Pre spin an empty PureLink Micro column in a collection tube at 10 000 x g for 1 minute before use 2 If not already prepared prepare the Binding and Wash Buffers a Add sufficient 100 isopropanol to Binding Buffer B2 L to prepare Binding Buffer B2 L with 40 isopropanol b Add sufficient 100 ethanol to the Wash Buffer W1 to prepare Wash Buffer W1 with 80 ethanol 3 To1 volume 65 uL of ligation reaction add 4 volumes 260 uL of Binding Buffer B2 L with isopropanol 40 Mix well 4 Load the DNA onto the PureLink Micro column a Apply all of the sample from step 3 to the PureLink Micro column in a collection tube b Spin the column at 10 000 x g for 1 minute at room temperature then discard the flow through dsDNA is bound t
65. ind the DNA to the resuspended ambient Agencourt AMPure XP Reagent a Prepare the bead suspension in the sample reaction Component Volume Ligation reaction 65 pL AMPure XP Reagent 39 uLt t Equal to 0 6 volumes of sample reaction Fragment Library Preparation 5500 Series SOLiD Systems User Guide 23 Chapter 2 Prepare a Single Fragment Library Ligate adaptors to the DNA 24 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 the 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 carefully remove and discard the supernatant Wash the DNA 3 times For each wash a Add 200 uL of freshly prepared 70 ethanol to the tube mix by inverting the tube a few times then pulse spin b Place the tube in the DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant 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 uL pipettor Open the tube then dry the beads at room temperature 20 25 C for 5 10 minutes Elute the DNA a Remove the tube from the DynaMag 2 magnetic rack then add 22 uL Low TE Buffer directly to the pellet to disperse the beads
66. mperature 2 Prepare 70 ethanol for N number of libraries Component Volume Nuclease Free Water 300 pL x N Ethanol Absolute 700 uL x N Total 1000 yL x N Fragment Library Preparation 5500 Series SOLID Systems User Guide 37 Chapter 3 Prepare Multiple Fragment Libraries Size select the DNA by Agencourt AMPure XP Reagent 3 Size select the DNA with resuspended ambient Agencourt AMPure XP Reagent a Combine sheared DNA and Agencourt AMPure XP Reagent Component Volume End polished DNA 200 uL Agencourt AMPure XP Reagent 100 Lt Total 300 uL t Equal to 0 5 volume of end polished DNA 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 the tube in a DynaMag 2 Magnetic Rack for at least 1 minute until the solution clears The solution is clear of brown tint when viewed at an angle as shown below 4 Without disturbing the pellet carefully transfer the supernatant of each library which contains the DNA of the desired size to anew 1 5 mL LoBind Tube labelled with the library name Discard the pellet 5 Bind the size selected DNA in the supernatant to the Agencourt AMPure XP Reagent a Combine Component Volume Supernatant 300 uL Agencourt AMPure XP Reagent 60 uL Total 360 uL t Equal to 0 3 volume of the end polish reaction volume of 200 uL b Vortex the bead
67. nent a library libraries 5X Reaction Buffer 10 uL 10 pL x 1 1 x N 10 mM dATP 1 0 uL 1 0 pL x 1 1 x N A Tailing Enzyme 5 0 uL 5 0 pL x 1 1 x N Total 16 pL 16 pL x 1 1 x N Label 1 5 mL LoBind Tubes one tube for each library Transfer 34 uL of each size selected DNA to the appropriately labeled tube Pipette 16 uL of the master mix from step 1 into each labeled tube While pipetting the master mix take care not to cross contaminate libraries Incubate each reaction at 68 C for 30 minutes then cool to room temperature Note While the reaction is incubating calculate the amount of adaptors needed for ligation see Calculate the amount of adaptor to use for ligation on page 41 Ligate adaptors to the DNA 40 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 lt 4 libraries are being barcoded Use the barcodes according to these conditions e If lt 4 libraries are prepared for sequencing then use multiple barcodes per library in equal ratios For example for 2 libraries use 2 barco
