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Ion PGM鈩?Sequencing 200 Kit v2 User Guide

1. following table Reference Description EU Directive 2006 95 EC European Union Low Voltage Directive IEC 61010 1 Safety requirements for electrical equipment EN 61010 1 for measurement control and laboratory UL 61010 1 use Part 1 General requirements CSA C22 2 No 61010 1 IEC 61010 2 010 Safety requirements for electrical equipment EN 6 1010 2 010 for measurement control and laboratory use Part 2 010 Particular requirements for laboratory equipment for the heating of materials lon PGM Sequencing 200 Kit v2 User Guide 67 Appendix F Safety EMC Environmental design Chemical safety 68 Reference Description Directive 2004 108 EC European Union EMC Directive EN 61326 1 Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Part 1 General Requirements FCC Part 18 47 CFR 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 2002 96 EC European Union WEEE Directive Waste electrical and electronic equipment WARNING General chemical handling To minimize hazards ensure laboratory personnel read and pract
2. calibration FAILED 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 lon PGM Sequencing 200 Kit v2 User Guide 51 Appendix B Troubleshooting Initialization Initialization General Initialization 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 the test continues to fail contact Technical Support Bottle leak check fails e Bottle seal i
3. AmpliSeq TargetSeq Whole Genome RNA Sequencing ETS Kits Field name Description Application Select the sequencing application you are performing Run Type Select Forward Template Kit Select the lon PGM Template OT2 200 Kit Sequencing Kit Select the lon PGM Sequencing 200 Kit v2 Flows Enter the appropriate number of flows for the read length e g 500 flows for 200 base read sequencing See Appendix D Sequencing run times on page 60 for a table listing the number of flows for different read lengths Barcode Set optional See Appendix A Barcoded libraries on page 45 for more information If you are using barcodes with e DNA libraries Select the lonXpress barcode set which includes all barcodes in the lon Xpress Barcode Adapters 1 96 Kits e RNA libraries prepared using the lon Total RNA Seq Kit v2 Select the lonXpressRNA barcode set which contains all 16 barcodes in the lon Xpress RNA BC01 16 Kit Cat no 4475485 If you are not using barcodes with e DNA libraries Leave the Barcode field blank e RNA libraries prepared using the lon 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 Monitor Set thresholds for Bead Loading Usable Sequence and Key Signal During a run in the Torrent Browser under the
4. 27 hours after initialization IMPORTANT The ISPs are difficult to see To avoid aspirating the particles in the following steps orient the PCR tube the same way each time when centrifuging so that it is easy to know where the pellet has formed and remove the supernatant from the top down lon PGM Sequencing 200 Kit v2 User Guide 26 Sequencing protocol lon 316 Chip v2 or lon 318 Chip v2 Optional Prepare lon Sphere Test Fragments If you are performing an installation or troubleshooting sequencing run 1 Vortex the Ion Sphere Test Fragments from the Ion PGM Controls Kit Cat no 4480449 and centrifuge for 2 seconds before taking aliquots TM Add 5 uL of Ion Sphere Test Fragments to 100 uL of Annealing Buffer in a 0 2 mL non polystyrene PCR tube Skip directly to Anneal the Sequencing Primer Prepare enriched template positive ISPs TM Vortex the Control Ion Sphere Particles and centrifuge for 2 seconds before taking aliquots TM 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 Proceed to Anneal the Sequencing Primer Anneal the Sequencing Primer Chip Check Mix the contents of the tube by thoroughly pipetting up and down Centrifuge for 2 minutes at 15 500 x g Carefully remove the supernatant without disturbing the pellet leaving 15 pL in the tube visually compare to 15
5. 7 Calibrate the pH meter using a three point calibration Rinse any buffering solution from the pH probe prior to preparing solutions 8 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 9 When the pH is stable turn off the gas remove the gas line and cap the Wash 2 Bottle lon PGM Sequencing 200 Kit v2 User Guide 58 Appendix C Manually adjust the W2 Solution pH 10 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 11 Secure the cap firmly Press Next to exit the automated pH check and continue with instrument initialization lon PGM Sequencing 200 Kit v2 User Guide 59 Appendix D Sequencing run times Number of flows Average read Number of runs per Average run time by Single read runs kit length initialization chip type 314v2 316v2 318v2 80 25 bp 3 35 40 60 minutes 12 128 50 bp 3 45 60 80 minutes 12 180 80 bp 3 1 1 2 1 7 hours 12 260 100 bp 3 1 3 1 7 2 4 hours 12 500 200 bp 2 2 4 3 1 4 5 hours 8 7 Read length may vary based on your library size Only 8 runs are
6. Bottle on page 21 Alternatively select lon PGM Sequencing 200 Kit v2 from the dropdown list lon PGM System ion torrent sg PE D tte EXD IDDN Scan or enter the W2 Solution barcode or select the Sequencing Kit below v lon PGM Sequencing 200 Kit v2 Enter Barcode IONPGM IMPORTANT Be careful to scan the correct bottle or select the correct kit type to ensure proper pH adjustment Press Next and confirm that the cleaning chip is on the instrument and the Reagent Bottle sipper tubes and collection trays are in place Press Next again The system will verify the gas pressure If the gas pressure is sufficient press Next to begin the initialization If the gas pressure is low press Yes to retry gas pressure verification If the gas pressure remains low contact Technical Support lon PGM Sequencing 200 Kit v2 User Guide 10 Clean and initialize the lon PGM System Wearing clean gloves firmly attach a new sipper tube long gray to the cap in the W2 position New sipper attachments are push on shown below whereas older models may be threaded Do not let the sipper touch any surfaces IMPORTANT Be careful to firmly attach the sipper to the port Loosely attached sippers may adversely affect results Immediately attach the prepared Wash 2 Bottle in the W2 position and tighten the cap Press Next Change gloves and firmly install new sipper tubes short gray in the caps in the W
7. Cords Use properly configured and line cords for the power supply in your facility A 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 decontamination 66 A CAUTION Cleaning and Decontamination Use only the cleaning and 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 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 lon PGM Sequencing 200 Kit v2 User Guide Laser Gas safety Appendix F Safety CAUTION LASER HAZARD Bar Code Scanner The bar code scanner included with the instrument system
8. F Safety e Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply WARNING Hazardous waste from instruments Waste produced by the instrument is potentially hazardous Follow the guidelines noted in the preceding General Chemical Handling warning WARNING 4L Reagent and Waste Bottle Safety Four liter reagent and waste bottles can crack and leak Each 4 liter bottle should be secured in a low density polyethylene safety container with the cover fastened and the handles locked in the upright position Biological hazard safety WARNING Potential Biohazard Depending on the samples used on this instrument the surface may be considered a biohazard Use appropriate decontamination methods when working with biohazards 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 Individua
9. Manually adjust the W2 Solution pH Materials and equipment needed e Orion 3 Star Plus pH Benchtop Meter Kit or equivalent e Nitrogen gas tank tube and flow meter e 100 mM NaOH prepared fresh daily e Pipette tips and pipette e Magnetic stirrer and stir bar e 100mM HCl Procedure 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 instrument 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 2 Remove the Wash 2 Bottle attached to the instrument and cap the bottle 3 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 4 Move the Wash 2 Bottle containing the W2 Solution to the stir plate near the nitrogen gas tube 5 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 6 Set the gas flow to 0 5 lpm Start mixing the W2 Solution fast enough for a small whirlpool to form
