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Ion PGM™ Hi‑Q™ Sequencing Kit User Guide (Pub. no

1. 37 Remove liquid from the chip 37 Load the chipni miiia anaa ha ean aye ence Saeed nan 39 Select the Planned Run and perform the run 41 Select the Planned Run 2 eee 41 Perform therdn 2e en nets eet Wha ode ene acs wld ac bt aca ee 42 CHAPTER 6 Sequencing protocol lon 314 Chip v2 43 Materials required ooc c op cache evans dhe wae Hoe teen Peano aoa R ia 43 Before starting a sree iae S705920 87 Dias ns ee a RRR TRR Sb E E A 43 Optional Prepare lon Sphere Test Fragments 44 Add controls to the enriched template positive ISPs 44 lon PGM Hi Q Sequencing Kit User Guide Contents Anneal the sequencing primer x e e e K K cece eee eee eee eee 45 Perform Chip Gheck 22 6 aie antenne ete E a ea elas eos alee estes 46 Bind the Sequencing Polymerase tothe ISPs 48 Prepare and load the chip 48 Remove liquid from the chip 48 Load the chip siiin 79 aig pente See ghd ide berth bes donnes Sod regen oe Zae 50 Select the Planned Run and perform the run 52 Select the Planned Run se c s x c e cece ee eens 52 Perform the fun pas
2. 73 Safety precautions 5 52 rimiti naa oan iia ann ae bat RTA den D diner ed dut 73 Voltage s l ction sauna ee Baten made R NR RE ne eed ent R Senet eae 74 Operatlont sc act a ct Aid plang aioe Skeet eee Taa a eee 74 Voltage R BN and ROF ea niei ha aeia ea Ronnie dg RR T dled ine needs 74 CLEANING veranda everest eed bate Hee ee en eel Lie RE ti ere et eee tee 75 lon PGM Hi Q Sequencing Kit User Guide 5 Contents APPENDIXF Additional instrument information 76 lon PGM Sequencer input and output connections 76 Power the lon PGM Sequencer on or off 77 Power ON ceive eee a E a Ne no eee RAR EURE VAE 77 Power Off olcesii Sr neod meet aoa Rte Soe Sate Hee RS ee ees 77 Update the lon PGM Sequencer software 78 APPENDIX G Safety sine sens tunes dem urootraaeseteean sun 79 Symbols on this Instrument e e e cece cece cee eee cee e eee eeeeeeeas 80 Safety alerts on this instrument 81 Safety information for instruments not manufactured by Thermo Fisher Scientific 81 Instrumentsaf ty erie tement pds TTS a Sia eR AEAEE ac Ha ated a 81 General ERKE Tr RKR A R RER EE nn a ee ee Mn Are 81 Physical injury 22200065 4 2a choad cba nck er seme ete et Senet 81 Electrical joes sak de Sha R T task le dada ea Aha N NA ay 82
3. Repeat the chip mixing in step 6 then spin for 30 seconds with the chip tab pointing in toward the center of the minifuge Template prepared with the Ion PGM Template OT2 400 Kit only Repeat the chip mixing step one more time except pipet the mixture in and out of the chip five times Tilt the chip at a 45 degree angle and slowly remove as much liquid as possible from the loading port by dialing the pipette Discard the liquid lon PGM Hi Q Sequencing Kit User Guide TM Chapter 5 Sequencing protocol lon 316 Chips or lon 318 Chips eg Select the Planned Run and perform the run CA 11 Ifsome liquid remains in the chip perform a 5 second quick spin with the chip tab pointing out and remove and discard any additional liquid Do not spin the chip upside down 12 Ifsome liquid remains in the chip after the quick spin lightly and rapidly tap the point of the chip tab against the benchtop a few times and remove and discard any collected liquid Do not flush the chip 13 When chip loading is complete press Next on the touchscreen and proceed immediately to performing the run Select the Planned Run and perform the run Select the 1 Press Browse next to the Planned Run field and select the name of the plan you Planned Run created then touch Next TM Note The lon PGM Sequencer automatically populates this field for barcoded Ion chips lon PGM System ion torrent Lf urns DIDIA 5 Select P
4. 5 Immediately attach the prepared Wash 2 Bottle in the W2 position and tighten the cap Press Next 6 Change gloves and firmly install new sipper tubes short gray in the caps in the W1 and W3 positions 7 Immediately attach the prepared Wash 1 and 3 Bottles and tighten the caps Press Next 8 The sequencer will begin adjusting the pH of the W2 Solution which takes 30 minutes After 15 minutes check the instrument touchscreen to confirm that initialization is proceeding normally Note If an error occurs during the automatic pH process note the error message and proceed to Initialization Auto pH errors on page 59 During the process you can begin preparing the Reagent Bottles with dNTPs as described in the next section Prepare the 1 Use the labels provided with the kit to label four new Reagent Bottles as dGTP Bottles with 2 Confirm that no ice crystals are visible in each thawed dNTP stock solution Vortex each tube to mix and centrifuge to collect the contents Keep the dNTP stock solutions on ice throughout this procedure dNTPs IMPORTANT To avoid cross contamination in the next step open only one dNTP stock tube at a time and use a fresh pipette tip for each aliquot lon PGM Hi Q Sequencing Kit User Guide 29 Chapter 4 Clean andinitialize Initialize the lon PGM System 3 Using separate filtered pipette tips and clean gloves carefully transfer 20 uL of each dNTP stock so
5. 15 Precautions before using the lon PGM SYSTEM en de dede haine n mars 15 Update the software 0 cc cece eee eens 15 Instrument installation by trained personnel only 15 Nucleic acid contamination 15 CO contamination 6 20 34 dene wian es Mange RR E ARR ia coe E n 15 Instrument vibration and clearances 16 Static electricity s ccc0 i042 en eis debe dant ees eae a pannes de ae eat 16 Ventilation requirements 200 K eee eee 16 Procedural guidelines 2 16 Chip handling and use guidelines 17 Gas cylinders 5 sss the E eee eee Ge ee ad ee a ete ea nv ae Se tee 17 Perform a leak test 0 0 ei aad ai aon apaa a eee eens 17 Protocol Workflow soricei 079 R Rr 09 he ee ow d c R a aa ence We cere 18 lon PGM Hi Q Sequencing Kit User Guide 3 Contents m CHAPTER 23 Create a Planned Run required 19 S CHAPTER4 Clean and initialize 22 Condition the Wash 2 Bottle for first use 22 CLEANING emi cuinn a a a aiaa annee donn es ina 22 Materials required e 0 0 cece eee eee eee 22 Cleaning SCheGUle 9 009 pele Grace da Pence a eine eee par 23 Cleaning T aT 45 285088 delai
6. Sequencing Kit User Guide 9 gua Chapter 1 Product information KI SA BU lon PGM Wash 2 Bottle Kit lon PGM Hi Q Sequencing Solutions Part no A25589 Component Label Quantity Volume Storage lon PGM Hi Q Sequencing W2 Black 4 bottles 126 25 mL 2 C to 8 C Solution store W2 E Solution lon PGM Cleaning Tablet 4 tablets protected Annealing Buffer 1 bottle 12 mL fror light lon PGM Sequencing W3 Solution 2 bottles 100 mL each lon PGM Hi Q Sequencing dNTPs Part no A25590 Component Cap Color Quantity Volume Storage lon PGM Hi Q Sequencing dGTP Black 1 tube 80 uL lon PGM Hi Q Sequencing dCTP Blue 1 tube 80 pL _30 C to lon PGM Hi Q Sequencing dATP Green 1 tube 80 UL e lon PGM Hi Q Sequencing dTTP Red 1 tube 80 UL lon PGM Wash 2 Bottle Kit The Ion PGM Wash 2 Bottle Kit Cat no A25591 is sold separately and includes the 10 following components Component Quantity Volume Storage Wash 2 Bottle w label 2 L 1 bottle Note Must be conditioned at least 8 hours before use as described in Condition the Wash 2 Bottle 15 to for first use on page 22 30 C Wash 2 Bottle Conditioning Solution 1 bottle 125 mL lon PGM Hi Q Sequencing Kit User Guide lon Chip Kits Chapter 1 Product information lon Chip Kits A fe snes The following Ion Chip Kits
7. Recommended action Error message W2 average not stable Try reseating replacing chip Reading for W2 solution is not stabilizing quickly enough Remove the waste bottle and gently wipe excess fluid from the waste lines with a lab wipe a Check for leaks and reseat the chip see troubleshooting for Chip Check and Chip calibration above Replace the chip with a new unused one if needed Note The new chip can be used for sequencing after initialization completes Loosen the cap in the W2 position and re tighten the sipper Since the gas flows when the cap Is loose tighten the sipper as quickly as possible The gas is not harmful and not a hazard After performing one or more above steps press Start to re start auto pH If auto pH fails even after replacing the chip contact Technical Support and manually adjust the pH of the Wash 2 Bottle as described in Appendix C Manually Adjust W2 pH Error message W2 out of range e Chip measurements very unstable e Chip is damaged See troubleshooting tips for W2 average not stable above Error message Chip reading inconsistent Please replace chip and try again e pH response of the chip is not uniform or reliable e Ran out of W3 Solution or volume too low 1 Verify that there is enough W3 Solution gt 25 mL in the Wash 3 Bottle and that the sipper is secure If necessary loosen the Wash 3 Bottle cap
8. tighten the sipper and add more W3 Solution to fill to 50 mL Since the gas flows when the cap is loose perform these operations as quickly as possible The gas is not harmful to the W3 Solution and is not a hazard If there is enough W3 Solution replace the chip with a new unused one Insert the chip in the socket then press Start Note The new chip can be used for sequencing after initialization completes 62 lon PGM Hi Q Sequencing Kit User Guide Appendix A Troubleshooting Initialization Auto pH errors Observation Possible cause Recommended action Error message Added too much W1 to W2 e Poor water quality e 18 MQ water exposed to air for too long e Incorrect solution added to the Wash 2 Bottle e Too little NaOH added to Wash 1 Bottle e Damaged chip Check whether the water meets the 18 MQ specification and 100 mM NaOH and W2 Solution were added correctly If solutions are incorrect or water does not meet specifications correctly prepare the solution s and or use high quality water Abort the initialization and restart using correct solutions water If solutions are correct and water meets specifications abort the initialization return to the main menu and proceed to the next steps Leave the Wash 2 Bottle on the instrument Remove the Wash 1 Bottle leaving the sipper on the W1 port Empty the bottle and rinse the bottle twice with 18 MQ water Add 350 uL
9. 257 ft of gas when full Use only prepurified nitrogen of 99 995 grade 4 5 or greater purity AN CAUTION Damage to the instrument and its products can result from using impure gas gases other than nitrogen or an inadequate amount of gas T WARNING EXPLOSION HAZARD Pressurized gas cylinders are potentially explosive Always cap the gas cylinder when it is not in use and attach it firmly to the wall or gas cylinder cart with approved brackets or chains T WARNING Gas cylinders are heavy and may topple over potentially causing personal injury and tank damage Cylinders should be firmly secured to a wall or work surface Please contact your environmental health and safety coordinator for guidance on the proper installation of a gas cylinder To perform a leak test on the gas cylinder 1 Open the main tank shutoff valve The gas tank regulator s high pressure gauge should read approximately 2000 2500 psi for a full tank Adjust the pressure to the instrument by slowly turning the pressure adjustment valve clockwise until the low pressure gauge reads 30 psi Close the needle valve then close the main tank valve Monitor the high pressure gas tank regulator gauge for 5 minutes There should be no noticeable drop in pressure If the pressure Then Drops in 5 minutes There may be a leak at either the needle valve or the gas tank regulator itself Check the fittings and resolve any problems
10. e Chip not seated in socket correctly e Damaged chip e Loose Sipper Follow the procedure for Error message Please insert a chip and press Start Follow the procedure for Error message Wash 2 average not stable lon PGM Hi Q Sequencing Kit User Guide 59 Appendix A Troubleshooting nitialization Auto pH errors Observation Possible cause Recommended action Error message The system did not reach the target W2 pH and or has a clog The waste lines may be clogged Press the Troubleshoot button Note You may choose to skip the Troubleshoot button and change the chip to restart the Auto pH routine Remove the waste bottle Place lab wipes under the waste arm Gently wipe the waste arm with a lab wipe to clear liquid from around the waste line ET Press Next to begin buffer flow Observe flow rates from both waste lines One line should drip slightly faster than the other Following the flow rate check one of 3 results is possible a If flow rate appears normal press Cancel and test another chip If Auto pH failure persists contact Technical Support b If flow is blocked press Line Clear to run the standard Line Clear procedure If the line is unable to clear contact Technical Support c Ifthe result of the flow rate check are uncertain press Re flow to re flow the buffer and re test the flow 60 lon PGM Hi Q Sequencing
11. 18 MQ water Wipe the bottom of the chip on this wipe to remove salts from the chip contacts Remove the wipe dry the grounding plate and place chip on grounding plate Confirm that there is no condensation outside the flow cell Replace the chip with a new unused one if needed Note The new chip can be used for sequencing after initialization completes Press Run to restart the experiment When prompted to install the new chip make certain that the chip clamp is fully closed If the chip leaks again clean the chip socket as described above Continued leaking even with new chips may indicate a chip clamp or socket problem Contact Technical support lon PGM Hi Q Sequencing Kit User Guide 55 Appendix A Troubleshooting Chip calibration before loading sample Observation Possible cause Recommended action Error message Calibration e Chip not seated in socket Remove the chip and confirm that there is no FAILED correctly leakage or debris on the chip socket If e Chip is damaged leaking or debris is seen follow the procedure for inspecting the chip and clearing debris as described under Chip Check fails and or Leak of unknown origin above If no leaking or debris is seen reseat the chip in the socket Press Calibrate to repeat the calibration If the chip passes press Next If the chip still fails return to the main menu and restart the experiment with a new chip If you