68. o the column c Ensure that the entire sample has been loaded onto the column 5 Wash the column a Return the PureLink Micro column to the same collection tube b Add 650 uL of Wash Buffer W1 with ethanol to wash the column c Spin the column at 10 000 x g for 1 minute at room temperature then discard the flow through 44 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 3 Prepare Multiple Fragment Libraries Quantitate the ligated DNA d Spin the column at 14 000 x g for 1 minute at room temperature to remove residual wash buffer and dry the silica membrane then discard the flow through and collection tube 6 Elute the DNA a Transfer the column to a clean Elution Tube b Add 22 uL of Elution Buffer E1 to the center of the column to elute the DNA then let the column stand for 1 minute at room temperature c Spin the column at 14 000 x g for 1 minute at room temperature 7 Optional To potentially improve recovery of DNA a Add the eluate from the last spin back to the column then let the column stand for 1 minute b Spin the column s at 14 000 x g for 1 minute at room temperature STOPPING POINT Store the purified DNA in Elution Buffer E1 at 4 C or proceed directly to Quantitate the ligated DNA Quantitate the ligated DNA Measure the DNA concentration by using e 2 uL of sample with the Qubit dsDNA HS Assay Kit Invitrogen Part no Q32851 and the Qubit
69. ocumentation 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 e Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Order Applied Biosystems user documents SDSs certificates of analysis and other related documents e Download PDF documents e Obtain information about customer training e Download software updates and patches Fragment Library Preparation 5500 Series SOLiD Systems User Guide 91 Documentation and Support Obtaining support 92 Fragment Library Preparation 5500 Series SOLID Systems User Guide barcode Barcoded Adaptor barcoded library fragment library internal adaptor IA library Library PCR Primer 1 Library PCR Primer 2 mate paired library multiplex sequencing Glossary A short unique sequence that is incorporated into a library that enables identification of the library during multiplex sequencing 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 A library that has a unique barc
70. ode sequence incorporated that enables identification of the library during multiplex sequencing 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 The internal adaptor sequence is incorporated into the template during library construction and provides a common hybridization target for SOLiD sequencing primers See the 5500 Series SOLiD Systems Sequencing Products Ordering Guide for a schematic of sequencing primers compatible with each type of SOLiD library e 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 E5 tag e The IA containing adaptors used during mate paired library preparation are different from the adaptors used for fragment library preparation but the SOLiD FWD2 Seq Primers are used for all forward reads originating in the IA sequence generating the R3 and BC tags A set of DNA or cDNA molecules prepared from the same biological specimen and prepared for sequencing on the SOLiD System Single stranded oligonucleotide used in library amplification and corresponding to the P1 T Adaptor sequence Single stranded oligonucleotide used in library amplification and corresponding to the P2 Adaptor sequence
71. ollow the manufacturer s cleanup procedures as recommended in the SDS e 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 The SDS for any chemical supplied by Applied Biosystems is available to you free 24 SDSs hours a day To obtain SDSs 1 Goto 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 e Open To view the document e Print Target To print the document e 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
