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Mate-Paired Library Preparation 5500 Series SOLiD™ Systems

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1. 3 Combine Component Volume DNA VuL 10X Plasmid Safe Buffer 7 10 uL Nuclease free Water T T 10 V uL Total T uL 4 Incubate the reaction in the heat block at 70 C for 5 minutes then place the reaction on ice for 5 minutes IMPORTANT The incubation time is critical Keep the time as close to 5 minutes as possible then proceed to the next immediately to Isolate the circularized DNA Isolate the circularized DNA Plasmid Safe DNase is used to eliminate uncircularized DNA After Plasmid Safe DNase treatment the DNA is purified using the Agencourt AMPure XP Reagent Treat the DNA with 1 Combine the following where T the total volume of the circularization Plasmid Safe reaction uL DNase Component Volume Circularized DNA T uL ATP 100 mM 7 100 uL Plasmid Safe DNase 10 U uL 7 100 uL 24 Fragment Library Preparation 5500 Series SOLID Systems User Guide Purify the DNA using Agencourt AMPure XP Reagent Fragment Library Preparation 5500 Series SOLiD Systems User Guide and Example If T 800 uL then Chapter 2 2 x 60 bp Mate Paired Library Preparation Isolate the circularized DNA Component Volume ATP 100 mM 8 0 uL Plasmid Safe DNase 10 U uL 8 0 uL Incubate the reaction mixture at 37 C for 40 minutes Resuspend the Agencourt AMPure XP Reagent beads Bind the DNA to the Agencourt AMPure XP Reagent a
2. 2000200 cece ee eens 28 Add a dA Tail to the digested DNA 2 2 eee eee e aes 30 Bind the library molecules to streptavidin beads 2 0000 cece eee 30 Ligate P1 T and P2 T Adaptors to the DNA 2 02 cece tects 32 Nick translate and trial amplify the library n nnan annann annan cece cece ee eeee 33 Nick translate and amplify the library 02 0200 e cece eens 35 Evaluate the library i ce Ad eee 37 Optional Size select the library with a SOLID Library Size Selection gel 38 Check the size distribution of the library 0000 ee rnrn rnnr 43 Quantitate the library by performing quantitative PCR qPCR 2 20 0000 43 roa bleshooHlg ote s aan donen iA x MPO Punt PT 44 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide 3 Contents APPENDIX A APPENDIX B APPENDIX C APPENDIX D APPENDIX E APPENDIX F Ordering Information 4256 sega sica hw eters Eres 47 Required Applied Biosystems reagent kit n nuanua nunnu annn eee ee 47 Required equipment o 49 Optional equipment e g ele bh ve Eur bh RR deER dened AG eee ta 51 Required consumables 000 c cece cece eee ee eect en 52 Optional consumables i 22d eed phe um bbe tae eee deeds cede 54 Supplemental Procedures eees 55 Shear the DNA for inserts lt 1 kb with the Covaris System 0 0c cece cece e ee 55 Load
3. Component Volume Nuclease free Water 17 0 uL 5X Reaction Buffer 20 uL 10 mM dNTP 4 0 uL End Polishing E1 4 0 uL End Polishing E2 5 0 uL Size selected DNA 50 0 uL Total 100 pL 2 Incubate the mixture at room temperature 20 25 C for 30 minutes 3 Heat inactivate the enzymes at 75 C for 20 minutes 4 Put the DNA on ice Ligate MP Adaptors to the DNA Calculate the amount of adaptor to use This ligation step adds the MP Adaptors to the sheared end repaired DNA The MP Adaptors are missing a5 phosphate at the non joining end as a result there is a nick on each strand when the DNA is circularized The MP Adaptors are included in double stranded form in the SOLiD Mate Paired Library Standard Adaptors module Calculate the amount of adaptor needed Y for the reaction based on the amount of DNA before the end repair step ug to pmol 00pg pmo 1 conversion factor 1 ug 660 pg Average insert size ug to pmol 1 uL adaptor needed Y uL adaptor needed fug DNA conversion factor 20 25 pmol For example if you have 1 ug of purified size selected DNA and an average insert size of 1 5 kb to 6 1 ugio pmo _ _10 pg_ pmo 4 0 pmolug DNA conversion factor 1 ug 660 pg 1500 1 uL adaptor needed Y uL adaptor needed 1HgDNA x 1 0pmolligDNA x 50 x 25 pmol 2 uL adaptor needed Note If Y 1 uL use 1 uL in the reaction Fragment Library Preparation 5500 Series SOLiD Systems User G
4. 1 4 uL Total 72 8 uL t Platinum PCR Amplification Mix contains a proofreading enzyme for high fidelity amplification 2 Vortex the PCR master mix For the negative control transfer 23 uL of the PCR master mix to a PCR tube Label the tube PCR 0 3 Add 4 uL of DNA bead complex solution to the remaining 49 8 uL of PCR master mix Vortex the mix then divide evenly 25 uL between two PCR tubes labelled PCR 1 and PCR 2 4 Use two different thermocyclers or run PCR sequentially for these numbers of cycles as follows Number of Sample no cycles 0 14 cycles 1 10 cycles 2 14 cycles Fragment Library Preparation 5500 Series SOLiD Systems User Guide 33 Chapter 2 2 x 60 bp Mate Paired Library Preparation Nick translate and trial amplify the library 5 Run Stage Step Temp Time Holding Nick translation TANG 20 min Holding Denature 94 C 3 min Cyclingt Denature 94 C 15 sec Anneal 62 C 15 sec Extend 70 C 1 min Holding Extend 70 C 5 min Holding 4 C oo t Tube 1 10 cycles Tubes 0 and 2 14 cycles Confirm library 1 Mix 0 5 uL of 1 ug uL 100 bp DNA Ladder Invitrogen Part no 10628 050 or amplification with a 0 5 uL of 1 ug uL 50 bp DNA Ladder Invitrogen Part no 10416 014 with 40 uL 2 E Gel EX Gel of Nuclease free Water 2 Load 20 uL of PCR 0 PCR 1 and PCR 2 into separate wells of a 2 E Gel EX Gel Load 2
5. 17 Chapter 2 2 x 60 bp Mate Paired Library Preparation Size select the DNA Size select the DNA Prepare an agarose gel Run the agarose gel Excise the sample from the agarose gel 18 1 TM The correctly sized products are excised and purified using the SOLID Library Quick Gel Extraction Kit Prepare a 1 agarose gel with 1X SYBR Safe Gel Stain and 1X TAE buffer Component Volume 1X TAE 100 mL Agaroset 1g 10 000 SYBR Safe gel staint 10 uL Total 100 mL t Use either Agarose LE Applied Biosystems AM9040 or UltraPure Agarose 1000 Invitrogen 10975 035 t Invitrogen Part no 533102 Add 10X BlueJuice Gel Loading Buffer to the purified sheared DNA 1 uL of 10X Gel Loading Buffer for every 10 uL of mate paired library Load the 1 Kb Plus DNA Ladder Invitrogen 10787 018 to one well Use these guidelines Load dye mixed sample per well according to the well capacity into remaining wells e Use the minimum number of wells possible There should be at least one lane between the ladder well and the sample wells to avoid contamination of the sample with ladder Run the gel at the appropriate voltage to achieve optimal separation of the size of interest IMPORTANT To obtain maximum resolution of DNA fragments run agarose gels at lt 5 V cm The distance is measured as the shortest path between the electrodes not the agarose gel length itself 1 Visualize the ge
6. C Spin the column at 10 000 x g for 1 minute at room temperature then discard the flow through d Spin the column at 14 000 x g to remove residual wash buffer 5 Elute the DNA a Transfer the column to clean 1 5 mL LoBind Tube b Add 25 uL of Elution Buffer E1 to the center of the column to elute the DNA then let the column stand for 1 minute C Spin the column at 14 000 x g for 1 minute at room temperature d Add the eluate from the last spin back to the column then let the column stand for 1 minute e 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 to Evaluate the library Evaluate the library 1 Run1 uL of the concentrated library on a High Sensitivity DNA Chip in the Agilent Technologies 2100 Bioanalyzer to confirm amplification quality Bioanalyzer electropherogram of library without PCR by products that can be quantitated by qPCR FU 5UG_3K8 35 100 200 300 400 500 700 2000 10380 b Fragment Library Preparation 5500 Series SOLID Systems User Guide 37 Chapter 2 2 x 60 bp Mate Paired Library Preparation Optional Size select the library with a SOLiD Library Size Selection gel Bioanalyzer electropherogram of library with PCR by products that needs to be gel purified with a SizeSelect gel Q4 HuRef 1 ug SOLID 150 gt 100 50 35 100 200 300 400 500 700
7. Components SOLID Library Quick Gel Extraction Kit 4443733 Wash Buffer W1 Elution Buffer E5 Gel Solubilization Buffer L3 Quick Gel Extraction Columns Wash Tubes Recovery Tubes eo Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance For the SDS of any chemical not distributed by Applied Biosystems or Invitrogen contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions Invitrogen products can be ordered at www invitrogen com Required equipment Product Namet Vendor HydroShear DNA Shearing Device from Genomic Solutions 918 Applied Biosystems 4392889 115 V Applied Biosystems 4392890 230 V Microcentrifuge 5417R refrigerated without e Eppendorftt rotor 022621807 120 V 60 Hz Eppendorf 022621840 230 V 50 Hz FA 45 24 11 fixed angle rotor Eppendorf 24 x 1 5 2 mL including aluminum lid 022636006 aerosol tight 96 well GeneAmp PCR System 9700 thermal cycler Applied Biosystems N8050200 Base e Applied Biosystems 4314443 Block t Labquake Rotisserie Rotator Barnstead Thermolyne VWR 56264 312 6 Tube Magnetic Stand or DynaMag 2 Magnet magnetic rack e Applied Biosystems AM10055 Invitrogen 123 21D Mate Paired Library Pr
8. Library Size Selection gel When a full length sequencing tag is not critical such as for clonal coverage then the size selecting the library is not necessary Proceed to Check the size distribution of the library on page 43 If you intend to maximize sequencing coverage then you may prefer to size select the library For sequencing of 2 x 60 bp libraries size select the library to remove fragments lt 240 bp For sequencing of 2 x 50 bp libraries size select the library to remove fragments lt 220 bp Limit the size of the library to lt 350 bp Size selection may reduce the final library yield over not size selecting the library by 3 4 fold You must take into account the reduced yield when determining the optimal number of PCR cycles for final library amplification The library is run on an SOLiD Library Size Selection gel Extract and desalt the library band 250 350 bp using the SOLiD Library Micro Column Purification Kit Load the library 1 Plug the adapter plug of the E Gel iBase Power System into an electrical outlet 2 Remove the SOLiD Library Size Selection gel from its package then insert the gel with its combs into the iBase system a Slide the gel into the two electrode connections on the iBase system Ensure that the two electrodes on the right side of the cassette touch the two contacts on the iBase system The Invitrogen logo should be at the bottom of the base b Press the gel firmly at
9. STOPPING POINT Store the DNA Bead complexes in Elution Buffer E1 at 4 C or proceed directly to Nick translate and amplify the library Nick translate and amplify the library The library is amplified using Library PCR Primers 1 and 2 with the Platinum PCR Amplification Mix which includes a proofreading enzyme for high fidelity amplification Reduce the number of cycles as much as possible and use the entire nick translated DNA complex for amplification to get maximum representation of the library and to avoid PCR related biases due to differential amplification of library molecules Fragment Library Preparation 5500 Series SOLiD Systems User Guide 35 Chapter 2 2 x 60 bp Mate Paired Library Preparation Nick translate and amplify the library Perform PCR on 1 the library 2 3 4 5 6 Purify the DNA 1 with the SOLiD Library Micro 2 Column Purification Kit 36 Prepare a master mix for amplification reactions Component Volume Platinum PCR Amplification Mix 100 0 uL Library PCR Primer 1 50 uM 2 0 uL Library PCR Primer 2 50 uM 2 0 uL Total 104 uL t Platinum PCR Amplification Mix contains a proofreading enzyme for high fidelity amplification Place the tube of P1 T P2 T ligated DNA beads in the DynaMag 2 magnetic rack for 21 minute until the solution clears see Ligate P1 T and P2 T Adaptors to the DNA on page 32 With a 20 uL pipettor carefully remove and dispose
10. gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories http www cdc gov biosafety publications index htm Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 www access gpo gov nara cfr waisidx 01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov 78 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide Documentation and Support Related documentation For related documents refer to the 5500 Series SOLiD Systems User Documentation Quick Reference Part no 4465102 Obtaining support For the latest services and support information for all locations go to www appliedbiosystems com At the Applied Biosystems website you can e Access worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales fac
11. 2 In a Covaris microTUBE mix Component Amount DNA 1 5 ug 1X TE Buffer Variable uL Total 120 uL Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 57 Appendix B Supplemental Procedures Shear the DNA for inserts lt 1 kb with the Covaris System 3 Shear the DNA using the Covaris 2 System shearing according to these conditions IMPORTANT Ensure that the Bath Temperature Limit is set to 15 C and keep the bath temperature to lt 10 C Covaris S2 System shearing Target insert peak condition 700 bp e Duty cycle 5 e Intensity 3 Cycles per burst 200 Time 60 seconds Number of cycles 1 e Waterbath temperature 6 8 C e Bath Temperature Limit 15 C Power mode Frequency sweeping e Degassing mode Continuous Water level 12 Water Quality Testing Function Off AFA intensifier Yes 1000 bp Duty cycle 196 e Intensity 5 e Cycles per burst 200 Time 45 seconds e Number of cycles 2 e Waterbath temperature 6 8 C Bath Temperature Limit 15 C Power mode Frequency sweeping e Degassing mode Continuous Water level 12 Water Quality Testing Function Off AFA intensifier Yes 4 Transfer 120 uL of sheared DNA into a clean 1 5 mL LoBind tube 5 Reduce the volume of the sheared DNA to 40 60 uL in order to load the sample into a 1 cm wide well on a size selection gel Use a SpeedVac Concentrator or equivalen
12. E Gel iBase Power System over a Safe Imager Real Time Transilluminator Use the orange cover or orange goggles to view the bands and to avoid overexposure of your eyes to the blue light 2 Run the gel On the iBase system a Select SizeSelect 2 refer to the iBase Power System manual for instructions b Press Go The red light turns green Fragment Library Preparation 5500 Series SOLiD Systems User Guide Chapter 2 2 x 60 bp Mate Paired Library Preparation Optional Size select the library with a SOLID Library Size Selection gel 3 Monitor the gel At the end of a run the iBase system flashes a red light and beeps rapidly Ifthe front line of library products has not reached the reference line run the gel for about 1 2 more minutes until the band reaches the line IMPORTANT The ideal size of a library is from 275 325 bp but a library ranging from 240 380 bp is acceptable To generate an optimal number of full length mate tags in 2 x 60 bp sequencing the minimum library size must be 2240 bp When the front line of library products reaches the reference line press Go to stop the run 4 When the front line of library products reaches the reference line refill the bottom row again with Nuclease free Water until each well is full Some pre filled water is lost during the run 5 Press Go to run the gel until the library products enter the collection well For optimal results monitor the run in
13. 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 appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing For additional safety guidelines consult the SDS Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safe
14. Prepare the bead suspension in the sample reaction Component Volume Sample reaction TuL Bead Dilution Buffer 0 7x TuL Agencourt AMPure XP Reagent 0 3 x TuL b Vortex the beads for 15 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 magnetic rack Wash the DNA 2 times For each wash keep the tube in the DynaMag 2 a Add 600 uL of 70 ethanol to the tube without disturbing the beads b Keep the tube ina DynaMag 2 magnetic rack for at least 1 minute then remove and discard the supernatant without disturbing the beads 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 3 minutes to dry the sample Mix Component Volume Nuclease free Water 84 uL Nick Translation Buffer 10 uL Elute the DNA a Remove the tube from the DynaMag 2 magnetic rack then add the 94 uL pre mixed solution of Nick Translation Buffer to the tube of DNA b Gently vortex the beads for 15 seconds pulse spin then incubate the beads at room temperature 20 25 C for 3 minutes 25 Chapter 2 2 x 60 bp Mate Paired
15. Safe Imager Blue Light O BlueJuice Gel Loading Buffer o Transilluminator O 1 Kb Plus DNA Ladder O Gel imaging system O SYBR Safe gel stain 9 O Qubit 2 0 Fluorometer O Invitrogen Library Quick Gel o O Razor blades Extraction Kit vd O 15 mL conical O Isopropyl alcohol N polypropylene tubes n O DynaMag 2 Magnetic Rack o O Qubit amp 2 0 Fluorometer O 5x TA DNALigase Buffer End repair reagents on ice O dNTP 10 mM Seq O T4 Polynucleotide Kinase Q 10 U uL in O T4 DNA Polymerase 5 U uL O Nuclease free Water O DynaMag 2 Magnetic O MPR Adaptor 25 uM Thaw adaptors on ice S Rack O MPL Adaptor 25 uM 25 O T4 DNA Ligase 5U uL rrt O 5x T4 DNA Ligase Buffer 5 20 O T4 DNA Ligase 189 O AMPure XP Beads z O Ethanol Absolute O Elution Buffer E1 O Qubit 2 0 Fluorometer O Quant iT dsDNA HS Assay I Kit t 3 ej Pe O 10x Plasmid Safe Buffer Thaw buffer on ice 02 O NO ss SZ585 BOELS zo 5 os 2 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide Appendix E Checklist and workflow tracking form Workflow checklists prepare a 2 x 60 bp mate paired library Equipment Reagents Preparation steps a O Qubit 2 0 Fluorometer O ATP 25 mM Thaw Plasmid Safe 2 9 O Incubator 37 C O 10x Plasmid Safe Buffer ATP Dependent DNase 2 E O Agencourt AMPure XP Kit reagents on ice S50 O Ethanol Absolute 2 O Elution Buffer E1 O Thermal cycler O
16. Source Agencourt AMPure9 XP 5 mL Kit Beckman Coulter oF Genomics A63880 or Agencourt AMPure XP 60 mL Kit A63881 Invitrogen Qubit dsDNA HS Assay Kit Invitrogen Q32851 or Q32854 or Invitrogen Qubit dsDNA BR Assay Kit Invitrogen Q32850 or Q32853 or Invitrogen Quant iT PicoGreen dsDNA Invitrogen Assay Kit P7589 E Gel EX Gel 2 10 Pak Invitrogen G4010 02 UltraPure DNA Typing Grade 50X TAE Invitrogen Suner 24710 030 Agarose LE Applied Biosystems AM9040 or UltraPure Agarose 1000 Invitrogen 10975 035 SYBR Safe DNA Gel Stain 10 000X Invitrogen 33102 10X BlueJuice Gel Loading Buffer Invitrogen 10816 015 50 bp DNA Ladder Invitrogen 10416 014 100 bp DNA Ladder Invitrogen 15628 050 1 Kb Plus DNA Ladder Invitrogen 10787 018 Covaris Tubes and Caps 125 Applied Biosystems 4399054 Ethanol Sigma Aldrich E7023 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide Appendix A Ordering Information Required consumables Item Source 2 Propanol Sigma Aldrich 19516 Ethylene glycol American Bioanalytical AB00455 01000 1 5 mL LoBind Tubes Eppendorf 022431021 2 0 mL LoBind Tubes Eppendorf 022431048 MicroAmp Optical 8 Tube Strip 0 2 mL Applied Biosystems 4316567 Hydrochloric Acid 0 20 N VWR VW8888 0 Sodium Hydroxide 0 20 N VWR VW8889 0 CF 1 Calibration Fluid Kit Thermo Scientific CF 1 PR 1 Conditioning Kitt Thermo Sc
17. a darkened room 6 Collect the sample 250 bp 350 bp from the collection well every 10 seconds using a 20 uL pipettor fitted with a tip After each collection flush the collection well with 20 uL of Nuclease Free Water then collect this wash For example Before After Fragment Library Preparation 5500 Series SOLiD Systems User Guide 41 Chapter 2 2 x 60 bp Mate Paired Library Preparation Optional Size select the library with a SOLiD Library Size Selection gel Purify the DNA with the SOLiD Library Micro Column Purification Kit 42 Do not perforate the bottom of the agarose collection well Due to migration of the DNA into the bottom of the well some residual DNA remains underneath the well IMPORTANT If the library products overrun the collection well and reenter the gel select REVERSE E Gel on the iBase Power System to run the library products backward into the collection well Collect all of the DNA Note If a concentrated sample is not necessary skip this purification step and proceed to Quantitate the library by performing quantitative PCR qPCR on page 43 1 Pre spin empty PureLink Micro columns in collection tubes at 10 000 x g for 1 minute before use Add 4 volumes of Binding Buffer B2 S with isopropanol 55 to 1 volume of sample Mix well Load the DNA onto the PureLink Micro columns a Apply the sample in the binding buffer to the PureLink Micro column s in collection tube
18. and evaluate the stringency of the first size selection Life Technologies recommends gt 10 of the target insert size lt 50 ng Quantitate and assess sample DNA quality on page 16 to start mate paired library preparation again If the starting material is 1 ug and the recovery remains low use more starting material STOPPING POINT Store the purified DNA in Elution Buffer E1 at 4 C or proceed directly to Circularize the DNA by intra molecular hybridization Circularize the DNA by intra molecular hybridization The mate paired adaptor contains a blocking oligonucleotide to protect the 3 overhangs of the MP Adaptors from self annealing At circularization heat denaturation removes the blocking oligonucleotide The DNA circularizes through intramolecular hybridization at low concentrations Fragment Library Preparation 5500 Series SOLiD Systems User Guide 23 Chapter 2 2 x 60 bp Mate Paired Library Preparation Isolate the circularized DNA 1 Fill all of the holes to be used in a heat block with water then pre heat the block to 70 C 2 Calculate the total volume of the circularization reaction T uL so that for a known concentration of DNA DNA ng uL and known volume of DNA V the final concentration of DNA in the reaction is 0 5 ng uL T DNA x V 0 5 Example If DNA 5 ng uL and V 50 LL then T 500 uL Note If the total volume of hybridization is 21000 uL use T 1000 uL
19. before loading on the size selection gel or With a SpeedVac Concentrator or equivalent concentrate the sheared DNA sample sufficiently to load in 1 well of a size selection gel so that 1 gel extraction column is sufficient for purification 45 Chapter 2 2 x 60 bp Mate Paired Library Preparation Troubleshooting 46 Fragment Library Preparation 5500 Series SOLID Systems User Guide APPENDIX A Ordering Information This appendix covers materials for 2 x 60 bp mate paired library preparation Required Applied Biosystems reagent kit Required equipment Optional equipment Required consumables Optional consumables Sufficient reagents are supplied in the 5500 Series SOLiD System kits to prepare up to 12 libraries at 5 ug input DNA for high throughput 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 kit Item part no Components 5500 SOLiD Mate Paired Library Kit 4464418 5500 SOLID Mate Paired Library Enzyme Module 5500 SOLID Amplification Module 5500 SOLID Mate Paired Bead amp Buffer Module SOLID Mate Paired Library Oligo Module SOLID Library Micro Column Purification Kit SOLiD Library Quick Gel Extraction Kit Mate Paired Library Preparation 5
20. dNTP Mix 10 mM each Thaw dNTP Mix and Nick 2 3 O Timer O DNA Polymerase Translation Buffer on ice E p N 10 U uL ons E O Nick Translation Buffer 223 O SOLID Library Micro 3 Column Purification Kit O Isopropyl alcohol o O Incubator 37 C O T7 exonuclease 10 U uL Thaw Buffer 4 Buffer on g o Incubator 70 C O 10x Buffer 4 ice On 9 O S1 Nuclease Dilution Buffer i b O S1 Nuclease 25 U uL z 3 9 O Agencourt AMPure XP Kit o EJ O Ethanol Absolute a x O Elution Buffer E1 o O Ice oc O 0 5M EDTA Thaw end repair reagents Bou O 1x Nick Translation Buffer on ice O dA dNTP 50 mM 5324 O Klenow Exo 5U uL e 2 z O Bead Binding Buffer O 6 Tube Magnetic Rack O 100x BSA Thaw 100x BSA and 5x 8 O Rotator O Dynabeads MyOne Ligase Buffer on ice gt 28 Streptavidin C1 beads 8292 O Bead Wash Buffer oss O Bead Binding Buffer 592 O 5x Ligase Buffer per E 7 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 71 Appendix E Checklist and workflow tracking form Workflow checklists prepare a 2 x 60 bp mate paired library Equipment Reagents Preparation steps O Rotator O T4 DNA Ligase 5 U uL Thaw P1 T Adaptor ds and ex O DynaMag 2 Magnetic O P1 T Adaptor ds P2 T Adaptor ds on ice aN 5 Rack O P2 T Adaptor ds 2580 O Bead Wash Buffer DR ys 4 qa Nick translate and trial amplify the Nick translate and Amplify the library Thermal cycler E Gel i
