Ion Sequencing Kit v2.0 User Guide (PN 4469714B)
Contents
1. Headquarters a 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 Ea For support visit www appliedbiosystems com support technologies www lifetechnologies com2. lon 316 Chip Begin the experiment 26 1 2 Follow the touch screen prompts to begin calibrating the chip The instrument will automatically flush any loose ISPs from the chip and begin calibrating the chip Be sure to check the chip for leaks before closing the lid When the calibration is complete 1 minute the PGM Sequencer will indicate whether calibration was successful Note If calibration fails press Abort reseat the chip then press Calibrate If the chip then passes calibration press Next to proceed with the sequencing run If the chip still fails proceed with the run anyway and contact Technical Support after the run is complete See also Appendix B Troubleshooting After 90 seconds the run automatically begins or press Next to begin the run immediately CAUTION During a run avoid touching the dNTP Conical Tubes and wash bottles or lifting the chip tray lid Touching these components while the instrument is sequencing may reduce the quality of the measurements When the run is complete the PGM Sequencer will return to the Main Menu You can then proceed with another run or perform a cleaning initialization if required Note The instrument should be cleaned and initialized after 130 cycles two runs for the standard 65 cycle workflow or if the reagents have been on the instrument gt 10 hours lon Sequencing Kit User Guide v2 0 Appendix A Sequencing barcoded libraries
3. 10 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 e Chip not seated in 1 Remove the chip and check for leaks and or calibration FAILED socket correctly debris on the chip socket following the e Chip is damaged 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 2 Press Calibrate 3 Ifthe 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 4 Ifyou continue to have chip calibration issues there may be an issue with the chip or chip socket Contact Technical Support 32 lon Sequencing Kit User Guide v2 0 Initialization General Observation Possible cause Recommended action Error message Confirm instrument has gas pressure Argon cylinder may be turned off or empty 1 Verify that the argon cylinder has at least 500 PSI and 40 PSI at the outlet of the regulator Confirm that all valves between the cylinder and PGM are open 2 Once you confirm argon pressure leading into the instrument press Yes to retry verification of gas pressure
4. 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 blockage or no NaOH in W1 Please check W1 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 completes a 36 lon Sequencing Kit User Guide v2 0 Initialization Auto pH errors continued Observation Possible cause Recommended action Error message Auto pH added more Wash Open the bottle of Wash 2 Solution just enough OVERSHOT TARGET 1 to the Wash 2 bottle than to pipette 10 uL of 100 mM HCl for every PH W2 pH n nn was needed and reports 0 1 pH unit gt 7 5 into the bottle Note Argon Failed the pH value will be flowing out of the cap while doing this this is not a hazard and will not cause any issues Press Start to re start auto pH If the pH is still high replace the chip with a new unused one Wash the new chip once with 100 isopropanol and twice with Annealing Buffer insert the chip in the socket then press Start Note The new chip can be used for sequencing after initialization completes If the pH is consistent with the
5. If the test continues to fail contact Technical Support Bottle leak check fails e Bottle seal is 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 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 3 Look for liquid outside the flow cell of the chip 4 Ifyou see liquid replace the chip with a new unused one Wash the new chip once with 100 isopropanol and twice with Annealing 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 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 Follow the procedure for Error message Please insert a chip and press St
6. Stir until the solution is fully dissolved usually in less than 1 minute While the solution is mixing proceed to preparing the Wash 1 and 3 bottles Prepare the Wash 1 and Wash 3 bottles Rinse the Wash 1 and Wash 3 bottles three times with 50 mL of 18 MO water Add 350 uL of freshly prepared 100 mM NaOH solution not 1 M NaOH to the Wash 1 bottle Cap the bottle Add 5 mL of the 10X W3 Solution from the kit to the Wash 3 bottle then add 45 mL of 18 MO water measured using 50 mL conical tube or serological pipette Cap the bottle Begin the initialization 12 IMPORTANT Do not remove the old dNTP sippers until instructed to do so Do not let the new sippers touch any surfaces IMPORTANT Load the bottles as quickly as possible to prevent atmospheric carbon dioxide from reducing the pH of the Wash 2 bottle solution Confirm that a chip is in place on the PGM Sequencer This should be the same chip used to clean the instrument Note The chip type used for cleaning initialization can be different than the chip type used for sequencing For example an Ion 314 chip can be used for cleaning initialization prior to running an Ion 316 Chip From the main menu press Init PGM 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 Technica
7. half of the sample into the large port of the chip dialing down the pipettor to gently and slowly deposit the ISPs A good loading speed is 1 uL second Stop the flow at the 3 uL setting Avoid introducing bubbles into the chip Remove the displaced liquid from the other port of the chip using the same pipette tip Transfer the Ion Chip to the centrifuge with the custom Ion centrifuge adapter rotor If preparing one Ion Chip at a time always balance the centrifuge adapter with a used chip Mark the used chip with a laboratory marker to differentiate it from the new chip for the current experiment Note For the following steps ignore the 10 minute timer on the PGM System screen Centrifuge for 4 minutes 5 Centrifuge the chip for 4 minutes using the custom centrifuge adapter rotor 6 After the first 4 minute spin use a Rainin SR L200F pipette tip to mix the remaining sample by pipetting up and down being careful to not introduce bubbles 7 Use the same pipette tip to carefully deposit the remainder of the sample into the large port of the chip dialing down the pipettor to gently and slowly deposit the ISPs A good loading speed is 1 pL second Stop the flow at the 3 uL setting Avoid introducing bubbles into the chip 8 Remove the displaced liquid from the other port of the chip using the same pipette tip 9 Centrifuge the chip for 4 minutes using the custom centrifuge adapter rotor 10 While the chip is spinning enter the
8. Kit to label four new Conical Tubes as dGTP dCTP dATP and dTTP 3 Using filtered pipette tips and clean gloves carefully transfer 20 uL of each dNTP stock solution into its respective Conical Tube Attach the dNTP sippers and Conical Tubes 1 After the Wash solutions have initialized follow the on screen prompts to remove the four used dNTP sippers and collection trays from the PGM Sequencer Change gloves 2 Using fresh gloves attach new sippers blue to each of the dNTP ports Do not let the sippers touch any surfaces Note The dNTP sippers do not lock they only need to be pushed on securely 13 lon Sequencing Kit User Guide v2 0 3 Attach each Conical Tube to the PGM Sequencer and tighten firmly until snug The correct order of the Conical Tubes on the PGM Sequencer is dGTP dCTP dATP and dTTP left to right when facing the instrument Note The PGM Sequencer will check the pressure of the dNTP tubes and Wash bottles If a bottle leaks you will be prompted to check that it is tightly attached to the instrument If it continues to leak there may be a crack and it should be replaced If you replace the bottle but the instrument still does not pass the leak check contact Technical Support 4 Follow the on screen prompts to complete initialization The PGM Sequencer will fill each Conical Tube with 40 mL of Wash 2 solution Note To conserve time the following ISP preparation procedure can be sta