72. om EVO instruments can use the SOLID 48 library core kits For more information contact your local representative To prepare 6 12 fragment libraries it takes 5 6 h without amplification and 6 8 h with amplification Quantitate the DNA page 33 y Shear the DNA page 33 y End polish the DNA page 36 4 Size select the DNA by Agencourt AMPure XP Reagent page 37 Stopping point Fragment Library Preparation 5500 Series SOLID Systems User Guide 31 Chapter 3 Prepare Multiple Fragment Libraries Workflow 32 Quantitate the size selected DNA page 39 Stopping point Add a dA tail to the size selected DNA page 40 Ligate adaptors to the DNA Calculate the amount of adaptor to use for ligation page 41 Ligate adaptors to the DNA page 41 Purify the DNA page 42 Stopping point Quantitate the ligated DNA page 45 Stopping point Optional Amplify the libraries Amplify the libraries page 46 Purify the DNA page 47 Stopping point Quantitate the DNA page 48 Stopping point Check the size distribution of the libraries page 48 Stopping point Optional Pool equal molar barcoded libraries page 49 Stopping point Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 3 Prepare Multiple Fragment Libraries Procedural guidelines Procedural guidelines Quantitate the Shear the DNA e The protocol is designed for 10 ng 5 ug of genomic DNA or ligated PCR pro
73. on 5500 Series SOLID Systems User Guide Appendix D Oligonucleotide Sequences Library construction oligonucleotides Barcoded adaptor sequence Length nt Barcode T 065 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGGGATCAAGCCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 066 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGATGTAATGT CTGCTGTACGGCCAAGGCGT 3 53 Barcode T 067 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGTCCTTAGGGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 068 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGCATTGACGACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 069 50 uM 5 CGCCTTGGCCGTACAGCAG3 19 5 CTGCCCCGGGTTCCTCATTCTCTGATATGCTTTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 070 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGCCCTACAGACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 071 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTACAGGGAACGCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 072 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAAGTGAATACCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 073 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTGCAATGACGTCTGCTGTACGGCCAAGGCGT 3 53 Barcode T 074 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTCCTCATTCTCTAGGACGCTGACTGCTGTACGGCCAAGGCGT 3 53 Barcode T 075 50 uM 5 CGCCTTGGCCGTACAGCAG 3 19 5 CTGCCCCGGGTTC
74. ontact your field application specialist 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 before use but thaw Shear Buffer at room temperature Fragment Library Preparation 5500 Series SOLID Systems User Guide Quantitate the Shear the DNA Shear the DNA with the Covaris 220 System Chapter 2 Prepare a Single Fragment Library Quantitate the DNA DNA For accuracy determine sample DNA concentration using a double stranded DNA specific fluorescence assay Use the HS Assay Kit to measure dsDNA concentrations from 10 pg uL to 100 ng uL For samples outside this range use the dsDNA BR Assay Kit for higher concentrations of DNA or PicoGreen dsDNA Assay Kit for lower concentrations e Invitrogen Qubit dsDNA HS Assay Kit Invitrogen Part no Q32851 or Q32854 or e Invitrogen Qubit dsDNA BR Assay Kit Invitrogen Part no Q32850 or Q32853 or e Invitrogen Quant iT PicoGreen dsDNA Assay Kit Invitrogen Part no P7589 This step involves sonicating the input DNA into small fragments with a mean fragment size of 160 bp and a f
75. otometer is properly calibrated Use the CF 1 Calibration Fluid Kit if necessary 2 Open the NanoDrop ND 1000 Spectrophotometer software to display a dialog box ND 1000 3 3 3 Select the Nucleic Acid button Fragment Library Preparation 5500 Series SOLiD Systems User Guide 63 Appendix B Supplemental Procedures Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer 4 Lift the sampling arm and load 2 uL of Nuclease free Water onto the lower measurement pedestal and lower the sampling arm Sampling Arm Measurement Pedestal 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 uL 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 ATATEN HFH Uns Petra E TO DOC SENTE ECO rem Abs 0 000 Lier Cuter A 120 nm Abs 0 000 EEEE ds do ds de du so de lo el al ds od ds x LINE 1 AAA 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 64 Fragment Library Preparation 5500 Series SOLID Systems User Guide Appendix B Supplemental Procedures Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer
76. pair and input DNA before shearing is lt 100 ng use 20 06 uL of adaptor or an equivalent quantity of diluted adaptor Incomplete nick translation Ensure that the incubation temperature after ligation is raised to 72 C for 20 minutes After adaptor ligation bioanalyzer trace shows multiple peaks in the library Narrow peaks shorter than library peaks are adaptors Repeat purification with the Agencourt AMPure XP Reagent using 1 5X volume of beads to sample volume or repeat purification with the SOLiD Micro Column Purification Kit Two broad peaks or a peak and a shoulder in the main library population of peaks 100 300 bp are due to lower ligation efficiency Quantitate the library by qPCR If needed amplify the library see Optional Amplify the library on page 26 or Optional Amplify the libraries on page 45 52 Fragment Library Preparation 5500 Series SOLID Systems User Guide APPENDIX A Ordering Information This appendix covers Required Applied Biosystems reagent kits for library preparation 53 REQUITERDMEGUIPIMENE id A Decne Gene hd oo dd 57 Optional equipment 5 666 ram 58 Required consumables 58 Optional consumables 60 Sufficient reagents are supplied in the 5500 Series SOLiD System kits to prepare up to 12 or up to 48 libraries for high throughp
77. potentially improve recovery of DNA a Add the eluate from the last spin back to the column then let the column stand for 1 minute b Spin the column s at 14 000 x g for 1 minute at room temperature STOPPING POINT Store the purified DNA in Elution Buffer E1 at 4 C or proceed directly to Quantitate the ligated DNA Quantitate the ligated DNA Measure the DNA concentration using e 2 uL of sample with the Qubit dsDNA HS Assay Kit Invitrogen Part no Q32851 and the Qubit 2 0 Fluorometer Invitrogen Part no Q32866 or e 2 uL of sample in the NanoDrop ND 1000 Spectrophotometer see Quantitate the DNA with the NanoDrop ND 1000 Spectrophotometer on page 62 or e 1uL of sample in the Agilent Technologies 2100 Bioanalyzer and or e The appropriate volume in qPCR refer to the Applied Biosystems SOLiD Library TagMan Quantitation Kit protocol Invitrogen Part no A12120 STOPPING POINT Store the purified DNA in its current buffer at 4 C or proceed directly to Optional Amplify the library Optional 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 26 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 Prepare a Single Fragment Library Optional Amplify the library IMPORTANT
78. priate 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 5500 Series SOLiD Systems User Guide 89 Appendix F Safety Biological hazard safety Biological hazard safety General biohazard WARNING BIOHAZARD Biological samples such as tissues
79. ragment size range of 100 250 bp before adaptor ligation using the Covaris System The conditions have been tested for shearing 10 ng 5 ug DNA in a total volume of 120 uL For certain DNA samples optimizing the shearing protocol may be necessary You can shear the DNA with two supported shearing systems e The Covaris 220 System see Shear the DNA with the Covaris 220 System or e The Covaris S2 System see Shear the DNA with the Covaris S2 System on page 16 IMPORTANT Ensure that the bath temperature during shearing is 5 10 C Higher shearing temperatures can be harmful to DNA 1 Dilute the components below ina 1 5 mL LoBind Tube Shear Buffer reduces DNA damage from sonication Component Amount DNA 10 ng 5 ug 1X Low TE Buffer Variable uL Shear Buffer 1 2 uL Total 120 uL 2 Prepare the Covaris 220 Tank a Ensure that the water in the Covaris 220 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 b Set the chiller temperature to 2 5 C to ensure that the temperature reading in the water bath displays 5 C Fragment Library Preparation 5500 Series SOLiD Systems User Guide 15 Chapter 2 Prepare a Single Fragment Library Shear the DNA Shear the DNA with the Covaris S2 System 16 The circulated water chiller should be supplemented with 20 ethylene
