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MiSeq FGx Instrument Reference Guide - Support

1. Allow the reagent cartridge to thaw in the room temperature water bath for approximately 60 90 minutes or until it is thawed completely Remove the cartridge from the water bath and gently tap it on the bench to dislodge water from the base of the cartridge Dry the base of the cartridge Make sure that no water has splashed on the top of the reagent cartridge Inspect the Reagent Cartridge 1 Invert the reagent cartridge ten times to mix the thawed reagents and then visually inspect that all positions are thawed NOTE It is critical that the reagents in the cartridge are thoroughly thawed and mixed to ensure proper sequencing Visually inspect the reagents in positions 1 2 and 4 to make sure that they are fully mixed and free of precipitates Gently tap the cartridge on the bench to reduce air bubbles in the reagents E NOTE e The MiSeq FGx sipper tubes go to the bottom of each reservoir to aspirate the reagents so it is important that the reservoirs are free of air bubbles Place the reagent cartridge on ice or set aside at 2 C to 8 C up to 6 hours until ready to set up the run For best results proceed directly to loading the sample and setting up the run MiSeq FGx Instrument Reference Guide 3 9 oDpUUED juebesy ou eyedojg Performing a Run Load Sample Libraries onto Cartridge 40 NOTE If you are performin
2. TGCAAC A AACC GACG BC CATTAAGAGCT TIN TGAI TCCACTGATTCAACGTACCGTAACGAACGTATCAATT GTTAACOTTAA CGAAAAGAATGATAACA TAACGAACGTATCAATTGAGACTAGCAACGACG TTAACGTACCA GAGACTAAATAI AACCTTAAGATTACTTGATCCACTGATTCAA GTATCAATTGAGACTAGCAAC CGAAAA AGAC TAAATAT TAACG RE TRA ACTAAATATTAAGG IAG TTAAGA CTTAAGATTACTTGA TCTG GTACCATTAAGAGCTACC j CC GACC GAATGATAACAGT ITCTGT ARES y TAGGAAC TACCATTAAGAG AC CGTACCGTAACGA GCTACC CAAC ACCGIA IES CATT GATCCACTGATTCAACGT CGAACGTATCAAT TGAGACTAACGACGA i TGCAACGACGAAAAGAAT GATAACAG TAACAC AA CTTGATCCACTGAT TCAACC AACCT TAAGAT TA iIACTAAGCTACCGTGCAACGACGAAAAGAATGA AGCTACCGTGCAACGACGAAAA ITAAGAGCTACC
3. NOTE Make sure to insert the MiSeq tube with sodium hypochlorite into tray position 17 only Inserting the tube in another position can make cluster generation fail in subsequent runs and can damage the fluidic system of the MiSeq FGx instrument From the post run wash screen select Start Wash The software automatically raises the sippers in the reagent chiller Wait several seconds to make sure that the sippers are fully raised before continuing Open the reagent compartment door and reagent chiller door and slide the used reagent cartridge from the chiller Slide the wash tray into the reagent chiller until it stops and then close the reagent chiller door Raise the sipper handle in front of the SBS Solution PR2 bottle and waste bottle until it locks into place Remove the SBS Solution PR2 bottle and replace it with the wash bottle E NOTE Discard the SBS Solution PR2 bottle after each run Do not reuse any remaining SBS Solution PR2 Remove the waste bottle and discard the contents appropriately Return the waste bottle to the reagent compartment q WARNING This set of reagents contains formamide an aliphatic amide that is a probable 11 12 13 reproductive toxin Personal injury can occur through inhalation ingestion skin contact and eye contact Dispose of containers and any unused contents in accordance with the governmental safety standards for your region For more information see the SDS for this
4. Introduction 52 Performing a Post Run Wash 53 Performing a Maintenance Wash 56 Performing a Standby Wash 59 Shutting Down the Instrument 61 m E kr d a Q SA eT mu m aa is D F A TRS a nee har d aca Z nacos egen gen sonent MiSeq FGx ATE Ea r da c rn ogagrcttH oe Instrument Reference Guide 51 y Jedeuo Maintenance Procedures Introduction Always perform an instrument wash following the completion of a sequencing run Regular instrument washes ensure continued performance in the following ways Flushes any remaining reagents from the fluidics lines and sippers Prevents salt accumulation and crystallization in the fluidics lines and sippers Prevents cross contamination from the previous run Maintenance Frequency Perform the maintenance activities described in this chapter at the intervals shown in the following tables Table 1 Maintenance During Normal Operation Activity Daily Monthly As Needed Post Run Wash After every run Maintenance X Wash Standby Wash To prepare for idle 2 7 days unused