9. 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 After removing the chip from the socket use a Rainin SR L200F pipette tip to slowly and steadily pipet 50 uL of Annealing Buffer into the large port on the chip With the same pipette tip aspirate any displaced solution from the other port lon Sequencing Kit User Guide v2 0 lon 314 Chip Load the ISPs and configure the experiment Materials provided in the kit Other materials and equipment Sequencing Polymerase e 1 5 mL microcentrifuge tube e Annealing Buffer e Rainin SR L20F pipette tips e Sonication bath 40 KHz e Vortex mixer e Microcentrifuge Bind Sequencing Polymerase to the ISPs 1 After annealing the Sequencing Primer to the ISPs remove the ISPs from the thermal cycler and add 1 uL of Sequencing Polymerase Pipet the sample up and down to mix Incubate the ISPs with polymerase at room temperature for 5 minutes Sonicate the ISPs for 10 seconds using a standard 40 KHz sonication bath Note Make sure the water level in the sonicator is filled to the appropriate mark Load the ISPs on the chip 8 9 IMPORTANT To ensure successful loading of the ISPs on the chip Press the pipette tip firmly into the circular loading port on the chip e Keep the pipette tip at a 90 angle to the chip e Load at a consistent speed and
10. This appendix describes how to create and select barcode sets for sequencing barcoded libraries on the PGM Sequencer Pre installed barcode sets Ion Torrent provides customers with the pre installed barcode set IonSet1 on the Torrent Server Release 1 4 or later This set contains all 16 barcodes in the lon DNA Barcoding 1 16 Kit Part no 4468654 and is viewable using the Torrent Browser Barcodes in ionSeti Sequence CGTGTCGCAC GATGATTGCC CGATAATCTT CTTACACCAC AGCCAAGTAC GACATTACTT GCCTTACCGC ACCGAGGCAC GCAAGCCTTC ACATTACATC CAAGCACCGC AGCTTACCGC CATGATCAAC GACCGCATCC GGTGTAGCAC ACTCACGATA Custom barcode sets You can create custom barcodes sets as comma separated value csv files then load these sets onto the Torrent Server for use during sequencing runs Note To access the Torrent Server you must have a username and password Note For more information on working with custom barcode sets refer to the Torrent Browser User Interface Guide On the Torrent Browser click the References tab then click Working with Barcodes Create a custom barcode set 1 Using Microsoft Excel Notepad or similar software create a custom barcode set with each barcode sequence listed on a separate line in a single column format as shown below A barcode set list can contain up to 384 barcodes Note You can run fewer than 384 barcodes in a sequencing run the PGM Sequencer will automatically detect and select the barco
11. added to Wash 1 bottle e Damaged chip and try again e Ran out of W3 Solution 2 If necessary loosen the Wash 3 bottle cap volume too low 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 3 If there is enough W3 Solution replace the chip with a new unused one Wash the new chip once with 100 isopropanol and twice with Annealing Buffer insert the chip in the socket then press Start Note The new chip can be used for sequencing after initialization completes Error message e Poor water quality 1 Confirm high water quality and correct Added too much W1 e 18 MQ water exposed preparation of the 100 mM NaOH and W2 to W2 to air for too long Solution e Too much W2 2 If solution preparation is incorrect or water concentrate added to quality is poor correctly prepare the prepare the W2 solution s and or use high quality water Solution 3 Clean the instrument e Incorrect solution 4 Repeat instrument initialization with fresh reagents and a new unused chip Note The new chip can be used for sequencing after initialization completes Note Once the system has added too much NaOH the only recourse is to clean the PGM and restart initialization or manually pH the Wash 2 solution see Appendix C Manually adjust the pH of the Wash 2 solution
12. and flow meter 100 mM NaOH prepared fresh daily Pipette tips and pipettor Magnetic stirrer Magnetic stir bar e 100 mM HCl Procedure 1 Before proceeding rinse an empty Wash 2 bottle and have it ready next to the instrument 2 Remove and cap the Wash 2 bottle attached to the instrument Gas will be flowing out of the sipper in the W2 position Note The gas will be flowing out of the Wash 2 cap so work as quickly as possible flowing gas will not harm the Wash 2 solution and is not a hazard 3 Secure the empty Wash 2 bottle from step 1 to the instrument do not remove the sipper This bottle will keep gas from flowing out of the instrument while you pH the Wash 2 solution and protect the sipper from contamination 4 Move the Wash 2 solution to the stir plate near the argon gas tube Secure the argon gas tube so that it extends inside the mouth of the bottle but not below the surface of the water 6 Set the argon flow to 0 5 LPM Start mixing the water fast enough for a small whirlpool to form 7 Calibrate the pH meter using a three point calibration Rinse any buffering solution from the pH probe prior to preparing solutions 8 Using the calibrated pH meter adjust the pH of the Wash 2 bottle to 7 5 0 1 by adding freshly prepared 100 mM NaOH Add small aliquots and allow the pH to equilibrate before adding more Note If the pH rises above 7 6 use 100 mM hydrochloric acid HCl to readjust the pH to 7 5 0 1 9 When
13. cleaning bottles are provided with the instrument This will be attached to the W1 W2 and W3 positions on the instrument when prompted After you have used the Wash bottles provided with the Sequencing Kit for the specified number of sequencing runs you can then use them as cleaning bottles be sure to mark them as for cleaning use only e Make sure 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 can be used for cleaning initialization prior to TM running an Ion 316 Chip MQ water cleaning In general perform an 18 MQ water cleaning on a daily basis after every 130 cycles or if the system has been left with reagents for more than 10 hours 1 Empty any remaining solution from each cleaning bottle and rinse each bottle twice with 100 mL of 18 MO water 2 Select Clean PGM on the touch screen 3 Add 250 mL of 18 MO water to a 250 mL cleaning bottle and attach the bottle to the W1 position on the PGM Sequencer 4 Follow the instructions on the touch screen to perform the cleaning procedure When prompted remove the W1 cleaning bottle rinse the outside of the sipper with a squirt bottle containing fresh 18 MQ water then rinse and reinstall the 250 mL cleaning bottle filled with 250 mL of fresh 18 MQ water 6 When cleaning is finished press Next to return to the Mai
14. in Microbiological and Biomedical Laboratories found at www cdc gov biosafety 40 Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx_01 29cfr1910a_01 html Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO_CDS_CSR_LYO_2004_11 en lon Sequencing Kit User Guide v2 0 Appendix E Documentation and Support Obtaining SDSs Safety Data Sheets SDSs are available from www appliedbiosystems 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 application notes and other product support documents 41 ISbT 269bb
15. may be impaired The Personal Genome Machine System is For Research Use Only Not intended for any animal or human therapeutic or diagnostic use lon Sequencing Kit User Guide v2 0 Contamination CAUTION A primary source of contamination is spurious DNA fragments from previous AN sample processing steps Do not introduce amplified DNA into the library preparation laboratory or work area CAUTION Possible contamination can occur during the transfer of dNTPs into their respective N reagent bottles Care must be taken to avoid cross contamination of dNTP stocks Barrier tips are required for all pipetting steps Gloves should be changed after handling concentrated dNTP stocks TM or sources of molecular biology grade water for the 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 PGM System A CAUTION Dry ice solid CO should be kept away from areas where buffers wash solutions Instrument vibration and clearances CAUTION Install the PGM Sequencer on a bench that is free from vibrations or in contact J with equipment that can cause vibrations to the bench freezers pumps and other similar equipment Significant vibration during sequencing may add noise and reduce the quality of the measurements Ion PGM Sequencer Space Requ