12. Add the entire bottle of Ion PGM Hi Q Sequencing W2 Solution to the Wash 2 Bottle wy Note Keep the Ion PGM Hi Q Sequencing W2 Solution bottle to scan the barcode during the initialization procedure lon PGM Hi Q Sequencing Kit User Guide 27 AS ZS Chapter 4 Clean and initialize fey Initialize the lon PGM System Prepare the Wash 1 and Wash 3 Bottles Begin the initialization 28 6 Using a P200 pipette add 70 uL of 100 mM NaOH to the Wash 2 Bottle Note Different sites may require adding different volumes of 100 mM NaOH Some sites for example may require doubling the volume to 140 uL See Error message Added too much W1 to W2 on page 63 for information on determining the volume of 100 mM NaOH to add 7 Cap the bottle and invert five times to mix and immediately proceed through the rest of the initialization procedure IMPORTANT Do not store the mixed Wash 2 Bottle Note For the following steps label the Wash 1 and Wash 3 Bottles to avoid confusion 1 Rinse the Wash 1 and Wash 3 Bottles three times with 50 mL of 18 MQ water 2 Wash 1 Bottle Add 350 uL of freshly prepared 100 mM NaOH to the Wash 1 Bottle and cap the bottle 3 Wash 3 Bottle Add Ion PGM Sequencing W3 Solution to the 50 mL line marked on the Wash 3 Bottle and cap the bottle Note Do not remove the old sipper tubes from the dNTP ports until instructed to do so Load the bottles
13. Cleaning and decontamination 82 S pein 225 gree eastside pea eee ee ree e anes abe eR aD Ea a 82 Safety and electromagnetic compatibility EMC standards 82 Safety aea a WEES A Read L Ra S 83 EME orr me O A aE R men mais DUT ONE maman dora ox 83 Environmental design 2 83 Ch rmical saf ty 221 er pntie sn date tant amant E nent E S ee ee es 84 Biological hazard safety 2 85 APPENDIXH Documentation and support 86 Customer and technical support e e e e cee eee eens 86 Limited product warranty s e e x e e eee eens 86 lon PGM Hi Q Sequencing Kit User Guide About this guide IMPORTANT Before using this kit with the lon PGM System read and understand the information in Appendix G Safety Revision history Revision Date Description of change B 0 4 September 2014 Full release version A 0 6 December 2013 Technology access version Purpose of the guide The Ion PGM Hi Q Sequencing Kit User Guide Pub no MAN0009816 provides protocols and reference information for using the Ion PGM Hi Q Sequencing Kit with Ion 314 Ion 316 and Ion 318 Chips on the Ion PGM System lon PGM Hi Q Sequencing Kit User Guide Product information Product description I
14. Kit User Guide Appendix A Troubleshooting Initialization Auto pH errors Observation Possible cause Recommended action Error message The system did not reach the target W2 pH continued Wash 1 or Wash 2 sipper may be loose Loosen the Wash 1 cap and re tighten the sipper Since the gas flows when the cap is loose tighten the sipper as quickly as possible The gas is not harmful to the NaOH solution and is not a hazard Loosen the Wash 2 cap and re tighten the sipper Since the gas flows when the cap is loose tighten the sipper as quickly as possible The gas is not harmful to the W2 Solution and is not a hazard Press Start to re start the auto pH process Forgot to add NaOH to the Wash 1 Bottle If there is no NaOH in the Wash 1 Bottle loosen the cap and add 350 uL of 100 mM NaOH to the Wash 1 Bottle The flowing gas is not harmful to the NaOH solution and is not a hazard Recap the bottle and shake gently to mix Press Start to restart auto pH Damaged chip Replace the chip with a new unused one Insert the chip in the socket then press Start Note The new chip can be used for sequencing after initialization completes If the error persists there could be a problem with the chip clamp Contact Technical Support lon PGM Hi Q Sequencing Kit User Guide 61 Appendix A Troubleshooting nitialization Auto pH errors Observation Possible cause
15. MLS Varies Varies lon PGM Hi Q Sequencing Kit User Guide Chapter 1 Product information a Required materials and equipment P he Catalog Description Supplier uantit P PP number S y Syringe 10 CC Female Luer Lock used for clearing lines Provided with the lon PGM Sequencer or MLS lon PGM 2 5 L Waste Bottlel2l Life 4482565 1 Technologies Optional materials lon PGM Controls Kit v2 3 Life 4482010 1 Technologies lon PGM Sequencing Sippers Kitl l Life 4478682 1 Technologies Uninterruptable Power Supply UPS MLS Varies 1 Materials needed if manual adjustment of W2 Solution pH is required see Appendix C Manually Adjust W2 pH Orion 3 Star Plus pH Benchtop Meter Kit Thermo 1112003 1 with electrode electrode stand and Scientific calibration buffers or equivalent Magnetic stirrer must hold 2 L bottle MLS Varies 1 Magnetic stir bar 4 cm MLS N A 1 1NHCL MLS N A Varies 21 Required one time purchase for older Blue versions of the lon PGM System BI Not commonly needed but available for troubleshooting Kl Contains additional sipper tubes not commonly needed 1 Ensure tips from any vendors are low binding tips Required for loading samples onto the lon PGM Chips I5 We recommend using an uninterruptable power supply UPS for laboratories that experience frequent power outages or
16. No decontamination or cleaning agents are used that could cause a HAZARD as a result of a reaction with parts of the equipment or with material contained in the equipment The instrument is properly decontaminated a if hazardous material is spilled onto or into the equipment and or b prior to having the instrument serviced at your facility or sending the instrument for repair maintenance trade in disposal or termination of a loan decontamination forms may be requested from customer service Before using any cleaning or decontamination methods except those recommended by the manufacturer users should confirm with the manufacturer that the proposed method will not damage the equipment Laser CAUTION LASER HAZARD Bar Code Scanner The bar code scanner included with the instrument system is a Class 2 laser To avoid damage to eyes do not stare directly into the beam or point into another person s eyes Safety and electromagnetic compatibility EMC standards The instrument design and manufacture complies with the standards and requirements for safety and electromagnetic compatibility as noted in the following table 82 lon PGM Hi Q Sequencing Kit User Guide Safety EMC Environmental design Appendix G Safety Safety and electromagnetic compatibility EMC standards Reference Description EU Directive European Union Low Voltage Directive 2006 95 EC IEC 61010 1 Safety requirements for electri
17. Note Do not remove the old sippers from the dNTP ports until instructed to do so Load the bottles as quickly as possible to prevent atmospheric CO from reducing the pH of the Wash 2 solution For all the following steps pour the 18 MO water directly from the purification system into the Wash 2 Bottle Do not use water that has been collected or measured in any other containers IMPORTANT Do not let the new sippers touch any surfaces 1 Rinse the Wash 2 Bottle 2 L three times with 200 mL of 18 MQ water 2 Prepare 500 uL of 100 mM NaOH by diluting 50 uL of 1 M NaOH in 450 uL of nuclease free water lon PGM Hi Q Sequencing Kit User Guide Chapter 4 Clean andinitialize Initialize the lon PGM System AR 3 If your 18 MO water system has a spigot extend it into but not below the neck of the Wash 2 Bottle Otherwise position the nozzle as close to the mouth of the bottle as possible E Note If your water system has a digital display make sure it reads 18 MQ throughout filling the bottle If not see Appendix A Troubleshooting 4 Fill the bottle to the mold line with 18 MQ water The volume of water will be 2 liters You can mark the mold line on the bottle for clarity as shown in the image above Note If you are preparing bottles for multiple sequencers cap each bottle immediately after filling and leave capped until you are ready to add Ion PGM Hi Q Sequencing W2 Solution TM 5
18. Quantity Choose from one of the following systems 1 ELGA PURELAB Flex 3 Water Purification Life 4474524 System Technologies Varies Equivalent 18 MQ water purification system MLS Microcentrifuge capable of gt 15 500 x g fits MLS Varies 1 1 5 mL and 0 2 mL microcentrifuge tubes 0 22 um or 0 45 um vacuum filtration system MLS Varies 1 and filters nylon or PVDF filters 1 L volume Rainin Pipet Lite XLS with RFID LTS 10 to 100 Rainin SR L200F 1 1 pl Gilson F167203 Alternatives from Gilson and Eppendorf may be Eppendorf 022493030 used Rainin Pipet Lite XLS with RFID LTS 2 to 20 Rainin L 20XLS 1 1 pi Gilson F21023 Alternatives from Gilson and Eppendorf may be Eppendorf 022470159 used Rainin pipette tips Rainin SR L200F 1 case Alternatives from Gilson and Eppendorf may be Gilson F167203 used Eppendorf 022493030 Rainin barrier pipette tips Rainin SR L10F 1 case Alternatives from Gilson and Eppendorf may be Gilson F171303 used Eppendorf 022493028 PCR tubes Flat Cap 0 2 mL do not use VWR 10011 780 1 box polystyrene tubes Vortexer with a rubber platform MLS Varies 1 Thermal cycler with a heated lid MLS Varies 1 Graduated cylinders 1 L or 2 L volume MLS Varies 1 Glass bottle 1 L MLS Varies 1 15 mL conical tubes MLS Varies Varies NaOH 10 M molecular biology grade MLS Varies Varies Pipette set and filtered tips P2 P20 P200 and MLS Varies 1 set P1000 uL Microcentrifuge tubes 1 5 mL or 1 7 mL
19. and adda custom barcode set on lon PGM Torrent Server You can create custom sets of barcodes as comma separated value csv files then load these sets onto the Torrent Server for use during sequencing runs To access the Torrent Server you must have a username and password For more information on working with custom barcode sets refer to the Torrent Browser User Interface Guide 1 Create a comma separated variable CSV text file for your custom barcode set The CSV file can contain up to 384 barcodes TM Note You can run fewer than 384 barcodes in a sequencing run the Ion PGM System automatically detects and selects the barcodes used in the run from the selected set 2 To add the file to the Torrent Server go to Ion PGM Torrent Browser and click the Gear button on the right side of the window then select References m gs Completed Runs amp Results Completed Runs amp Results List View Table View Go Any project Any sample Any Reference All Flows Any chip Any instrument AllResult Status Reports new to old Maar 3 In the left navigation bar select Barcodes lon PGM Hi Q Sequencing Kit User Guide 67 Appendix B Barcoded libraries Custom barcode sets 4 Click the Add new DNA Barcodes button gt Reference Sequences DNA Barcodes gt Obsolete Reference Sequences gt Target Regions gt Hotspots ae aan Test Fragments lo
20. ea eee Wal en ein Ghee bee eases 23 18 MO water cleaning 4 82 23 Chlorite cleaning 0 6 cece eee eee eee 24 Initialize the lon PGM System e eeeeeeeee 25 Materials required c svg aas 00 aimi aeai eee eee 25 Initialization guidelines e e e c cece ete eee 26 Before initialization 26 Prepare the Wash 2 Bottle 26 Prepare the Wash 1 and Wash 3 Bottles 28 Begin the initialization 0 0 0 R e R ee R RR eee 28 Prepare the 50 mL Reagent Bottles with dNTPs 29 Attach the sipper tubes and Reagent Bottles 30 CHAPTER5 Sequencing protocol lon 316 Chips or lon SI CHES READ REAN 32 Materials required 5 sock eter cea ana an GH eae e i eee wa wee 32 Before Starting 53280848 eh dee ee hase eRe Se Re he o ee wee Sogn hel pee sait 32 Optional Prepare lon Sphere Test Fragments 33 Add controls to the enriched template positive ISPs 33 Anneal the Sequencing Primer 2 2 33 Perform Chip CHECK TTT 34 Bind the Sequencing Polymerase tothe ISPs 36 Prepare and load the chip
21. i e the side with the tab on the tube lid Discard the supernatant Leave 3 uL in the tube visually compare to 3 uL of liquid in a separate tube Ensure that the Sequencing Primer is completely thawed prior to use no ice crystals should be visible Vortex the primer for 5 seconds then pulse spin in a picofuge for 3 5 seconds to collect the contents Leave on ice until ready to use Add 3 uL of Sequencing Primer to the ISPs and confirm that the total volume is 6 uL add Annealing Buffer if necessary Pipet the mixture up and down thoroughly to disrupt the pellet IMPORTANT Make sure that the pipette tip is at the bottom of the tube during mixing to avoid introducing air bubbles into the sample Program a thermal cycler for 95 C for 2 minutes and then 37 C for 2 minutes using the heated lid option Place the tube in the thermal cycler and run the program After cycling the reaction can remain in the cycler at room temperature 20 30 C while you proceed with Chip Check lon PGM Hi Q Sequencing Kit User Guide 45 Chapter 6 Sequencing protocol lon 314 Chip v2 Perform Chip Check Perform Chip Check Chip Check tests the chip and ensures that it is functioning properly prior to loading the sample IMPORTANT To avoid damage due to electrostatic discharge ESD do not place the chip directly on the bench or any other surface Always place the chip either on the grounding plate on the Ion PGM S
22. line voltage fluctuations The UPS must be compatible with 1500 W output or higher The 1500 VA unit from APC provides 11 minutes of backup power for an lon PGM System lon PGM Hi Q Sequencing Kit User Guide 13 1 Chapter 1 Product information lon PGM System with Reagent and Wash Bottles attached lon PGM System with Reagent and Wash Bottles attached Label Component A Touchscreen B Chip clamp C Grounding plate D Power button E Reagent bottles F Wash 1 Bottle W1 position G Wash 2 Bottle W2 position H Wash 3 Bottle W3 position Waste Bottle 14 lon PGM Hi Q Sequencing Kit User Guide S et rlia R Before you begin Precautions before using the lon PGM System Update the software Instrument installation by trained personnel only Nucleic acid contamination CO contamination For additional safety information see Appendix G Safety IMPORTANT Before proceeding make sure that you have updated the Torrent Suite and Ion PGM System software to Version 4 2 or later See Update the Ion PGM Sequencer software on page 78 IMPORTANT The Ion PGM System is installed by trained service personnel and must not be relocated without assistance from trained service personnel See Customer and technical support on page 86 IMPORTANT A primary source of contamination is DNA fragments from previously processed samples