10. is a Class 2 laser To avoid damage to eyes do not stare directly into the beam or point into another person s eyes Verify that your installation room can accommodate gas cylinders WARNING Ion instrumentation should 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 Ion instruments will be installed and operated in an environment with sufficient ventilation AN WARNING 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 A 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 EHS coordinator for guidance on the proper installation of a gas cylinder Safety and electromagnetic compatibility EMC standards Safety The instrument design and manufacture complies with the standards and requirements for safety and electromagnetic compatibility as noted in the
11. 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 Scroll down to the Barcodes section and click the name of the barcode set that you want to delete In the barcode set page click Delete Barcode Set then click Yes to confirm the deletion Add a barcode to a custom barcode set Open the Torrent Browser and click the References tab Scroll down to the Barcodes section and click the name of the barcode set to be edited Click Add Barcode You see the new barcode window lon PGM Sequencing 200 Kit v2 User Guide 47 Appendix A Barcoded libraries ion torrent LOKX04 Add new barcode in set barcode_test Barcode id Sequence Adapter Floworder Score Mode Score Cutoff Annotation Save 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 48 lon PGM Sequencing 200 Kit v2 User Guide Chip Check Appendix B Tro
12. supported at 2 runs initialization for any read length from a single kit 12 runs are supported only if using lt 260 flows at 3 runs initialization within a period of 27 hours 8 For best results begin the first run within 1 hour after initialization lon PGM Sequencing 200 Kit v2 User Guide 60 E Appendix E Additional instrument information lon PGM Sequencer input and output connections 61 Label Component Description A Instrument fan cover IMPORTANT The fan cover must be unobstructed to ensure adequate cooling and proper functioning of the lon PGM Sequencer B On off switch Power switch where the states are on or off 0 C Power port 100 240VAC port that provides power to the instrument 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 lon PGM Sequencing 200 Kit v2 User Guide Appendix E Additional instrument information Power the lon PGM Sequencer on or off Power on Power off Note If the Ion 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 Pr
13. uL of liquid in a separate tube Add 12 uL of the Sequencing Primer and confirm that the total volume is 27 uL add Annealing Buffer if necessary Pipet the sample up and down thoroughly to disrupt the pellet 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 while you proceed with 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 System or in the custom Ion centrifuge adapter rotor bucket IMPORTANT To avoid ESD damage do not wear gloves when transferring chips on and off the instrument lon PGM Sequencing 200 Kit v2 User Guide 27 Sequencing protocol lon 316 Chip v2 or lon 318 Chip v2 1 Remove a new chip from its packaging and label it to identify the experiment Save the chip package to scan the barcode later 2 Place the chip on the Ion PGM Sequencer grounding plate or in the Ion centrifuge adapter rotor bucket 3 Press Run on the main menu and follow the touchscreen prompts to prepare the Ion PGM System to test a new Ion PGM Chip Note When p
14. updated the Torrent Suite and Ion PGM System software to Version 3 6 or later Before initialization 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 3 Note the mold line on the Wash 2 bottle If there two mold lines on the bottle mark the lower line to indicate that it is the correct one Mold line marked Prepare the Wash 2 Bottle IMPORTANT For all the following steps pour the 18 MQ 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 1 Rinse the Wash 2 Bottle 2 L three times with 200 mL of 18 MO water 2 If your 18 MQ water system has a spigot extend the water spigot into but not below the neck of the Wash 2 Bottle 20 lon PGM Sequencing 200 Kit v2 User Guide Clean and initialize the lon PGM System 3 Fill the bottle to the mold line Volume of water will be 2 liters 4 Add the entire bottle of lon PGM Sequencing 200 v2 W2 Solution to the Wash 2 Bottle Note Keep the W2 Solution bottle to scan the barcode during the initialization procedure 5 Add 70 pL of freshly prepared 100 mM NaOH solution not 1 M NaOH to the Wash 2 Bottle Note Different sites may require adding different volumes of 100 mM NaOH Some sites for example may require doubli
15. 1 and W3 positions IMPORTANT Be careful to firmly attach each sipper to the port Loosely attached sippers may adversely affect results Immediately attach the prepared Wash 1 and 3 Bottles and tighten the caps Press Next The Ion PGM System will test the bottles for leaks fill the Wash 1 Bottle and then adjust the pH of the W2 Solution This procedure takes 30 minutes Note If awash bottle leaks or if an error occurs during the automatic pH process see Appendix B Troubleshooting If an error message indicates problems adjusting the pH of the prepared W2 Solution see Initialization Auto pH errors on page 52 in the Troubleshooting section Prepare the 50 mL Reagent Bottles with dNTP solutions IMPORTANT In the following steps handle the nucleotides carefully to avoid cross contamination and ensure that the correct dNTP solution is installed in each position on the Ion PGM System 1 After each dNTP stock solution has thawed vortex to mix and centrifuge to collect the contents Keep dNTP stock solutions on ice throughout this procedure Use the labels provided with the kit to label four new Reagent Bottles as dGTP dCTP dATP and dTTP Using filtered pipette tips and clean gloves carefully transfer 20 uL of each dNTP stock solution into its respective Reagent Bottle lon PGM Sequencing 200 Kit v2 User Guide 23 Clean and initialize the lon PGM System Attach the sipper tubes and Reagent Bottl
16. 64 lon PGM Sequencing 200 Kit v2 User Guide Appendix F Safety Conformity symbols on this instrument Symbol Description Fm S Indicates conformity with safety requirements for Canada and U S A TH C Indicates conformity with European Union requirements for safety and electromagnetic compatibility Indicates conformity with Australian standards for electromagnetic compatibility 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 A CAUTION Hazardous ATTENTION Produits chimiques dangereux chemicals Read the Safety Data Lire les fiches signal tiques FS avant de Sheets SDSs before handling manipuler les produits A CAUTION Hazardous waste ATTENTION D chets dangereux Lire les Refer to SDS s and local fiches signal tiques FS et la r glementation regulations for handling and locale associ es la manipulation et disposal l limination des d chets Safety information for instruments not manufactured by Life Technologies Some of the accessories provided as part of the instrument system are not designed or built by Life Technologies Consult the manufacturer s documentation for the information needed for the safe use of these products Instrument safet
17. 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 Ion PGM Chip back in the centrifuge adapter bucket and place the bucket on a flat stable surface such as a benchtop 2 Following polymerase incubation collect the entire sample 30 uL into a Rainin SR L200F pipette tip and insert the tip firmly into the loading port of the chip 3 Dial down the pipette as shown below to gently and slowly deposit the ISPs at a rate of 1 pL per second To avoid introducing bubbles into the chip leave a small amount of sample in the pipette tip 0 5 pL 4 Remove and discard any displaced liquid from the other port of the chip lon PGM Sequencing 200 Kit v2 User Guide 31 Sequencing protocol lon 316 Chip v2 or lon 318 Chip v2 5 Transfer the chip to the MiniFuge with the chip tab pointing in toward the center of the MiniFuge HA om 6 Centrifuge for 30 seconds then remove the chip from the centrifuge bucket 7 Mix the sample in the chip a Set the pipette volume to 30 pL 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 8 Centrifuge the chip for 30 seconds with the chip tab poi