16. pH of the previous chip add more HCL if needed If auto pH fails manually adjust the pH of the Wash 2 Solution see Appendix C Manually adjust the pH of the Wash 2 solution Initialization Reagent pH Verification Observation Possible cause Recommended action Red failure screen One or more reagents are reagent pH displayed not within the target pH Press Start to repeat the pH measurements to confirm the measurement 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 If any reagents still fail something likely went wrong with the initialization and you should clean and re initialize the instrument with fresh reagents and a new chip Red failure screen Chip did not calibrate reagent pH not displayed Replace the chip with a new unused one Wash the new chip once with 100 isopropanol and twice with Annealing Buffer before using Note The new chip can be used for sequencing after initialization completes Press Start to restart the pH measurement If the second test fails there may be a problem with the chip socket Contact Technical Support 37 lon Sequencing Kit User Guide v2 0 Appendix C Manually adjust the pH of the Wash 2 solution Materials and equipment needed Orion 3 Star Plus pH Benchtop Meter Kit or equivalent Argon gas tank tube
17. the Control Ion Spheres and centrifuge for 2 seconds before taking aliquots Add 5 uL of Control Ion Spheres to the PCR tube containing the enriched particles Note If you are using Ion Sphere Test Fragments for installation or troubleshooting add the 5 uL of Test Fragments directly to the Annealing Buffer in the next step Note 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 Add 100 uL of Annealing Buffer Mix by vortexing or pipetting up and down Centrifuge the tube for 2 minutes at 15 500 x g Carefully remove the supernatant with a pipette tip leaving 20 uL of supernatant in the bottom of the tube Add 150 uL of Annealing Buffer Mix by vortexing or pipetting up and down Collect the ISPs by centrifuging the tube for 2 minutes at 15 500 x g 21 lon Sequencing Kit User Guide v2 0 9 Carefully remove the supernatant with a pipette tip leaving slightly less than 15 pL of supernatant in the bottom of the tube Visually compare the volume to a separate tube containing 15 uL of liquid 10 Measure the volume of supernatant If needed use Annealing Buffer to adjust the volume up to 15 pL 11 Add 12 pL of Sequencing Primer to the ISP sample then pipet the sample up and down IMPORTANT Pipette thoroughly to
18. the pH is stable turn off the argon remove the gas line and cap the Wash 2 bottle 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 38 initialization lon Sequencing Kit User Guide v2 0 Appendix D Safety Safety Information 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 the Documentation and Support section in this document Chemical safety WARNING General chemical handling To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions a
19. to a set 1 Open the Torrent Browser and click the References tab 2 Inthe Barcodes panel click the file name of the barcode set to be edited 3 Enter the new barcode sequence in the edit window then click Add Barcode For example Add Barcode facctacetac Delete a barcode from a set 1 Open the Torrent Browser and click on the References tab 2 Inthe Barcodes panel click the file name of the barcode set to be edited 3 Click the Delete button next to the barcode sequence to be deleted You will be prompted to confirm the deletion Selecting a barcode set on the PGM Sequencer 1 During the experimental setup of a sequencing run follow the touch screen prompts to the Run Info screen 2 On the Run Info screen select the appropriate reference library from the library reference drop down menu 3 Click the barcode drop down menu and select the barcode set containing the barcodes used in the sequencing run as shown below Note A barcode set can contain up to 384 barcodes The barcodes used in the run may be a subset of the selected barcode set as long as the set contains all of the barcodes used in the run 28 lon Sequencing Kit User Guide v2 0 ION tOrrenf re Enter Experiment Information SpinDown 7 45 4 Enter the remaining information as needed on the Run Info screen Chip Barcode AA0032774 5 Click Next and follow the remaining prompts to start the run View a report of the barcoded lib
20. 11 lon Sequencing Kit User Guide v2 0 Prepare the Wash 2 bottle 1 Rinse the Wash 2 bottle 2 L three times with 200 mL of 18 MQ water Note Ensure the 18 MO water is fresh from a purification system and has not been stored for more than 3 4 hours in a container For the first use of the bottle add 1 978 mL of 18 MQ water to the Wash 2 bottle mark the fill line and empty the bottle This prevents having to measure the water separately each time Place a clean magnetic stir bar into the empty Wash 2 bottle Insert the argon gas tube into the bottle set the argon flow meter to 0 5 liters per minute LPM and flow argon into the Wash 2 bottle for 5 minutes This will purge carbon dioxide from the container Note For the next steps if your 18 MO water source is not next to your argon source cap the bottle before moving it between locations Remove the argon gas tube and add 18 MQ water to the fill line marked on the Wash 2 bottle 1 978 mL Be careful to extend the water spigot into the bottle to minimize air exchange Place the Wash 2 bottle on a stir plate Secure the argon gas tube so that it extends inside the mouth of the bottle but not below the surface of the water Set the argon flow to 0 5 LPM Start mixing the water fast enough for a small whirlpool to form Note For the following steps be careful to distinguish the W2 and W3 solution bottles to avoid confusion Add 22 mL of W2 Solution to the Wash 2 bottle
21. 18 MQ water supplied fresh not boxed from MLS N A 1 an appropriate system i e MilliQ or equivalent lon Sequencing Kit User Guide v2 0 v Description Supplier Part number Quantity NaOH 10 M molecular biology grade MLS N A Varies Isopropanol 100 MLS N A Varies Magnetic stirrer must hold 2 L bottle MLS N A 1 Magnetic stir bar 4 cm MLS N A 1 P2 P20 P200 P1000 uL pipette set and MLS N A 1 set filtered tips Barrier pipette tips MLS N A Varies Serological pipettor MLS N A 1 25 mL serological pipettes MLS N A Varies Rainin Pipet Lite LTS L 20 2 20 uL Rainin L 20 1 Rainin Pipet Lite LTS L 100 10 100 uL Rainin L 100 1 Rainin SR L20F pipette tips Rainin SR L20F Rainin SR L200F pipette tips Rainin SR L200F 0 2 mL MAXYMum Recovery Thin Wall PCR Axygen PCR 02 L C Varies Tubes Flat Cap do not use polystyrene tubes 1 5 mL or 1 7 mL microcentrifuge tubes MLS N A Varies Graduated cylinders 1 L or 2 L volume MLS N A Glass bottles 1 L MLS N A 15 mL conical tubes MLS N A Ice buckets and ice N A N A Sonicating bath MLS N A 1 2 3 L tank 80 160 W 40 kHz transducer Branson or Fisher supported Branson part no no heater necessary with floating PCR tube 952 118 holder Fisher part no 15 335 20 Squirt bottle N A 1 Vortex mixer with a rubber platform MLS N A 1 Thermal cycler with a heated li
22. 601 1001 01 8 for 250 mL bottle and 4 for 2 L bottle Conical Tube Sippers blue 601 1002 01 16 R TT Conical Tubes and labels 601 1003 01 25 15 25 C Wash 1 Bottle 250 mL and label 601 1004 01 1 Wash 2 Bottle 2 L and label 601 1005 01 1 Wash 3 Bottle 250 mL and label 601 1004 01 1 lon Sequencing Reagents Kit Part no 4468995 Component Cap Color Part Number Per Kit Volume Storage dGTP Black 602 1015 03 1 100 uL dCTP Blue 602 1016 03 1 100 uL dATP Green 602 1017 03 1 100 uL dTTP Red 602 1018 03 1 100 uL 20 C Sequencing Polymerase Yellow 602 1074 01 1 32 uL Sequencing Primer White 602 1073 03 1 96 uL Control lon Spheres Clear 603 1088 01 1 40 uL lon PGM Reagents Kit Part no 4468994 Component Part Number Per Kit Volume Storage W2 Solution round black label 603 1031 02 1 88 mL Annealing Buffer 603 1048 01 1 80 mL gt PGM Cleaning Tablet 603 1051 01 4 a 10X W3 Solution 603 1062 01 1 20 mL lon Sequencing Kit User Guide v2 0 Description The Ion Sequencing Kit provides reagents and materials for performing sequencing runs on the PGM System It also includes reagents and materials for cleaning and initializing the instrument TM This kit is designed to sequence templates that have been amplified on Ion Sphere Particles ISPs using the Ion Xpress Template Kit The kit includes reagents and materials for performing eight runs and four initializations This provides two runs per i
23. GM Sequencer or in the custom Ion centrifuge adapter rotor bucket To avoid ESD damage do not wear gloves when transferring chips onto and off the instrument lon Sequencing Kit User Guide v2 0 Wash the chip IMPORTANT Use Rainin SR L200F pipette tips to enable slow steady washing of the chip Do not let the chip dry out IMPORTANT When loading liquid into the chip press the pipette tip firmly into the circular loading port on the chip not into the slit and keep the pipette tip at a 90 angle to the chip Do not introduce bubbles into the chip 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 PGM Sequencer grounding plate or in the Ion centrifuge adapter rotor bucket 3 Using a Rainin SR L200F pipette tip slowly and steadily pipet 50 uL of 100 isopropanol into the large port of the chip 4 Aspirate any displaced solution from the other port Proceed immediately to the next step Wash the chip two times with Annealing Buffer as follows a Slowly and steadily pipet 50 uL of Annealing Buffer into the large port on the chip b Aspirate any displaced solution from the other port 17 lon Sequencing Kit User Guide v2 0 Chip check on the instrument 18 1 Press Experiment on the main menu and follow the prompts to prepare the PGM Sequencer to test a new Ion Chip When prompted grou