23. one chip at a time balance the chip in the Ion PGM Chip Minifuge rotor with a used chip of the same chip type and orientation Be careful to balance an upside down chip with another upside down chip Mark the used chip with a laboratory marker to differentiate it from the new chip lon PGM Hi Q Sequencing Kit User Guide 37 5 Chapter 5 Sequencing protocol lon 316 Chips or lon 318 Chips Prepare and load the chip 4 Place the chip upside down in the minifuge bucket and transfer the bucket to the with the chip tab pointing in toward the center of the minifuge as shown below Balance the bucket with another chip un LU a a J a a ig LU L LLE 5 Centrifuge for 5 seconds to completely empty the chip CAUTION Allow the minifuge to come to a complete stop before opening the lid 6 Remove the chip from the bucket and wipe the bucket with a disposable wipe to remove any liquid Place the chip right side up in the bucket 38 lon PGM Hi Q Sequencing Kit User Guide Chapter 5 Sequencing protocol lon 316 Chips or lon 318 Chips 5 Prepare and load the chip Load the chip Note When loading liquid into the chip keep the pipette tip at a 90 angle to the chip press the tip firmly into the circular loading port and apply gentle pressure between the pipette tip and chip Centrifuge adapter bucket 1 Place the chip in the bucket on a firm flat surface Following polymerase incubation co
24. the test continues to fail contact Technical Support Bottle leak check fails e Bottle seal is not tight s Bottle may be damaged defective 1 Finger tighten the bottles 2 If the bottle continues to leak replace the bottle 3 If leak check continues to fail contact Technical Support 58 lon PGM Hi Q Sequencing Kit User Guide Initialization Auto pH errors Appendix A Troubleshooting nitialization Auto pH errors Observation Possible cause Recommended action Error message Please insert a chip and press Start Instrument cannot detect the chip in chip socket 1 Open the chip clamp and remove the chip 2 Check for debris under the chip or in the chip socket Remove any debris by rinsing with 18 MQ water and gently dabbing the socket with a lab wipe tissue IMPORTANT Never rub or wipe the socket Rubbing the socket can damage it and cause it to fail 3 Look for liquid outside the flow cell of the chip 4 If you see liquid replace the chip with a new unused one Wash the new chip once with 100 isopropanol and twice with SEQ Sample Buffer before using Note The new chip can be used for sequencing after initialization completes 5 Close the clamp then press Start to restart the process 6 If the new chip also fails there could be a problem with the chip socket Contact Technical Support Error message Chip calibration failed
25. then continue with the following steps Does not drop in The instrument passes the leak test Re open the main tank 5 minutes valve and skip the following steps lon PGM Hi Q Sequencing Kit User Guide 17 z NO S S Chapter 2 Before you begin Protocol workflow De 5 Open the main tank valve and the needle valve for at least 15 seconds to pressurize the instrument 6 Close the main tank valve 7 Monitor the high pressure gas tank regulator gauge There should be no more than a 100 psi drop in pressure after 5 minutes Locate and resolve any leaks Turn the main tank valve back on Protocol workflow The Ion PGM System uses the following workflow when performing sequencing runs lon 316 lon 318 Chip v2 lon 314 Chip v2 Re initialization required before next run 18 lon PGM Hi Q Sequencing Kit User Guide lan N 4 O ye z 7 Q 4 L E Create a Planned Run required IMPORTANT Before proceeding make sure that you have updated the Torrent Suite and Ion PGM System software to Version 4 2 or later Planned Runs contain all the settings used in a sequencing run including application type gDNA RNA Ion AmpliSeq etc kits used number of flows barcodes if any and reference file You create and save Planned Runs in the lon PGM Torrent Browser and then select the plan in the Ion PGM Sequencer touchscreen as part of the Run workf
26. to the enriched template positive ISPs Note The Ion 314 Chip uses only half the volume of enriched template positive ISPs prepared using the template kit If you are performing an installation or troubleshooting sequencing run 1 Transfer half the volume of enriched template positive ISPs to a new 0 2 mL non polystyrene PCR tube and store at 2 8 C for up to 1 week They may be used for another sequencing run 2 Vortex the Control Ion Sphere Particles and pulse spin in a picofuge for 2 seconds before taking aliquots 3 Add 5 uL of Control Ion Sphere Particles directly to the entire volume of enriched template positive ISPs in a 0 2 mL non polystyrene PCR tube 4 Proceed to annealing the sequencing primer 44 lon PGM Hi Q Sequencing Kit User Guide TM Chapter 6 Sequencing protocol lon 314 Chip v2 Anneal the sequencing primer U Anneal the sequencing primer 1 10 Mix the tube containing the ISPs or test fragments by thoroughly pipetting up and down Place the tube in a microcentrifuge with an appropriate tube adapter Orient the tab of the tube lid so that it is pointing away from the center of the centrifuge to indicate where the pellet will be formed Centrifuge for 2 minutes at 15 500 x g Keeping the pipette plunger depressed insert a pipette tip into the tube containing the pelleted ISPs and carefully remove the supernatant from the top down avoiding the side of the tube with the pellet
27. 7 uL 3 Pipet the sample up and down to mix and incubate at room temperature for 5 minutes Prepare and load the chip Remove liquid from the chip lon 314 Chip v2 Loading port 1 Following Chip Check remove the new chip from the Ion PGM Sequencer Insert a used chip in the chip clamp while loading the new chip 2 Tilt the new chip 45 degrees so that the loading port is the lower port as shown below 48 lon PGM Hi Q Sequencing Kit User Guide Chapter 6 Sequencing protocol lon 314 Chip v2 Prepare and load the chip 3 Insert the pipette tip firmly into the loading port and remove as much liquid as possible from the loading port Discard the liquid IMPORTANT For the next steps if you are preparing one chip at a time balance the chip in the Ion PGM Chip Minifuge rotor with a used chip of the same chip type and orientation Be careful to balance an upside down chip with another upside down chip Mark the used chip with a laboratory marker to differentiate it from the new chip 4 Place the chip upside down in the minifuge bucket and transfer the bucket to the with the chip tab pointing in toward the center of the minifuge as shown below Balance the bucket with another chip 5 Centrifuge for 5 seconds to completely empty the chip A CAUTION Allow the minifuge to come to a complete stop before opening the lid 6 Remove the chip from the bucket and wipe the bucket with a disposable w
28. Do not introduce amplified DNA into the library preparation laboratory or work area IMPORTANT Possible contamination can occur during the transfer of dNTPs into Reagent Tubes Be careful to avoid cross contamination of dNTP stocks Barrier tips are required for all pipetting steps Change gloves after handling concentrated dNTP stocks IMPORTANT Dry ice solid CO must be kept away from areas where buffers wash solutions or sources of molecular biology grade water for the Ion PGM System are used High air concentrations of subliming CO may change the pH of such buffers during or after their preparation The stability of the pH of these buffers is a critical factor in the performance of the lon PGM System lon PGM Hi Q Sequencing Kit User Guide 15 SE Procedural guidelines Instrument vibration and clearances Static electricity Ventilation requirements Chapter 2 Before you begin IMPORTANT Significant vibration during sequencing may add noise and reduce the quality of the measurements The Ion PGM System must be installed on a bench that is free from vibrations or in contact with equipment that can cause vibrations to the bench freezers pumps and other similar equipment TM IMPORTANT Position the lon PGM System so that the front bezel is a minimum of 12 in 30 5 cm and the Reagent Tubes containing dNTPs are a minimum of 8 in 20 3 cm from the front of the laboratory bench Place t
29. Suite software and Ion PGM System software To view the current software version log in to the lon PGM Torrent Browser click on the Z Gear tab then select About TM To update the software see Update the lon PGM Sequencer software on page 78 Version specific information about the software is provided in the software Release Notes lon PGM Chip compatibility TM This sequencing kit is compatible with the Ion 314 Chip v2 Cat no 4482261 Ion 316 Chip v2 Cat nos 4483188 and 4483324 and Ion 318 Chip v2 Cat nos 4484354 and 4484355 lon PGM Hi Q Sequencing Kit contents and storage TM TM The Ion PGM Hi Q Sequencing Kit Cat no A25592 includes the following components lon PGM Sequencing Supplies Part no A25587 Component Color Quantity Storage Wash 1 Bottle w label 250 mL Green 1 bottle Wash 3 Bottle w label 250 mL Green 1 bottle lon PGM Reagent Bottle Sipper Blue 16 tubes Tubes 15 C to lon PGM Wash Bottle Sipper Tubes Gray 8 tubes for 250 mL taie bottles 4 tubes for 2 L bottles Reagent Bottles w labels 50 mL 25 bottles lon PGM Hi Q Sequencing Reagents Part no A25588 Component CapColor Quantity Volume Storage lon BGM Hi Q Sequencing Yellow 1 tube 36 UL Polymerase 30 C to Sequencing Primer White 1 tube 144 uL 10 C Control lon Sphere Particles Clear 1 tube 60pL lon PGM Hi Q
30. USER GUIDE ion torrent Aile technologies lon PGM Hi Q Sequencing Kit for use with lon PGM System lon 318 Chip v2 lon 316 Chip v2 lon 314 Chip v2 Catalog Number A25592 Publication Number MANO009816 Revision B 0 o For Research Use Only Not for use in diagnostic procedures technologies The information in this guide is subject to change without notice DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR MPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S 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 Important Licensing Information This product may be covered by one or more Limited Use Label Licenses By use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses Trademarks All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified Rainin and Pipet Lite are registered trademarks of Rainin Instrument LLC ELGA and PURELAB are registered trademarks of VWS UK Ltd Microsoft and E
31. able interlock devices you may be exposed to serious hazards including but not limited to severe electrical shock laser exposure crushing or chemical exposure Physical injury A CAUTION Moving Parts Moving parts can crush pinch and cut Keep hands clear of moving parts while operating the instrument Disconnect power before servicing lon PGM Hi Q Sequencing Kit User Guide 81 Appendix G Safety Safety and electromagnetic compatibility EMC standards Electrical AM WARNING Ensure appropriate electrical supply For safe operation of the instrument Plug the system into a properly grounded receptacle with adequate current capacity Ensure the electrical supply is of suitable voltage Never operate the instrument with the ground disconnected Grounding continuity is required for safe operation of the instrument AM WARNING Power Supply Line Cords Use properly configured and approved line cords for the power supply in your facility AN WARNING Disconnecting Power To fully disconnect power either detach or unplug the power cord positioning the instrument such that the power cord is accessible Cleaning and AM CAUTION Cleaning and Decontamination Use only the cleaning and decontamination decontamination methods specified in the manufacturer s user documentation It is the responsibility of the operator or other responsible person to ensure the following requirements are met
32. are compatible with this sequencing kit and are sold separately Component Quantity Catalog no Storage lon 318 Chip Kit v2 4 pack 4484354 8 pack 4484355 etches 15 C to lon 316 Chip Kit v2 4 pack 4483188 30 C 8 pack 4483324 lon 314 Chip Kit v2 8 pack 4482261 Instruments and server This sequencing kit is designed for use with the following instruments and server ordered separately Components Catalog no lon PGM System and accessories 4462921 lon PGM Torrent Server 4462918 lon PGM Chip Minifuge 120 VAC 4479672 230 VAC 4479673 Required materials and equipment This kit uses common molecular biology equipment supplies and reagents MLS Fisher Scientific www fisherscientific com or other major laboratory supplier Life Technologies website www lifetechnologies com Note The procedures in this guide have been validated using these specific materials Substitution may adversely affect system performance lon PGM Hi Q Sequencing Kit User Guide wee Catalog E Description Supplier number Quantity Tank of compressed nitrogen grade 4 5 MLS Varies 1 99 995 or better Multistage dual stage gas regulator 0 50 PSI VWR 55850 422 1 2 3 Bar output International 11 Chapter 1 Product information Required materials and equipment te S Catalog Description Supplier number