18. Chlorite solution e Once a week unless the instrument has not been used since the last chlorite cleaning in which case clean with 18 MQ water before using e If the instrument has been left with reagents for more than 48 hours for example over the weekend 17 lon PGM Sequencing 200 Kit v2 User Guide Clean and initialize the lon PGM System Cleaning setup 18 MQ water cleaning Chlorite cleaning 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 e Remove any bottles that are attached to the Ion PGM System e Do not remove old sipper tubes before cleaning The sipper tubes are used as part of the cleaning procedure e Separate cleaning bottles are provided with the instrument After you have used the Wash Bottles provided with the kit for the specified number of runs you can use them as extra cleaning bottles Mark them for cleaning use only e Ensure that an old chip is in position on the instrument before cleaning Note The chip type used for cleaning initialization can be different than the chip type used for sequencing For example an Ion 314 Chip v2 can be used for TM cleaning initialization prior to running an Ion 318 Chip v2 See Cleaning schedule on page 17 1 Empty any remaining solution from each cleaning bottle two 250 mL bottles and one 2 L bottle an
19. Monitor tab the Runs in Progress screen displays an alert if the values for a run fall below the selected thresholds Reference Library Select the reference library on the Torrent Server if any BED files Select the appropriate BED files on the Torrent Server if any lon PGM Sequencing 200 Kit v2 User Guide 15 Create a Planned Run 16 Field name Description Plugins Select the appropriate plugins for your application See your lon library kit guide for recommended plugins based on library type Project Projects can be used to group your run data Select or adda project for the current run if desired You can include runs in multiple projects and remove runs from a project at any time Export If lon Reporter Uploader is installed you can enable it in this screen Planned Run Name Enter a name for the run Sample name e If lon Reporter Uploader is enabled enter each sample name and select the appropriate values for workflow relation relation role and set ID e If lon Reporter Uploader is not enabled enter the sample name or names Separated by commas 4 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 Note You can then select the plan when you are setting up the run on the Ion TM PGM Sequencer see Select the Planned Run and perform
20. NT Do not edit delete or modify the pre installed barcode sets Select a barcode set for a sequencing run To select a barcode set e Recommended Select the barcode set in the Torrent Browser when planning the run See Create a Planned Run on page 14 e Optional Select the barcode set in the Ion PGM Sequencer touchscreen when setting up the sequencing run See Select the Planned Run and perform the run on page 33 or 42 depending on your chip type 45 lon PGM Sequencing 200 Kit v2 User Guide Appendix A Barcoded libraries lon PGM System ion torrent 4 EE TER IR Ei Rinvio DATA IR ASE Confirm or enter the run information A then press Next Barcode selection Abort Data Mngt IONPGM Custom barcode sets 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 Create and add a custom barcode set on the Torrent Server 1 Create the Comma Separated Variable csv text file of the custom barcode set The csv text file can contain up to 384 barcodes 2 To add the custom barcode set to the Torrent Server go to the Torrent Browser and click the Settings button on the right side of the window then select References mm i
21. Product information lon PGM System with Reagent and Wash Bottles attached Ion PGM System rc w o o pe Component Touchscreen Chip clamp Grounding plate Power button Reagent bottles Wash 1 Bottle W1 position Wash 2 Bottle W2 position Wash 3 Bottle W3 position Waste Bottle TTIOnImIo Om gt lon PGM Sequencing 200 Kit v2 User Guide 11 Product information Precautions before using the lon PGM System Update the software For additional safety information see Appendix F Safety starting on page 63 IMPORTANT Before proceeding make sure that you have updated the Torrent Suite and Ion PGM System software to Version 3 6 or later See Update the Ion PGM Sequencer software on page 62 Instrument should only be moved by trained personnel IMPORTANT The Ion PGM System is installed by trained Life Technologies service personnel and should not be moved Nucleic acid contamination CO contamination IMPORTANT A primary source of contamination is spurious DNA fragments from previous sample processing steps 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 Bottles Be careful to avoid cross contamination of dNTP stocks Barrier tips are required for all pipetting steps Change gloves after handling concent
22. RTANT Be sure to completely empty and return the waste container before each run Proceed immediately through the following steps to load the chip Bind Sequencing Polymerase to the ISPs 38 After annealing the Sequencing Primer remove the ISPs from the thermal cycler and add 1 uL of Ion PGM Sequencing 200 v2 Polymerase to the ISPs for a total final volume of 7 uL Pipet the sample up and down to mix and incubate at room temperature for 5 minutes lon PGM Sequencing 200 Kit v2 User Guide Sequencing protocol lon 314 Chip v2 Load the chip Ion 314 Chip v2 Loading port Alternate chip loading method for lon PGM Chip loading with the lon PGM Weighted Chip Bucket For an alternate chip loading method with fewer handling steps see the Ion PGM Chip Loading with the Ion PGM Weighted Chip Bucket User Bulletin Pub no MAN0007517 which is available for download from the Ion Community at ioncommunity lifetechnologies com This protocol requires the use of the Ion PGM Weighted Chip Bucket Cat no 4480283 which can be ordered by contacting Life Technologies customer service as described on page 70 Remove liquid from the chip 1 Tilt the chip 45 degrees so that the loading port is the lower port as shown below 2 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 ba
23. ails 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 a new chip See also Appendix B Troubleshooting Note To return damaged chips contact Life Technologies Technical Support Following a successful Chip Check remove the new chip and place it on the grounding plate Insert a used chip in the socket and close the clamp Completely empty the waste bottle as instructed in the touchscreen IMPORTANT Be sure to completely empty and return the waste container before each run Proceed immediately through the following steps to load the chip Bind Sequencing Polymerase to the ISPs Load the chip After annealing the Sequencing Primer remove the ISPs from the thermal cycler and add 3 uL of Ion PGM Sequencing 200 v2 Polymerase to the ISPs for a total final volume of 30 pL Pipet the sample up and down to mix and incubate at room temperature for 5 minutes lon 316 or lon 318 Chip v2 Loading port lon PGM Sequencing 200 Kit v2 User Guide 29 Sequencing protocol lon 316 Chip v2 or lon 318 Chip v2 Alternate chip loading method lon PGM Chip loading with the lon PGM Weighted Chip Bucket For an alternate chip loading method with fewer handling steps see the lon PGM Chip Loading with the Ion PGM Weighted Chip Bucket User Bul
24. ap 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 Error message Added too much W1 to W2 Poor water quality 18 MO water exposed to air for too long Incorrect solution added to the W2 Solution Too little NaOH added to Wash 1 Bottle Damaged chip Check whether the water meets the 18M0 specification and 100 mM NaOH and W2 Solution were prepared 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 W2 bottle on the instrument Remove the W1 bottle leaving the sipper on the W1 port Empty the bottle and rinse the bottle twice with 18MQ water Add 350 uL of 100 mM NaOH to the W1 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 initial
25. d rinse each bottle twice with 100 mL of fresh 18 MO water 2 Press Clean on the touchscreen and select the 18 MOhm water cleaning checkbox Add 250 mL of 18 MO water to a 250 mL cleaning bottle 4 Rinse the outside of the sipper tube in the W1 position on the instrument with a squirt bottle containing 18 MQ water and attach the bottle to the W1 position 5 Following the touchscreen instructions place the empty 2 L cleaning bottle in the W2 position and the empty 250 mL bottle in the W3 position Place collection trays below the sipper tubes in the dNTP positions Press Next to begin cleaning 6 When cleaning is complete remove all bottles and sipper tubes from the W1 W2 and W3 positions Leave the reagent sipper tubes and collection tray s in place Press Next to return to the Main Menu and proceed to initialization See Cleaning schedule on page 17 Note Prepare a stock of 1 M NaOH each week by diluting 10 M NaOH with 18 MQ water 1 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 2 Filla 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 3 When the tablet 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 solu