24. USER GUIDE ion torrent 6 AOXO by Life technologies lon Sequencing Kit v2 0 User Guide For use with the Personal Genome Machine PGM System Catalog Number 4468997 Publication Part Number 4469714 Rev B Revision Date July 2011 0 technologies For research use only Not intended for any animal or human therapeutic or diagnostic use COPYRIGHT 2011 Life Technologies Corporation All rights reserved Beyond a single download for personal use no part of this publication may be reproduced transmitted transcribed stored in retrieval systems or translated into any language or computer language in any form or by any means electronic mechanical magnetic optical chemical manual or otherwise without prior written permission from lon Torrent Systems Inc The information in this guide is subject to change without notice Life Technologies Corporation reserves the right to change its products and services at any time to incorporate technological developments Although this guide has been prepared with every precaution to ensure accuracy Life Technologies Corporation hereinafter Life Technologies and its affiliate lon Torrent Systems hereinafter lon Torrent assume no liability for any errors or omissions nor for any damages resulting from the application or use of this information Life Technologies welcomes customer input on corrections and suggestions for improvement TRADEMARKS The trademark
25. active 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 39 lon Sequencing Kit User Guide v2 0 Biological hazard safety AN 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 ZN WARNING Biohazard Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety equipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the information below In the U S e U S Department of Health and Human Services guidelines published in Biosafety
26. apply gentle pressure between the pipette tip and chip e Do not suction the mixture back into the pipette tip Place the chip on the PGM Sequencer grounding plate or in the bucket of the Ion centrifuge adapter rotor Use a Rainin SR L20F pipette tip to carefully deposit 7 uL of the sample half of the sample into the large port of the chip Avoid introducing bubbles into the chip by leaving a small amount of sample in the pipette 0 5 pL Remove the displaced liquid from the other port of the chip using the same pipette tip Transfer the Ion Chip to the centrifuge with the custom Ion centrifuge adapter rotor If preparing one Ion Chip at a time always balance the centrifuge adapter with a used chip Mark the used chip with a laboratory marker to differentiate it from the new chip for the current experiment Note For the following steps ignore the 10 minute timer on the PGM System screen Centrifuge for 4 minutes Centrifuge the chip for 4 minutes using the custom centrifuge adapter rotor Use a fresh Rainin SR L20F pipette tip to mix the remaining sample by pipetting up and down being careful to not introduce bubbles Use the same pipette tip to carefully deposit the remainder of the sample into the loading port on the chip Avoid introducing bubbles into the chip Remove the displaced liquid from the other port of the chip using the same pipette tip Centrifuge the chip for 4 minutes using the custom centrifuge ada
27. art 33 lon Sequencing Kit User Guide v2 0 Initialization Auto pH errors continued Observation Possible cause Recommended action 34 Error message e There may bea blockage or no NaOH e in W1 Please check W1 and run line clear then try again The waste lines may be blocked Wash 1 or Wash 2 sipper may be loose Chip does not detect large enough pH difference between Wash 1 and Wash 2 solutions e Damaged chip Check for blockage 1 Remove the waste bottle 2 Place lab wipes under the waste arm 3 Gently wipe the waste arm with a lab wipe to clear liquid from around the waste line 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 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 Ifthe line s remain blocked contact Technical Support Otherwise press Start to restart auto pH Check Wash 1 and Wash 2 bottles for loose sippers 1 Loosen the Wash 1 cap and re tighten the sipper Since the gas flow
28. d MLS N A 1 Optional Materials and Equipment The following may be required to verify and adjust the pH of the Wash 2 solution during Initialization if prompted by the PGM Sequencer x Description Supplier Part number Quantity Orion 3 Star Plus pH Benchtop Meter Kit Thermo Scientific 1112003 1 with electrode electrode stand and calibration buffers or equivalent 1N HCl MLS N A Varies Note on Power Supply We recommend using an uninterruptable power supply UPS for laboratories that experience frequent power outages or line voltage fluctuations The UPS must be compatible with 500 W output or higher The 1500 VA unit from APCC provides approximately 11 minutes of backup power for an lon PGM Sequencer lon Sequencing Kit User Guide v2 0 Protocol Overview The following protocol describes the steps necessary to prepare a sequencing run or control run The PGM System uses the following workflow when performing sequencing runs Start new experiment Experiments done clean up lon Sequencing Kit User Guide v2 0 PGM Sequencer with Bottles and Conical Tubes Attached Wash 3 Bottle W3 position Wash 2 Bottle W2 position p Waste Bottle dNTP conical tubes L Wash 1 Bottle W1 position Cleaning and Initialization Before Starting Always use freshly prepared NaOH solution Weekly Prepare a stock of 1 M NaOH by diluting 10 M NaOH with fresh 18 MO water on a w
29. 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 continue to have chip calibration issues there may be an issue with the chip socket Contact Technical Support 31 lon Sequencing Kit User Guide v2 0 Chip calibration with sample already loaded Observation Possible cause Recommended action Leak of unknown e Chip leak 1 Press the Abort button origin e Chip clamp not 2 Openthe chip clamp remove the chip and gently closed properly dab the chip socket with a lab wipe tissue to absorb any fluid Do not rub or wipe the chip socket 3 Rinse the socket with 18 MA water and gently absorb most of the water with the lab wipe tissue 4 Repeat the rinse then gently dab the chip socket until dry 5 Place a lab wipe tissue on the grounding plate and dampen it with 18 MQ 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 the chip on the grounding plate Check for condensation outside the flow cell 7 f there is condensation or fluid the chip is damaged and cannot be run 8 If there is no condensation or fluid press Calibrate to restart the calibration procedure 9 lf calibration passes and no leaks are visible press Next to begin the experiment
30. des used in the run from the selected set On aw I gt MyBarcodeSet c uay Home Insert Page Layout Formula amp Calibri j it fa Alignme A7 X B c D CGTGTCGCAC GATGATTGCC CGATAATCTT CTTACACCAC AGCCAAGTAC GACATTACTT 2 Save the file as a csv file and transfer the file to the Torrent Server as described below Clipboard DW mw iN H 27 lon Sequencing Kit User Guide v2 0 Add a custom barcode set to the Torrent Server 1 To add the custom barcode set to the Torrent Server go to the Torrent Browser and click on the References tab In the Barcodes panel click Add to display the Add new DNA barcodes window Enter the barcode set name in the edit window Click Browse to select the file you created ao fF ON Click Upload amp Save The barcode set file name will be displayed in the Barcode panel View a barcode set 1 To view a barcode set go to the Torrent Browser and click on the References tab 2 Inthe Barcodes panel click on the barcode set name to display it Delete a barcode set from the Torrent Server 1 To view the barcode set names go to the Torrent Browser and click the References tab 2 Inthe Barcodes panel click the name of the barcode set that you want to delete The Torrent Browser will display the barcode set 3 At the bottom of the page click Delete Barcode Set A dialog will prompt you to confirm the deletion Add a barcode