21. 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 Open To view the document 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 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 76 Mate Paired
22. of the supernatant until 5 uL of Elution Buffer E1 remains above the beads Do not draw beads into the pipettor tip Add the master mix from step 1 to the beads Vortex the beads for 15 seconds then transfer the suspension to a new PCR tube Run Stage Step Temp Time Holding Nick translation TAG 20 min Holding Denature 94 C 3 min Cyclingt Denature 94 C 15 sec Anneal 62 C 15 sec Extend 70 C 1min Holding Extend 70 C 5 min Holding 4 C oo Cycling number determined by trial amplification See Nick translate and trial amplify the library on page 33 Pre spin empty PureLink Micro column in collection tubes at 10 000 x g for 1 minute before use Add 4 volumes of Binding Buffer B2 L with isopropanol 40 to 1 volume of sample Mix well Load the DNA onto the PureLink Micro column a Apply all of the PCR sample with beads to the PureLink Micro column in 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 PCR sample has been loaded onto the column s Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2 x 60 bp Mate Paired Library Preparation Evaluate the library 4 Wash the column s 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
23. one of its oligonucleotides Nick translation using E coli DNA polymerase I pushes the nick into the genomic DNA region in 5 3 direction The length of nick translated DNA Mate Paired Library Preparation 5500 Series SOLID Systems User Guide Appendix C Overview Preparation of mate paired libraries can be controlled by adjusting reaction temperature and time T7 Exonuclease and S1 Nuclease digestion cuts the DNA at the position opposite to the nick and releases the DNA mate pair P1 T and P2 T Adaptors are then ligated to the ends of the mate paired library for subsequent amplification by PCR see Figure 3 on page 64 6 Figure 2 Basic 2 x 60 bp mate paired library preparation workflow gt 72 gt I2 A 6 A i n B QQ MP Adaptors Genomic DNA Sheared DNA MPR and MPL ligated to Circularization by sheared DNA hybridization C gt Oe S ae QQ P1 T P2 T Ligated Biotinylated MP Library Molecule adaptors with Nick translated genomic DNA tags circularized DNA with biotinylated MP adaptors Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 63 Appendix C Overview Preparation of mate paired libraries Figure 3 Mate paired library design P1 T Adaptor ds 41 42 bp UGEUDOGSUDDODSUDDDUBDEBDEBDBUDDUDBSG EEBEDEBBUDEDIGEDLE JOOUBCBUSVCOOUSCUBUEUEOECBBCCUUeEBUSUEOUUe After P1 T and P2 T Adaptors are ligated to the sheared DNA t
24. reverse reads F5 tag e ThelA 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 T Adaptor sequence 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 The double stranded oligonucleotides that are ligated to a sheared DNA insert to form the internal adaptor sequence during mate paired library construction 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 A T tailed double stranded oligonucleotide containing the P2 sequence that is ligated to A tailed DNA segments during library construction the result is that the P2 sequence is attached to the 3 end of the template stra
25. 0 uL of diluted 100 bp DNA Ladder in an adjacent well for reference Do not add any loading dye to the samples or DNA Ladder 3 Run the E Gel EX Gel on an E Gel iBase Power System according to the manufacturer s instructions for 10 minutes 4 Take a picture of the gel see Figure 1 on page 35 Choose a PCR cycle where amplified library products are faintly visible on the trial PCR gel Determine the number of PCR cycles for final library amplification Size select the DNA after Then determine the number of PCR amplification cycles based on No The intensity of the products on the gel only Yes The intensity of the products on the gel and add at least one more PCR cycle to compensate for sample loss Note For a 2x60 mate paired library the ideal peak size of an amplified library is just over 300 bp A peak size of amplified library from 280 350 bp is acceptable 34 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2 60 bp Mate Paired Library Preparation Nick translate and amplify the library Figure 1 Mate paired library trial amplification sample run on a 2 E Gel EX Gel Lane 1 50 bp DNA Ladder Lane 2 PCR 0 negative control Lane 3 PCR 1 10 cycles Lane 4 PCR 2 14 cycles Based on this picture if there is no size selection after amplification use 9 cycles for final library amplification If there is size selection after amplification use 10 or 11 cycles
26. 2000 10380 bp 2 Proceed as follows If you need Then Greatest sequencing output for 2x 60 bp Remove fragments 240 bp Proceed to mate paired sequencing run Optional Size select the library with a SOLiD Library Size Selection gel IMPORTANT Size selection reduces library yield by 3 5 fold Maximum number of mate paired Skip size selection and proceed to molecules and there are no PCR by Quantitate the library by performing products in a library of acceptable size quantitative PCR qPCR on page 43 Size selection is optional STOPPING POINT Store the purified DNA in Elution Buffer E1 at 4 C or proceed to Optional Size select the library with a SOLiD Library Size Selection gel or Quantitate the library by performing quantitative PCR qPCR on page 43 as required Optional Size select the library with a SOLiD Library Size Selection gel Choosing to size select the library or not depends on the purity and size of the amplified library There must be no visible small PCR by products in the final amplified library after column purification The library peak size must be acceptable for the type of sequencing to see acceptable library peak sizes for 2 x 60 bp libraries see Evaluate the library on page 37 38 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2 x 60 bp Mate Paired Library Preparation Optional Size select the library with a SOLiD
27. 5 mL LoBind Tube containing Binding Buffer B2 S Binding Buffer B2 S denatures the enzyme and stops the reaction Pre spin an empty PureLink Micro columns in collection tubes at 10 000 x g for 1 minute Verify the column membranes are intact and are not lifted or folded after the spin Load the DNA onto the PureLink Micro columns a Mix the nick translated DNA well in Binding Buffer B2 S with isopropanol 55 b Apply all of the mix to the PureLink Micro column s in collection tube s C Spin the column s at 10 000 x g for 1 minute at room temperature then discard the flow through dsDNA is bound to the column Wash the column s a Return the PureLink Micro column s to the same collection tube s b Add 650 uL of Wash Buffer W1 with ethanol to wash the column s c Spin the column s at 10 000 x g for 1 minute at room temperature then discard the flow through d Spin the column s at 14 000 x g at room temperature to remove residual wash buffer Elute the DNA a Transfer the column s to clean 1 5 mL LoBind tube s b Add 25 uL of Elution Buffer E1 to the center of the column s to elute the DNA then let the column s stand for 1 minute c Spin the column s at 14 000 x g for 1 minute at room temperature d Add the eluate from the last spin back to the column s then let the column s stand for 1 minute e Spin the column s at 14 000 x g for 1 minute at room temperature If necessary
28. 500 Series SOLID Systems User Guide 47 Appendix A Ordering Information Required Applied Biosystems reagent kit Item part no Components 5500 SOLiD Mate Paired Library Enzyme Module 4464419 8 e 10mM dNTP dA dNTP Mix e End Polishing E1 e End Polishing E2 e A tailing Enzyme Il e 5X Reaction Buffer e T4 DNA Ligase 5 U uL DNA Polymerase e Nick Translation Buffer e EDTA 0 5 M e 100X BSA e 10X Plasmid Safe Buffer e Plasmid Safe DNase 10 U uL ATP 100 mM e T7 Exonuclease 10X Buffer 4 e S1 Nuclease e 3M NaCl e S1 Nuc Dilution Buffer 5500 SOLID Mate Paired Amplification Module 4464421 Platinum PCR Amplification Mix SOLID Mate Paired Library Bead amp Buffer Module 4464420 e Dynabeads MyOne Streptavidin C1 Bead Wash Buffer Bead Binding Buffer Bead Dilution Buffer SOLiD Mate Paired Library Oligo Module 4464422 e P1 T Adaptor ds 10 uM e P2 T Adaptor ds 10 uM e Library PCR Primer 1 50 uM Library PCR Primer 2 50 uM MPR Adaptor ds 25 uM e MPL Adaptor ds 25 uM SOLID Library Micro Column Purification Kit 4443751 e Binding Buffer B2 L e Binding Buffer B2 S Wash Buffer Elution Buffer Micro Spin Columns e Elution Tubes 48 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide Appendix A Ordering Information Required equipment Item part no
29. Base Power System Gel imaging system PCR strip tubes Thermal cycler E Gel iBase Power System Microcentrifuge DynaMag 2 Magnetic Rack 2100 Bioanalyzer PCR strip tubes OO 000 00 000 Library PCR Primer 1 Library PCR Primer 2 Platinum PCR Amplification Mix 2 E Gel EX Gel 100 bp DNA ladder Library P1 PCR Primer Library P2 PCR Primer Platinum PCR Amplification Mix DNA 1000 Chip SOLiD Library Micro Column Purification Kit O Thaw Library P1 and P2 PCR Primers on ice O Thaw Platinum PCR Amplification Mix on ice O Thaw Library P1 and P2 PCR Primers on ice O Thaw Platinum PCR Amplification Mix on ice O Thaw DNA 1000 kit reagents on ice E Gel iBase Power System Safe Imager Blue Light E Gel SizeSelect 296 Gel 100 bp DNA ladder Gel Loading Solution Dmm Transilluminator Gel imaging system SOLID Library Micro Column Purification Kit O O O O O O O O O Optional Size select the O Real time thermal cycler O SOLID Library TaqMan Quantitation Kit Quantitate the library by qPCR Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide Appendix E Checklist and workflow tracking form Workflow tracking prepare a 2 x 60 bp mate paired library Workflow tracking prepare a 2 x 60 bp mate paired library Sample Quantitation Lot number Step Quantity of DNA Step Lot number Start
30. DNA a Remove the tube from the DynaMag 2 magnetic rack then add the 50 uL of Elution Buffer E1 to the tube of DNA b Vortex the beads for 15 seconds pulse spin then incubate the beads at room temperature 20 25 C for 23 minutes C Place the tube ina DynaMag 2 magnetic rack for at least 1 minute until the solution clears d Transfer the supernatant to a new 1 5 mL LoBind Tube STOPPING POINT Store the purified DNA in Elution Buffer E1 at 4 C or proceed directly to Add a dA Tail to the digested DNA on page 30 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 29 Chapter 2 2 x 60 bp Mate Paired Library Preparation Add a dA Tail to the digested DNA Add a dA Tail to the digested DNA Adding a dA tail to the S1 nuclease treated DNA by A Tailing Enzyme II increases the Prewash the beads efficiency of ligation to P1 T and P2 T Adaptors 1 Combine to prepare the dA tailing mix Component Amount T7 S1 digested DNA 50 uL Nick Translation Buffer 10 uL dA dNTP Mix 1 0 uL A Tailing Enzyme II 3 0 uL Nuclease free Water 36 0 uL Total 100 pL 2 Incubate the reaction mix at 37 C for 30 minutes Note During incubation you can pre wash the streptavidin beads see Prewash the beads 3 Add 5 0 uL of 0 5 M EDTA to the dA tailing mix to stop the reaction 4 Combine Component Volume Stopped dA tailing mix 105 ul Bead Binding Buffer 200 0 uL Nuc