26. d the volume of 100 mM NaOH added to the W2 Solution as described under Error message Added too much W1 to W2 perform the following steps and then return to adding 70 uL of 100 mM NaOH for subsequent initializations 1 Open the Wash 2 Bottle just enough to pipette 10 uL of 100 mM HCI for every 0 1 pH unit gt 7 55 into the bottle The flowing gas is not a hazard 2 Press Start to re start auto pH If the pH is still high 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 3 If the pH is consistent with the pH of the previous chip add more HCI if needed If auto pH fails manually adjust the pH of the W2 Solution see Appendix C Manually adjust the pH of the W2 Solution W2 pH consistently overshoots target pH of water is too low before any NaOH is added When preparing the Wash 2 Bottle see Prepare the Wash 2 Bottle on page 20 add more than the recommended 70 uL of 100 mM NaOH After adding the NaOH the Wash 2 Bottle should be in the range of pH 6 0 6 5 before you begin initialization 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
27. e a bench can result in damage due to static electric discharge lon PGM Sequencing 200 Kit v2 User Guide Product information Gas safety WARNING Ion instrumentation should 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 Ion instruments will be installed and operated in an environment with sufficient ventilation 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 lon PGM Sequencing 200 Kit v2 User Guide 13 Create a Planned Run IMPORTANT Before proceeding make sure that you have updated the Torrent Suite and Ion PGM System software to Version 3 6 or later See Update the Ion PGM Sequencer software on page 62 Planned Runs contain all the settings used in a sequencing run including application type gDNA RNA Ion AmpliSeq etc kit names number of flows barcodes if any and reference file They are created and saved in the Torrent Brow
28. e 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 Note Not all the liquid can be removed from the chip Remove as much liquid as possible using the methods above and then proceed with the run 13 Immediately proceed to Select the Planned Run and perform the run Select the Planned Run and perform the run Select the Planned Run or enter settings manually In the touchscreen we recommend that you select a Planned Run see Create a Planned Run on page 14 Alternatively you can make the run selections manually on the following screen 1 Press the Browse button next to the Planned Run field and select the name of the plan you created then press Next 42 lon PGM Sequencing 200 Kit v2 User Guide Sequencing protocol lon 314 Chip v2 lon PGM System ion torrent Le PSD I TD XD LE n Db aa Recommended Select a Planned Run created in the Torrent Suite Software Press Next Tanned Run ENBSR Mage Rus IONPGM 2 The run settings will be automatically populated based on the Planned Run Confirm that these settings are correct Make any changes using the buttons and dropdown lists if necessary lon PGM System ion torrent ifinia DDD Rani IDIA then press Next e BD Fleas 160 Cycles ame ION test_quoject User Notes Abort Data Mngt IONPGM Note If the number of flows cycles to be ru
29. e to have chip calibration issues there may be an issue with the chip socket Contact Technical Support lon PGM Sequencing 400 Kit User Guide 50 Appendix B Troubleshooting Chip Calibration Chip calibration with sample already loaded Observation Possible cause Recommended action Leak of unknown origin e Chip leak e Chip clamp not closed properly 10 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 Contact Technical Support Error message
30. elect a Planned Run created in the Torrent Suite Software Press Next Pronned Run ENDSR T pu IONPGM frre Goce gens OB rwioics 2 The run settings will be automatically populated based on the Planned Run Confirm that these settings are correct Make any changes using the buttons and dropdown lists if necessary lon PGM System ion torrent u D ID LR XD LB IDR en IDI Confirm or enter the run information then press Next BNO Flouus 160 Cycles ame IONAHtest_punyect User Notes Abort Data Mngt IONPGM 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 Press the Data Mngt button to start the Data Management application this can also be accessed TM from the Tools Menu and delete old runs from the Ion PGM System lon PGM Sequencing 200 Kit v2 User Guide 33 Sequencing protocol lon 316 Chip v2 or lon 318 Chip v2 Perform the run 34 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 com
31. ents Other materials and equipment e Jon 314 Chip v2 e Enriched template positive ISPs e 0 2 mL PCR tube non polystyrene e Rainin SR L10F and SR L200F pipette and tips e Vortex mixer e MiniFuge e Thermal cycler with heated lid programmed at 95 C for 2 minutes and 37 C for 2 minutes TM e Barcode scanner included with the Ion PGM System e Thaw the Sequencing Primer on ice e Make sure that you have updated the Torrent Suite and Ion PGM System software to Version 3 6 or later IMPORTANT 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 in the following steps orient the PCR tube the same way each time when centrifuging so that it is easy to know where the pellet has formed and remove the supernatant from the top down lon PGM Sequencing 200 Kit v2 User Guide Sequencing protocol lon 314 Chip v2 Optional Prepare lon Sphere Test Fragments If you are performing an installation or troubleshooting sequencing run 1 Vortex the Ion Sphere Test Fragments from the Ion PGM Controls Kit Cat no 4480449 and centrifuge for 2 seconds before taking aliquots Add 5 pL of Ion Sphere Test Fragments to 100 uL of Annealing Buffer in a 0 2 mL non polystyrene PCR tube Skip directly to Anneal t
32. es 1 After the wash solutions have initialized follow the touchscreen prompts to remove the used sipper tubes and collection trays from the dNTP ports Change gloves 2 Using new gloves 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 3 Attach each Reagent Bottle to the correct dNTP port and tighten firmly until snug The correct order of the Reagent Bottles on the Ion PGM System is dGTP dCTP dATP and dTTP left to right when facing the instrument Note The Ion PGM System checks the pressure of the Reagent Bottles and Wash Bottles If a bottle leaks you are prompted to check that it is tightly attached to the instrument If it continues to leak it should be replaced If the instrument still does not pass the leak check contact Technical Support 4 Follow the touchscreen prompts to complete initialization The Ion PGM System will fill each Reagent Bottle with 40 mL of W2 Solution Note You can create Planned Runs and or prepare the ISPs while the Ion PGM System is initializing See the following sections 24 lon PGM Sequencing 200 Kit v2 User Guide Clean and initialize the lon PGM System 5 At the end of initialization the lon PGM System will measure the pH of the reagents e If every reagent is in the target pH range a
33. ess the power button on the front of the instrument The switch should illuminate When the instrument touchscreen Main Menu appears the instrument is ready for use 3 See Cleaning schedule on page 17 for when to perform 18 MQ water or chlorite solution cleaning after powering on 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 In the Main Menu select Tools gt Shut Down 2 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 4 After you exit the main touchscreen press the Halt button then OK when prompted The instrument will power down Update the lon PGM Sequencer software 62 IMPORTANT After updates are installed the instrument must be restarted TM If an update to the Ion PGM Sequencer software 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 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 appea