31. e adapter bucket 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 PGM Sequencer grounding plate or in the bucket of the Ion centrifuge adapter rotor Using a Rainin SR L200F pipette tip slowly and steadily pipet 100 uL of 100 isopropanol into the large port of the chip Aspirate any displaced solution from the other port Proceed immediately to the next step Wash the chip two times with Annealing Buffer as follows a Slowly and steadily pipet 100 uL of Annealing Buffer into the large port on the chip b Aspirate any displaced solution from the other port Chip check on the instrument 1 Press Experiment on the main menu and follow the prompts to prepare the PGM Sequencer to test a new Ion Chip When prompted ground yourself by touching the grounding pad next to the chip clamp on the instrument and replace the old chip with the new one for the experiment Do not wear gloves when transferring the chips on and off the instrument Chip in position on the PGM Sequencer Use the barcode scanner to scan the chip barcode located on the chip package Note To enter the barcode manually press the Change button next to the barcode field A chip cannot be run without scanning or entering the barcode After the barcode is entered press the Chip Check button Chip Check tests the chip and ensures that it is functio
32. eekly basis Daily Prepare fresh 100 mM NaOH by diluting the 1 M stock in fresh 18 MQ water Cleaning the PGM System Materials provided in the kit Other materials and equipment e Chlorite cleaning PGM e 18 MQ water Cleaning Tablet e Cleaning bottles and collection trays provided with the PGM System e Used chip leave on the instrument during cleaning e Used sippers from previous run or provided with the instrument e Squirt bottle e Chlorite cleaning 1 M NaOH e Chlorite cleaning 0 22 um or 0 45 um vacuum filtration system and filters lon Sequencing Kit User Guide v2 0 Cleaning schedule Cle 18 Ch 10 In general clean the PGM System with 18 MQ water on a daily basis or after 130 cycles and with a chlorite solution once a week e The instrument should be cleaned with 18 MQ water and initialized after every 130 cycles e g two 65 cycle runs Cleaning must be run every time the PGM Sequencer is initialized e If more than 10 hours have elapsed since the previous cleaning initialization re clean with 18 MQ water and initialize before starting a run e Perform a chlorite cleaning once a week or if the instrument has been left with reagents for more than 48 hours e g over the weekend aning setup e Remove any bottles that may be attached to the PGM Sequencer e Do not remove old sippers before performing a cleaning The sippers are used as part of the procedure e Separate
33. eka ated eae a Lakes hehe deh ace cand oa occa oe ian hatnaty 39 Chemical Satety i eenean tanaoa aana eane aea enaa aaa aeaa a EED Ea e e AAAA 39 lon Sequencing Kit User Guide v2 0 Bidlkogical hazard pote No cscs a A LA A 40 Appendix E Documentation and SUPPOrt cccccceseceseceseeeceeeceneeeneecneeceeeceeesenssonesonesoes 41 OBtaIMING SDSS es cs Fats selehenchcnchcnencaches chee cheschenench E st yesadaua senses A E 41 OBtaIMING SUPPOML cc ccecccescececeeeceesctencneectescneecnescneeenencneaeneecneaensneseneneeesenes tan senacesatenaceretenasesmemnecmndts 41 lon Sequencing Kit User Guide v2 0 Preface IMPORTANT Before using this product read and understand the information in Safety in this document IMPORTANT Ifthe Personal Genome Machine PGM System is used or installed in a manner not specified in the installation guide the protection provided by the equipment may be impaired Safety alert words Four safety alert words appear in Life Technologies user documentation at points in the document where you need to be aware of relevant hazards Each alert word IMPORTANT CAUTION WARNING DANGER implies a particular level of observation or action as defined below IMPORTANT Indicates 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 CAUTION Indicates a potentially hazardous situati
34. encing Protocol c cccssssscesesssscesesesseeesesessneesesssneeeesesseneesesseeeeesnseaes 21 lon 316 Chip Prepare the template positive ISPs for SCQUENCING ccccccccsseeseesseeseeteeeseenes 21 lon 316 Chip Test a new lon Chip Chip Cheek 2ccc cs cssccesctonetevetensceseesiiscansetanetenlensusonisanencies 22 lon 316 Chip Load the ISPs on the chip and configure the experiment c ceceeeseeeeeeteees 24 lon 316 Chip Begin the experiment cave euecsrearenresrecsenausseuuacutactucttaasruartus teuttadtMauvers feces 26 Appendix A Sequencing barcoded libraries ccssccesceseeeceeeceeeeeneesneecneeseeeseeeeenesenesens 27 Presinstalled parcode SETS negu a a ee nlenabas 27 Custom barcode Sets ccccicccsssstscecanstacececanenadenstansnananadananananaceeacaeaceeaeaeaceeacndeaneceneseewseteceesedeecceenees 27 Selecting a barcode set on the PGM Sequencer ccccccesceessecseecescesssecseeceseesseecseeceseesseeeseee 28 View a report of the barcoded library SEQUENCING FUN i ittie EEEE EEEE EEEE EEEE ennnen 29 Appendix B Troubleshooting cccessccesccesccnsecenesonseceeeeenenenseoueecasecnsessneesnseonessnesoes 30 Appendix C Manually adjust the pH of the Wash 2 Solution ssscccseeeceeseecenneeseenees 38 Appendix D Safety cu ccccccccsveteccs ve veces en dccee tented tection feos Aatapee tes Senta eter ee a ee released cee 39 Safety ILO rrmatlons 3 x cs tage oleate hate ak
35. experimental configuration on the PGM screen 11 Using the table below determine the number of cycles you want to run based on the average base pair read length Press to specify the number of cycles if it is different than the default default 65 cycles Application Number of cycles Average read Number of Runs per Run time length Initialization Small RNA miRNA 20 cycles 25 bp 5 45 minutes All other supported 65 cycles 100 bp 2 2 hours applications Within a 10 hour time frame 12 Note If the number of cycles to be run cannot be selected there may not be enough disk space to store the experiment data Press the Data Mgnt button to start the Data Management application this can also be accessed from the Tools Menu and delete old runs from the PGM Sequencer Ensure that Autoanalysis and Pre Analysis are selected Note For barcoded libraries select the appropriate barcode set on this screen See Appendix A Sequencing barcoded libraries for more information 25 lon Sequencing Kit User Guide v2 0 Load the chip on the PGM Sequencer 1 When the final spin is complete press Next to verify the experimental setup You do not have to wait for the timer to finish counting down Press OK to confirm the settings or press Cancel to return to the Experimental Configuration Screen to adjust the configuration TM When prompted by the instrument load and clamp the chip on the PGM Sequencer
36. ing enough replacing chip a 2 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 Wash the new chip once with 100 isopropanol and twice with Annealing Buffer before using Note The new chip can be used for sequencing after initialization completes 3 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 flowing gas is not harmful to the Wash 2 Solution and is not a hazard 4 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 Wash 2 solution Error message W2 e out of range Chip measurements very unstable e Chip is damaged See troubleshooting tips for W2 average not stable above 35 lon Sequencing Kit User Guide v2 0 Initialization Auto pH errors continued Observation Possible cause Recommended action Error message Chip reading inconsistent Please replace chip pH response of the chip is not uniform or reliable Verify that there is enough W3 Solution gt 25 mL in the Wash 3 bottle and that the sipper is secure added to the W2 Solution e Too little NaOH
37. ip socket with a lab wipe tissue to absorb any fluid Do not rub or wipe the chip socket Rinse the socket with 18 MO 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 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 chip on grounding plate Confirm that there is no condensation outside the flow cell Replace the chip with a new unused one if needed Wash the new chip once with 100 isopropanol and twice with Annealing Buffer before using Note The new chip can be used for sequencing after initialization completes Press Experiment 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 socket problem Contact Technical support Error message e Calibration FAILED Chip not seated in socket correctly e Chipis damaged Remove the chip and confirm that there are no leaks and no debris on the chip socket If 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