80. rcode Adaptors 4464404 Library Builder Fragment Core Kit for 5500 SOLiD 4463763 SOLiD 4 System Library Kits mm Adaptors SOLiD Fragment Library Construction Kit SOLiD Fragment Library Oligos Kit 4443473 4401151 Library Builder Fragment Core Kit for SOLiD 4 0 SOLiD Fragment Library Barcoding Kit 1 96 4463762 4449637 How to use library This user guide describes how to use the 5500 SOLiD Fragment Library Core Kit core kits with with the 5500 SOLiD Fragment Library Standard Adaptors or the 5500 SOLiD adaptors 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 Library Builder Core Kit for 5500 SOLiD with the adaptors refer to Fragment Library Preparation Using the AB Library Builder System 5500 Series SOLiD Systems User Guide Use the 5500 SOLiD Fragment Library Core Kit and the adaptors to e Prepare a single fragment library for forward and reverse reads 100 250 bp before adaptor ligation for sequencing on the 5500 Series SOLiD Sequencers A fragment library consists of a sheared DNA fragment with a P1 Adaptor and a Standard Adaptor ligated to the 5 end and 3 end respectively e Prepare multiple fragment libraries in parallel for multiplex or non multiplex sequencing 10 Fragm
81. s for 10 seconds then pulse spin 38 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 3 Prepare Multiple Fragment Libraries Quantitate the size selected DNA c Incubate the mixture at room temperature 20 25 C for 5 minutes d Place the tube in a DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant Save the pellet which contains the DNA 6 Wash the DNA bead complex 3 times For each wash a Add 200 uL of freshly prepared 70 ethanol to the tube mix by inverting the tube a few times then pulse spin b Place the tube in the DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant 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 uL pipettor 8 Open the tube then dry the beads at room temperature 20 25 C for 5 10 minutes 9 Elute the DNA a Remove the tube from the DynaMag 2 magnetic rack then add 36 uL Low TE Buffer directly to the pellet to disperse the beads b Pipette the suspension up and down to mix c Vortex the beads for 10 seconds then pulse spin d Place the tube in a DynaMag 2 magnetic rack for at least 1 minute until the solution clears e Transfer the supernatant containing the size selected DNA to a new 1 5 mL LoBind Tube
82. say Library PCR Primer 2 Standard Adaptor Barcode OXX Sample Barcode Quantitation Lot Number Step Quantity of DNA Step Lot number Starting Amount P1 Adaptor End Repair Library PCR Primer 1 Quantitative assay Library PCR Primer 2 Standard Adaptor Barcode 0XX Fragment Library Preparation 5500 Series SOLiD Systems User Guide 85 86 Appendix E Checklist and workflow tracking form Workflow tracking prepare a fragment library Fragment Library Preparation 5500 Series SOLID Systems User Guide APPENDIX F Safety This appendix covers General chemical safety cc 66 sek eee bebe Ne eee ek ee ee ea eS 87 SDSS ss iine inne EE EA uw died di 88 Chemical waste safety 5 454 ie saw rr diia nii Ei A 88 Biological hazard safety 0 eee eee 90 General chemical safety Chemical safety guidelines WARNING CHEMICAL HAZARD Before handling any chemicals refer to the Safety Data Sheet SDS provided by the manufacturer and observe all relevant precautions A 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 A WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secure
83. sequenced on the 5500 Series SOLiD Sequencers see Choosing the appropriate library type on page 67 For a graphical overview of fragment library preparation see Overview on page 67 Preparing a single fragment library takes 4 h without amplification and 5 h with amplification Quantitate the DNA page 15 y Shear the DNA page 15 y End polish the DNA page 18 4 Size select the DNA by Agencourt AMPure XP Reagent page 18 Stopping point Quantitate the size selected DNA page 21 Stopping point Add a dA tail to the size selected DNA page 21 4 Fragment Library Preparation 5500 Series SOLID Systems User Guide 13 Chapter 2 Prepare a Single Fragment Library Procedural guidelines Ligate adaptors to the DNA Calculate the amount of adaptor to use for ligation page 22 Ligate adaptors to the DNA page 22 Purify the DNA page 23 Stopping point Quantitate the ligated DNA page 26 Stopping point Optional Amplify the library Amplify the library page 27 Purify the DNA page 27 Stopping point Quantitate the DNA page 29 Stopping point Check the size distribution of the library page 29 Stopping point Procedural guidelines 14 The protocol is designed for 10 ng 5 ug of genomic DNA To construct a targeted resequencing library with small sized PCR products lt 500 bp first perform a PCR product ligation step to concatenate the DNA For a concatenation protocol c