34. for use of this instrument If the equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired TM IMPORTANT If you use and or install the lon PGM System in an unspecified manner you may impair the protection provided by the equipment Symbols on this instrument 63 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 that 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 lon PGM Sequencing 200 Kit v2 User Guide Appendix F Safety English Francais 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 Caution hot surface Attention surface chaude Sa gt A S ar Caution risk of electrical shock Attention risque de choc lectrique Potential biohazard Danger biologique potentiel Lase
35. green Passed screen will be displayed e Jfa red failure screen appears see Appendix B 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 Sequencing 200 Kit v2 User Guide 25 Materials required Before starting Sequencing protocol lon 316 Chip v2 or lon 318 Chip v2 TM Use the following sequencing protocol with the Ion 316 Chip v2 or Ion 318 Chip v2 For the Ion 314 Chip v2 see page 35 Materials provided in the kit e Sequencing Primer e Control Ion Sphere Particles e Annealing Buffer e Jon PGM Sequencing 200 v2 Polymerase Materials provided in the lon PGM Controls Kit e Optional Ion Sphere Test Fragments Other materials and equipment e Ton 316 Chip v2 or Ion 318 Chip v2 e Enriched template positive ISPs e 0 2 mL PCR tube non polystyrene e Rainin SR L200F pipette and tips e Vortex mixer e MiniFuge e Thermal cycler with heated lid programmed at 95 C for 2 minutes and 37 C for 2 minutes TM e Barcode scanner included with the Ion PGM System e Thaw the Sequencing Primer on ice e Make sure that you have updated the Torrent Suite and Ion PGM System software to Version 3 6 or later IMPORTANT For each initialization the first run should be started within 1 hour after initialization and the last run must be started within
36. he Sequencing Primer Prepare enriched template positive ISPs TM Note The Ion 314 Chip v2 uses only half the volume of enriched template positive ISPs prepared using the template kit 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 Vortex the Control Ion Sphere Particles and centrifuge for 2 seconds before taking aliquots Add 5 uL of Control Ion Sphere Particles directly to the half volume of enriched template positive ISPs in a 0 2 mL non polystyrene PCR tube Proceed to Anneal the Sequencing Primer Anneal the Sequencing Primer 36 Mix the contents of the tube by thoroughly pipetting up and down Centrifuge for 2 minutes at 15 500 x g Carefully remove the supernatant without disturbing the pellet leaving 3 pL in the tube visually compare to 3 pL of liquid in a separate tube Add 3 uL of Sequencing Primer and confirm that the total volume is 6 uL add Annealing Buffer if necessary Pipet the sample up and down thoroughly to disrupt the pellet 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 while you proceed with Chip Check lon PGM Sequencing 200 Kit v2 User Guide Ch
37. ice the general safety guidelines for chemical 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 Appendix G Documentation and support on page 70 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 lon PGM Sequencing 200 Kit v2 User Guide Appendix
38. ip Check Sequencing protocol lon 314 Chip v2 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 System or in the custom Ion centrifuge adapter rotor bucket IMPORTANT To avoid ESD damage do not wear gloves when transferring chips on and off the instrument 1 Remove a new chip from its packaging and label it to identify the experiment Save the chip package to scan the barcode later Place the chip on the Ion PGM Sequencer grounding plate or in the Ion centrifuge adapter rotor bucket Press Run on the main menu and follow the touchscreen prompts to prepare the Ion PGM System to test a new Ion PGM Chip Note When prompted to insert a cleaning chip you can use the same used chip that was used for initialization When prompted ground yourself by touching the grounding pad next to the chip clamp on the instrument and replace the old chip in the chip socket with the new one for the experiment Do not wear gloves when transferring the chips on and off the instrument Close the chip clamp lon PGM Sequencing 200 Kit v2 User Guide 37 Sequencing protocol lon 314 Chip v2 5 10 11 When prompted use the barcode scanner to scan the barcode loca
39. ization The next time you initialize the instrument add 140 uL of 100 mM NaOH to the W2 Solution instead of 70 pL 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 on page 56 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 lon PGM Sequencing 200 Kit v2 User Guide 55 Appendix B Troubleshooting Initialization Initialization Auto pH errors continued Observation Possible cause Recommended action Error message Auto pH couldn t add UNDERSHOT TARGET enough Wash 1 to the PH W2 pH n nn Wash 2 before the Failed maximum iterations 10 occurred 1 A blockage may have occurred Follow the procedure for Error message There may be a blockage or no NaOH in W1 Please check W1 and run line clear then try again 2 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 completes Error message Auto pH added more OVERSHOT TARGET NaOH from the Wash 1 PH W2 pH n nn Bottle to the Wash 2 Failed Bottle than was needed and reports the pH value Note If you increase
40. lance the centrifuge adapter with a used chip of the same chip type and orientation if you are preparing one Ion PGM Chip at a time 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 containing the sample lon PGM Sequencing 200 Kit v2 User Guide 39 Sequencing protocol lon 314 Chip v2 3 Place the chip upside down in the centrifuge adapter bucket as shown below and transfer the bucket to the MiniFuge with the chip tab pointing in toward the center of the MiniFuge 4 Centrifuge for 5 seconds to completely empty the chip Remove the chip from the bucket and wipe off any liquid on the bucket Load the sample on the chip IMPORTANT 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 Ion PGM Chip back in the centrifuge adapter bucket and place the bucket on a flat stable surface such as a benchtop 2 Following polymerase incubation collect the entire sample 7 uL into a Rainin SR L10F pipette tip and insert the tip firmly into the loading port of the chip 40 lon PGM Sequencing 200 Kit v2 User Guide Sequencing protocol lon 314 Chip v2 3 Dial down the pipette as shown below to gently and slowly deposit
41. letin Pub no MAN0007517 which is available for download from the Ion Community at ioncommunity lifetechnologies com This protocol requires the use of the Ion PGM Weighted Chip Bucket Cat no 4480283 which can be ordered by contacting Life Technologies customer service as described on page 70 Remove liquid from the chip 30 1 Tilt the chip 45 degrees so that the loading port is the lower port as shown below i 2 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 balance the centrifuge adapter with a used chip of the same chip type and orientation if you are preparing one Ion PGM Chip at a time 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 containing the sample 3 Place the chip upside down in the centrifuge adapter bucket and transfer the bucket to the MiniFuge with the chip tab pointing in toward the center of the MiniFuge as shown below 4 Centrifuge for 5 seconds to completely empty the chip Remove the chip from the bucket and wipe off any liquid on the bucket lon PGM Sequencing 200 Kit v2 User Guide Sequencing protocol lon 316 Chip v2 or lon 318 Chip v2 Load the sample on the chip IMPORTANT When loading liquid into the chip keep the pipette tip at a
42. ls 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 e 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 bmb15 BMBL pdf e World Health Organization Laboratory Biosafety Manual 3rd Edition WHO CDS CSR LYO 2004 11 found at www who int csr resources publications biosafety Biosafety7 pdf lon PGM Sequencing 200 Kit v2 User Guide 69 G Appendix G Documentation and support Obtaining SDSs Safety Data Sheets SDSs are available from www lifetechnologies com sds For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufacturer Obtaining support For the latest services and support information for all locations go to www iontorrent com support At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities e Search through frequently asked questions FAQs e Submit a question directly to Technical Support e Search for user documents SDSs vector maps and sequences applicatio