33. as quickly as possible to prevent atmospheric CO from reducing the pH of the Wash 2 Bottle solution IMPORTANT Do not let the new sipper tubes touch any surfaces 1 On the main menu press Initialize 2 In the next screen click Enter barcode to scan or enter the barcode on the Ion PGM Hi Q Sequencing W2 Solution bottle or the 2D barcode on the Ion PGM Hi Q Sequencing Solutions box Alternatively select lon PGM Hi Q Sequencing Kit from the dropdown list Press Next lon PGM System Scan or enter the W2 Solution barcode or select the Sequencing Kit below Vv lon PGM Hi Sequencing Kit Y Enter Barcode IMPORTANT Be careful to scan the correct barcode or select the correct kit type to ensure proper pH adjustment lon PGM Hi Q Sequencing Kit User Guide Chapter 4 Clean andinitialize Initialize the lon PGM System ae 3 The system will check the gas pressure If the pressure is sufficient confirm that the cleaning chip reagent sipper tubes and collection trays are in place and press Next to begin the initialization If the gas pressure is low press Yes to re check the pressure If the pressure remains low contact Technical Support 4 Wearing clean gloves firmly attach a new long gray sipper to the cap in the W2 position Do not let the sipper touch any surfaces IMPORTANT Be careful to firmly attach sippers to the ports Loosely attached sippers may adversely affect results
34. ate a new run without an existing template or e Select an existing template from the list click on the Gear button for that template and select Plan Run to create a run from that template We Favorites 3 Recently Created D Generic Sequencing AmpliSeq DNA Template Name Instr OTAC Barcode Kit Reference lon Reporter Account lon Reporter Workflow Date Source Baiser 201 Rapar system Generic Seq Template ae tas E eneric sequencing RNA Seq TargetSeq Dae Genome 20 lon PGM Hi Q Sequencing Kit User Guide Chapter 3 Create a Planned Run required 4 Inthe wizard review each screen and make your selections On the Kits screen select the appropriate library template and sequencing kits as well as chip and other information Select the sequencing kits and then hit next Sample Preparation Kit optional Control Sequence optional v v Library Kit Type Details Chip Type required lon Xpress Plus Fragment Library Kit Y lon 318 Chip v2 Template Kit OneTouch lonChef Barcode Set optional lon PGM Template OT2 400 Kit M lonXpress_1 16 N Sequencing Kit Mark as Duplicates Reads lon PGM Hi Q Sequencing Kit Y Enable Base Recalibration lt Enable Realignment Flows 850 Note For a complete description of each field see the Torrent Browser User Interface Guide 5 We recommend using the default 850 flows for 400 base read sequencing with the Ion PGM Hi Q Sequenc
35. cal equipment for measurement EN 61010 1 control and laboratory use Part 1 General requirements UL 61010 1 CSA C22 2 No 61010 1 IEC 61010 2 010 EN 61010 2 010 Safety requirements for electrical equipment for measurement control and laboratory use Part 2 010 Particular requirements for laboratory equipment for the heating of materials Reference Description Directive European Union EMC Directive 2004 108 EC EN 61326 1 Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Part 1 General Requirements FCC Part 15 U S Standard Industrial Scientific and Medical Equipment AS NZS 2064 Limits and Methods of Measurement of Electromagnetic Disturbance Characteristics of Industrial Scientific and Medical ISM Radiofrequency Equipment ICES 001 Issue 3 Industrial Scientific and Medical ISM Radio Frequency Generators Reference Description Directive 2012 19 EU European Union WEEE Directive Waste electrical and electronic equipment Directive 2011 65 EU European Union RoHS Directive Restriction of hazardous substances in electrical and electronic equipment lon PGM Hi Q Sequencing Kit User Guide 83 Chemical safety 84 Appendix G Safety WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemica
36. ceed with the sequencing run e Ifthe chip fails calibration see Error message Calibration FAILED on page 58 After 60 seconds the run will automatically begin or press Next to begin the run immediately IMPORTANT During a run avoid touching the instrument and any of the attached bottles or tubes as this may reduce the quality of the measurements When the run is complete leave the chip in place then touch Next to return to the Main Menu You can then remove the chip and proceed with another run or perform a cleaning initializing if required Note See Cleaning schedule on page 23 to determine whether cleaning is required after the run lon PGM Hi Q Sequencing Kit User Guide Sequencing protocol lon 314 Chip nanan v2 TM TM Use the following sequencing protocol with the Ion 314 Chip v2 For the Ion 316 Chip v2 or Ion 318 Chip v2 see Chapter 5 Sequencing protocol Ion 316 Chips or Ion 318 Chips Materials required Materials provided in the lon PGM Hi Q Sequencing Kit s Ion PGM Hi Q Sequencing Polymerase e Sequencing Primer e Control Ion Sphere Particles e Annealing Buffer Materials provided in the lon PGM Controls Kit v2 e Optional lon PGM Ion Sphere Test Fragments Other materials and equipment e Ton 314 Chip v2 e Enriched template positive ISPs e 0 2 mL PCR tube non polystyrene s Rainin SR L200F pipette and tips e Vortex mixer s Ton PGM Chip Minifuge e The
37. cessary you can balance a loaded chip with a used chip of any type Turn the power switch on To begin centrifugation close the lid of the minifuge The centrifugation time will vary depending on the step in the chip loading protocol To stop centrifugation press down on the lid release tab on the front of the minifuge AM CAUTION Do not attempt to open the lid or remove the chips until the unit has come to a complete stop After the rotor has stopped open the lid by grabbing it with the thumb on the front and fingers on the back then lifting the lid back on the hinge RCF The following tables list the revolutions per minute RPM and relative centrifugal force RCF at different voltages 120 50 VAC 60 Hz RPM RCF 90 4100 836 100 4550 1030 110 4960 1224 74 lon PGM Hi Q Sequencing Kit User Guide Appendix E lon PGM Chip Minifuge Cleaning 120 50 VAC 60 Hz RPM RCF 120 5330 1424 130 5710 1628 230 50 VAC 60 Hz RPM RCF 210 5070 1279 220 5310 1403 230 5515 1513 240 5705 1619 250 5900 1732 Cleaning To clean the minifuge use a damp cloth and a mild noncorrosive detergent pH lt 8 After cleaning ensure that all parts are dried thoroughly before attempting to operate the unit Do not immerse the centrifuge in liquid or pour liquids over it Note Use only the cleaning protocol described above lon PGM Hi Q Seq
38. continue to have chip calibration issues there may be an issue with the chip socket Contact Technical Support 56 lon PGM Hi Q Sequencing Kit User Guide Chip calibration after loading sample Appendix A Troubleshooting Chip calibration after loading sample Observation Possible cause Recommended action Leak of unknown origin Chip leak Chip clamp not closed properly Press the Abort button Open the chip clamp remove the chip and gently dab the chip socket with a lab wipe tissue to absorb any fluid Do not rub or wipe the chip socket Rinse the socket with 18 MQ water and gently absorb most of the water with the lab wipe tissue Repeat the rinse then gently dab the chip socket until dry Place a lab wipe tissue on the grounding plate and dampen it with 18 MO water Wipe the bottom of the chip on this wipe to remove salts from the chip contacts Remove the wipe dry the grounding plate and place the chip on the grounding plate Check for condensation outside the flow cell If there is condensation or fluid the chip is damaged and cannot be run If there is no condensation or fluid press Calibrate to restart the calibration procedure If calibration passes and no leaks are visible press Next to begin the experiment If the chip leaks again clean the chip and chip socket as described above Continued leaking may indicate a chip clamp or socket problem Conta
39. ct Technical Support lon PGM Hi Q Sequencing Kit User Guide 57 Appendix Troubleshooting nitialization General errors Observation Possible cause Recommended action Error message Calibration FAILED e Chip not seated in socket correctly e Chip is damaged Remove the chip and check for leaks and or debris on the chip socket following the procedures described in Chip Check fails and or Leak of unknown origin above If no leaks or debris are visible reseat the chip in the socket Press Calibrate If the chip passes press Next to start the experiment If the chip still fails you can try reseating the chip multiple times and pressing Calibrate If you are still unable to pass calibration press Next to start the run anyhow you may still get some data on your sample If you continue to have chip calibration issues there may be an issue with the chip or chip socket Contact Technical Support Initialization General errors Observation Possible cause Recommended action Error message Confirm instrument has gas pressure Gas cylinder may be turned off or empty 1 Verify that the cylinder has at least 500 PSI and 30 PSI at the outlet of the regulator Confirm that all valves between the cylinder and the lon PGM Sequencer are open 2 Once you confirm gas pressure leading into the instrument press Yes to retry verification of gas pressure If
40. d introducing air bubbles into the sample Program a thermal cycler for 95 C for 2 minutes and then 37 C for 2 minutes using the heated lid option Place the tube in the thermal cycler and run the program After cycling the reaction can remain in the cycler at room temperature 20 30 C while you proceed with Chip Check Perform Chip Check Chip Check tests the chip and ensures that it is functioning properly prior to loading the sample IMPORTANT To avoid damage due to electrostatic discharge ESD do not place the chip directly on the bench or any other surface Always place the chip either on the grounding plate on the lon PGM Sequencer or in the lon PGM Chip Minifuge adapter bucket To avoid ESD damage do not wear gloves when transferring chips on and off the instrument 1 34 On the main menu of the Ion PGM Sequencer touchscreen press Run Remove the waste bottle and completely empty it Press Next When prompted to insert a cleaning chip use the same used chip that was used for initialization Press Next to clean the fluid lines Remove gloves and ground yourself by touching the grounding pad on the sequencer Remove a new chip from its packaging and label it to identify the experiment save the chip package Press Next lon PGM Hi Q Sequencing Kit User Guide Chapter 5 Sequencing protocol lon 316 Chips or lon 318 Chips b Perform Chip Check A 4 Replace the old chip
41. e the bottle containing the chlorite solution from the W1 position Rinse the outside of the W1 sipper tube with a squirt bottle containing 18 MO water Fill a clean 250 mL bottle with 250 mL of 18 MO water and attach the bottle in the W1 position Make sure the cap is tight Press Next to begin the water rinse When cleaning is complete remove the bottles and sippers from the W1 W2 and W3 positions Leave the reagent sippers and collection trays in place Press Next to return to the main menu and proceed to initialization Initialize the lon PGM System Initialization takes 1 hour As part of the initialization process first prepare the Wash and Reagent Bottles as described in this section Materials Materials provided in the kit required s Ion PGM Hi Q Sequencing dGTP Ion PGM Hi Q Sequencing dCTP Ion PGM Hi Q Sequencing dATP Ion PGM Hi Q Sequencing dTTP Ion PGM Hi Q Sequencing W2 Solution stored protected from light Ion PGM Sequencing W3 Solution Wash 1 and Wash 3 Bottles and sipper tubes Wash 2 Bottle and sipper tubes bottle must be conditioned prior to first use as described in Condition the Wash 2 Bottle for first use on page 22 Wash 2 Bottle Conditioning Solution Reagent Bottles and sipper tubes Other materials and equipment Used chip leave chip on the instrument during initialization 18 MQ water 100 mM NaOH prepared daily Ice 5 mL and 25 mL pipettes Filte
42. ed to annealing the sequencing primer Anneal the Sequencing Primer 1 Mix the tube containing the ISPs or test fragments by thoroughly pipetting up and down 2 Place the tube in a microcentrifuge with an appropriate tube adapter Orient the tab of the tube lid so that it is pointing away from the center of the centrifuge to indicate where the pellet will be formed 3 Centrifuge for 2 minutes at 15 500 x g 4 Keeping the pipette plunger depressed insert a pipette tip into the tube containing the pelleted ISPs and carefully remove the supernatant from the top down avoiding the side of the tube with the pellet i e the side with the tab on the tube lid Discard the supernatant Leave 15 uL in the tube visually compare to 15 uL of liquid in a separate tube lon PGM Hi Q Sequencing Kit User Guide 33 ba SEE Perform Chip Check TM Chapter 5 Sequencing protocol lon 316 Chips or lon 318 Chips Ensure that the Sequencing Primer is completely thawed prior to use no ice crystals should be visible Vortex the primer for 5 seconds then pulse spin in a picofuge for 3 5 seconds to collect the contents Leave on ice until ready to use Add 12 uL of Sequencing Primer to the ISPs and confirm that the total volume is 27 uL add Annealing Buffer if necessary Pipet the mixture up and down thoroughly to disrupt the pellet IMPORTANT Make sure that the pipette tip is at the bottom of the tube during mixing to avoi
43. en the chip clamp remove the chip and look for signs of water outside the flow cell If the chip appears damaged replace it with a new one Look for debris on the chip socket Remove any debris by rinsing with 18 MO water and gently dabbing the socket with a lab wipe tissue IMPORTANT Never rub or wipe the socket Rubbing the socket can damage it and cause it to fail Close the clamp and repeat the Chip Check If the chip passes click Next If the chip fails replace it with a new chip scan the new chip s barcode then press Chip Check If Chip Check continues to fail there could be a problem with the chip socket Contact Technical Support 54 lon PGM Hi Q Sequencing Kit User Guide Chip calibration before loading sample Appendix A Troubleshooting Chip calibration before loading sample Observation Possible cause Recommended action Leak of unknown origin Chip leak Chip clamp not closed properly Problem with the chip clamp or socket Press Main Menu Open the chip clamp remove the chip and gently dab the chip socket with a lab wipe tissue to absorb any fluid IMPORTANT Never rub or wipe the socket Rubbing the socket can damage it and cause it to fail Rinse the socket with 18 MQ water and gently absorb most of the water with the lab wipe Repeat the rinse then gently dab the chip socket until dry Place a lab wipe on the grounding plate and dampen it with