31. DNA on page 23 Mass of sample DNA too low e Use a fluorescence assay specific for dsDNA to measure the starting DNA see Quantitate and assess sample DNA quality on page 16 Use gt 1 ug DNA sample for library construction Initial size selection too narrow Size select DNA in the gel gt 10 of the targeted insert size For example for a 1 kb insert cut at least 0 9 1 1 kb A gel band of 0 9 1 3 kb is cut routinely Poor genomic DNA quality Check the integrity and purity of the DNA sample by running a small fraction on an agarose gel Need to be familiar with the procedure or refine techniques Practice the protocol with 5 ug of high quality DNA sample Substantial loss of DNA during concentration or purification e f micro columns are used check the column filters to ensure they are seated in the columns and pre spin the columns before use f the Agencourt AMPure XP Reagent Kit is used avoid vortexing the DNA bound beads vigorously during 7096 ethanol washes Unknown Repeat the protocol with more DNA sample For a 1 3 kb insert use as close to 5 ug as possible After 14 cycle amplification no trial PCR product visible Circularized DNA break before nick translation Do not stop between the DNase reaction and the nick translation Mate paired library insert size is gt gt 3 kb Use gt 5 ug genomic DNA or amplify the library beads for gt 14 cyc
32. Library Preparation Optional Quantitate the circularized DNA c Place the tube in a DynaMag 2 magnetic rack for at least 1 minute until the solution clears d Transfer the supernatant to a new 0 2 mL PCR tube IMPORTANT Proceed to the next step immediately Optional Quantitate the circularized DNA Nick translate Nick translate the circularized DNA 26 IMPORTANT If the starting material is 1 2 ug skip this step and proceed immediately to Nick translate the circularized DNA Quantitate the purified DNA using 1 uL of sample with the Qubit dsDNA HS Assay Kits Invitrogen Part no Q32851 and the Qubit 2 0 Fluorometer Invitrogen Part no Q32866 IMPORTANT Proceed to the next step immediately the circularized DNA Nick translation using E coli DNA polymerase I translates the nick into the genomic DNA region The size of the mate paired tags to be produced can be controlled by adjusting the reaction temperature and time For convenience for different mate tag sizes change the reaction time but keep the temperature constant IMPORTANT Incubate the nick translation reaction at 5 C on a thermal cycler using the No heated lid feature DNA polymerase I is very sensitive to slight changes in temperature If your thermocycler does not have a No heated lid feature leave the lid off Before adding enzyme to the reaction mix for nick translation chill the enzyme and the reaction mix separately in a
33. SOLID Systems User Guide 73 74 Appendix E Checklist and workflow tracking form Workflow tracking prepare a 2 x 60 bp mate paired library Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide APPENDIX F Safety This appendix covers General chemical safety cc 66 cc kee eee bee Re ER ead ea ETE 75 SDSS Em 76 Chemical waste safety oi ccs ee e eerte eee diau s RPG Pd eaten 76 Biological hazard safety 0 eee 78 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 WARNING CHEMICAL HAZARD AII chemicals in the instrument including liquid in the lines are potentially hazardous Always determine what chemicals have been used in the instrument before changing reagents or instrument components Wear appropriate eyewear protective clothing and gloves when working on the instrument WARNING CHEMICAL HAZARD Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Wear appropriate eyewear clothing and gloves when handling reagent and waste bottles WARNING CHEMICAL STORAGE HAZARD Never collect or store waste in a glass container because of the risk of br
34. SSIBILITY OF SUCH DAMAGES NOTICE TO PURCHASER DISCLAIMER OF LICENSE The products in this User Guide may be covered by one or more Limited Use Label License s Please refer to the respective product documentation or the Applied Biosystems website under www appliedbiosystems com for the comprehensive license information By use of these products the purchaser accepts the terms and conditions of all applicable Limited Use Label Licenses These products are sold for research use only and are not intended for human or animal diagnostic or therapeutic uses unless otherwise specifically indicated in the applicable product documentation or the respective Limited Use Label License s For information on obtaining additional rights please contact outlicensing alifetech 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 Plasmid Safe is a trademark of EPICENTRE Biotechnologies Covaris is a registered trademark of Covaris Inc HydroShear is a registered trademark of Genomic Solutions Inc NanoDrop is a registered trademark of NanoDrop Technologies SpeedVac is a registered trademark of Thermo Fisher Scientific TaqMan is a registered trademark of Roche Molecular Systems Inc Copyright 2011 Life Technologies Corporation All rights rese
35. USER GUIDE applied biosystems by Life technologies Mate Paired Library Preparation 5500 Series SOLiD Systems Publication Part Number 4460958 Rev A Revision Date March 2011 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 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 PO
36. ace the tube in the DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant Add the entire 400 uL of solution of library DNA in Bead Binding Buffer see Add a dA Tail to the digested DNA on page 30 to the pre washed beads then vortex for 15 seconds Rotate the solution at room temperature 20 25 C for 30 minutes then pulse spin Prepare 1X Reaction Buffer For one sample Component Volume 5X Reaction Buffer 120 uL Nuclease free Water 480 uL Total 600 uL Place the tube with the bead DNA complex in the DynaMag 2 magnetic rack for atleast 1 minute until the solution clears then remove and discard the supernatant Wash the beads 3 times For each wash a Resuspend the beads in 500 uL of Bead Wash Buffer Vortex the beads for 15 seconds 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 Wash and resuspend the beads a Resuspend the beads in 500 uL of 1X Reaction Buffer Vortex the beads for 15 seconds 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 c Resuspend the beads in 86 uL of 1X Reaction Buffer Fragment Library Preparation 5500 Series SOLiD Systems User Guide 31 Chapter 2 2 x 60 bp Mate Paired Lib
37. and unload Covaris microTUBE vials 0 annerer rnnr rrr rrer 59 Overview siria E 61 Choose the appropriate library type 0 0 cece eee eee ete eeeeee 61 Preparation of mate paired libraries 0 000 cece eee cece eeee 62 Sequence orientation from source DNA to sequence Map 0 0000ee cece eee 66 Oligonucleotide Sequences 67 Library construction oligonucleotides 0000 c cece eect eeeees 67 Checklist and workflow tracking form 69 Workflow checklists prepare a 2 x 60 bp mate paired library 2 00 69 Workflow tracking prepare a 2 x 60 bp mate paired library 2222000055 73 c r AAA 75 General chemical safety 2 2 0 cece cece eee eee en 75 II uM C UI tee Regt eee LE 76 Chemical waste safety 1 dg a athe uvis Re Whee 76 Biological haza rd safety ica a REEL EA PLAIRE 78 Documentation and Support 79 Related documentation aia reRPpESCIPUSRPeMD ReRnDerlesetnsed tE 79 Obtaining support occ 79 GlOSSAly iranis aa anaa bane cdccnsiateadtecetane s E esses 81 MAER c m D EE 83 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide Contents Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide 5 Contents 6 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide About This Guide Safety information Note For i
38. aring system 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 You can shear the DNA with two supported shearing systems The Covaris S220 System see Shear the DNA with the Covaris S220 System on page 56 or e The Covaris S2 System see Shear the DNA with the Covaris S2 System on page 57 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 55 Appendix B Supplemental Procedures Shear the DNA for inserts lt 1 kb with the Covaris System Shear the DNA with the Covaris 220 System 1 In a Covaris microTUBE mix Component Amount DNA 1 5 ug 1X TE Buffer Variable uL Total 120 uL 2 Shear the DNA using the Covaris S220 System shearing according to these conditions IMPORTANT Ensure that the Bath Temperature Limit is set to 15 C and keep the bath temperature to lt 10 C Target insert peak Covaris S220 System shearing conditions 700 bp Duty Factor 5 Peak Incident Power PIP 105 Watts Cycles per burst 200 Time 60 seconds Number of cycles 1 Waterbath temperature 6 8 C Bath Temperature Limit 15 C Power mode Frequency sweeping Degassing mode Continuous Water level 12 Water Quality Testing Function Off AFA intensifier Yes 1000 bp Duty Factor 1 Peak Incident Power PIP 175 Watts Cycles per burst 200 T
39. ce At 50 C the DNA denatures and short insert libraries form heteroduplexes Heteroduplexes are deleterious to the library c Add 1 gel volume of isopropanol to the dissolved gel slice For example add 10 uL of isopropanol to 10 mg of gel Mix well 3 Load the DNA onto the column s a Apply the dissolved gel mixture to the Quick Gel Extraction column s in Wash Tube s Use one column per 400 mg agarose or load 2000 uL of dissolved gel mixture per column Use more columns if necessary Fragment Library Preparation 5500 Series SOLiD Systems User Guide 19 Chapter 2 2 x 60 bp Mate Paired Library Preparation Quantitate the sheared purified DNA b Spin the column s at gt 12 000 x g for 1 minute at room temperature then discard the flow through and place the column back on the Wash Tube s 4 Wash the column s a Add 500 uL of Wash Buffer W1 with ethanol to the Quick Gel Extraction column s b Spin the column s at gt 12 000 x g for 1 minute at room temperature then discard the flow through c Spin the Quick Gel Extraction column s again at maximum speed for 2 minutes to remove any residual Wash Buffer 5 Elute the DNA a Transfer the Quick Gel Extraction column s to clean 1 5 mL LoBind tube s b Add 50 uL of Elution Buffer E5 and incubate the sample at room temperature for 5 minutes c Spin the column s at gt 12 000 x g for 1 minute at room temperature The 1 5 mL LoBind tube s conta
40. e Selection gel Load the library page 28 Run the SOLiD Library Size Selection gel and collect the library fragment page 40 Purify the DNA with the SOLID Library Micro Column Purification Kit SUM page 42 Check the size distribution of the library page 43 Stopping point Quantitate the library by performing quantitative PCR qPCR page 43 Varies with laboratory practice Stopping point Procedural guidelines The protocol is designed for 1 5 ug starting genomic DNA For optimal complexity the required amount of starting DNA depends on the size of the genome and the required sequencing depth For high 30X or more sequence coverage of a human genome 3 Gb use 5 ug starting DNA For low sequence coverage such as 2 3X of a human genome or 30X sequence coverage of a 500 Mb genome use 1 2 ug DNA and about the same number of amplification cycles as for 5 ug input e 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 just before use Fragment Library Preparation 5500 Series SOLiD Systems User Guide 15 Chapter 2 2 x 60 bp Mate Paired Library Preparation Quantitate and assess sam
41. e handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions 54 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide APPENDIX B Supplemental Procedures This appendix covers Shear the DNA for inserts lt 1 kb with the Covaris System ce eee eee 55 Load and unload Covaris microTUBE Vials cceeeeeeeeeeeueees 59 Shear the DNA for inserts lt 1 kb with the Covaris System Shea ring Perform a small scale shearing trial before large scale shearing if DNA is guidelines available e Adjust shearing conditions according to different organisms e Calibrate the shearing run to assess the shearing efficacy of the device before starting the first library preparation Quantitate the 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 Quant iT dsDNA HS Assay Kit Invitrogen Part no Q32851 or Q32854 or e Invitrogen Quant iT dsDNA BR Assay Kit Invitrogen Part no Q32850 or 32853 or Invitrogen Quant iT PicoGreen dsDNA Assay Kit Invitrogen Part no P7589 Choose the IMPORTANT If you are using the Covaris System set the chiller temperature to she