84. size selection 0 3 ug of size selected DNA ug to pmol 10 pg 1 pmol 1 z conversion factor Tug 660 pg x ie 9 2 pmol ug DNA 1 uL adaptor needed Y uL adaptor needed 0 3 Hg DNA x 9 2 pmol ug DNA x 10 x 50 pmol 0 55 uL adaptor needed IMPORTANT Do not use P1 and P2 Adaptors that are designed for fragment library preparation and sequencing on the SOLiD 4 System These adaptors are not compatible with reverse read sequencing on the 5500 Series SOLiD Sequencers Use only P1 T and Barcode T 0XX Adaptors that are designed for the 5500 Series SOLiD Sequencers 1 In anew 1 5 mL LoBind Tube combine for a ligation master mix Component Volume per Master mix for N library libraries 5X Reaction Buffer 3 0 uL 3 0 pL x 1 1 x N P1 T Adaptor 50 uM Y uL Yul x 1 1 x N Barcode T 0XX 50 uM Y uL YuLx 1 1 x N T4 DNA Ligase 5 U uL 6 5 uL 6 5 uL x 1 1 x N 10 mM dNTP 1 2 uL 1 2 uL x 1 1 x N Nuclease free Water Variable Variable Total 15 pL 15 uL x 1 1 x N 2 Add 15 uL of the ligation master mix from step 1 above to the reaction mixture from Add a dA tail to the size selected DNA on page 40 for a total of 61 uL total volume in each tube Fragment Library Preparation 5500 Series SOLID Systems User Guide 41 Chapter 3 Prepare Multiple Fragment Libraries Ligate adaptors to the DNA 3 Vortex each reaction for 5 seconds then pulse spin 4 Incubate each rea
85. t Library Standard Adaptors are used Barcode T OXX is Barcode T 001 22 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 Prepare a Single Fragment Library Ligate adaptors to the DNA Add the entire 15 uL of ligation master mix from step 1 above to the reaction mixture from Add a dA tail to the size selected DNA on page 21 for a total of 61 uL total volume Vortex the reaction for 5 seconds then pulse spin Incubate the reaction in a thermocycler with the lid heater on IMPORTANT Incubation nick translates the DNA Purify the DNA Stage Temp Time Holding 20 C 30 min Holding 72 C 20 min Holding 4 C oo If you want to Then Purify the ligated DNA quickly with high yield Proceed to Purify the DNA using Agencourt AMPure XP Reagent Use 39 uL of Agencourt AMPure XP Reagent with 65 uL of ligated DNA equal to 0 6X of Agencourt AMPure XP Reagent per sample volume Purify the ligated DNA with a column for convenience Proceed to Purify the DNA with the SOLiD Library Micro Column Purification Kit on page 25 Purify the DNA using Agencourt AMPure XP Reagent 1 Resuspend the Agencourt AMPure XP Reagent beads and allow the mixture to come to room temperature 2 Prepare 70 ethanol Component Volume Nuclease Free Water 300 uL Ethanol Absolute 700 uL Total 1000 pL 3 B
86. te adaptors to the DNA d Place the 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 carefully remove and discard the supernatant 4 Wash the DNA 3 times For each wash a Add 200 uL of freshly prepared 70 ethanol to the tube mix by inverting the tube a few times then pulse spin b Place the tube in the DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant 5 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 uL pipettor 6 Open the tube then dry the beads at room temperature 20 25 C for 5 10 minutes 7 Elute the DNA a Remove the tube from the DynaMag 2 magnetic rack then add 22 uL Low TE Buffer directly to the pellet to disperse the beads b Pipette the suspension up and down to mix c Vortex the beads for 10 seconds then pulse spin d Place the tube in the magnetic rack for at least 1 minute until the solution clears e Transfer the supernatant containing the size selected DNA to a new 1 5 mL LoBind Tube STOPPING POINT Store the purified DNA in Low TE Buffer at 4 C 8 Proceed as follows If you want to Then Further remove residual adaptors from the DNA Repeat Purify the DNA using Agencourt AMPure XP Rea