44. ent is ready for use 3 See Cleaning schedule on page 23 for when to perform 18 MQ water or chlorite solution cleaning after powering on Power off It is not necessary to power off the instrument overnight or over the weekend If the instrument will not be used for more than 3 days power off the instrument as follows 1 2 In the Main Menu select Tools gt Shut Down If you have not already cleaned the instrument select 18 MQ water cleaning then press Next to start the cleaning process When cleaning is complete press Shut Down After you exit the main touchscreen press the Halt button then OK when prompted The instrument will power down lon PGM Hi Q Sequencing Kit User Guide 77 Appendix F Additional instrument information Update the lon PGM Sequencer software Update the lon PGM Sequencer software IMPORTANT After updates are installed the instrument must be restarted If an update to the lon PGM Sequencersoftware is available the red Alarms and Events pop up appears in the touchscreen Main Menu to alert you Click the red pop up to see the detailed messages If a message states New Software Available update the software as follows L 1 In the Main Menu select Options gt Updates 2 Select the Released Updates checkbox then press Check 3 When the message Press Update to begin update process appears press Update Note If the message All Software Current appears pre
45. equencer or in the lon PGM Chip Minifuge adapter bucket To avoid ESD damage do not wear gloves when transferring chips on and off the instrument 1 On the main menu of the Ion PGM Sequencer touchscreen press Run Remove the waste bottle and completely empty it Press Next 2 When prompted to insert a cleaning chip use the same used chip that was used for initialization Press Next to clean the fluid lines 3 Remove gloves and ground yourself by touching the grounding pad on the sequencer Remove a new chip from its packaging and label it to identify the experiment save the chip package Press Next 4 Replace the old chip in the chip socket with the new one for the experiment Close the chip clamp then press Next 46 lon PGM Hi Q Sequencing Kit User Guide TM Chapter 6 Sequencing protocol lon 314 Chip v2 ged ee Perform Chip Check en 5 When prompted use the scanner to scan the barcode located on the chip package or press Change to enter the barcode manually Optionally you can also enter the library kit catalog number Note A chip cannot be run without scanning or entering the barcode lon PGM System ion torrent gg REDD Cru DIDIDIDIDIDID a Scan the chip barcode or enter manually Optional Enter the library kit catalog number Press Chip Check Chip Check sn10c011203 fso Gs Sas GB riuiics 6 Press Chip Check During the initial part of Chip Check visually inspect the chi
46. er before using e Ifthe instrument has been left with reagents for more than 48 hours for example over the weekend IMPORTANT For all the following steps use 18 MQ water directly from the purification system Do not use water that has been collected or stored in any other containers TM Remove any wash and reagent bottles that are attached to the lon PGM System before cleaning Do not remove old sippers before cleaning The sippers are used as part of the cleaning procedure Old chips that have been used for sequencing can be marked and used in the cleaning procedure Wash bottles 250 mL and 2 L provided as part of instrument installation can be marked and used for cleaning After you have used the wash bottles provided with the sequencing kit for the specified number of runs you can use them as extra cleaning bottles Mark them for cleaning use only Empty any remaining solution from each cleaning bottle two 250 mL bottles and one 2 L bottle and rinse each bottle twice with 100 mL of 18 MO water Press Clean on the touchscreen and select the 18 MOhm water cleaning checkbox Press Next Using ungloved hands secure a used chip designated for cleaning in the chip clamp IMPORTANT Always make sure that both red rubber gasket port fittings are securely in place when securing chips with the chip clamp Failure to do so can result in a spill hazard and instrument damage Remove al
47. gas remove the gas line and cap the Wash 2 Bottle Move the bottle to the instrument remove the empty Wash 2 Bottle from the instrument and place the sipper inside the Wash 2 Bottle whose pH adjusted Secure the cap firmly Press Next to exit the automated pH check and continue with instrument initialization lon PGM Hi Q Sequencing Kit User Guide 71 72 Sequencing run times Number of flows Average read Average run time by chip Single read length 1 type 314 316 318 runs kit 21 850 400 bp 4 8 6 3 9 4 hours 4 500 200 bp 2 4 3 1 4 5 hours 8 260 100 bp 1 3 1 7 2 4hours 12 1 Read length may vary based on library size 21 Only 4 runs are supported for any read length For best results run should be started within 1 hour after initialization lon PGM Hi Q Sequencing Kit User Guide The Ion lon PGM Chip Minifuge PGM Chip Minifuge is supplied with one custom rotor and two buckets The buckets are designed to hold two chips one in each bucket The rotor and bucket design enables effective and efficient reagent loading of chips Safety precautions C AUTION Make sure your supply voltage matches the voltage label on the minifuge i e never plug a 120V minifuge into an 220 240 VAC outlet Operating the minifuge with a supply voltage outside the specified range may cause a fire or electric shock Do not run the minifuge for more than 30 seconds Never
48. gi veoh eee opp debe et a a a E ts ane nine hs 53 APPENDIXA Troubleshooting 54 Chip Checks 58e Sil Se ws i Sele ea oe Paid So 54 Chip calibration before loading sampel nannan rannen rnnr nnmnnn 55 Chip calibration after loading sample 57 Initialization General errors 58 Initialization Auto pH errors x e x e e e cece ete eee 59 Initialization Reagent pH verification 64 Troubleshooting using the controls 65 APPENDIXB Barcoded libraries 66 Pre installed barcode sets 66 Select a barcode set for a sequencing run 67 Custom barcode sets 2 67 Create and add a custom barcode set on lon PGM Torrent Server 67 Other barcode set operations 68 APPENDIXC Manually Adjust W2 pH 70 Materials and equipment needed 70 Proc dure lmi ree ernst tee ete et ent nn ne Re trente en Beene 70 APPENDIX D Sequencing run times 72 APPENDIXE lon PGM Chip Minifuge
49. has dissolved add 1 mL of 1 M NaOH and filter the solution using a 0 22 um or 0 45 um filter Use the chlorite solution within 2 3 hours Discard any unused solution after this time Press Clean on the touchscreen and select the Chlorite cleaning checkbox Press Next Using ungloved hands secure a used chip designated for cleaning in the chip clamp IMPORTANT Always make sure that both red rubber gasket port fittings are securely in place when securing chips with the chip clamp Failure to do so can result in a spill hazard and instrument damage Remove all wash and reagent bottles attached to the instrument Keep the sippers in place at all positions Press Next Add 250 mL of the filtered chlorite solution to an empty 250 mL cleaning bottle Rinse the outside of the sipper tube in the W1 position on the instrument with a squirt bottle containing 18 MQ water Attach the 250 mL bottle with the filtered chlorite solution to the W1 position Make sure that the W1 cap is tight Press Next Place the empty 2 L cleaning bottle in the W2 position and the empty 250 mL bottle in the W3 position and insert the sippers into the bottles Do not screw on the caps lon PGM Hi Q Sequencing Kit User Guide 11 12 13 14 15 Chapter 4 Clean andinitialize ene Initialize the lon PGM System CS Place collection trays below the reagent sippers in the dNTP positions Press Next to begin cleaning When prompted remov
50. he IonXpress barcode set which includes all barcodes in the Ion Xpress Barcode Adapters 1 96 Kits e RNA libraries prepared using the Ion Total RNA Seq Kit v2 Select the IonXpressRNA barcode set which contains all 16 barcodes in the Ion Xpress RNA BC01 16 Kit Cat no 4475485 If you are not using barcodes e DNA libraries Leave the Barcode field blank e RNA libraries prepared using the Ion Total RNA Seq Kit v2 Select RNA_Barcode_None from the dropdown list This will ensure that the proper trimming is performed on the resulting sequence when the RNA library does not have a barcode IMPORTANT Do not edit delete or modify the pre installed barcode sets 66 lon PGM Hi Q Sequencing Kit User Guide Select a barcode set for a sequencing run Appendix B Barcoded libraries Select a barcode set for a sequencing run Select the barcode set in the Torrent Browser when planning the run Select the sequencing kits and then hit next Sample Preparation Kit optional Library Kit Type Details lon Xpress Plus Fragment Library Kit Template Kit OneTouch lonChef lon PGM Template OT2 400 Kit Control Sequence optional Chip Type required lon 318 Chip v2 v Barcode Set optional lonXpress_1 16 U Sequencing Kit Mark as Duplicates Reads lon PGM Hi Q Sequencing Kit Y Enable Base Recalibration lt Enable Realignment Flows 850 Custom barcode sets Create
51. he instrument at least 40 in 1 meter away from major sources of electronic noise such as refrigerators or microwaves IMPORTANT To avoid possible damage to the chip from static electricity you must ground yourself on the grounding plate located next to the chip clamp prior to handling chips and do not place chips on non grounded surfaces such as a bench Always use the grounding plate to hold chips that are not in the package or inserted TM in the chip clamp or the Ion PGM Chip Minifuge bucket WARNING Instrumentation must be installed and operated in a well ventilated environment as defined as having a minimum airflow of 6 10 air changes per hour Assess the need for ventilation or atmospheric monitoring to avoid asphyxiation accidents from inert gases and or oxygen depletion and take measures to clearly identify potentially hazardous areas through training or signage Please contact your Environmental Health and Safety Coordinator to confirm that the instruments will be installed and operated in an environment with sufficient ventilation Procedural guidelines 16 e Use good laboratory practices to minimize cross contamination of products When designing the laboratory layout consider the need for space separation of pre and post amplification activities Dedicate laboratory supplies and or equipment to the appropriate space to significantly reduce the potential for contamination e Pipet viscous solutions
52. i Q Sequencing Kit User Guide TM Chapter 5 Sequencing protocol lon 316 Chips or lon 318 Chips EX Optional Prepare lon Sphere Test Fragments FA Note For each initialization the first run should be started within 1 hour after initialization and the last run must be started within 27 hours after initialization IMPORTANT The ISPs are difficult to see To avoid aspirating the particles When centrifuging the ISPs orient the tab of the tube lid so that it is pointing away from the center of the centrifuge to indicate where the pellet will be formed Always remove supernatant from the tube from the top down Optional Prepare lon Sphere Test Fragments If you are performing an installation or troubleshooting sequencing run 1 Vortex the lon PGM Ion Sphere Test Fragments from the Ion PGM Controls Kit v2 Cat no 4482010 and pulse spin in a picofuge for 2 seconds before taking aliquots 2 Add 5 uL of lon PGM Ion Sphere Test Fragments to 100 uL of Annealing Buffer in a 0 2 mL non polystyrene PCR tube 3 Skip directly to annealing the sequencing primer Add controls to the enriched template positive ISPs 1 Vortex the Control Ion Sphere Particles and pulse spin in a picofuge for 2 seconds before taking aliquots 2 Add 5 uL of Control ISPs directly to the entire volume of enriched template positive ISPs prepared using your template preparation method in a 0 2 mL non polystyrene PCR tube 3 Proce
53. in the chip socket with the new one for the experiment Close the chip clamp then press Next 5 When prompted use the scanner to scan the barcode located on the chip package or press Change to enter the barcode manually Optionally you can also enter the library kit catalog number Note A chip cannot be run without scanning or entering the barcode lon PGM System ion torrent sorsero REID on III IDI Scan the chip barcode or enter manually Optional Enter the library kit catalog number Press Chip Check Chip Check sn10c011203 220 gsm S2 HH FLuipics 6 Press Chip Check During the initial part of Chip Check visually inspect the chip in the clamp for leaks Note If there is a leak press the Abort button immediately to stop the flow to the chip Proceed to Appendix A Troubleshooting The chip socket can be damaged by rubbing or wiping its surface Never rub or wipe the socket to clean up leaks See Appendix A Troubleshooting for more information lon PGM Hi Q Sequencing Kit User Guide 35 TM me Chapter 5 Sequencing protocol lon 316 Chips or lon 318 Chips L Bind the Sequencing Polymerase to the ISPs 7 When Chip Check is complete e Ifthe chip passes press Next e Ifthe chip fails open the chip clamp re seat the chip in the socket close the clamp and press Calibrate to repeat the procedure If the chip passes press Next If the chip still fails press Main Menu and resta