42. e safety Index IMPORTANT description 7 isolate the circularize the DNA 24 L library preparation 11 ligate P1 T and P2 T Adaptors to the DNA 32 M MSDS See SDS multiple fragment libraries shear the DNA 16 55 N nick translate and trial amplify the library 33 nick translate the circularized DNA 26 0 oligonucleotide sequences 67 ordering information 47 P product information 9 product purpose of 9 Q quantitate the library by qPCR 43 R radioactive waste handling 77 S safety 75 biological hazards 78 chemical 75 chemical waste 76 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide 83 Index guidelines 75 76 77 SDSs about 8 description 76 obtaining 76 79 size select the DNA 18 size select the library 39 supplemental procedures 55 1 training information on 79 W WARNING description 7 waste disposal guidelines 77 waste profiles description 77 84 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide V8560977 Headquarters a 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 o For support visit www appliedbiosystems com support technologies www lifetechnologies com
43. eaking 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 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 76 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 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 75 Appendix F Safety SDSs Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood For additional safety guidelines consult the SDS Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS 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
44. ence assay page 16 4 Varies with laboratory Run an agarose gel to assess sample DNA quality page 18 practice Fragment Library Preparation 5500 Series SOLiD Systems User Guide 11 Chapter 2 2 x 60 bp Mate Paired Library Preparation Workflow Shear the DNA with the HydroShear DNA Shearing Device Shearing conditions page 17 Shear the DNA page 17 Size select the DNA Prepare an agarose gel page 18 Run the agarose gel page 18 Excise the sample from the agarose gel page 18 Elute the DNA using the SOLID Library Quick Gel Extraction Kit page 19 Quantitate the sheared purified DNA page 20 Stopping point End repair the DNA page 20 Ligate MP Adaptors to the DNA Calculate the amount of adaptor to use page 21 i Ligate the MP Adaptors to the DNA page 22 Purify the DNA using Agencourt AMPure XP Reagent page 22 Quantitate the ligated purified DNA page 23 y Assess the recovery of DNA page 23 Stopping point 12 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2 x 60 bp Mate Paired Library Preparation Workflow Circularize the DNA by intra molecular hybridization page 23 y Isolate the circularized DNA Treat the DNA with Plasmid Safe DNase page 24 y Purify the DNA using Agencourt AMPure XP Reagent page 25 Optional Quantitate the circularized DNA page 26 2h y Nick translate the circularized DNA Nick t
45. entical to the hazard symbols that are affixed to Applied Biosystems instruments Mate Paired 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 76 IMPORTANT For the SDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer 8 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide CHAPTER 1 About the Products For a more detailed overview of library types that can be sequenced on the 5500 Series SOLiD Sequencers see Choose the appropriate library type on page 61 Library preparation Library preparation is the first step in which samples are adapted for sequencing on the 5500 Series SOLiD Sequencers During library preparation forward and reverse adaptors are added to the ends of DNA inserts The bead is for illustration purposes only and is not added until the bead preparation step Mate Paired Library Sequence up to 60 bp gt Sequence up to 60 bp P1 T Adaptor Mate Pair Tag E Mate Pair Tag j ptor Fragment Library mm Sequence up to 75 bp Sequence up to 35 bp fa gt Sequence 5 or 10 bp P1 T Adaptor DNA Fragment Bor Product information Purpose of the To prepare mate paired libraries for sequencing
46. eparation 5500 Series SOLID Systems User Guide 49 Appendix A Ordering Information Required equipment Product Namet Vendor Safe Imager 2 0 Blue Light Invitrogen Transilluminator 66600 id Invitrogen Safe Imager Blue Light Transilluminator 537102 Qubit 2 0 Fluorometer Invitrogen Q32866 Gel imaging system Major Laboratory Supplier MLS Tabletop Centrifuge MLS Sample concentrator SpeedVac MLS Concentrator Gel boxes and power supplies for agarose MLS gels Vortexer MLS PicoFuge Microcentrifuge MLS Incubator 37 C MLS Incubator 70 C MLS Incubator 75 C MLS Scale MLS Timer 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 i For more information on the HydroShear DNA Shearing Device and materials refer to the manufacturer s documentation 8 Optional if used for all DNA fragmentation 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 Mate Paired Library Preparation 5500 Series SOLiD Systems User G
47. he library is amplified using primers specific to the P1 T and P2 T Adaptors see Figure 4 on page 65 These primers can be used only for library amplification and not for alternative or modified library construction adaptor design because they do not have 3 sequences compatible with the sequencing primers 64 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide Appendix C Overview C Preparation of mate paired libraries Figure 4 Mate paired library amplification design AT JUBEPVECPBUBECUCOCEUDCOCCECOVCwO LUCO JB ULULUEVUCECDVLUODUBLUCUUCGUBECLUVLUNLUUJOVLLO P1 T Adaptor P1 T Adaptor fag 1 Tag 2 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide 65 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 Um v Circularization with Internal Adaptor and Nick Translation Nick translation goes beyond the length of the read Sequencing of the F3 a M fragment on a bead P1 Ja LEER 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 The reads are expected to map at a distance equal to the average fragment length plus the average nick translation distance The sequencing direction for mate pair libraries i
48. ications Go to Mate paired e Two DNA insert tags e De novo sequencing 2x 60 bp Mate Paired 700 bp 3 kb apart Separated by an internal adaptor More input DNA required 1 5 ug 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 primary library Genomic resequencing primary library Methylation analysis Library Preparation on page 11 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 61 Appendix C Overview Preparation of mate paired libraries Library type Features Applications Go to Fragment Appropriate for Targeted Refer to the Fragment sequence lengths resequencing Library Preparation lt 300 bp primary library 5500 Series SOLiD Adaptors on each end of sheared DNA Genomic resequencing Systems User Guide Part no 4460960 insert e Methylation analysis e 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 The type of library used depends on the application and info
49. ientific PR 1 Filtered pipettor tips8 Major Laboratory Supplier MLS Razor blades MLS 15 mL conical polypropylene tubes 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 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 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 53 Appendix A Ordering Information Optional consumables Optional consumables Product namett Vendor 1X TE Buffer Invitrogen 12090 015 SOLID Library Size Selection Gel Applied Biosystems 4443733 Agilent High Sensitivity DNA Kit Agilent Technologies 5067 4626 t Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance t For the SDS of any chemical not distributed by Applied Biosystems contact the chemical manufacturer Befor
50. ilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents SDSs certificates of analysis and other related documents e Download PDF documents Obtain information about customer training Download software updates and patches Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 79 Documentation and Support Obtaining support 80 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide fragment library internal adaptor IA library Library PCR Primer 1 Library PCR Primer 2 mate paired library MP Adaptor Adaptors MPR and MPL P1 T Adaptor P2 T Adaptor Glossary 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 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
51. ime 45 seconds Number of cycles 2 Waterbath temperature 6 89C Bath Temperature Limit 15 C Power mode Frequency sweeping Degassing mode Continuous Water level 12 Water Quality Testing Function Off AFA intensifier Yes 3 Transfer 120 uL of sheared DNA into a clean 1 5 mL LoBind tube 56 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide Appendix B Supplemental Procedures Shear the DNA for inserts lt 1 kb with the Covaris System 4 Reduce the volume of the sheared DNA to 40 60 uL in order to load the sample into a 1 cm wide well on a size selection gel Use a Speed Vac Concentrator or equivalent instrument Note Ifa SpeedVac Concentrator is not available and there is 22 ug of DNA to concentrate then use the SOLiD Library Micro Column Purification Kit with B2 S Buffer After the wash elute the purified sheared DNA with 50 uL Elution Buffer E1 5 Proceed to Measure the amount of sheared purified DNA on page 59 Shear the DNA with the Covaris S2 System 1 Prepare the Covaris S2 Tank a Ensure that the water in the Covaris S2 tank is filled with fresh deionized water to fill line level 12 on the graduated fill line label The water should cover the visible glass part of the tube 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 2076 ethylene glycol
52. in the purified DNA d Add the eluate from the last spin back to the Quick Gel Extraction column s then let the column s stand for 5 minutes e Spin the column s at gt 12 000 x g for 1 minute at room temperature 6 If more than one gel extraction column is used pool the eluted DNA then reduce the total volume to 70 uL with a SpeedVac Concentrator or equivalent method Quantitate the sheared purified DNA Quantitate the purified DNA using 1 uL of sample with the Qubit dsDNA HS Assay Kits Invitrogen Part no Q32851 and the Qubit 2 0 Invitrogen Part no Q32866 STOPPING POINT Store the purified DNA in Elution Buffer E5 at 4 C or proceed directly to End repair the DNA End repair the DNA For fast and efficient blunt ended ligation End Polishing E2 enzyme is used to convert DNA with damaged or incompatible 5 protruding and or 3 protruding ends to 5 phosphorylated blunt ended DNA End Polishing E1 enzyme and ATP are also included for phosphorylation of the 5 ends of the blunt ended DNA for subsequent ligation 1 For S ug of starting material combine and mix the components below in a LoBind tube If gt 5 ug of starting material for mate paired libraries with 1 3 kb inserts scale up or set up parallel reactions 20 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2 x 60 bp Mate Paired Library Preparation Ligate MP Adaptors to the DNA
53. ing Amount 5500 SOLID Mate Paired Library Standard Adaptors Shearing the DNA 5500 SOLID Mate Paired Library Kit Size selection Platinum PCR Amplification Mix Ligation of NIC Adaptors Circularization Quantitative PCR Sample Quantitation Lot number Step Quantity of DNA Step Lot number Starting Amount 5500 SOLiD Mate Paired Library Standard Adaptors Shearing the DNA 5500 SOLID Mate Paired Library Kit Size selection Platinum PCR Amplification Mix Ligation of NIC Adaptors Circularization Quantitative PCR Sample Quantitation Lot number Step Quantity of DNA Step Lot number Starting Amount 5500 SOLID M Mate Paired Library Standard Adaptors Shearing the DNA Size selection 5500 SOLiD Mate Paired Library Kit Platinum PCR Amplification Mix Ligation of NIC Adaptors Circularization Quantitative PCR Sample Quantitation Lot number Step Quantity of DNA Step Lot number Starting Amount Shearing the DNA 5500 SOLiD Mate Paired Library Standard Adaptors 5500 SOLID Mate Paired Library Kit Size selection Platinum PCR Amplification Mix Ligation of NIC Adaptors Circularization Quantitative PCR Mate Paired Library Preparation 5500 Series