38. irements and Clearances Position the lon PGM sequencer so that the front bezel of the sequencer is a minimum of 12 in 30 5 cm and the tubes containing nucleotides are a minimum of 8 in 20 3 cm from the front of the laboratory bench Place the sequencer at least 40 in 1 meter away from major sources of electronic noise such as refrigerators or microwaves Static electricity 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 Placing the chip on other non grounded surfaces like a bench can result in damage due to static electric discharge Components and Storage Conditions lon Sequencing Kit User Guide v2 0 The Ion Sequencing Kit Part no 4468997 provides reagents and materials for performing sequencing runs on the PGM System The kit includes reagents and materials for performing eight runs and four initializations This provides two runs per initialization if you are performing 65 cycle runs Some applications allow more runs per initialization reagents are provided for up to eight runs for all applications lon PGM Supplies Kit Part no 4468996 Component Part Number Per Kit Storage Wash Bottle Sippers gray
39. l Support Wearing clean gloves follow the on screen prompts to install a new sipper long gray in the cap on the W2 position on the PGM Sequencer Do not let the sipper touch any surfaces lon Sequencing Kit User Guide v2 0 Note Use two hands to attach the sipper as shown below Be careful to avoid cross threading and make sure the sipper is sealed properly 4 Immediately attach the prepared Wash 2 bottle Press Next 5 Install new sippers short gray in the caps on the W1 and W3 positions following the same procedure used for the W2 sipper 6 Attach the prepared Wash 1 and 3 bottles and tighten the caps on the bottles Press Next 7 The PGM Sequencer will test the bottles for leaks fill the Wash 1 bottle and then adjust the pH of the Wash 2 solution This procedure takes approximately 30 minutes Meanwhile proceed to preparing the dNTP Conical Tubes If a wash bottle leaks or if an error occurs during the automatic pH process see Appendix B Troubleshooting Prepare the 50 mL dNTP Conical Tubes CAUTION In the following steps handle the nucleotides carefully to avoid cross contamination and ensure that the correct dNTP tubes are installed in each position on the PGM Sequencer 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 2 Use the labels provided with the Ion PGM Supplies
40. make sure the pellet is completely disrupted before proceeding 12 Program a thermal cycler for 95 C for 2 minutes and then 37 C for 2 minutes using the heated lid option 13 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 316 Chip Test a new lon Chip Chip Check Materials provided in the kit Other materials and equipment Annealing Buffer e New Ion 316 Chip e 100 isopropanol e Rainin SR L200F pipette tips e 1 5 mL microcentrifuge tube e Barcode scanner included with the PGM Sequencer Handling lon Chips 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 PGM Sequencer or in the custom Ion centrifuge adapter rotor bucket To avoid ESD damage do not wear gloves when transferring chips onto and off the instrument Wash the chip 22 IMPORTANT Use Rainin SR L200F pipette tips to enable slow steady washing of the chip Do not let the chip dry out IMPORTANT When loading liquid into the chip press the pipette tip firmly into the circular loading port on the chip not into the slit and keep the pipette tip at a 90 angle to the chip Do not introduce bubbles into the chip Loading port lon Sequencing Kit User Guide v2 0 Centrifug
41. n Menu and proceed to initialization lorite cleaning Clean with a chlorite solution once a week or if the system has been left with reagents for more than 48 hours e g over the weekend Note Ona weekly basis prepare a fresh stock of 1 M NaOH by diluting 10 M NaOH with fresh 18 MO water 1 Fill a container with 1 L of 18 MO water and add a PGM Cleaning Tablet chlorite tablet Wait 10 minutes for the tablet to completely dissolve 2 After 10 minutes 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 lon Sequencing Kit User Guide v2 0 3 Add 250 mL of filtered chlorite solution to a 250 mL cleaning bottle and attach the bottle to the W1 position on the PGM Sequencer 4 Follow the instructions on the touch screen to perform the cleaning procedure When prompted remove the W1 cleaning bottle with chlorite solution rinse the outside of the sipper with a squirt bottle containing fresh 18 MQ water then install a fresh 250 mL cleaning bottle filled with 250 mL of fresh 18 MO water Note The second cleaning bottle is different than the one used for chlorite solution 6 When cleaning is finished press Next to return to the Main Menu and proceed to initialization Initializing the PGM Sequencer Initialization takes 1 hour As part of the initialization process prepare the wash and dNTP solutions a
42. n prompted by the instrument load and clamp the chip on the PGM Sequencer lon 314 Chip Begin the experiment 20 1 2 Follow the touch screen prompts to begin calibrating the chip The instrument will automatically flush any loose ISPs from the chip and begin calibrating the chip Be sure to check the chip for leaks before closing the lid TM When the calibration is complete 1 minute the PGM Sequencer will indicate whether calibration was successful Note If calibration fails press Abort reseat the chip then press Calibrate If the chip then passes calibration press Next to proceed with the sequencing run If the 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 CAUTION During a run avoid touching the dNTP Conical Tubes and wash bottles or lifting the chip tray lid Touching these components while the instrument is sequencing may reduce the quality of the measurements When the run is complete the PGM Sequencer will return to the Main Menu You can then proceed with another run or perform a cleaning initialization if required Note The instrument should be cleaned and initialized after 130 cycles two runs for the standard 65 cycle workflow or if the reagents have been on the instrument gt 10 hou
43. nd instructions e Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document e Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing e Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood e Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures as recommended in the SDS e Handle chemical wastes in a fume hood e 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 e After emptying a waste container seal it with the cap provided e Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory e Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radio
44. nd yourself by touching the grounding pad next to the chip clamp on the instrument and replace the old chip with the new one for the experiment Do not wear gloves when transferring the chips on and off the instrument Chip in position on the PGM Sequencer Use the barcode scanner to scan the chip barcode located on the chip package Note To enter the barcode manually press the Change button next to the barcode field A chip cannot be run without scanning or entering the barcode After the barcode is entered press the Chip Check button Chip Check tests the chip and ensures that it is functioning properly prior to loading the sample After Chip Check is complete press Next to proceed to calibration The chip will be flushed with the solution from the Wash 2 bottle prior to calibration Check for leaks on the chip If there is a leak press the Abort button immediately to stop the flow to the chip Then proceed to Appendix B Troubleshooting CAUTION The chip socket can be damaged by rubbing or wiping its surface When cleaning up spills always gently dab the socket without rubbing After calibration is complete the system indicates if the chip passed or failed e If the chip passed press Next and follow the prompts to remove the Ion Chip e Ifthe chip failed open the chip clamp re seat the chip in the socket close the clamp and repeat the Chip Check If the chip passes press Next If the chip still fails press