54. ing Kit Otherwise see Appendix D Sequencing run times for a table listing the number of flows for different read lengths 6 When you have completed your selections click on Plan Run at the end of the workflow The plan will appear listed on the Planned Runs page under the name you specified Planned Runs All by Template by Sample Run Plan N H Select Run Code un Pian ame Barcodes Application Project sample Last Modified v Status a 2013 10 30 10 54 0R0O9 test1 aaa lanned s o an p S TM 7 You can then select the plan when you are setting up the run on the Ion PGM Sequencer see Select the Planned Run and perform the run on page 41 lon PGM Hi Q Sequencing Kit User Guide 21 a lt lt M ay R Clean and initialize E E Condition the Wash 2 Bottle for first use New Wash 2 Bottles must be conditioned with Wash 2 Bottle Conditioning Solution for at least 8 hours before first use The Wash 2 Bottle Kit cat no A25591 includes both a Wash 2 Bottle and Conditioning Solution Note If necessary you can reuse an existing Wash 2 Bottle while you condition a new bottle Bottles can be used for sequencing up to 40 times before they must be replaced To condition the Wash 2 Bottle 1 Fill the bottle to the mold line with 18 MQ water add the entire container of Wash 2 Bottle Conditioning Solution then cap the bottle and invert it five times to mix 2 Allow the bottle to sit at room tem
55. ipe to remove any liquid Place the chip right side up in the bucket lon PGM Hi Q Sequencing Kit User Guide 49 Chapter 6 Sequencing protocol lon 314 Chip v2 Prepare and load the chip Load the chip Note When loading liquid into the chip keep the pipette tip at a 90 angle to the chip press the tip firmly into the circular loading port and apply gentle pressure between the pipette tip and chip 1 Place the chip in the bucket on a firm flat surface Following polymerase incubation collect the entire sample 7 uL into a Rainin SR L200F pipette tip and insert the tip firmly into the loading port of the chip 2 With the pipette unlocked apply gentle pressure between the tip and chip and slowly dial down the pipette 1 uL per second to deposit the ISPs To avoid introducing bubbles into the chip leave a small amount of sample in the pipette tip 0 5 uL 50 lon PGM Hi Q Sequencing Kit User Guide Chapter 6 Sequencing protocol lon 314 Chip v2 Prepare and load the chip Note Do not remove the pipette tip from the port during the dial down process since this can introduce air bubbles and inhibit loading 3 Remove and discard any displaced liquid from the other port of the chip 4 Transfer the chip in the bucket to the minifuge right side up with the chip tab pointing in toward the center of the minifuge 5 Centrifuge for 30 seconds then remove the chip from the centrifuge bucket 6 Remo
56. l 3rd Edition WHO CDS CSR LYO 2004 11 found at www who int csr resources publications biosafety Biosafety7 pdf lon PGM Hi Q Sequencing Kit User Guide 85 Documentation and support Customer and technical support Visit www lifetechnologies com support for the latest in services and support including e Worldwide contact telephone numbers e Product support including Product FAQs Software patches and updates e Order and web support e Product documentation including User guides manuals and protocols Certificates of Analysis Safety Data Sheets SDSs also known as MSDSs Note For SDSs for reagents and chemicals from other manufacturers contact the manufacturer Limited product warranty Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support 86 lon PGM Hi Q Sequencing Kit User Guide For support visit lifetechnologies com support or email techsupportfdlifetech com lifetechnologies com technologies 4 September 2014 A Thermo Fisher Scientific Brand
57. l usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions 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 To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS Handle chemical wastes in a fume hood Ensure use of 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 After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported a
58. l wash and reagent bottles attached to the instrument Keep the sippers in place at all positions Press Next Add 250 mL of 18 MO water to an empty 250 mL cleaning bottle lon PGM Hi Q Sequencing Kit User Guide 23 Chapter 4 Clean andinitialize far Cleaning 6 Rinse the outside of the sipper tube in the W1 position on the instrument with a squirt bottle containing 18 MO water Attach the 250 mL bottle containing 18 MO water to the W1 position ensuring that the W1 cap is screwed on tightly Press Next Place the empty 2 L cleaning bottle in the W2 position and the empty 250 mL bottle in the W3 position and insert the sippers into the bottles Do not screw on the caps Place collection trays below the reagent sippers in the dNTP positions Press Next to begin cleaning When cleaning is complete remove the bottles and sippers from the W1 W2 and W3 positions Leave the reagent sippers and collection trays in place Press Next to return to the main menu and proceed to initialization Chlorite cleaning Note Prepare a stock of 1 M NaOH each week by diluting 10 M NaOH with 18 MO water 1 24 Empty any remaining solution from each cleaning bottle two 250 mL bottles and one 2 L bottle and rinse each bottle twice with 100 mL of 18 MQ water Fill a glass bottle with 1 L of 18 MO water and add an Ion PGM Cleaning Tablet chlorite tablet Allow the tablet to completely dissolve 10 minutes When the tablet
59. lanned Run IDAS Recommended Select a Planned Run created in the Torrent Suite Software IONPGM lon PGM Hi Q Sequencing Kit User Guide 41 L E Perform the run 42 ZS Chapter 5 Sequencing protocol lon 316 Chips or lon 318 Chips v Select the Planned Run and perform the run Confirm that the settings are correct If necessary make any changes using the touchscreen controls lon PGM System ion torrent 44 SSO Confirm or enter the run information then press Next Abort Data Mngt Next gt IONPGM EE S7 unis Note If the number of flows cycles to be run cannot be selected there may not be enough disk space to store the experiment data Touch Data Mngt to start the Data Management application this can also be accessed from the Tools Menu and delete old runs from the Ion PGM System After you enter the Planned Run press Next to verify the experimental setup Press OK to confirm the settings or press Cancel to return to the touchscreen to adjust the settings When prompted by the instrument load and clamp the chip then press Next At the beginning of the run visually inspect the chip in the clamp for leaks before closing the cover The instrument will flush any loose ISPs from the chip and begin calibrating the chip When the calibration is complete 1 minute the touchscreen will indicate whether calibration was successful e Ifthe chip passes calibration press Next to pro
60. letes lon PGM Hi Q Sequencing Kit User Guide 63 Appendix A Troubleshooting Initialization Reagent pH verification Observation Possible cause Recommended action Error message The system overshot the target W2 pH Auto pH added more NaOH from the Wash 1 Bottle to the Wash 2 Bottle than was needed and reports the pH value Press the Overshoot button to proceed with W2 pH adjustment Unscrew the cap of the Wash 2 Bottle Without removing the sipper from the bottle lift the cap high enough to pipette 15 uL of 100 mM HCl into the Wash 2 Bottle close and tighten cap Press Next to re pressurize the Wash 2 Bottle and mix the W2 solution Press Start to retry auto pH Initialization Reagent pH verification Observation Possible cause Recommended action Red failure screen reagent pH displayed One or more reagents are not within the target pH 1 Press Start to repeat the pH measurements to confirm the measurement 2 If any reagents still fail try replacing the chip with a new unused chip and repeating Note The new chip can be used for sequencing after initialization completes 3 If any reagents still fail clean and re initialize the instrument with fresh reagents and a new chip Red failure screen reagent pH not displayed Chip did not calibrate 1 Replace the chip with a new unused one Note The new chip can be used f
61. llect the entire sample 30 uL into a Rainin SR L200F pipette tip and insert the tip firmly into the loading port of the chip 2 With the pipette unlocked apply gentle pressure between the tip and chip and slowly dial down the pipette 1 uL per second to deposit the ISPs To avoid introducing bubbles into the chip leave a small amount of sample in the pipette tip 0 5 uL Note Do not remove the pipette tip from the port during the dial down process since this can introduce air bubbles and inhibit loading 3 Remove and discard any displaced liquid from the other port of the chip lon PGM Hi Q Sequencing Kit User Guide 39 TM Chapter 5 Sequencing protocol lon 316 Chips or lon 318 Chips Prepare and load the chip 4 40 Transfer the chip in the bucket to the minifuge right side up with the chip tab pointing in toward the center of the minifuge Centrifuge for 30 seconds Remove the bucket from the minifuge and place it on a flat surface Mix the sample in the chip as follows a Set the pipette volume to 30 uL b Tilt the chip 45 degrees so that the loading port is the lower port and insert the pipette tip into the loading port c Without removing the tip slowly pipet the sample in and out of the chip three times Pipet slowly to avoid creating bubbles Return the bucket to the minifuge with the chip tab pointing out away from the center of the minifuge Centrifuge for 30 seconds
62. low as described in the sequencing protocol for your chip type later in this guide For additional information see the Torrent Browser User Interface Guide available on the Ion Community at http ioncommunity lifetechnologies com 1 To create a Planned Run log into the Torrent Browser for the Torrent Server connected to your Ion PGM System ion torrent EE PRES Sequencing forall OR Register for a new account TORRENT B ROWSE R MONITOR Plan instrument runs using lon Torrent View the progress of the instrument run templates or create your own and assess run metrics in real time ER CT OR REVIEW EXI Search and review data across Automatically package and all runs and drill down to see group data and export it data in the run report to other applications lon PGM Hi Q Sequencing Kit User Guide 19 Chapter 3 Create a Planned Run required 2 Select the Plan tab select Plan Template Run and locate the type of experiment you want to perform e g lon AmpliSeq DNA DNA and Fusions Generic Sequencing etc Plan Data s aen izn an maana He Favorites Pian Runs Recently Created WARNING RAID storage disk error Visit Services Tab AmpliSeq DNA AmpliSeq RNA B na ana Fusions K g Generic Sequencing p Rra sea 2 TargetSeq Plan Plan Sample Type Template Run EB whole Genome B 16S Target Sequencing 3 To plan a new run for that experiment type e Click Plan New Run to cre
63. lution into its respective Reagent Bottle 4 Cap each Reagent Bottle and store on ice until you are ready to attach it to the instrument Place the remaining dNTP stocks back into 20 C for storage Attach the sipper 1 After the wash solutions have initialized follow the touchscreen prompts to tubes and Reagent remove the used sipper tubes and collection trays from the dNTP ports Bottles 2 Change gloves then firmly insert a new sipper tube blue into each dNTP port Do not let the sipper touch any surfaces IMPORTANT Be careful to firmly push each sipper onto the port Loosely attached sippers may adversely affect results 30 lon PGM Hi Q Sequencing Kit User Guide Chapter 4 Clean andinitialize Initialize the lon PGM System 3 Attach each prepared Reagent Bottle to the correct dNTP port e g the dGTP tube on the port marked G as shown below and tighten firmly by hand until snug Press Next Note The instrument checks the pressure of the Reagent Bottles and Wash Bottles If a bottle leaks check that it is tightly attached to the instrument If it continues to leak replace it If the instrument still does not pass the leak check contact Technical Support 4 Follow the touchscreen prompts to complete initialization The instrument will fill each Reagent Bottle with 40 mL of W2 Solution 5 At the end of initialization lon PGM System will measure the pH of the reagents e If every reagent is i
64. n the target pH range a green Passed screen will be displayed e Ifa red failure screen appears see Appendix A Troubleshooting 6 Press Next to finish the initialization process and return to the main menu 7 Proceed to the appropriate sequencing protocol for your chip type lon PGM Hi Q Sequencing Kit User Guide 31 5 EN n 9 Ce ZA E w EE x Sequencing protocol lon 316 Chips or lon 318 Chips Use the following sequencing protocol with Ion 316 Chips or Ion 318 Chips For the Ion 314 Chips see Chapter 6 Sequencing protocol Ion 314 Chip v2 Materials required Before starting 32 Materials provided in the lon PGM Hi Q Sequencing Kit Ion PGM Hi Q Sequencing Polymerase Sequencing Primer Control Ion Sphere Particles ISPs Annealing Buffer Materials provided in the lon PGM Controls Kit v2 Optional lon PGM Ion Sphere Test Fragments Other materials and equipment Ion 316 Chip v2 or Ion 318 Chip v2 Enriched template positive ISPs 0 2 mL PCR tube non polystyrene Rainin SR L200F pipette and tips Vortex mixer Ion PGM Chip Minifuge Thermal cycler with heated lid programmed at 95 C for 2 minutes and 37 C for 2 minutes Barcode scanner included with the Ion PGM System Thaw the Sequencing Primer on ice Make sure that you have updated the Ion PGM Torrent Suite System and Ion TM PGM System software to Version 4 2 or later lon PGM H
65. nXpressRNA ae lonXpressPlusCtrl ae 5 In the popup box click on the Download the example file link for an example file showing the correct CSV format Edit your own CSV barcode list to match this format and save the CSV file on your computer Add New DNA Barcodes Barcode Set Name Descriptive name of the Result Barcode CSV File Choose File CSV File of barcodes Download the example file fequence column is required Cancel Upload amp Save 6 Enter the Barcode Set Name and click on Choose File to select your formatted barcode CSV file Then click Upload amp Save 7 The barcode set file name is displayed in the list Other barcode set operations View a barcode set 1 To view a barcode set go to the Torrent Browser and click the References tab 2 Scroll down to the Barcodes section and click on the barcode set name to display the list of barcodes in the set Delete a custom barcode set from the Torrent Server 1 To view the barcode set names click the References tab in the Torrent Browser 2 Scroll down to the Barcodes section and click the name of the barcode set that you want to delete 3 In the barcode set page click Delete Barcode Set then click Yes to confirm the deletion Add a barcode to a custom barcode set 1 Open the Torrent Browser and click the References tab 2 Scroll down to the Barcodes section and click the name of the barcode set to be edited 68 lon PGM Hi Q Se