54. it contents and storage temperatures Kit contents The 5500 SOLiD Mate Paired Library Construction Kit contains materials sufficient to prepare 12 mate paired libraries Part Description Storage temperature 5500 SOLiD Mate Paired Library One each 20 C Enzyme Module 5500 SOLiD Mate Paired Library One each 20 C Amplification Module 5500 SOLID Mate Paired Library One each Lo Bead amp Buffer Module SOLID Mate Paired Library Oligo One each 20 C Module SOLID Library Micro Column One each Room temperature Purification Kit SOLID Library Quick Gel Extraction One each Room temperature Kit 10 Fragment Library Preparation 5500 Series SOLID Systems User Guide CHAPTER 2 2 x 60 bp Mate Paired Library Preparation For an overview of library types that can be sequenced on the 5500 Series SOLiD Sequencers see Choose the appropriate library type on page 61 Workflow This chapter describes the method to prepare a mate paired library with a 1 3 kb insert size Mate paired libraries with a 600 bp 1 kb insert size can also be prepared without modification But mate paired libraries with gt 3 kb inserts may need modification for optimal library construction For a graphical overview of mate paired library preparation see Preparation of mate paired libraries on page 62 Total Steps estimated time Quantitate and assess sample DNA quality Quantitate the DNA with a fluoresc
55. l on a Safe Imager Blue Light Transilluminator with a ruler lying on top of the transilluminator IMPORTANT Exposing DNA to UV light may damage the DNA Using SYBR Safe gel stain and the Safe Imager Blue Light Transilluminator eliminates the risk of UV damage to DNA during size selection Using the ladder bands and the ruler for reference cut the band from the gel with a clean razor blade Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2x 60 bp Mate Paired Library Preparation Size select the DNA For example cut a band 0 9 1 3 kb for a 1 kb insert from a 1 agarose gel 1 6Kb gt 1 6Kb gt 1Kb 1Kb For a narrower size selection make a tighter cut For a wider size distribution make a broader cut such as 0 8 1 4 kb 3 Ifthe gel piece is large then slice it into smaller pieces Elute the DNA 1 Weigh the gel slice If the gel slice 2 200 mg elute the gel slice in a 15 mL using the SO Lip polypropylene conical colorless tube If the gel slice is lt 200 mg elute the gel slice Library Quick Gel in a 1 5 mL LoBind Tube Extraction Kit 2 Dissolve the gel a Add 30 uL of Gel Solubilization Buffer L3 for every 10 mg of gel b Dissolve the gel slice by vortexing the tube a few times during incubation at room temperature until the gel slice has dissolved completely The gel slice dissolves in 15 minutes IMPORTANT Do not heat the gel to dissolve the gel sli
56. lease free Water 95 0 uL Total 400 uL Bind the library molecules to streptavidin beads Dynabeads MyOne Streptavidin C1 specifically bind to the biotin labeled MP Adaptor in the library molecules to purify the library from side products 1 Prepare 1X BSA solution Component Volume 100X BSA 5uL Nuclease free Water 495 uL Total 500 uL 2 Vortex the tube of Dynabeads MyOne Streptavidin C1 then transfer 50 uL of the beads into a 1 5 mL LoBind Tube Fragment Library Preparation 5500 Series SOLID Systems User Guide Bind the library DNA molecules to the beads Wash the bead DNA complex Chapter 2 2 60 bp Mate Paired Library Preparation Bind the library molecules to streptavidin beads Add 500 uL of Bead Wash Buffer to the 50 uL of solution of beads vortex the beads for 15 seconds then pulse spin Place the tube in the DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant Add 500 uL of 1X BSA and vortex for 15 seconds then pulse spin the tube Place the tube in the DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant Add 500 uL of Bead Binding Buffer Vortex the beads for 15 seconds then pulse spin IMPORTANT Proceed to step 8 only after the A tailing of the DNA is stopped see step 4 of Add a dA Tail to the digested DNA on page 30 Pl
57. les Need to be familiar with the procedure or refine techniques Practice the protocol with 5 ug of high quality DNA sample Substantial loss of DNA during concentration or purification e f micro columns are used check the column filters to ensure they are seated in the columns and pre spin the columns before use f the Agencourt AMPure XP Reagent Kit is used avoid vortexing the DNA bound beads vigorously during 7096 ethanol washes Unknown Repeat the protocol with more DNA sample For a 1 3 kb insert use as close to 5 ug as possible 44 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2 x 60 bp Mate Paired Library Preparation Troubleshooting Observation Possible cause Recommended action A library from a trial PCR shows large molecular weight gt 1 kb products in addition to mostly the expected size PCR products Carry over of linear fragment or of non mate paired fragments by streptavidin beads Proceed to final PCR amplification The large molecular weight by products usually are reduced in the final PCR and a small fraction of large molecular weight PCR by products does not affect emulsion PCR ePCR and sequencing results Trial PCR library size is too small 250 bp Nick translation did not work as expected e f the PCR library size is 200 250 bp consider 2 x 50 mate paired sequencing ncrease the nick tran
58. m storage or at 20 C for long term storage Or proceed to Check the size distribution of the library 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 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 Quantitate the library by performing quantitative PCR qPCR For accurate library quantitation quantitative PCR is strongly recommended For a protocol using the SOLiD Library TaqMan Quantitation Kit Part no 4449639 refer to the Applied Biosystems SOLiD Library TaqMan Quantitation Kit protocol Invitrogen Part no A12120 STOPPING POINT Store the purified DNA in Elution Buffer E1 at 20 C or proceed directly to emulsion PCR as describe in the SOLiD EZ Bead Emulsifier Getting Started Guide Part no 4441486 Fragment Library Preparation 5500 Series SOLiD Systems User Guide 43 Chapter 2 2 x 60 bp Mate Paired Library Preparation Troubleshooting Troubleshooting Observation Possible cause Recommended action lt 50 ng DNA recovered before circularization see Assess the recovery of
59. mportant instrument safety information refer to the 5500 Series SOLiD Sequencers User Guide Part no 4456991 For general safety information see this section and Appendix F Safety on page 75 When a hazard symbol and hazard type appear by a chemical name or instrument hazard see the Safety Appendix for the complete alert on the chemical or instrument Safety alert words Four safety alert words appear in Applied Biosystems user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of Observation or action as defined below IMPORTANT Indicates information that is necessary for proper instrument operation accurate chemistry kit use or safe use of a chemical CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANTS each safety alert word in an Applied Biosystems document appears with an open triangle figure that contains a hazard symbol These hazard symbols are id
60. n Shear the DNA for inserts lt 1 kb with the Covaris System on page 55 1 kb HydroShear Standard Shearing e SC2 Assembly e 20 cycles 2 kb HydroShear Standard Shearing e SC9 Assembly e 20 cycles 3 kb HydroShear Standard Shearing e SC13 Assembly e 20 cycles Speed code SC 2 Shear the DNA 1 Fragment Library Preparation 5500 Series SOLID Systems User Guide In 1 5 mL LoBind Tubes dilute 1 5 ug of DNA to 150 uL with Nuclease free Water For better coverage of large and complex genomes use 5 ug of DNA On the Edit Wash Scheme tab specify the solution and cycles e 2 cycles of WS1 0 2 N HCl 2 cycles of WS2 0 2 N NaOH e 3 cycles of Nuclease free Water Run the wash scheme on the HydroShear DNA Shearing Device Adjust the speed code SC and number of cycles according to the table in Shearing conditions on page 17 and adjust the volume setting to 150 uL Shear the DNA Run the wash scheme after DNA shearing is complete to clean the shearing device Reduce the volume of the sheared DNA to 40 60 uL in order to load the sample into a 1 cm wide well on a size selection gel Use a Speed Vac Concentrator or equivalent instrument Note If a SpeedVac Concentrator is not available and there is 22 ug of DNA to concentrate then use the SOLiD Library Micro Column Purification Kit with B2 S Buffer After the wash elute the purified sheared DNA with 50 uL Elution Buffer E1
61. nd Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 81 Glossary tag There are two uses for this term Sequencing data from a single bead with a single primer set sometimes used interchangeably with read 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 respectively templated bead Process of covalently attaching and clonally amplifying template strands to beads by preparation 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 82 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide A about the kit 9 11 Add a dA Tail to the digested DNA 30 amplify the library 35 bind the library molecules to streptavidin beads 30 biohazardous waste handling 78 C CAUTION description 7 checklists and workflow tracking forms 69 chemical hazard warning 75 chemical safety 75 chemical waste safety 76 77 circularize the DNA 23 D DANGER description 7 digest the DNA 28 documentation related 79 E end repair the DNA 20 G glossary 81 guidelines chemical safety 75 chemical waste disposal 76 chemical waste safety 77 H hazard warning chemical 75 hazards Se
62. nded see Shear the DNA for inserts lt 1 kb with the Covaris System on page 55 To shear for a mate paired library with insert sizes between 1 kb and 6 kb the HydroShear DNA Shearing Device is recommended Optional Use the Covaris System instead of the HydroShear DNA Shearing Device to generate kb DNA fragments see Shear the DNA for inserts lt 1 kb with the Covaris System on page 55 Follow the manufacturer s guidelines for shearing Use the appropriate tubes for the targeted size fragments Concentrate the sheared DNA to 40 60 uL with a SpeedVac Concentrator or SOLiD Library Micro Column Purification Kit to load the concentrated DNA into a single 1 cm lane of a size selection gel see the volume reducing conditions in Shear the DNA Fragment Library Preparation 5500 Series SOLID Systems User Guide Shear the DNA with the HydroShear DNA Shearing Device Chapter 2 2 x 60 bp Mate Paired Library Preparation Shear the DNA Shearing guidelines Perform a small scale shearing trial before large scale shearing if DNA is available Adjust shearing conditions as needed especially when working with organisms whose DNA have high or low GC content Calibrate the shearing run to assess the shearing efficacy of the device before starting the first library preparation Shearing conditions Insert size Shearing method Shearing conditions lt 1 kb Follow the shearing conditions i
63. nt 3 Wash and resuspend the DNA bead complex a Resuspend the DNA bead complex in 500 uL of Elution Buffer E1 Vortex the DNA bead complex for 15 seconds then pulse spin b Place the tube in the DynaMag 2 magnetic rack for 21 minute until the solution clears then remove and discard the supernatant c Resuspend the DNA bead complex in 30 uL of Elution Buffer E1 STOPPING POINT Store the purified DNA in Elution Buffer E1 at 4 C or proceed directly to Nick translate and trial amplify the library on page 33 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2 x 60 bp Mate Paired Library Preparation Nick translate and trial amplify the library Nick translate and trial amplify the library The ligated purified DNA undergoes nick translation during PCR with Platinum PCR Amplification Mix Next the library is trial amplified using Library PCR Primers 1 and 2 with the Platinum PCR Amplification Mix Trial amplification determines the number of PCR cycles to be used for final library amplification without overamplification Choose the number of PCR cycles from the trial PCR so that the amplified library is just visible on 2 E Gel EX Gel Perform trial PCR 1 Prepare a PCR master mix for amplification reactions on the library Component Volume Platinum PCR Amplification Mix 70 0 uL Library PCR Primer 1 50 uM 1 4 uL Library PCR Primer 2 50 uM