45. ning properly prior to loading the sample 23 lon Sequencing Kit User Guide v2 0 After Chip Check is complete press Next to proceed to calibration The chip will be flushed with the solution from the Wash 2 bottle prior to calibration Check for leaks on the chip If there is a leak press the Abort button immediately to stop the flow to the chip Then proceed to Appendix B Troubleshooting CAUTION The chip socket can be damaged by rubbing or wiping its surface When cleaning up spills always gently dab the socket without rubbing After calibration is complete the system indicates if the chip passed or failed e Ifthe chip passed press Next and follow the prompts to remove the Ion Chip e Ifthe chip failed open the chip clamp re seat the chip in the socket close the clamp and repeat the Chip Check 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 After removing the chip from the socket use a Rainin SR L200F pipette tip to slowly and steadily pipet 100 uL of Annealing Buffer into the large port on the chip 10 With the same pipette tip aspirate any displaced solution from the other port lon 316 Chip Load the ISPs on the chip and configure the experiment Materials provided in the kit Other materials and equipment Sequencing Polymerase e 18 MQ water e Annealing Buffer e 0 2 mL PCR tube non
46. nitialization if you are performing 65 cycle runs Some applications allow more runs per initialization reagents are provided for up to eight runs for all applications The Control Ion Spheres provided in this kit are spike in controls used to assess the quality of a sequencing run Materials and Equipment Required The Ion Sequencing Kit uses common molecular biology equipment supplies and reagents Where noted supplies are available from Major Laboratory Suppliers MLS The following table lists required materials and equipment v Description Supplier Part Quantity number lon 314 Chip Kit or Life Technologies 4462923 8 pack lon 316 Chip Kit Life Technologies 4466616 4 pack 4469496 8 pack lon Torrent PGM Sequencer and all included Life Technologies 4462917 1 accessories Torrent Server Life Technologies 4462918 1 lon Control Materials Kit Life Technologies 4466465 1 Tank of Compressed Argon 99 9 industrial MLS N A N A grade or better Multistage dual stage gas regulator VWR International 55850 422 1or2 0 50 PSI 2 3 Bar output 1 8 x 1 4 stem reducing coupler McMaster 5779K699 1 per only required if using a separate tank for the wash wash station Microcentrifuge capable of gt 15 500 x g fits VWR International 93000 196 1 1 5 mL and 0 2 mL microcentrifuge tubes Uninterruptable Power Supply UPS see note MLS N A 1 below Major Laboratory Supplier for example APCC
47. on that if not avoided may result in minor or moderate injury It may also be used to alert against unsafe practices WARNING Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANGER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This signal word is to be limited to the most extreme situations Except for IMPORTANT notes each safety alert word in a Life Technologies document appears with an open triangle figure that contains a hazard symbol These hazard symbols are identical to the hazard symbols that are affixed to Life Technologies instruments SDSs The SDSs for any chemicals supplied by Applied Biosystems or Ambion are available to you free 24 hours a day For instructions on obtaining SDSs see Obtaining SDSs on page 41 IMPORTANT For the SDSs of chemicals not distributed by Applied Biosystems or Ambion contact the chemical manufacturer Equipment intended use The Personal Genome Machine PGM System is intended for performing genomic sequencing of amplified DNA and should only be used for life science research applications The PGM System should only be used by professionals trained in laboratory techniques and who have studied the instructions for use of this instrument If the equipment is used in a manner not specified by the manufacturer the protection provided by the equipment
48. polystyrene e 1 5 mL microcentrifuge tube e Rainin SR L200F pipette tips e Sonication bath 40 KHz e Custom centrifuge adapter rotor with chip spinning bucket included with the PGM Sequencer e Vortex mixer e Microcentrifuge Bind the Sequencing Polymerase to the ISPs 24 1 After annealing the Sequencing Primer remove the ISPs from the thermal cycler and add 3 uL of Sequencing Polymerase to the ISPs Pipet the sample up and down to mix Incubate the sample at room temperature for 5 minutes Add 30 uL of 50 Annealing Buffer 50 water mixture to sample bringing the total to 60 uL Sonicate the ISPs for 10 seconds using a standard 40 KHz sonication bath Note Make sure the water level in the sonicator is filled to the appropriate mark Load lon Sequencing Kit User Guide v2 0 the ISPs on the chip IMPORTANT To ensure successful loading of the ISPs on the chip Press the pipette tip firmly into the circular loading port on the chip e Keep the pipette tip at a 90 angle to the chip e Load at a consistent speed by dialing down the pipettor e Apply gentle pressure between the pipette tip and chip e Do not suction the mixture back into the pipette tip 1 When loading the chip place the chip on the grounding plate on the PGM Sequencer or remove the custom Ion centrifuge adapter rotor bucket and place the chip in it 2 Use a Rainin SR L200F pipette tip to carefully deposit 30 uL of the sample
49. pter rotor 10 While the chip is spinning enter the experimental configuration on the PGM screen 19 lon Sequencing Kit User Guide v2 0 11 Using the table below determine the number of cycles you want to run based on the average base pair read length Press to specify the number of cycles if it is different than the default default 65 cycles Application Number of Average read Number of Runs Run time cycles length per Initialization Small RNA miRNA 20 cycles 25 bp 5 45 minutes All other supported 65 cycles 100 bp 2 2 hours applications Within a 10 hour time frame Note If the number of cycles to be run cannot be selected there may not be enough disk space to store the experiment data Press the Data Mgnt button to start the Data Management application this can also be accessed from the Tools Menu and delete old runs from the PGM Sequencer 12 Ensure that Autoanalysis and Pre Analysis are selected Note For barcoded libraries select the appropriate barcode set on this screen See Appendix A Sequencing barcoded libraries for more information Load the chip on the PGM Sequencer 1 2 When the final spin is complete press Next to verify the experimental setup You do not have to wait for the timer to finish counting down Press OK to confirm the settings or press Cancel to return to the Experimental Configuration Screen to adjust the configuration TM Whe
50. rary sequencing run 1 To view a report of the barcoded library sequencing run go to the Torrent Browser and click the Reports tab 2 Click the report with the name of the selected barcode set 29 lon Sequencing Kit User Guide v2 0 Appendix B Troubleshooting 30 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 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 lon Sequencing Kit User Guide v2 0 Chip calibration before loading sample Observation Possible cause Recommended action Leak of unknown origin e Chip leak Chip clamp not closed properly e Problem with the chip clamp or socket Press Main Menu Open the chip clamp remove the chip and gently dab the ch
51. rs lon Sequencing Kit User Guide v2 0 lon 316 Chip Sequencing Protocol TM Note For a protocol using the Ion 314 Chip see page 15 lon 316 Chip Prepare the template positive ISPs for sequencing Materials provided in the kit Other materials and equipment Control Ion Spheres Annealing Buffer Sequencing Primer thawed on ice e Enriched template positive ISPs prepared as described in the Ion Template Kit User Guide v2 0 Part no 4469004 e 0 2 mL PCR tube non polystyrene e Pipette tips and pipettor e Vortex mixer e Microcentrifuge e Thermal cycler with heated lid programmed at 95 C for 2 minutes and 37 C for 2 minutes Before starting e Use the template positive ISPs that were enriched using the protocol provided in the Ion Xpress Template Kit User Guide v2 0 Thaw the Sequencing Primer on ice Using lon Sphere Test Fragments for installation or troubleshooting If you are performing an installation or troubleshooting sequencing run skip directly to step 2 below and substitute 5 pL of Ion Sphere Test Fragments from the Ion Control Material Kit Part no 4466465 for the 5 uL of Control Ion Spheres in the procedure Add Control lon Spheres and anneal Sequencing Primer to the enriched ISPs 1 TM Transfer the entire volume of enriched ISPs prepared as described in the Ion Xpress Template Kit User Guide v2 0 to a new 0 2 mL PCR tube Do not use polystyrene tubes Vortex