5. KJ01SIH UOISIASY Part 15050524 Rev C Table of Contents Chapter 1 Getting Started Intended Use Components Starting the MiSeq FGx Required Consumables Chapter 2 MiSeq FGx Software Introduction MiSeq FGx Control Software Analysis Software Options for RUO Runs Chapter 3 Performing a Run Introduction MiSeq FGx Workflow Prepare the Reagent Cartridge Log In and Select Run Type Cleaning the Flow Cell Loading the Flow Cell Loading Reagents Starting the Run Monitoring the Run Chapter 4 Maintenance Procedures Introduction Performing a Post Run Wash Performing a Maintenance Wash Performing a Standby Wash Shutting Down the Instrument Chapter 5 Troubleshooting Introduction Resolving Run Setup Errors Resolving RFID Read Failure Preventing Reboots During a Run Troubleshooting Flow Rate Error Performing a Volume Test Measuring Expected Wash Volumes MiSeq FGx Instrument Reference Guide Part 15050524 Rev C Getting Started Intended Use Components Starting the MiSeq FGx Required Consumables MiSeq FGx Instrument Reference Guide LJe1deuo Getting Started Intended Use The MiSeq FGx is a sequencing instrument that measures fluorescence signals of labeled nucleotides through the use of instrument specific reagents and flow cells imaging hardware and data analysis software The MiSeq FGx as part of the MiSeq Forensic Genomics System is
6. Please select BaseSpace option Z Use BaseSpace for storage and analysis Y all raw data from this run will also be stored on this instrument Enter your Mylllumina account Email Password If you do not have an account please go to https my illumina com to sign up y A Part 15050524 Rev C Load Flow Cell Screen The Load Flow Cell screen appears when it is time to load the flow cell After loading the flow cell close the flow cell latch and flow cell compartment door Both the latch and compartment door must be closed before beginning the run Figure 21 Load Flow Cell Screen Please load the flow cell and walt for the RFID to be read When the flow cell is loaded the software reads and records the RFID A confirmation that the RFID was successfully read appears in the lower right corner of the screen If the REID cannot be read identifying information can be entered manually However the software allows only one of the three RFID labeled components flow cell reagent cartridge SBS Solution PR2 to fail on a run For more information see Resolving REID Read Failure on page 66 Select the help icon in the upper right corner of the screen The help file contains a video that illustrates the steps for loading the flow cell Load Reagents Screen The Load Reagents screen has two steps first load the SBS Solution PR2 bottle and empty waste bottle and then load the reagent cartridge When the SBS Sol
7. 15050524 Rev C d When prompted slowly lower the sipper handle completely making sure that the sippers lower into the wash bottle and waste bottle Figure 49 Remove Droplet from Sipper Please lift the PR2 sipper Remove any large droplets on the tip by dipping the sipper slightly back into water Surface tension should pull the droplet off Insert PR2 and waste bottle Insert Wash Tray Insert MiSeq Flow Cell Conduct Volume Test x Stop Test 4 f de N mi 4 P one 1se sunjon e BUIUUJOLIS9J 8 Select Next The volume test begins When the volume test is complete the results appear on the screen Figure 50 Volume Test Results lumina MiSeq FGx System Check is complete Results are displayed below Conduct Volume Test Show Details Export Results 7 e 00000 400 C nae If the test did not pass perform a maintenance wash See Procedure on page 56 9 When the maintenance wash is complete repeat the volume test MiSeq FGx Instrument Reference Guide 71 Troubleshooting Measuring Expected Wash Volumes 12 Measuring expected wash volumes confirms that wash fluidics are performing efficiently 1 2 Before beginning a wash empty the waste bottle When the wash is complete measure the wash volume in the waste bottle Wash Type Post Run Wash Standby Wash Maintenance Wash Expected Wash Volume 25 5 ml 46 ml 17 25 ml Part 15050524 Rev C Index A activity