64. on the 5500 Series SOLID Sequencers product or SOLiD 4 System use the 5500 SOLiD Mate Paired Library Construction Kit Part no 4464418 Fragment Library Preparation 5500 Series SOLID Systems User Guide 9 Chapter 1 About the Products Kit contents and storage temperatures Use the 5500 SOLiD Mate Paired Library Construction Kit and the adaptors to Prepare a 2 x 60 bp mate paired DNA library The mate paired library is sequenced on the SOLiD 3 and 4 Systems and the 5500 Series SOLiD Sequencers A mate paired library consists of pairs of DNA fragments that are mates because they originated from the two ends of the same genomic DNA fragment Mate paired adaptors MPR and MPL adaptors form an internal adaptor to connect the DNA mate pair together Prepare mate paired libraries with shorter mate tags such as 2 x 35 bp and 2 x 50 bp For shorter mate paired libraries use shorter nick translation times The longer 2 x 60 bp mate paired library can however be sequenced as a 2 x 35 bp or 2 x 50 bp mate paired library Prepare mate paired libraries with 1 3 kb insert size Mate paired libraries with a 600 bp 1 kb insert size can also be prepared without modification But mate paired libraries with 3 kb inserts may need modification for optimal library construction Increase mapping specificity over standard fragment library sequencing Detect large structural variations in the genome Bridge sequencing gaps K
65. ple DNA quality Quantitate and assess sample DNA quality Quantitate the DNA with a fluorescence assay Run an agarose gel to assess sample 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 Invitrogen Qubit dsDNA HS Assay Kit Invitrogen Part no Q32851 or Q32854 or Invitrogen Qubit dsDNA BR Assay Kit Invitrogen Part no Q32850 or Q32853 Or Invitrogen Quant iT PicoGreen dsDNA Assay Kit Invitrogen Part no P7589 1 Runa fraction of the sample DNA on an agarose gel 2 Inspect the gel bands for sample DNA quality DNA quality penne p D Observation Possible cause Mecommenten SCHONE pr consequences Smear at bottom of gel RNA Remove excess RNA Long smear below high molecular Severe damage to sample DNA e High risk of library preparation weight band failure Ladder pattern for genomic DNA sample e Lower library yield e Low coverage during sequencing Use another undamaged DNA sample Shear the DNA Shear the DNA with the Covaris System 16 The DNA is sheared to yield 700 bp to 3 kb fragments To shear for a mate paired library with insert sizes between 700 bp and 1 kb the Covaris System is recomme
66. pool the eluted DNA into one 1 5 mL LoBind Tube Fragment Library Preparation 5500 Series SOLiD Systems User Guide 27 Chapter 2 2 x 60 bp Mate Paired Library Preparation Digest the DNA with T7 Exonuclease and S1 Nuclease STOPPING POINT Store the purified DNA in Elution Buffer E1 at 4 C or proceed directly to Digest the DNA with T7 Exonuclease and S1 Nuclease Digest the DNA with T7 Exonuclease and S1 Nuclease Digest the DNA with T7 exonuclease Digest the circularized DNA with S1 Nuclease 28 T7 exonuclease recognizes the nicks within the circularized DNA With its 5 3 exonuclease activity T7 exonuclease digests the unligated strand away from the tags creating a gap in the sequence This gap creates an exposed single stranded region that is more easily recognized by 51 Nuclease so the mate paired tags can be cleaved from the circularized template 1 Combine Component DNA Amount 25 uL 10X Buffer 4 5 0 uL T7 Exonuclease 2 0 uL Nuclease free Water 18 0 uL Total 50 uL 2 Incubate the reaction mixture at 37 C for 15 minutes 3 Heat inactivate the T7 exonuclease at 70 C for 20 minutes 4 Chill the reaction on ice for 5 minutes 1 Freshly dilute 1 uL of S1 Nuclease to 50 U uL with S1 Nuc nuclease Dilution Buffer 2 Combine Component Amount DNA 50 uL 3 M NaCl 1 7 uL S1 Nuclease 2 0 uL Total 53 7 uL 3 Incubate the reac
67. 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 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 5 Series instrument Unload Covaris 1 Use a thumb to push the stainless steel plunger up into the body of the microTUBE vials microTUBE holder to relieve pressure on the cap Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 59 60 Appendix B Supplemental Procedures Load and unload Covaris microTUBE vials 2 Press against the side of the glass tube not against the cap to free the microTUBE from the grip of the holder Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide APPENDIX C Overview This appendix covers Choose the appropriate library type 1 6 eee 61 Preparation of mate paired libraries oooooooomommmmmmmmmmmoooo 62 Sequence orientation from source DNA to sequence MapP ooooooooooo o 66 Choose the appropriate library type TM These are the types of libraries that can be sequenced on the 5500 Series SOLiD Sequencers Library type Features Appl
68. ranslate the circularized DNA page 26 Purify the DNA with the SOLiD Library Micro Column Purification Kit page 27 Stopping point Digest the DNA with T7 Exonuclease and S1 Nuclease Digest the DNA with T7 exonuclease page 28 4 Digest the circularized DNA with S1 Nuclease page 28 i 2h Purify the DNA using Agencourt AMPure XP Reagent page 29 Stopping point Fragment Library Preparation 5500 Series SOLiD Systems User Guide 13 Chapter 2 2 x 60 bp Mate Paired Library Preparation Workflow Add a dA Tail to the digested DNA page 30 4 Bind the library molecules to streptavidin beads Prewash the beads page 30 Bind the library DNA molecules to the beads page 31 Wash the bead DNA complex page 31 Ligate P1 T and P2 T Adaptors to the DNA page 32 Stopping point Nick translate and trial amplify the library Perform trial PCR on the library page 33 lt 1 5h Confirm library amplification with a 2 E Gel9 EX Gel page 34 Stopping point Nick translate and amplify the library Perform PCR on the library page 36 Purify the DNA with the SOLiD Library Micro Column Purification Kit page 36 Stopping point Evaluate the library page 37 Stopping point 14 Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2 x 60 bp Mate Paired Library Preparation Procedural guidelines Optional Size select the library with a SOLiD Library Siz
69. rary Preparation Ligate P1 T and P2 T Adaptors to the DNA Ligate P1 T and P2 T Adaptors to the DNA Note P1 T and P2 T Adaptors have a 3 T overhang Previous SOLiD P1 and P2 Adaptors do not The adaptor sequences in the final ligated library with the P1 T and P2 T Adaptors are the same as the SOLiD P1 and P2 Adaptors which are used for preparing the library for sequencing with the SOLiD 4 System P1 T and P2 T Adaptors are ligated to the ends of the end repaired DNA The P1 T and P2 T Adaptors are included in double stranded form in the SOLiD Mate Paired Library Standard Adaptors module The ligated library molecules are bound to streptavidin beads washed and purified from ligation by products 1 Ligate the P1 T and P2 T Adaptors to the bead bound DNA a Combine Component Volume DNA bead complex 86 uL P1 T Adaptor ds 10 uM 2 0 uL P2 T Adaptor ds 10 uM 2 0 uL T4 DNA Ligase 5 U uL 10 0 uL Total 100 uL b Rotate the reaction mixture at room temperature 20 25 C for 30 minutes C Place the tube in the DynaMag 2 magnetic rack for 21 minute until the solution clears then remove and discard the supernatant 2 Wash the DNA beads 3 times For each wash a Resuspend the beads in 500 uL of Bead Wash Buffer Vortex the beads for 15 seconds then pulse spin b Place the tube in the DynaMag 2 magnetic rack for 21 minute until the solution clears then remove and discard the supernata
70. rmation 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 SOLiD System website www appliedbiosystems com solid5500 or contact your field applications specialist Preparation of mate paired libraries A mate paired library consists of pairs of DNA fragments that are mates because they originated from the two ends of the same genomic DNA fragment The mate paired ends are connected together through an internal adaptor MP Adaptor to form a circle For the 5500 Series SOLiD Sequencers the preparation of a mate paired library involves use of a circularization method that is different from the circularization method used for mate paired library preparation with previous versions of the SOLiD System The new circularization method improves circularization efficiency by several fold For long mate paired libraries for example to produce sequencing reads 2 x 60 nt size selected genomic DNA fragments are ligated to new intramolecular circularization MPR and MPL adaptors then self circularized by hybridization in a very dilute solution see Figure 2 on page 63 The resulting DNA circle has one nick in each strand because the MP Adaptor does not have the 5 phosphate in
71. rved Part Number 4460958 Rev A 03 2011 Contents About This GUIAS occ criticas 7 Safety information ie hse pea de dert A Tuo ae duo e ee obe R AA 7 CHAPTER 1 About the Products 2 eorr aa 9 Library preparation siere atopei EBD GER Ch een ala ale ale RD alo o a seme e o koe 9 Product iNforMAtION 2 45 vue RENE ped abd og elf ducati et dh 9 Kit contents and storage temperatures 0 0c cece cette ees 10 CHAPTER 2 2 x 60 bp Mate Paired Library Preparation 11 WOFKIIOW ies ones eee e Fa E re e medetur de Porn cu ues 11 Proced ral guidelines 2 od ds Ep WERDEN b un ERIS Pul ben 15 Quantitate and assess sample DNA quality 16 Shearithie DNA co deae re peior videa idee PI die acu tan ee fe iaa ds 16 Sizezselect Me DNA cuotas lied Ming ees v ee US Re 18 Quantitate the sheared purified DNA sssssssssssssss eee 20 End repair the DNA 0000 cece ccc m n 20 Ligate MP Adaptors to the DNA ssssssssssssssss eee eee 21 Quantitate the ligated purified DNA cece eee III 23 Assess the recovery of DNA 0 00 c eee sse 23 Circularize the DNA by intra molecular hybridization 2 20020000e eee eee 23 Isolate the circularized DNA sssssssssssssssss ee 24 Optional Quantitate the circularized DNA 0 0 6 cece eee eens 26 Nick translate the circularized DNA cc eee cee nett eens 26 Digest the DNA with T7 Exonuclease and S1 Nuclease
72. s b Spin the column s at 10 000 x g for 1 minute then discard the flow through dsDNA is bound to the column C If necessary apply the sample in the binding buffer to the PureLink Micro column s in collection tube s d Spin the column s at 10 000 x g for 1 minute then discard the flow through dsDNA is bound to the column e Ensure that the entire sample has been loaded onto the column s 4 Wash the column s a Return the PureLink Micro column s to the same collection tube s b Add 650 uL of Wash Buffer W1 with ethanol to wash the column s c Spin the column s at 10 000 x g for 1 minute then discard the flow through d Spin the column s at 14 000 x g to remove residual wash buffer 5 Elute the DNA a Transfer the column s to clean 1 5 mL LoBind tube s b Add 20 uL of Elution Buffer E1 to the center of the column s to elute the DNA then let the column s stand for 1 minute c Spin the column s at 14 000 x g for 1 minute d Add the eluate from the last spin back to the column s then let the column s stand for 1 minute e Spin the column s at 14 000 x g for 1 minute 6 If necessary pool the eluted DNA into one 1 5 mL LoBind Tube Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2 x 60 bp Mate Paired Library Preparation Check the size distribution of the library STOPPING POINT Store the purified DNA in Elution Buffer E1 at 4 C for short ter