43. n notes formulations handbooks certificates of analysis citations and other product support documents e Obtain information about customer training e Download software updates and patches lon contact information Web site www iontorrent com Ion community ioncommunity lifetechnologies com Support email ionsupport lifetech com Phone numbers In North America 1 87 SEQUENCE 1 877 378 3623 Outside of North America 1 203 458 8552 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 lon PGM Sequencing 200 Kit v2 User Guide 70 Headquarters d 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit lifetechnologies com support or email techsupport lifetech com technologies lifetechnologies com 7 August 2013
44. n cannot be selected there may not be enough disk space to store the experiment data Press the Data Mngt button to start the Data Management application this can also be accessed from the Tools Menu and delete old runs from the Ion PGM System Perform the run 1 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 2 When prompted by the instrument load and clamp the chip then press Next 3 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 lon PGM Sequencing 200 Kit v2 User Guide 43 Sequencing protocol lon 314 Chip v2 44 4 When the calibration is complete 1 minute the touchscreen will indicate whether calibration was successful e If the chip passes calibration press Next to proceed with the sequencing run e If the chip fails calibration press Abort reseat the chip then press Calibrate to re calibrate e Ifthe chip still fails calibration proceed with the run anyway and contact Technical Support after the run is complete See also Appendix B Troubleshooting After 90 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 a
45. ng the volume to 140 pL See Error message Added too much W1 to W2 on page 55 for information on determining the volume of 100 mM NaOH to add 6 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 lon PGM Sequencing 200 Kit v2 User Guide 21 Clean and initialize the lon PGM System Prepare the Wash 1 and Wash 3 Bottles Note For the following steps label the Wash 1 and Wash 3 Bottles to avoid Begin the initialization 22 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 200 v2 1X W3 Solution to the 50 mL line marked on the Wash 3 Bottle and cap the bottle IMPORTANT Do not remove the old sipper tubes from the dNTP ports until instructed to do so Do not let the new sipper tubes touch any surfaces IMPORTANT Load the bottles as quickly as possible to prevent atmospheric CO from reducing the pH of the Wash 2 Bottle solution 1 TM Confirm that the chip used to the clean the Ion PGM System is still in place on the instrument On the main menu press Initialize In the next screen scan or enter the barcode on the Ion PGM Sequencing 200 v2 W2 Solution bottle from Step 4 under Prepare the Wash 2
46. nitialization Auto pH errors continued Observation Possible cause Recommended action Error message There e The waste lines may Check for blockage may be a blockage or no be blocked 1 Remove the waste bottle NaOH in W1 Please e Wash 1 or Wash 2 2 Place lab wipes under the waste arm check W1 and run line sipper may be loose 3 Gently wipe the waste arm with a lab wipe to clear clear then try again e Chip does not detect liquid from around the waste line large enough pH difference between the i NaOH and W2 Solution e Damaged chip A LT 4 Press Flow check one or more times to observe the flow rates from both lines One line should drip slightly faster than the other If one or both lines are blocked no flow or the drip rates are significantly different go to the next step If the flow rates are normal see Check for a damaged chip below 5 Press Line Clear Follow the prompts and use the syringe supplied with the lon PGM System 6 After Line Clear press Flow check and check for normal flow rates from the waste lines 7 Ifthe flow rates are still not normal perform Line Clear one more time 8 If the line s remain blocked contact Technical Support Otherwise press Start to restart auto pH Check Wash 1 and Wash 2 Bottles for loose sipper tubes 1 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 n
47. ns mshi Completed Runs amp Results About References m enices Completed Runs amp Results Plugins List View Table View camane Date Search names Go Any project x Any Sample x Any Reference x All Flows x Any Chip X Any Instrument X All Result Status X Results new to old x Clear Scroll down to the DNA Barcodes section 4 Click Add new DNA Barcodes Click the Download the example file link to download an example file to your computer 46 lon PGM Sequencing 200 Kit v2 User Guide Appendix A Barcoded libraries 6 On your computer edit the csv example file directly or use Microsoft Excel Notepad or similar software to create a custom barcode set in the same format with each barcode sequence listed on a separate line The barcode list can contain up to 384 barcodes Save the csv file on your computer 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 Back in the Torrent Browser enter a name for the barcode set and click Browse to select the csv file that you created Click Upload amp Save The barcode set file name is displayed in the Barcode panel Other barcode set operations View a barcode set To view a barcode set go to the Torrent Browser and click the References tab Scroll down to the Barcodes section and click on the barcode set name to display the
48. nting out away from the center of the MiniFuge 9 Repeat the chip mixing in step 7 one more time then spin for 30 seconds with the chip tab pointing in toward the center of the MiniFuge 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 If some 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 If some 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 Select the Planned Run and perform the run 32 lon PGM Sequencing 200 Kit v2 User Guide Sequencing protocol lon 316 Chip v2 or lon 318 Chip v2 Select the Planned Run and perform the run Select the Planned Run or enter settings manually In the touchscreen we recommend that you select a Planned Run see Create a Planned Run on page 14 Alternatively you can make the run selections manually on the following screen 1 Press the Browse button next to the Planned Run field and select the name of the plan you created then press Next lon PGM System ion torrent while DEED TD LD TD L DD ER Recommended S
49. ot 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 lon PGM Sequencing 200 Kit v2 User Guide 53 Appendix B Troubleshooting Initialization Initialization Auto pH errors continued Observation Possible cause Recommended action Continued Continued Check for a damaged chip or chip clamp pariposer 1 2 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 Forgot to add NaOH to the Wash 1 Bottle 1 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 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 4 ET Check for leaks and reseat the chip see tr
50. oubleshooting 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 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 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 W2 Solution as described in Appendix C Manually adjust the pH of the W2 Solution Error message W2 out of range Chip measurements very unstable e Chip is damaged See troubleshooting tips for W2 average not stable above 54 lon PGM Sequencing 200 Kit v2 User Guide Appendix B Troubleshooting Initialization Initialization Auto pH errors continued Observation Possible cause Recommended action Error message Chip reading inconsistent Please replace chip and try again pH response of the chip is not uniform or reliable Ran out of W3 Solution or volume too low 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 tighten the sipper and add more W3 Solution to fill to 50 mL Since the gas flows when the c