8. 900000 Contact the facility administrator to get information about what network settings to enter System Check Screen The System Check screen is typically used to connect with an Illumina Technical Support representative during a Live Help session Use of this feature is not required during normal operation or for instrument maintenance Some system checks can be performed before contacting Illumina Technical Support such as the Volume Test A volume test checks the health of the fluidics system by estimating the flow volume as bubbles pass by the sensors For more information see Performing a Volume Test on page 70 Upon completion of a system check the test results appear on the screen Select Show Details to see a summary of the results on the software interface Select Export Results to export the results in a csv file format to a USB drive MiSeq FGx Instrument Reference Guide 1 9 9JEMIJOS 0J1UO2 XJ DOSIWN MiSeq FGx Software Figure 13 System Check Options Please select the system check options to pertorm O Select All Motion System O Y Stage Test Or emm D rone O sueros Manual Update Screen The Manual Update feature is used to update instrument control software and analysis software This feature is not available for the MiSeq FGx All software updates are performed by Illumina personnel to maintain proper system configuration Review Screen The Review screen confirms that MiSeq
9. Access Code Instrument ID M99999 Machine Name CONFIG376VM0E MiSeq FGx Software 00000 e 2 Part 15050524 Rev C Analysis Software Options for RUO Runs After RTA analysis is complete MiSeq FGx sequencing data for ForenSeq runs require analysis on the ForenSeq Universal Analysis Software When performing an RUO run on the MiSeq FGx sequencing data can be analyzed on the cloud using BaseSpace or on the instrument computer using MiSeq Reporter BaseSpace and MiSeq Reporter produce information about alignment variants and contig assemblies for each genome requested and for each sample of a multi sample run BaseSpace Overview BaseSpace is the Illumina cloud computing environment Using BaseSpace to store and analyze your run data provides the following benefits Eliminates the need for onsite storage and computing Enables web based data management and analysis Allows a new sequencing run to be started while data are being analyzed Provides tools for global collaboration and sharing When you begin your sequencing run data files are encrypted in transit decrypted during analysis and encrypted again when stored You can log in to BaseSpace when you set up the sequencing run For more information see BaseSpace Option Screen on page 24 BaseSpace automatically disconnects from the MiSeq FGx at the end of the run or as soon as RTA analysis files have finished uploading If the internet connection is interrupted a
10. NextBio NextSeq Powered by Illumina SeqMonitor SureMDA TruGenome TruSeq TruSight Understand Your Genome UYG VeraCode verifi VeriSeq the pumpkin orange color and the streaming bases design are trademarks of Illumina Inc and or its affiliate s in the U S and or other countries All other names logos and other trademarks are the property of their respective owners Read Before Using this Product This Product and its use and disposition is subject to the following terms and conditions If Purchaser does not agree to these terms and conditions then Purchaser is not authorized by Illumina to use this Product and Purchaser must not use this Product 1 Definitions Application Specific IP means Illumina owned or controlled intellectual property rights that pertain to this Product and use thereof only with regard to specific field s or specific application s Application Specific IP excludes all Illumina owned or controlled intellectual property that cover aspects or features of this Product or use thereof that are common to this Product in all possible applications and all possible fields of use the Core IP Application Specific IP and Core IP are separate non overlapping subsets of all IIlumina owned or controlled intellectual property By way of non limiting example Illumina intellectual property rights for specific diagnostic methods for specific forensic methods or for specific nucleic acid biomarkers sequences or comb
11. Sippers m E Z 00000 The Perform Wash screen also includes a command that raises the sippers so the reagent cartridge can be removed from the instrument in the case of an error or unexpected run interruption a y CAUTION Always close the reagent chiller door after loading the wash tray and before starting a wash This prevents potential injury that could occur if your hands are in the path of the sippers when they lower Manage Files Screen Use the Manage Files feature to move upload or delete files on the instrument computer The screen is divided into five tabs Runs Sample Sheets Manifests Genomes Recipes and Bundle Logs Figure 7 Manage Files Screen 14 Eustomer yourlab com Manage Files Sample Sheets Mani ifests Genomes Directory p Milumina MiSeqF GxOutput O EZB gt Dato ResgortBarcodo enden mm ReuzanoroocDE Unknown O mme Q RG1ZM557 00CDE ven D wm nou2sasoroocpE Unknown D wm Rei23456r00cDE unknown Lp 12 Username 8 engraamin engracmin engradmin engradmin Part 15050524 Rev C Manage Files Options From any tab on the Manage Files screen select Browse to navigate to any files accessible to the instrument Tab Features Use Runs Delete or Move For forensic or RUO mode Sample Sheets Delete Upload or Rename For RUO mode Manifests Delete or Upload For RUO mode Genomes Delete or Upload For RUO mode Recipes Delete or Upload