66. nd 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 lon PGM Hi Q Sequencing Kit User Guide Appendix G Safety Biological hazard safety Biological hazard safety AX 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 All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Safety equipment also may include items for personal protection such as gloves coats gowns shoe covers boots respirators face shields safety glasses or goggles Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially biohazardous materials Follow all applicable local state provincial and or national regulations The following references provide general guidelines when handling biological samples in laboratory environment U S Department of Health and Human Services Biosafety in Microbiological and Biomedical Laboratories BMBL 5th Edition HHS Publication No CDC 21 1112 Revised December 2009 found at www cdc gov biosafety publications bmbl5 BMBL pdf World Health Organization Laboratory Biosafety Manua
67. ntended use The Ion PGM Hi Q Sequencing Kit Cat no A25592 includes reagents and materials for sequencing up to 400 bp inserts using the following chips on the Ion PGM System s Ion 314 Chip v2 e Jon 316 Chip v2 s Ion 318 Chip v2 The kit includes components for cleaning and initializing the instrument The number of sequencing runs per kit depends on the length of the base reads as shown in the table below see Appendix D Sequencing run times for more information Maximum number Number of runs per Sequencing type Number of flows kit of runs per initialization 400 base read 850 4 1 200 base read 500 8 2 lt 100 base read 260 12 3 11 For best results begin the run within 1 hour after initialization The Ion PGM Sequencer performs real time measurements of hydrogen ions produced during DNA replication Library kit compatibility This sequencing kit can be used with all library types prepared using any Ion library kit Template kit compatibility We recommend the following template preparation kits for optimal performance s lon PGM Template OT2 400 Kit Cat no 4479878 s Ion PGM Template OT2 200 Kit Cat no 4480974 lon PGM Hi Q Sequencing Kit User Guide Chapter 1 Product information Software compatibility ey O C ke Software compatibility The instruments and kits described in this guide are designed for use with Version 4 2 or later of the Torrent
68. o confirm the settings or press Cancel to return to the touchscreen to adjust the settings When prompted by the instrument load and clamp the chip then press Next At the beginning of the run visually inspect the chip in the clamp for leaks before closing the cover The instrument will flush any loose ISPs from the chip and begin calibrating the chip When the calibration is complete 1 minute the touchscreen will indicate whether calibration was successful e Ifthe chip passes calibration press Next to proceed with the sequencing run e Ifthe chip fails calibration see Error message Calibration FAILED on page 58 After 60 seconds the run will automatically begin or press Next to begin the run immediately IMPORTANT During a run avoid touching the instrument and any of the attached bottles or tubes as this may reduce the quality of the measurements When the run is complete leave the chip in place then touch Next to return to the Main Menu You can then remove the chip and proceed with another run or perform a cleaning initializing if required Note See Cleaning schedule on page 23 to determine whether cleaning is required after the run lon PGM Hi Q Sequencing Kit User Guide 53 Chip Check Troubleshooting Observation Possible cause Recommended action Chip Check fails Clamp not closed Chip not properly seated Debris on the chip socket Chip damaged 1 Op
69. of 100 mM NaOH to the Wash 1 Bottle and reinstall on the instrument Press Initialize select the kit type and keep pressing the Next button to skip all bottle prep steps until the instrument begins purging air from the bottle Then proceed through the touchscreens as normal to complete the initialization The next time you initialize the instrument add 140 uL of 100 mM NaOH to the Wash 2 Bottle instead of 70 uL Continue to use this larger volume for subsequent initializations until you receive an Overshot Target error message at the first auto pH iteration at which point follow the troubleshooting steps in Error message The system overshot the target W2 pH on page 64 on the following page and then return to adding 70 uL of 100 mM NaOH If you still receive the same initialization error Added too much W1 to W2 contact Technical Support Error message UNDERSHOT TARGET PH W2 pH n nn Failed Auto pH couldn t add enough Wash 1 to the Wash 2 before the maximum iterations 10 occurred A blockage may have occurred Follow the procedure for Error message There may be a blockage or no NaOH in W1 Please check WT and run line clear then try again Press Start to re start auto pH If you still get the Undershot target pH error try replacing the chip with a new unused chip and restarting auto pH Note The new chip can be used for sequencing after initialization comp
70. operate the minifuge without a rotor properly attached to the shaft Never operate with only one chip in place A chip must be present in each bucket to balance the rotor If necessary you can balance a loaded chip with a used chip of any type Never put hands in the rotor area unless the rotor is completely stopped Never move the minifuge while the rotor is spinning Do not leave the minifuge running when not in use lon PGM Hi Q Sequencing Kit User Guide 73 TM Appendix E lon PGM Chip Minifuge Voltage selection Voltage selection Two different minifuges are available depending on your supply voltage 120 VAC and 230 VAC Make sure that the voltage specification on the label of your minifuge matches the supply voltage If they do not match change your supply voltage or contact Customer Support to request the appropriate minifuge ZN CAUTION Never plug a 120V minifuge into an 220 240 VAC outlet or vice versa Operating the minifuge with a supply voltage outside the range specified on the label may cause a fire or electric shock Operation Voltage RPM and Place the lon PGM Chip Minifuge on a level clean surface near an accessible power outlet so that the cord and outlet are within easy reach of the operator Make sure the power switch on the minifuge is in the off position Load a chip into each bucket IMPORTANT A chip must be present in each bucket to balance the rotor If ne
71. or sequencing after initialization completes 2 Press Start to restart the pH measurement 3 If the second test fails contact Technical Support 64 lon PGM Hi Q Sequencing Kit User Guide Troubleshooting using the controls Appendix A Troubleshooting Troubleshooting using the controls Observation Possible cause Recommended action lon Sphere Test Fragments are not present in the Test Fragment Report section of the run report and library sequencing is poor e Poor chip loading e Control lon Sphere Particles were not added to the sample 1 Confirm that the Control lon Sphere Particles included in this sequencing kit were added 2 If controls were added contact Technical Support Control lon PGM lon Sphere Particles were added but sample loading is poor Problems with library or template preparation Verify the quantity and quality of the library and template preparations lon PGM Hi Q Sequencing Kit User Guide 65 Barcoded libraries TM This appendix describes how to select and create barcode sets on the Ion PGM System for sequencing barcoded libraries Pre installed barcode sets The Torrent Suite Software includes the pre installed barcode sets IonXpress and TonXpressRNA When setting up a Planned Run or performing a run select the appropriate barcode set for your library type as follows s DNA libraries Select t
72. p in the clamp for leaks Note If there is a leak press the Abort button immediately to stop the flow to the chip Proceed to Appendix A Troubleshooting The chip socket can be damaged by rubbing or wiping its surface Never rub or wipe the socket to clean up leaks See Appendix A Troubleshooting for more information 7 When Chip Check is complete e Ifthe chip passes press Next e Ifthe chip fails open the chip clamp re seat the chip in the socket close the clamp and press Calibrate to repeat the procedure If the chip passes press Next If the chip still fails press Main Menu and restart the experiment with anew chip See Appendix A Troubleshooting for more information Note To return damaged chips contact Technical Support 8 Following a successful Chip Check completely empty the waste bottle and select the Waste bottle is empty checkbox on the touchscreen Press Next lon PGM Hi Q Sequencing Kit User Guide 47 TM Chapter 6 Sequencing protocol lon 314 Chip v2 Bind the Sequencing Polymerase to the ISPs Bind the Sequencing Polymerase to the ISPs 1 Remove the Ion PGM Hi Q Sequencing Polymerase from storage and flick mix with your finger tip four times Pulse spin for 3 5 seconds Place on ice 2 After annealing the Sequencing Primer remove the ISPs from the thermal cycler and add 1 uL of lon PGM Hi Q Sequencing Polymerase to the ISPs for a total final volume of
73. perature for at least 8 hours and preferably overnight then dispose of the contents The bottle is now ready for use Cleaning Materials s 18 MQ water e g the ELGA PURELAB Flex Water Purification System required e Cleaning bottles and collection trays provided with the lon PGM System e Old chip that has been used for sequencing marked for cleaning e Used sipper tubes from the previous run e Squirt bottle e Chlorite cleaning Ion PGM Cleaning Tablet provided in the kit e Chlorite cleaning 1 M NaOH diluted fresh each week from 10 M NaOH e Chlorite cleaning Glass bottle 1 L e Chlorite cleaning 0 22 um or 0 45 um vacuum filtration system and filters 22 lon PGM Hi Q Sequencing Kit User Guide Cleaning schedule Cleaning setup 18 MU water cleaning Chapter 4 Clean andinitialize WE Cleaning a The Ion PGM Sequencer requires cleaning with either 18 MQ water or a chlorite solution every time the instrument is initialized Clean with Schedule 18 MO water e Daily when instrument is in use e g not necessary on weekends e After lt 1100 flows e If more than 27 hours but less than 48 hours have elapsed between the last cleaning initialization and the start of arun e If you cleaned with chlorite a week ago and have not used the instrument since then Chlorite e Once a week unless the instrument has not been used since the last solution chlorite cleaning in which case clean with 18 MO wat
74. quencing Kit User Guide Appendix B Barcoded libraries Custom barcode sets 3 Click Add Barcode You see the new barcode window ion torrent y a P Ces LT Sons seas L Ado new barcode in set barcode_test Barcode i4 Sequence LT Flenorder Score Mode Score Oton Sono Barcode 4 Complete the fields then click Save Barcode Edit or delete a barcode from a set 1 Open the Torrent Browser and click the Settings button on the right side of the window then select References 2 In the Barcodes panel click the file name of the barcode set to be edited 3 Click the button under Action to edit or delete the panel e To edit a barcode change the barcode in the edit window then click Save Barcode e To delete a barcode from a set click Delete Barcode then click Yes to confirm the deletion lon PGM Hi Q Sequencing Kit User Guide 69 Manually Adjust W2 pH Materials and equipment needed Procedure 70 Orion 3 Star Plus pH Benchtop Meter Kit or equivalent Nitrogen gas tank tube and flow meter 100 mM NaOH prepared fresh daily Pipette tips and pipette Magnetic stirrer and stir bar 100 mM HCl If an error message during the automatic pH process indicates that there is a problem adjusting the pH of the W2 Solution use the following procedure to manually adjust the pH of the W2 Solution in the Wash 2 Bottle 1 Before proceeding rinse an empty Wash 2 Bottle and have it ready next to the inst
75. r les dispositions d limination et communiquer avec le service la client le pour des renseignements sur les options d limination responsable 80 lon PGM Hi Q Sequencing Kit User Guide Appendix G Safety Safety alerts on this instrument Safety alerts on this instrument Additional text may be used with one of the symbols described above when more specific information is needed to avoid exposure to a hazard See the following table for safety alerts found on the instrument English French translation ATTENTION Produits chimiques dangereux Lire les fiches signal tiques FS avant de manipuler les produits VAN CAUTION Hazardous chemicals Read the Safety Data Sheets SDSs before handling ATTENTION D chets dangereux Lire Les fiches signal tiques FS et la r glementation locale associ es la manipulation et l limination des d chets AM CAUTION Hazardous waste Refer to SDS s and local regulations for handling and disposal Safety information for instruments not manufactured by Thermo Fisher Scientific Some of the accessories provided as part of the instrument system are not designed or built by Thermo Fisher Scientific Consult the manufacturer s documentation for the information needed for the safe use of these products Instrument safety General N CAUTION Do not remove instrument protective covers If you remove the protective instrument panels or dis