51. plete 1 minute the touchscreen will indicate whether calibration was successful e Ifthe chip passes calibration press Next to proceed with the sequencing run e If the chip fails calibration press Abort reseat the chip then press Calibrate to re calibrate e Ifthe chip still fails calibration proceed with the run anyway and contact Technical Support after the run is complete See also Appendix B Troubleshooting After 90 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 the touchscreen will return to the Main Menu You can then proceed with another run or perform a cleaning initializing if required Note See Cleaning schedule on page 17 to determine whether cleaning is required after the run lon PGM Sequencing 200 Kit v2 User Guide Materials required Before starting 35 Sequencing protocol lon 314 Chip v2 TM Use the following sequencing protocol with the Ion 314 Chip v2 For the Ion 316 Chip v2 or Ion 318 Chip v2 see page 26 Materials provided in the kit e Sequencing Primer e Control Ion Sphere Particles e Annealing Buffer TM e Ion PGM Sequencing 200 v2 Polymerase Materials provided in the lon PGM Controls Kit e Optional Ion Sphere Test Fragm
52. r radiation Rayonnement laser Caution piercing hazard Attention danger de perforation On On marche Off Off arret On Off On Off marche arr t Earth ground terminal Borne de mise la terre 9 0 BSE Protective conductor terminal main ground Borne de conducteur de protection mise a la terre principale l Terminal that can receive or supply alternating current or voltage Borne pouvant recevoir ou envoyer une tension ou un courant de type alternatif x Do not dispose of this product in unsorted municipal waste A 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 A 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 pour les dispositions d limination et communiquer avec le service la client le pour des renseignements sur les options d limination responsable
53. rated dNTP stocks IMPORTANT Dry ice solid CO2 should 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 influence 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 Instrument vibration and clearances Static electricity IMPORTANT Significant vibration during sequencing may add noise and reduce the quality of the measurements 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 IMPORTANT The Ion PGM System must be positioned so that the front bezel is a minimum of 12 in 30 5 cm and the Reagent Bottles containing dNTPs are a minimum of 8 in 20 3 cm from the front of the laboratory bench The instrument should be at least 40 in 1 meter away from major sources of electronic noise such as refrigerators or microwaves IMPORTANT Always ground yourself on the touch plate located next to the chip clamp prior to handling chips to avoid possible damage from static electricity Always use the touch plate to hold chips when they are not installed in the chip clamp or chip rotor bucket Placing the chip on other non grounded surfaces lik
54. 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 Chip did not calibrate 1 Replace the chip with a new unused one Note The new chip can be used for sequencing after displayed initialization completes 2 Press Start to restart the pH measurement 3 Ifthe second test fails contact Technical Support 56 lon PGM Sequencing 200 Kit v2 User Guide Appendix B Control lon Sphere Particles Control lon Sphere Particles Troubleshooting using the Control lon Sphere Particles 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 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 57 lon PGM Sequencing 200 Kit v2 User Guide Appendix C
55. rompted to insert a cleaning chip you can use the same used chip that was used for initialization 4 When prompted ground yourself by touching the grounding pad next to the chip clamp on the instrument and replace the old chip in the chip socket with the new one for the experiment Do not wear gloves when transferring the chips on and off the instrument Close the chip clamp 5 When prompted use the barcode scanner to scan the barcode located on the chip package or press Change to enter the barcode manually Note A chip cannot be run without scanning or entering the barcode lon PGM System ion torrent 4 RRS SSD 2 Duc Bi bDibiLiSi Dis aw Scan the chip barcode Optional Enter the library kit catalog number Press Chip Check Chip Check 5 riuinics 6 Press Chip Check on the touchscreen 7 During the initial part of Chip Check visually inspect the chip in the clamp for leaks If there is a leak press the Abort button immediately to stop the flow to the chip Then proceed to Appendix B Troubleshooting 28 lon PGM Sequencing 200 Kit v2 User Guide 10 11 Sequencing protocol lon 316 Chip v2 or lon 318 Chip v2 IMPORTANT The chip socket can be damaged by rubbing or wiping its surface If there is a spill or leak onto the chip socket contact technical support see Ion contact information on page 70 When Chip Check is complete e If the chip passes press Next e If the chip f
56. rs press 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 lon PGM Sequencing 200 Kit v2 User Guide Equipment use Appendix F Safety 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 Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document e 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 e 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 Appendix G Documentation and support on page 70 Note The Ion PGM Sequencer performs real time measurements of hydrogen ions produced during DNA replication TM The Ion PGM System is for performing sequencing of amplified DNA and should only be used for life science research applications The lon PGM System should only be used by professionals trained in laboratory techniques and who have studied the instructions
57. s not tight e Bottle may be damaged defective 1 Finger tighten the bottles 2 Ifthe bottle continues to leak replace the bottle 3 If leak check continues to fail contact Technical Support Initialization 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 MO water and gently dabbing the socket with a lab wipe tissue 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 Note The new chip can be used for sequencing after initialization completes 5 Close the clamp then press Start to restart the process 6 Ifthe new chip also fails there could be a problem with the chip socket Contact Technical Support Error message Chip calibration failed 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 Sequencing 200 Kit v2 User Guide 52 Appendix B Troubleshooting Initialization I
58. ser and provide a fast and convenient way to set up and organize your runs We recommend that you create a Planned Run using the Torrent Browser and then select the plan in the lon PGM Sequencer touchscreen as part of the Run workflow see Select the Planned Run and perform the run under the appropriate sequencing workflow for your chip type Note 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 torrent browser Member Name Password Monitor 2 Select the Plan tab select Templates locate the type of experiment you want to run for example Ion AmpliSeq then select one of the following e Plan Run to plana new run using an existing template e Plan New Run to plan a new run without using a template Ampliseq Add New Template Plan New Run lon AmpliSeq Cancer Panel Template Reviev lon PGM Sequencing 200 Kit v2 User Guide 14 Create a Planned Run 3 Inthe wizard review each screen and make your selections Key fields are described in the following table For a complete description of each field see the Torrent Browser User Interface Guide New AmpliSeq Run Plan Kits Monitor Reference Plugins Project Export Plan Application Run Type Generic Sequencing Forward default
59. sh 1 2 and 3 Bottles and sipper tubes Reagent Bottles and sipper tubes Ion PGM Sequencing 200 v2 dGTP Ion PGM Sequencing 200 v2 dCTP Ion PGM Sequencing 200 v2 dATP Ion PGM Sequencing 200 v2 dTTP Ion PGM Sequencing 200 v2 W2 Solution stored protected from light Ion PGM Sequencing 200 v2 1X W3 Solution Other materials and equipment Used chip leave chip on the instrument during initialization 18 MO water 100 mM NaOH prepared daily Ice 5 mL and 25 mL pipettes Filtered and unfiltered pipette tips and pipettes Vortex mixer Microcentrifuge Optional Ion PGM Sequencing Sippers Kit Cat no 4478682 lon PGM Sequencing 200 Kit v2 User Guide 19 Clean and initialize the lon PGM System IMPORTANT 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 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 After four initializations do not use the Wash 1 2 and 3 Bottles for initialization or sequencing to avoid breakage or leaking You can reuse the bottles in the cleaning procedure e Replace the Reagent Bottles and sipper tubes every time you initialize e Make sure that you have