73. s 5 to 3 Paired end ae Pus Sheared size selected DNA v Sequencing of the Bn F5 Bm 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 66 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide APPENDIX D Oligonucleotide Sequences Library construction oligonucleotides Adaptor sequences Note The internal adaptor used for DNA mate paired libraries is different from the internal adaptor used for RNA libraries Adaptor and primer sequences Length nt P1 T Adaptor 50 uM 5 CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT 3 41 5 TCACCGACTGC CCATAGAGAGGAAAGCGGAGGCGTAGTGGCC 3 42 P2 T Adaptor 50 uM 5 GAGAATGAGGAACCCGGGGCAGCC 3 24 5 CIGCCCCGGGTTCCTCATTCTCT 3 23 Library PCR Primer 1 50 uM 5 CCACTACGCCTCCGCTTTCCTCTCTATG 3 28 Library PCR Primer 2 50 uM 5 CTGCCCCGGGTTCCTCATTCT 3 21 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 67 68 Appendix D Oligonucleotide Sequences Library construction oligonucleotides Mate Paired Library Preparation 5500 Series SOLiD Systems U
74. ser Guide APPENDIX E Checklist and workflow tracking form Workflow checklists prepare a 2 x 60 bp mate paired library 69 Workflow tracking prepare a 2 x 60 bp mate paired library 73 Workflow checklists prepare a 2 x 60 bp mate paired 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 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 69 70 Appendix E Checklist and workflow tracking form Workflow checklists prepare a 2 x 60 bp mate paired library Equipment Reagents Preparation steps O Covaris S220 System O 1M Tris pH 8 0 Degas the water in the eg 9 gO Covaris Tubes and Caps O Ethylene glycol Covaris S220 System 30 22028490 Qubit 2 0 Fluorometer O UltraPure Glycerol minutes prior to use s A f O SpeedVac Concentrator Supplement the circulated 50 O water chiller with 20 ethylene glycol O HydroShear DNA O Nuclease free Water E eg Shearing Device O 0 2N HCI A Sg5 H Qubit 2 0 Fluorometer L1 0 2 N NaOH 25522 O 1 5 mL LoBind tubes 1300 O SOLID Library Column 35 Purification Kit o ra O Gel box and power supply O 1x TAE buffer Prepare 1x TAE buffer for agarose gel O Agarose Prepare 1 0 agarose gel 5 O
75. slation time Ensure that the nick translation incubation is at 5 C Genomic DNA sample is severely damaged for example genomic DNA from formalin fixed paraffin embedded sample FFPE Increase the nick translation time Too much sample DNA used for a 1 3 kb insert library Use as close to 5 ug sample DNA as possible If gt 5 ug is used scale up the the end repair and nick translation reactions Trial PCR library size is too big gt 350 bp Nick translation did not work as expected e Ensure that the nick translation incubation is at 5 C e Shorten the time for nick translation e A library peak of up to 400 bp can still be sequenced Limited sequencing performance data is available for a long mate paired library with a peak gt 400 bp Bioanalyzer electropherogram displays multiple peaks in the library after final size selection Incomplete or mis timed sample collection from the size selection collection well Collect the sample every 10 seconds then flush the well with 20 uL of Nuclease Free Water and collect the wash after each collection After gel extraction volume of sample for end repair is too large Used gt 1 gel extraction column and or did not concentrate the sheared DNA sample sufficiently in the SpeedVac Concentrator Fragment Library Preparation 5500 Series SOLiD Systems User Guide e Use a SOLiD Library Micro Column to micro concentrate the sample
76. t instrument Note Ifa SpeedVac Concentrator is not available and there is 22 ug of DNA to concentrate then use the SOLiD Library Micro Column Purification Kit with B2 S Buffer After the wash elute the purified sheared DNA with 50 uL Elution Buffer E1 Check the insert If the DNA source is not limiting ensure that the shearing conditions result in the sizes desired insert sizes Shear 5 ug DNA and run 150 ng sheared DNA on a 196 E Gel EX Gel according to the manufacturer s specifications 58 Mate Paired Library Preparation 5500 Series SOLiD Systems User Guide Appendix B Supplemental Procedures Load and unload Covaris microTUBE vials Measure the Quantitate the purified DNA using 1 uL of sample with the Quant iT dsDNA HS amountofsheared Assay Kits Invitrogen Part no Q32851 and the Qubit 2 0 Fluorometer Invitrogen purified DNA Part no 32866 STOPPING POINT Store the purified DNA in Elution Buffer E1 at 4 C or proceed directly to Size select the DNA on page 18 Load and unload Covaris microTUBE vials Load Covaris 1 Usea 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 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
77. the top and bottom to seat the gel in the iBase system If the gel is correctly inserted a red light glows 3 Remove the combs 4 Load lt 500 ng of sample in 25 uL volume without loading dye into the wells of the top row Use Nuclease free Water as diluent if necessary Skip the center well smaller well in the top center of the gel for the ladder and skip a single well to the right and left of the center top well Skip the two outermost wells to avoid edge effects Do not load more than 500 ng of DNA per lane 5 Mix 0 5 uL of 1 ug L of 100 bp DNA Ladder Invitrogen 15628 050 with 20 uL of Nuclease free Water Load 10 uL of the diluted DNA ladder into the small middle well of the top row 6 Load 25 uL of Nuclease free Water into remaining empty wells in the top row 7 Load 25 uL of Nuclease free Water into wells 1 8 in the middle of the gel and 10 uL of Nuclease free Water in the middle marker well of the bottom row Fragment Library Preparation 5500 Series SOLiD Systems User Guide 39 Chapter 2 2 x 60 bp Mate Paired Library Preparation Optional Size select the library with a SOLiD Library Size Selection gel Run the SOLiD Library Size Selection gel and collect the library fragment 40 Nuclease Nuclease Nuclease Nuclease free free free free Water DNA Water Ladder Water DNA Water 25 pL Nuclease Nuclease Nuclease free free free Water Water 4 1 Place the
78. the tube in a DynaMag 2 magnetic rack for at least 1 minute then remove and discard the supernatant without disturbing the beads 4 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 5 Open the tube then dry the beads at room temperature 20 25 C for 3 minutes Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2 x 60 bp Mate Paired Library Preparation Quantitate the ligated purified DNA 6 Elute the DNA a Remove the tube from the DynaMag 2 magnetic rack then add 50 uL Elution Buffer E1 b Vortex the beads for 15 seconds pulse spin then incubate the beads at room temperature 20 25 C for 3 minutes C Place the tube ina DynaMag 2 magnetic rack for at least 1 minute until the solution clears d Transfer the supernatant to a new 1 5 mL LoBind Tube Quantitate the ligated purified DNA Quantitate the purified DNA using 1 uL of sample with the Qubit dsDNA HS Assay Kits Invitrogen Part no Q32851 and the Qubit 2 0 Fluorometer Invitrogen Part no Q32866 Assess the recovery of DNA If the recovery compared to starting unsheared genomic DNA is Then goto gt 5 Circularize the DNA by intra molecular hybridization lt 5 Circularize the DNA by intra molecular hybridization Minimize loss in the following purification steps
79. thermocycler at 5 C for several minutes 1 Combine in the 0 2 mL PCR tube Component Volume DNAse treated purified DNA 93 94 uL 10 mM dNTP 5 uL 2 Vortex the mix then pulse spin 3 Incubate the mix without DNA polymerase I at 5 C in a thermocycler for for 2 3 minutes Use the no heated lid feature or leave the lid off 4 In another 0 2 mL PCR tube add 3 uL of DNA polymerase I then pulse spin 5 Incubate the DNA polymerase I at 5 C in a thermocycler for 21 minute Use the no heated lid feature or leave the lid off Fragment Library Preparation 5500 Series SOLID Systems User Guide 10 Purify the DNA 1 with the SOLiD Library Micro Column 2 Purification Kit 4 5 Chapter 2 2 x 60 bp Mate Paired Library Preparation Nick translate the circularized DNA Set the timer to 10 11 minutes Transfer all of the reaction mix to the tube containing the DNA polymerase I incubating at 5 C then pipet the total reaction mix up and down 5 times to mix Use the no heated lid feature or leave the lid off Start the timer Note The time for nick translation depends on laboratory practice and thermocycler conditions The ideal library peak for 2 x 60 bp mate paired library sequencing is just over 300 bp Prepare 400 uL of Binding Buffer B2 S with isopropanol 55 in a 1 5 mL LoBind Tube At the end of the incubation immediately transfer the nick translation reaction to the 1
80. tion mixture at 37 C for 45 minutes IMPORTANT Proceed to the next step immediately Fragment Library Preparation 5500 Series SOLID Systems User Guide Chapter 2 2 x 60 bp Mate Paired Library Preparation Digest the DNA with T7 Exonuclease and S1 Nuclease Purify the DNA 1 Resuspend the Agencourt AMPure XP Reagent beads using aN 2 Bind the DNA to the Agencourt AMPure XP Reagent n AE a Prepare the bead suspension in the sample reaction Component Volume Sample reaction 53 uL Agencourt AMPure XP Reagent 95 uL Total 148 uL t Equal to 1 8 volumes of sample b Vortex the beads for 15 seconds then pulse spin C Incubate the mixture at room temperature 20 25 C for 5 minutes d Place the tube ina DynaMag 2 magnetic rack for at least 1 minute until the solution clears then remove and discard the supernatant 3 Wash the DNA 2 times For each wash keep the tube in the DynaMag 2 magnetic rack a Add 600 uL of 70 ethanol to the tube without disturbing the beads b Keep the tube in a DynaMag 2 magnetic rack for at least 1 minute then remove and discard the supernatant without disturbing the beads 4 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 5 Open the tube of DNA then dry the beads at room temperature 20 25 C for 3 minutes to dry the sample 6 Elute the
81. ty 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 Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 77 Appendix F Safety Biological hazard safety Biological hazard safety General biohazard WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and
82. uide Appendix A Ordering Information Optional equipment Optional equipment Product namet Vendor Covaris S220 Systemt Applied Biosystems 4465653 110 V for U S customers 220 V for international customers The Covaris S220 System includes e Covaris S220 sonicator Universal Voltage Kit e Latitude laptop from Dell Inc e MultiTemp lll 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 microTUBE e Covaris microTUBE Prep Station e Covaris Water Tank Label Kit e Covaris microTUBEs 1 pack of 25 Covaris S2 System8 Note Mate paired libraries can be prepared with the Covaris9 S2 System 220 V for international customers New users should purchase the Covaris S220 System 110 V for U S customers E Gel iBase and E Gel Safe Imager Invitrogen Combo Kit 66465 2100 Bioanalyzer Agilent Technologies 62938C t Applied Biosystems has validated this protocol using this specific material Substitution may adversely affect system performance i Or the Covaris S2 System 8 Or the Covaris S220 System Mate Paired Library Preparation 5500 Series SOLID Systems User Guide 51 Appendix A Ordering Information Required consumables Required consumables Item
83. uide 21 Chapter 2 2 x 60 bp Mate Paired Library Preparation Ligate MP Adaptors to the DNA Ligate the MP Adaptors to the DNA Purify the DNA using Agencourt AMPure XP Reagent 22 1 Combine Component Volume End repaired DNA 100 uL 5X Reaction Buffer 10 uL ATP 100 mM Tul MPR Adaptor ds 25 uM YuLt MPL Adaptor ds 25 uM YuLt T4 DNA Ligase 5U uL 15uL Nuclease free Water Variable uL Total 150 uL t If Y lt 1 pL use 1 uL 2 Incubate the reaction mixture at room temperature 20 25 C for 30 minutes 1 Resuspend the Agencourt AMPure XP Reagent beads 2 Bind the DNA to the Agencourt AMPure XP Reagent a Prepare the bead suspension in the sample reaction Component Volume Sample reaction 150 uL Nuclease free Water 150 pL Agencourt AMPure XP Reagent 240 uL t Total 540 uL t Equal to 1 volume of sample reaction j Equal to 1 6 volumes of sample reaction b Vortex the beads for 15 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 3 Wash the DNA 2 times For each wash keep the tube in the DynaMag 2 magnetic rack a Add 600 uL of 70 ethanol to the tube without disturbing the beads b Keep

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