76. red and unfiltered pipette tips and pipettes Vortex mixer Microcentrifuge Optional Ion PGM Sequencing Sippers Kit Cat no 4478682 lon PGM Hi Q Sequencing Kit User Guide 25 Chapter 4 Clean andinitialize BG Initialize the lon PGM System Initialization guidelines Before initialization Prepare the Wash 2 Bottle 26 IMPORTANT Handle nucleotides carefully to avoid cross contamination Always change gloves after removing used sipper tubes from the lon PGM System to avoid cross contamination of the nucleotides Also change gloves after handling concentrated dNTP stocks e For each initialization the first run should be started within 1 hour after initialization and the last run must be started within 27 hours after initialization e Make sure that you have updated the Ion PGM Torrent Suite System and Ion PGM System software to Version 4 2 or later Bottle usage e Wash 2 Bottles may be used for up to 40 initializations after which you can use them in the cleaning procedure e Wash 1 and 3 Bottles may be used for up to four initializations after which you can reuse them in the cleaning procedure e Replace the Reagent Bottles and sipper tubes every time you initialize 1 Remove the dNTP stock solutions from the freezer and begin thawing on ice 2 Check the tank pressure for the nitrogen gas When the tank pressure drops below 500 psi change the tank see also Gas cylinders on page 17
77. rmal cycler with heated lid programmed at 95 C for 2 minutes and 37 C for 2 minutes s Barcode scanner included with the lon PGM System Before starting e Thaw the Sequencing Primer on ice e Make sure that you have updated the Ion PGM PGM System software to Version 4 2 or later TM Torrent Suite System and Ion lon PGM Hi Q Sequencing Kit User Guide 43 TM CE Chapter 6 Sequencing protocol lon 314 Chip v2 L Optional Prepare lon Sphere Test Fragments ika Note Foreachinitialization the first run should be started within 1 hour after initialization and the last run must be started within 27 hours after initialization IMPORTANT The ISPs are difficult to see To avoid aspirating the particles When centrifuging the ISPs orient the tab of the tube lid so that it is pointing away from the center of the centrifuge to indicate where the pellet will be formed Always remove supernatant from the tube from the top down Optional Prepare lon Sphere Test Fragments If you are performing an installation or troubleshooting sequencing run 1 Vortex the lon PGM Ion Sphere Test Fragments from the Ion PGM Controls Kit v2 Cat no 4482010 and pulse spin in a picofuge for 2 seconds before taking aliquots 2 Add 5 uL of lon PGM Ion Sphere Test Fragments to 100 uL of Annealing Buffer in a 0 2 mL non polystyrene PCR tube 3 Skip directly to annealing the sequencing primer Add controls
78. rt the experiment with anew chip See Appendix A Troubleshooting for more information Note To return damaged chips contact Technical Support 8 Following a successful Chip Check completely empty the waste bottle and select the Waste bottle is empty checkbox on the touchscreen Press Next Bind the Sequencing Polymerase to the ISPs 1 Remove the Ion PGM Hi Q Sequencing Polymerase from storage and flick mix with your finger tip four times Pulse spin for 3 5 seconds Place on ice 2 After annealing the Sequencing Primer remove the ISPs from the thermal cycler and add 3 uL of Ion PGM Hi Q Sequencing Polymerase to the ISPs for a total final volume of 30 UL 3 Pipet the sample up and down to mix and incubate at room temperature for 5 minutes 36 lon PGM Hi Q Sequencing Kit User Guide Chapter 5 Sequencing protocol lon 316 Chips or lon 318 Chips 5 Prepare and load the chip Prepare and load the chip Remove liquid from the chip lon 316 lon 318 Chip v2 Loading port 1 Following Chip Check remove the new chip from the Ion PGM Sequencer Insert a used chip in the chip clamp while loading the new chip 2 Tilt the new chip 45 degrees so that the loading port is the lower port as shown below 3 Insert the pipette tip firmly into the loading port and remove as much liquid as possible from the loading port Discard the liquid IMPORTANT For the next steps if you are preparing
79. rument Also have an additional Wash 2 Bottle cap ready Note Gas will be flowing out of the Wash 2 cap so perform the next steps as quickly as possible flowing gas will not harm the W2 Solution and is not a hazard Remove the Wash 2 Bottle attached to the instrument and cap the bottle Secure the empty Wash 2 Bottle from step 1 to the instrument do not remove the sipper This bottle will contain the gas flowing out of the instrument while you pH the W2 Solution and protect the sipper from contamination Move the Wash 2 Bottle containing the W2 Solution to the stir plate near the nitrogen gas tube Secure the gas tube so that it extends inside the mouth of the Wash 2 Bottle but not below the surface of the W2 Solution Set the gas flow to 0 5 lpm Start mixing the W2 Solution fast enough for a small whirlpool to form Calibrate the pH meter using a three point calibration Rinse any buffering solution from the pH probe prior to preparing solutions lon PGM Hi Q Sequencing Kit User Guide 10 11 Appendix C Manually Adjust W2 pH Procedure Adjust the pH of the W2 Solution to 7 55 0 1 by adding a small amount of freshly prepared 100 mM NaOH to the solution and then measuring the pH using the pH meter Add small aliquots and allow the pH to equilibrate before adding more Note If the pH rises above 7 75 use 100 mM hydrochloric acid HCl to readjust the pH to 7 55 0 1 When the pH is stable turn off the
80. slowly and ensure complete mixing e Ensure all reagents are completely thawed at room temperature i e no ice crystals are visible e Vortex all reagents except for enzymes for 5 seconds enzymes should be mixed by flicking the tube with your finger four times Pulse spin in a picofuge for 3 5 seconds before use s Ion OneTouch Instrument only Always change gloves after handling waste oil used amplification plates and cleaning adapters s Ion PGM Sequencer only When performing two sequencing runs from the same initialization the second run must be started within 27 hours after initialization lon PGM Hi Q Sequencing Kit User Guide Chapter 2 Before you begin aa Chip handling and use guidelines on Chip handling and use guidelines Gas cylinders Perform a leak test Always remove gloves before transferring chips on or off the instrument Hold chips by their edges when handling To avoid damage due to electrostatic discharge ESD do not place chips directly on the bench or any other surface Always place chips either on the grounding plate on the Ion PGM Sequencer or in the lon PGM Chip Minifuge bucket Used chips cannot be reused for sequencing Used chips may be marked for cleaning and initialization You must supply the required nitrogen gas cylinder and accessories for the installation This instrument requires a pressurized house line or one size 1 A nitrogen gas cylinder that holds approximately 7 2 m
81. ss Back to return to the Main Menu 4 When the message Installing Completed displays follow the onscreen prompts to restart the instrument Note In some cases the instrument restarts automatically after software installation 78 lon PGM Hi Q Sequencing Kit User Guide Safety AN WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before using an instrument or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document lon PGM Hi Q Sequencing Kit User Guide 79 Appendix G Safety Symbols on this instrument Symbols on this instrument Symbols may be found on the instrument to warn against potential hazards or convey important safety information In this document the hazard symbol is used along with one of the following user attention words e CAUTION Indicates a potentially hazardous situation tha
82. t if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices e WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury e DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury Symbol English Francais ZN Caution risk of danger Consult the manual for further safety information Attention risque de danger Consulter le manuel pour d autres renseignements de s curit Protective conductor terminal main ground Borne de conducteur de protection mise a la terre principale X Do not dispose of this product in unsorted municipal waste CAUTION To minimize negative environmental impact from disposal of electronic waste do not dispose of electronic waste in unsorted municipal waste Follow local municipal waste ordinances for proper disposal provision and contact customer service for information about responsible disposal options Ne pas liminer ce produit avec les d chets usuels non soumis au tri s lectif CAUTION Pour minimiser les cons quences n gatives sur l environnement la suite de l limination de d chets lectroniques ne pas liminer ce d chet lectronique avec les d chets usuels non soumis au tri s lectif Se conformer aux ordonnances locales sur les d chets municipaux pou
83. uencing Kit User Guide 75 ZR L a Ty mor PA FA AV E gt x 0 Additional instrument information lon PGM Sequencer input and output connections Label Component Description Instrument fan cover IMPORTANT The fan cover must be unobstructed to ensure adequate cooling and proper functioning of the lon PGM Sequencer On off switch Power switch where the states are on or off 0 Power port 100 240VAC port that provides power to the instrument 76 lon PGM Hi Q Sequencing Kit User Guide Appendix F Additional instrument information Power the lon PGM Sequencer on or off Label Component Description D USB ports Connects the barcode reader to the instrument E Ethernet port An RJ45 port that provides Ethernet Gigabit communication with the lon PGM Sequencer F RS232 port A diagnostic port G Gas inlet For nitrogen gas H iPod port A port for docking your iPod portable media player Power the lon PGM Sequencer on or off Power on Note If the lon PGM Sequencer is powered on and the touchscreen is blank touch the screen to wake the touchscreen 1 Locate the power switch on the back of the instrument and turn to the on position 2 Press the power button on the front of the instrument The switch should illuminate When the instrument touchscreen Main Menu appears the instrum
84. ve and discard any collected liquid Do not flush the chip 13 When chip loading is complete press Next on the touchscreen and proceed immediately to performing the run Select the Planned Run and perform the run Select the 1 Press Browse next to the Planned Run field and select the name of the plan you Planned Run created then touch Next TM Note The lon PGM Sequencer automatically populates this field for barcoded Ion chips lon PGM System ion torrent 4 _5 SelectPlonnedRun SD SDSS Recommended Select a Planned Run created in the Torrent Suite Software Next gt IONPGM gt c Ens 87 D FLUIDICS 52 lon PGM Hi Q Sequencing Kit User Guide Perform the run TM Chapter 6 Sequencing protocol lon 314 Chip v2 Select the Planned Run and perform the run U Confirm that the settings are correct If necessary make any changes using the touchscreen controls lon PGM System ion torrent sg Confirm or enter the run information then press Next Change Abort Data Mngt lt Prev Next gt IONPGM EE S7 eus Note If the number of flows cycles to be run cannot be selected there may not be enough disk space to store the experiment data Touch Data Mngt to start the Data Management application this can also be accessed from the Tools Menu and delete old runs from the Ion PGM System After you enter the Planned Run press Next to verify the experimental setup Press OK t
85. ve the bucket from the minifuge and place it on a flat surface Mix the sample in the chip as follows a Set the pipette volume to 5 uL b Tilt the chip 45 degrees so that the loading port is the lower port and insert the pipette tip into the loading port c Without removing the tip slowly pipet the sample in and out of the chip three times Pipet slowly to avoid creating bubbles 7 Return the bucket to the minifuge with the chip tab pointing out away from the center of the minifuge Centrifuge for 30 seconds 8 Repeat the chip mixing in step 6 then spin for 30 seconds with the chip tab pointing in toward the center of the minifuge lon PGM Hi Q Sequencing Kit User Guide 51 WA Chapter 6 Sequencing protocol lon 314 Chip v2 AS Select the Planned Run and perform the run 9 Template prepared with the Ion PGM Template OT2 400 Kit only Repeat the chip mixing step one more time except pipet the mixture in and out of the chip five times 10 Tilt the chip at a 45 degree angle and slowly remove as much liquid as possible from the loading port by dialing the pipette Discard the liquid 11 Ifsome liquid remains in the chip perform a 5 second quick spin with the chip tab pointing out and remove and discard any additional liquid Do not spin the chip upside down 12 Ifsome liquid remains in the chip after the quick spin lightly and rapidly tap the point of the chip tab against the benchtop a few times and remo
86. xcel are registered trademarks of Microsoft Corporation iPod is a registered trademark of Apple Inc Gilson is a registered trademark of Gilson Inc Eppendorf is a registered trademark of Eppendorf AG Orion is a registered trademark of Thermo Orion Inc 2014 Thermo Fisher Scientific Inc All rights reserved Contents ADOU THIS GUIDE 5 2 nd 2 eee es ne ES 7 Revision history 242 244us0urev del tasker EEA EE e E en dae dees ae sers 7 Purpose of the guide vca aR RRR RR unten Hoek eee ioe eee 7 S CHAPTER 1 Product information 4 444 2444ussessusesssss 8 Product description lt 4233242884 cee eee eee eee eae de be ne REG Re E i 8 Intended SG Ge 5 0 25 Sa ere EET RR Lane VERNA wae eae 8 Library kit compatibility x e e e e e 2e 8 Template kit compatibility e e e e 00 e e eee 8 Software compatibility 4444440442 2 9 lon PGM Chip compatibility e eue 9 lon PGM Hi Q Sequencing Kit contents and storage 9 lon PGM Wash 2 Bottle Kit 10 lon CMP KIS EEE aie Sito y ER AM NN mens denne 11 Instruments and server e e e cece eee eens 11 Required materials and equipment se e e e e e eee eee eee 11 lon PGM System with Reagent and Wash Bottles attached 14 M CHAPTER 2 Before you begin

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