60. st of the water with the lab wipe 4 Repeat the rinse then gently dab the chip socket until dry 5 Place a lab wipe 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 6 Remove the wipe dry the grounding plate and place chip on grounding plate Confirm that there is no condensation outside the flow cell 7 Replace the chip with a new unused one if needed Note The new chip can be used for sequencing after initialization completes 8 Press Run to restart the experiment 9 When prompted to install the new chip make certain that the chip clamp is fully closed 10 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 Error message e Chip not seated in 1 Remove the chip and confirm that there is no leakage Calibration FAILED socket correctly or debris on the chip socket If leaking or debris is e Chip is damaged 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 2 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 4 Ifyou continu
61. ted on the chip package or press Change to enter the barcode manually lon PGM System ion torrent 44 EEE A LL LD ii gt s gt Scan the chip barcode Optional Enter the library kit catalog number Press Chip Check Chip Check izPGM1 B2e TT 3 Fwivics Note A chip cannot be run without scanning or entering the barcode Press Chip Check on the touchscreen During the initial part of Chip Check visually inspect the chip in the clamp for leaks If there is a leak press the Abort button immediately to stop the flow to the chip Then proceed to Appendix B Troubleshooting IMPORTANT The chip socket can be damaged by rubbing or wiping its surface If there is a spill or leak onto the chip socket contact technical support see Ion contact information on page 70 When Chip Check is complete e If the chip passes press Next e If the 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 a new chip See also Appendix B Troubleshooting Note To return damaged chips contact Life Technologies Technical Support Following a successful Chip Check remove the new chip and place it on the grounding plate Insert a used chip in the socket and close the clamp Completely empty the waste bottle as instructed in the touchscreen IMPO
62. the ISPs at a rate of 1 pL per second To avoid introducing bubbles into the chip leave a small amount of sample in the pipette tip 0 5 uL 4 Remove and discard any displaced liquid from the other port of the chip 5 Transfer the chip to the MiniFuge with the chip tab pointing in toward the center of the MiniFuge 6 Centrifuge for 30 seconds then remove the chip from the centrifuge bucket 7 Mix the sample in the chip a Set the pipette volume to 5 pL 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 small bubbles lon PGM Sequencing 200 Kit v2 User Guide 41 Sequencing protocol lon 314 Chip v2 8 Centrifuge the chip for 30 seconds with the chip tab pointing out away from the center of the MiniFuge 9 Repeat the chip mixing in step 7 one more time then spin for 30 seconds with the chip tab pointing in toward the center of the MiniFuge 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 If some 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 If some liquid remains in the chip after th
63. the run on page 33 or 42 lon PGM Sequencing 200 Kit v2 User Guide 3 Clean and initialize the lon PGM System Before starting e Weekly Prepare a stock of 1 M NaOH by diluting 10 M NaOH with 18 MQ water e Daily Prepare 100 mM NaOH by diluting the 1 M stock in 18 MQ water You will need 500 uL of 100 mM NaOH per initialization Clean the lon PGM System Materials required e 18 MQ water e g Elga PURELAB Flex Water Purification System e Cleaning bottles and collection trays provided with the Ion PGM System e Used chip leave chip on the instrument during cleaning e Used sipper tubes from previous run or provided with the instrument e Squirt bottle e Chlorite cleaning Ion PGM Cleaning Tablet provided in the kit e Chlorite cleaning 1 M NaOH e Chlorite cleaning Glass bottle 1 L e Chlorite cleaning 0 22 um or 0 45 um vacuum filtration system and filters Cleaning schedule TM The Ion PGM Sequencer requires cleaning with either 18 MO water or a chlorite solution every time the instrument is initialized Clean with Schedule 18 MQ water e Daily when instrument is in use e g not necessary on weekends e After lt 1000 flows e g 2 x 200 base read runs e If more than 27 hours but less than 48 hours have elapsed between the last cleaning initialization and the start of a run e If you cleaned with chlorite a week ago and have not used the instrument since then
64. tion after this time lon PGM Sequencing 200 Kit v2 User Guide Clean and initialize the lon PGM System Press Clean on the touchscreen and select the Chlorite cleaning checkbox Add 250 mL of filtered chlorite solution to a 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 and attach the bottle to the WI position Following the touchscreen instructions place the empty 2 L cleaning bottle in the W2 position and the empty 250 mL bottle in the W3 position Place collection trays below the sipper tubes in the dNTP positions Press Next to begin cleaning When prompted remove the W1 cleaning bottle with chlorite solution rinse the outside of the sipper with a squirt bottle containing 18 MO water then install a clean 250 mL cleaning bottle filled with 250 mL of 18 MQ water in the W1 position Note The second cleaning bottle is different than the one used for chlorite solution When cleaning is complete remove all bottles and sipper tubes from the W1 W2 and W3 positions Leave the reagent sipper tubes and collection tray s 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 required Materials provided in the kit Wa
65. ttached bottles or tubes as this may reduce the quality of the measurements When the run is complete the touchscreen will return to the Main Menu You can then proceed with another run or perform a cleaning initializing if required Note See Cleaning schedule on page 17 to determine whether cleaning is required after the run lon PGM Sequencing 200 Kit v2 User Guide Appendix A Barcoded libraries 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 IonXpressRNA When setting up a Planned Run or performing a run select the appropriate barcode set for your library type as follows e DNA libraries Select the 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 TonXpressRNA 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 IMPORTA
66. ubleshooting Chip Check Observation Possible cause Recommended action Chip Check fails Clamp not closed Chip not properly seated Debris on the chip socket Chip damaged Open 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 MQ water and gently dabbing the socket with a lab wipe tissue 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 the press Chip Check If Chip Check continues to fail there could be a problem with the chip socket Contact Technical Support 49 lon PGM Sequencing 400 Kit User Guide Appendix B Troubleshooting Chip Calibration Chip calibration Chip calibration before loading sample Observation Possible cause Recommended action Leak of unknown e Chip leak 1 Press Main Menu origin e Chip clamp not 2 Open the chip clamp remove the chip and gently dab closed properly the chip socket with a lab wipe tissue to absorb any e Problem with the fluid Never rub or wipe the socket Rubbing the chip clamp or socket socket can damage it and cause it to fail 3 Rinse the socket with 18 MQ water and gently absorb mo
67. y General CAUTION Do not remove instrument protective covers If you remove the protective instrument panels or disable interlock devices you may be exposed to serious hazards including but not limited to severe electrical shock laser exposure crushing or chemical exposure lon PGM Sequencing 200 Kit v2 User Guide 65 Appendix F Safety Electrical CAUTION Solvents and Pressurized fluids Wear eye protection when working with any pressurized fluids Use caution when working with any polymeric tubing that is under pressure Extinguish any nearby flames if you use flammable solvents Do not use polymeric tubing that has been severely stressed or kinked Do not use polymeric tubing with tetrahydrofuran or nitric and sulfuric acids Be aware that methylene chloride and dimethyl sulfoxide cause polymeric tubing to swell and greatly reduce the rupture pressure of the tubing Be aware that high solvent flow rates 40mL min may cause a static charge to build up on the surface of the tubing and electrical sparks may result 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 A WARNING Power Supply Line

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