87. 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 e If 24 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 Quantitate the libraries to be pooled by qPCR refer to the Applied Biosystems SOLiD Library TagMan Quantitation Kit protocol Invitrogen Part no A12120 2 Mix together equal molar amounts of each barcoded library of similar size in an appropriately sized LoBind Tube Vortex the tube STOPPING POINT Store the library DNA in Elution Buffer E1 at 4 C or proceed directly to emulsion PCR as describe in the SOLiD EZ Bead Emulsifier Getting Started Guide PN 4441486 Troubleshooting See Troubleshooting on page 51 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 49 Chapter 3 Prepare Multiple Fragment Libraries Troubleshooting 50 Fragment Library Preparation 5500 Series SOLID Systems User Guide CHAPTER 4 Troubleshooting Observation Possible cause Recommended action Quantities of DNA for the same Quantities differ due to different e Measure duplicates of sample or sample do not match between properties of DNA measured
88. to a labelled 0 2 mL PCR tube Less than 20 uL volume is acceptable Do not adjust the volume to 20uL 2 Inanew 1 5 mL LoBind Tube combine for a PCR master mix Volume per Master mix for N Component ie be amplification libraries Platinum PCR Amplification Mix 100 pL 100 uL x 1 1 x N Library PCR Primer 1 50 uM 2 5 uL 2 5 uL x 1 1 x N Library PCR Primer 2 50 uM 2 5 uL 2 5 uL x 1 1 x N Total 105 pL 105 uL x 1 1 x N 3 Pipette 105 uL of the master mix from step 2 into each labelled tube While pipetting the master mix take care not to cross contaminate libraries 4 Vortex the reaction for 5 seconds then pulse spin 5 Determine the number of PCR cycles Starting amount of DNA er ol 10 100 ng 10 cycles 100 ng 1 ug 6 8 cycles 1 2 ug 4 6 cycles 2 5 ug 0 3 cycles 6 Run the PCR Stage Step Temp Time Holding Denature 252C 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 co Fragment Library Preparation 5500 Series SOLID Systems User Guide Purify the DNA come to room temperature Chapter 3 Prepare Multiple Fragment Libraries Optional Amplify the libraries Resuspend the Agencourt AMPure XP Reagent beads and allow the mixture to Prepare 70 ethanol for N number of libraries Component Volume Nuclease Free Water 300 uL x N
89. ulsifier Getting Started Guide Part no 4441486 If you do not see the expected size distribution troubleshoot or contact your Life Technologies Applications Specialist STOPPING POINT Store the DNA in Low TE Buffer at 4 C for short term storage or at 20 C for long term storage Troubleshooting See Troubleshooting on page 51 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 29 Chapter 2 Prepare a Single Fragment Library Troubleshooting 30 Fragment Library Preparation 5500 Series SOLID Systems User Guide Workflow CHAPTER 3 Prepare Multiple Fragment Libraries Follow this chapter if you are preparing libraries by the non automated method e Pooled barcoded libraries for multiplexed sequencing or e Multiple libraries in parallel that are not pooled For an overview of library types that can be sequenced on the 5500 Series SOLiD Sequencers see Choosing the appropriate library type on page 67 For a graphical overview of fragment library preparation see Overview on page 67 IMPORTANT Customers who have purchased the AB Library Builder System and who wish to prepare a fragment library with an automated system refer to the Fragment Library Preparation Using the AB Library Builder System 5500 Series SOLiD Systems User Guide Part no 4460965 Customers who have access to automated liquid handling systems such as the Beckman Coulter Biomek FXP and Tecan Freed