52. rted while the PGM System is initializing When initialization is complete the experiment can begin At the end of initialization the PGM Sequencer will take pH measurements of all the reagents If every reagent is in the target pH range a green Passed screen will be displayed If a red failure screen appears see Appendix B Troubleshooting ION Orrent prosor menn 5 Press Next to finish the initialization process and return to the main menu 14 lon Sequencing Kit User Guide v2 0 lon 314 Chip Sequencing Protocol TM Note For a protocol using the Ion 316 Chip see page 21 lon 314 Chip Prepare the template positive ISPs for sequencing Materials provided in the kit Other materials and equipment e Sequencing Primer thawed e Enriched template positive ISPs prepared as described in the on ice Ion Xpress Template Kit User Guide v2 0 Part no 4469004 e Control Ion Spheres e 0 2 mL PCR tube non polystyrene e Annealing Buffer e Pipette tips and pipettor e Vortex mixer e Microcentrifuge e Thermal cycler with heated lid Before starting e Use the enriched template positive ISPs that were enriched using the protocol provided in the Ion Xpress Template Kit User Guide v2 0 e Thaw the Sequencing Primer on ice Using lon Sphere Test Fragments for installation or troubleshooting If you are performing an installation or troubleshooting sequencing run skip directly to step 3 below and subs
53. s described in this section IMPORTANT Sequencing kits are designed for four PGM initializations After four initializations the Wash 1 2 and 3 bottles should not be used for sequencing to avoid breakage or leaking Save the bottles for use in the cleaning procedure if desired IMPORTANT The dNTP Conical Tubes and sippers must be replaced after every run Materials provided in the kit Other materials and equipment e Wash 1 2 and 3 bottles and e Used chip leave on the instrument during initialization sippar e 18 MQ water aN conical tubes and e 100 mM NaOH prepared fresh daily sippers e Ice e dGTP dCTP dATP and dTTP e W2 Solution e W3 Solution e Argon gas tank tube and flow meter e Magnetic stirrer e Magnetic stir bar e Serological pipette and 25 mL pipettes e Filtered and unfiltered pipette tips and pipettors e Vortex mixer e Microcentrifuge e Optional pH meter multipoint pH calibration reagents pH probe and probe stand Before initialization e Remove the dNTP stock solutions from the freezer and begin thawing on ice CAUTION Handle nucleotides carefully to avoid cross contamination Always discard gloves after removing used sippers from the PGM Sequencer in order to avoid cross contamination of the nucleotides Gloves should also be changed after handling concentrated dNTP stocks e Check the argon tank pressure When the tank pressure drops below 500 psi change the tank
54. s mentioned herein are the property of Life Technologies Corporation or their respective owners KimWipes is a registered trademark of Kimberly Clark Corporation Pipet Lite and Rainin are registered trademarks of Rainin Instrument LLC MAXYMum Recovery is a registered trademark of Axygen Inc WEB SITE www iontorrent com IMPORTANT PHONE NUMBERS If you are located in North America please contact lon Torrent at 1 87 SEQUENCE 1 877 378 3623 If you are located outside of North America please contact lon Torrent at 1 203 458 8552 ION COMMUNITY ioncommunity iontorrent com SERVICE EMAIL ionsupport alifetech com ADDRESS lon Torrent lon Torrent 246 Goose Lane 7000 Shoreline Court Suite 100 Suite 201 Guilford CT 06437 South San Francisco CA 94080 USA USA Part Number 4469714 Rev B 07 2011 lon Sequencing Kit User Guide v2 0 Table of Contents PROTACC soe eee ease ee ts es ee eat cect aes eee acca ean te tae eee 3 Safety alert WOrdS cccccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeaaaeaeeeeeeeeeecaaaaaceeeeeeeeeaaaaeeeeeeessaaaaceeeeeeesesaaaeeeeeeeess 3 oS Oe EE 3 Equipment intended Usni ii E TE babe be ee te eees bane decey E E eves ended E 3 CONANA TON 2s ae he De a a a a a a a be NN 4 Instrument vibration and clearances eeeeeeececeeeceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeteeeeeeetttetess 4 Static electri etty cz tcccgcasee E E sees pede cteenepe yess yep ob pane aembeenetadeenecenatitetadeee
55. s when the cap is loose tighten the sipper as quickly as possible The flowing gas is not harmful to the Wash 1 Solution and is not a hazard 2 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 flowing gas is not harmful to the Wash 2 Solution and is not a hazard 3 Press Start to re start the auto pH process lon Sequencing Kit User Guide v2 0 Initialization Auto pH errors continued Observation Possible cause Recommended action Continued Continued Check for a damaged chip or chip clamp pariposer 1 Replace the chip with a new unused one Wash the new chip once with 100 isopropanol and twice with Annealing Buffer 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 a hazard 2 Recap the bottle and shake gently to mix 3 Press Start to restart auto pH Error message W2 Reading for W2 Solution is 1 Remove the waste bottle and gently wipe excess average not stable not stabilizing quickly fluid from the waste lines with a lab wipe Try reseat
56. teae 4 Components and Storage Conditions cssccceeeceeeceeecensceenecenecenesonseenneeenesenseeesesenesenens 5 DESCrIPUON si eccivcsecccdvesesvwis sd aranwap ninina iu anaa iha ekaia sebeis a aaa ekaa oceeduvyweuseWayccanibeiNweurweutvoeden 6 Materials and Equipment Required cccsccsseesceseeeceeeesneeeneecneeceeecosecenesenssensesenesenesones 6 Ar T A TT 8 PGM Sequencer with Bottles and Conical Tubes Attached cscsssssscssessssseesessseeeeeeeeas 9 Cleaning and Initialization icccciceccceeieeceecedecicecedeedacdecvedavsueuudeaveensdenueetaleeduveavedanvenusdeuvensedes 9 Bere Star a a a eae ee E os a an aaa alana ERLEEN 9 Cleaning the POM SyS E fel tat a Sol tot Sa laa ae ot te ee A 9 Initializing the PGM Sequencer cccccccccessccesseececeeeeseecesseaeeseecenseecesseecnuseeenseecetseeeeetseeeseeees 11 lon 314 Chip Sequencing Protocol ssccccecsssssssscecceceeeesesssnseceeesesesnessssnsnceseesaessessanees 15 lon 314 Chip Prepare the template positive ISPs for SEQUENCING cc cccccceseeeseeesteeeteeeeees 15 lon 314 Chip Test a new lon Chip Chip Check ccccccccescceseecseecesceeseecseeesseeeseecneeenseens 16 lon 314 Chip Load the ISPs and configure the experiment cccccceceeseeseeeseeeseeeteeeeeeeeees 19 lon 314 Chip Begin the experiment cec ctsccnccctaccscscdatecstesansdacsiacasensbaccpecatacagetascnssesteeeiatedanecouce 20 lon 316 Chip Sequ
57. the ISPs by centrifuging the tube for 2 minutes at 15 500 x g 15 lon Sequencing Kit User Guide v2 0 Carefully remove the supernatant with a pipette tip leaving slightly less than 9 uL of supernatant in the bottom of the tube Visually compare the volume to a separate tube containing 9 uL of 7 liquid 8 9 uL 9 Measure the volume of supernatant If needed add Annealing Buffer to increase the volume to Add 5 uL of Sequencing Primer to the ISP sample then pipet the sample up and down IMPORTANT Pipette thoroughly to make sure the pellet is completely disrupted before proceeding 10 Program a thermal cycler for 95 C for 2 minutes and then 37 C for 2 minutes using the heated lid option 11 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 314 Chip Test a new lon Chip Chip Check Materials provided in the kit Annealing Buffer e Handling lon Chips 16 Other materials and equipment New Ion 314 Chip 100 isopropanol Rainin SR L200F pipette tips 1 5 mL microcentrifuge tube Barcode scanner included with the PGM System Ion centrifuge adapter rotor included with the PGM System 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 P
58. titute 5 pL of Ion Sphere Test Fragments from the Ion Control Material Kit Part no 4466465 for the 5 uL of Control Ion Spheres in the procedure Add Control lon Spheres and anneal Sequencing Primer to the enriched ISPs 1 Transfer half of the volume of enriched template positive ISPs prepared as described in the Ion Xpress Template Kit User Guide v2 0 to a new 0 2 mL PCR tube Do not use polystyrene tubes Note Store the remaining unused enriched ISPs at 4 C for up to 1 week They may be used for another sequencing run 2 If the transferred volume of ISPs in step 1 was e lt 50 uL Proceed to step 3 e gt 50 pL Collect the ISPs by centrifuging the tube for 2 minutes at 15 500 x g Remove the supernatant leaving 50 uL at the bottom of the tube then proceed to step 3 Vortex the Control Ion Spheres and centrifuge for 2 seconds before taking aliquots Add 5 pL of Control Ion Spheres to the PCR tube containing the enriched particles Note For installation or troubleshooting add the 5 uL of Ion Sphere Test Fragments directly to the Annealing Buffer in the next step Note 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 5 Add 150 uL of Annealing Buffer Mix by vortexing or pipetting up and down 6 Collect
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