90. ure 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 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 Prepare a Single Fragment Library Shear the DNA 2 Dilute the desired amount of DNA to 100 uL in 1X Low TE Buffer in a LoBind tube Component Amount DNA 10 ng to 5 ug 1X Low TE Buffer Variable uL Shear Buffer 1 2 uL Total 120 uL 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 100 uL of DNA sample through the pre split septa Be careful not to introduce a bubble into the bottom of the tube 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 61 4 Shear the DNA using the following Covaris S2 System conditions IMPORTANT Ensure that the bath temperature limit is set to 15 C and keep the bath temperature to lt 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 Pla
91. ut sequencing with the 5500 Series SOLiD System Upon receipt of the 5500 Series SOLiD System kits immediately store each components at the temperature specified on the label Required Applied Biosystems reagent kits for library preparation Item part no t Components 5500 SOLID Fragment Library Core Kit 4464412 5500 SOLID Fragment Library Enzyme Module 5500 SOLID Fragment Library Amplification Module 5500 SOLiD Fragment Library Enzyme Module 4464413 10 mM dNTP End Polishing E1 End Polishing E2 5X Reaction Buffer A Tailing Enzyme T4 DNA Ligase 5 U uL 10 mM dATP Shear Buffer 5500 SOLiD Fragment Library Amplification Module 4464414 Platinum PCR Amplification Mix Fragment Library Preparation 5500 Series SOLiD Systems User Guide 53 AppendixA Ordering Information Required Applied Biosystems reagent kits for library preparation Item part no t Components 5500 SOLID Fragment Library Standard Barcode T 001 50 uM Adaptors P1 T Adaptor 50 uM Gen Library PCR Primer 1 50 uM Library PCR Primer 2 50 uM 5500 SOLiD Fragment Library Barcode 5500 SOLID Fragment Adaptors 1 96 4464404 Library Barcode Adaptors 1 16 50 uM each 5500 SOLID Fragment Library Barcode Adaptors 17 32 50 uM each 5500 SOLID Fragment Library Barcode Adaptors 33 48 50 uM each 5500 SOLiD Fragment Library Barcode Adaptors 49 6
92. y preparation workflows see Overview on page 67 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 sheared DNA fragments The bead is for illustration purposes only and is not added until the bead preparation step Fragment Library mah Sequence up to 75 bp Sequence up to 35 bp Sequence 5 or 10 bp P1 T Adaptor DNA Fragment Bor de Adaptor Mate Paired Library Sequence up to 60 bp gt Sequence up to 60 bp P1 T Adaptor Mate Pair Tag or Mate Pair Tag Product information Purpose of the To prepare fragment and barcoded fragment libraries for sequencing on the product 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 5500 Series SOLID Systems User Guide 9 Chapter 1 About the Products Product information 5500 Series SOLID System Library Core Kits Rs Adaptors 5500 SOLiD Fragment Library Standard Adaptors 4464411 5500 SOLiD Fragment Library Core Kit 4464412 5500 SOLiD 48 Fragment Library Core Kit 4464415 5500 SOLiD Fragment Library Ba
93. ycol System 30 minutes prior to a a y y y O Covaris microTube use o loading station O Supplement the circulated 3 O Covaris microTube water chiller with 20 2 ethylene glycol Y O Thaw Shear Buffer at room temperature O NanoDrop ND 1000 O 5x Reaction Buffer O Thaw buffers on ice 8 lt Spectrophotometer O dA dNTP Mix 10 mM E O DynaMag 2 Magnetic O End Polishing E1 50 Rack O End Polishing E 2 O A Tailing Enzyme 1 Due O0 Agencourt AMPure XP O Warm AMPure XP Bead 32XxX E Reagent mixture to room E s g O Ethanol Absolute temperature 1Q o amp N CE Le x O Qubit 2 Fluorometer or O Quant iT kit 8 O NanoDrop ND 1000 23 lt Spectrophotometer or o 36 l So O Agilent Technologies eu 2100 Bioanalyzer O NanoDrop ND 1000 O 5x Reaction Buffer O Thaw buffers on ice lt 2 3 Spectrophotometer O 10 mM dATP Sob S O A Tailing Enzyme 1 at 2 a O Nuclease free Water Du Y a O DynaMag 2 magnetic O 5x Reaction Buffer O Thaw adaptors on ice 2 lt rack O P1 T Adaptor 50 uM O Thaw 5x T4 Ligase Buffer 2 E 2 O Barcoded T 0XX adaptor 50 on ice a D uM Ige O 10 mM dNTP lt O Agencourt AMPure XP Reagent O Ethanol Absolute O Thermocycler O Library PCR Primer 1 Thaw Library PCR Primers as S O Library PCR Primer 2 1 and 2 on ice S26 O Platinum PCR Amplification O Thaw Platinum PCR 25 Mix Amplification Mix on ice o 7 O Agencourt AMPure XP Reagent O Ethanol A

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