12. To perform a run on the MiSeq FGx follow the setup steps described in this chapter After the run begins no other user intervention is required After the sequencing run is complete perform an instrument wash 3 6 Part 15050524 Rev C MiSeq FGx Workflow Make sure that the sample libraries have already been loaded onto the reagent cartridge before setting up the run These steps apply to any assay protocol Prepare the reagent cartridge and then load the pooled library into the designated reservoir From the software interface select Sequence to start the run set up steps Optional Connect to BaseSpace if performing an RUO run Wash and thoroughly dry the flow cell Follow the software prompts to load the flow cell Follow the software prompts to load the SBS Solution PR2 bottle make sure that the waste bottle is empty and load the reagent cartridge For an RUO run select a sample sheet Review run parameters and pre run check results Select Start Run As desired monitor the run from the Sequencing screen Perform a post run wash using nuclease free water mixed with sodium hypochlorite Cluster Generation During cluster generation single DNA molecules are bound to the surface of the flow cell and then bridge amplified to form clusters Sequencing Following cluster generation clusters are imaged using LED and filter combinations specific to each of the four fluorescently labeled dideoxynu
13. and ii pay all settlements entered into and all final judgments and costs including reasonable attorneys fees awarded against Purchaser in connection with such infringement claim If this Product or any part thereof becomes or in Illumina s opinion may become the subject of an infringement claim Ilumina shall have the right at its option to A procure for Purchaser the right to continue using this Product B modify or replace this Product with a substantially equivalent non infringing substitute or C require the return of this Product and terminate the rights license and any other permissions provided to Purchaser with respect this Product and refund to Purchaser the depreciated value as shown in Purchaser s official records of the returned Product at the time of such return provided that no refund will be given for used up or expired Consumables This Section states the entire liability of Illumina for any infringement of third party intellectual property rights Exclusions to Illumina Indemnification Obligations Illumina has no obligation to defend indemnify or hold harmless Purchaser for any Illumina Infringement Claim to the extent such infringement arises from i the use of this Product in any manner or for any purpose outside the scope of research use purposes ii the use of this Product in any manner not in accordance with its Specifications its Documentation the rights expressly granted to Purchaser hereunder or any br
14. gt gt e 256 10 o9 Sro loo a gt SQ 65 E gt o gt O m D A Q el gt o EI gt 3 o gt o o o ej E gt o D AI EDGAR NG CTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACT TGATCCACTGAT TCAACG TACCGTAACGAACGTATCAAT TGAGACTAAATAT T CTGTIAACCTTAAGATTACTTGT RE e ACE AA ECT e AC TAAATAT IAACGTACCAT TAAGAGCTACCG fi GATAACAGTAACACACT TCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACG IACCGTAACGAACG TAT CAAT T TGAGACTAAATATTAACGTTGTTAACCTTAAGATTACT TGATCCACTGAT TCAACG TACCGTAACGAACGTAT CAAT TGAGAC TAAATAT T TCTGTIAACCTTAAGATTACT AT AERA DE AG TAA ACA AES AUN EEN AT TAACGTACCAT TAAGAGC TACAACCT TAAGAT TACT TGAT CC GATTCAACGTACCGTAACGAA o e gt 3 z O o z O gt O 8 iG E 93 O ACTAAAT GATAACAGTAACACACTI TCTGTTAACCTTAAGATTAC GATCCAGT GAT CAAGGTACCGTAACCAACGL ATCAATTG O7 GO x GAGACTAAATATTAACGTACCAT TAA AGACTAAATAT TAACG ACCAT LAC TACO OTORAAGTI Ee TRAD ACA TAT CAAT TOA A 3CIACCGTGCAACGAAAATAACCTTAAGATTACT TGATCCACTGAT TCAACGTACTTCTGTTAACCTTAAGAT TACTTGAT f ASE AE AS CAC TAA A CI TI CTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCG TAAAGATTACT TGATO GA PRG A CTGTTAACCTTAAGATTACTTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT Taca ACCAT TAAGAGCTACCGTGCAACAGTAACACACTICTGT TAACCTIAAG e A GATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGT
15. label directly below the barcode Select Done 8 Select Next to proceed to the next run setup step SBS Solution PR2 Bottle 1 Always retry the RFID read before proceeding To do so raise and then lower the reagent sipper handle If the RFID fails a second time open the reagent compartment door Raise the sipper handle and remove the SBS Solution PR2 bottle Check the SBS Solution PR2 bottle and the reagent compartment for debris or salts that can stop the REID read Replace the SBS Solution PR2 bottle and slowly lower the sipper handle until it locks into place Make sure that the sippers lower into the SBS Solution PR2 and waste bottles If the RFID continues to fail select the keyboard icon to open the on screen keyboard Enter the barcode number of the SBS Solution PR2 bottle which is located on the SBS Solution PR2 bottle label directly below the barcode Select Done Select Next to proceed to the next run setup step Part 15050524 Rev C Reagent Cartridge 1 8 9 Always retry the RFID read before proceeding To do so open and then close the reagent chiller door If the RFID fails a second time open the reagent chiller door and remove the cartridge Check the reagent cartridge and reagent chiller for debris or salts that can stop the RFID read Replace the reagent cartridge and close the reagent chiller door If the RFID continues to fail select Enter Reagent Kit Barcode Enter the
16. 8 Part 15050524 Rev C Starting the Run After loading the flow cell and reagents review the run parameters and perform a pre run check before starting the run Review Run Parameters 1 Review Worklist Name Analysis Workflow and Read Length In RUO mode these parameters are specified in the sample sheet 2 When in RUO mode review the folder locations in the lower left corner If any changes are needed select Change Folders When the changes are complete select Save and then select Next 3 Select Next The Pre Run Check screen opens Review Pre Run Check The system performs a check of all run components disk space and network connections before starting the run If any items do not pass the pre run check a message appears on the screen with instructions on how to correct the error For more information see Resoloing Run Setup Errors on page 65 When all items successfully pass the pre run check select Start Run Important Note Before Starting the Run Y WARNING The MiSeq FGx is sensitive to vibration Touching the instrument after starting a run could adversely affect sequencing results After selecting Start Run do not open the flow cell compartment or the reagent compartment doors or touch the instrument monitor except to pause the run For more information see Pausing a Run on page 30 MiSeq FGx Instrument Reference Guide A 9 ung eui 6unjeis Performing a Run Monitoring the Run 1 During the run mo
17. E GAACTTC y Q 6 gt IAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAA d o D gt A D A gt Di gt gt gt gt O J 3 e 3 gt O o A 8 583 o gt d gt o e e D gt D A Q O g o S gt o O Hole gt Q Q Bg SES O D EE iQ io A E ER 22 2 z O 3 D A O O gt A 3 O gt Q O gt Q O D S Q gt gt gt gt Q e gt n 3 D gt C gt O m gt 3 gt Q S Es O 9 ps SE o gt CH Q gt O Q O A OOIT gt 00 BE o ze gt gt Ie 2 a ES O D Q 3 o fo gt D E O 3 2 gt gt gt e gt Ol O D o gt D O 9 Q e o gt CTG TAA CG C C C CATTAAGAG GIAGCGTGCAACAGTAAGACACTICTGT TAACCTTAAGAT TACT GAT CCACTGAT CAACGTACCGTAACGAAGGIATCAATI GAGACTAAATAT AGE AG TT ITGATCCACTGATTCAACGTTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGCT TCTGTTAACCTTAAGATTACT TGATCCACTGAT AATTGAGACTAAATAT TAACGTACCAT TAAGAG TCTGTTAACCT TAAGAT TACT TGATCCACTGAT TCAACGTACCGTAACGAACG TATCAAT TGAGAC TAAATAT TAACG TACCAT T WES Ce CAACGTIAAGATTACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAATTGAGCTTCTGTTAACCTTAAGAT TACT TGATCCACTGAT m CM EI
18. FGx Control Software received the run information from the ForenSeq Universal Analysis Software Figure 14 Review Screen Reagents Review Please review the run setup Run Name MockRun1 Workflow Type FG Read 1 Index 1 Index2 Read2 351 8 8 31 User customer yourlab com Read Length m Z e 00000 ELI Run Setup Screens 20 After selecting Sequence on the Welcome screen select a run type When you select Forensic Genomics a series of run setup screens open in the following order Select Run Load Flow Cell Load Reagents Review and Pre Run Check When you select a Research Use Only Run a different series of run setup screens opens See Run Setup Screens for an RUO Run on page 24 Part 15050524 Rev C Select A Run Screen Select a run for sequencing using the drop down menu Runs are set up in the ForenSeq Universal Analysis Software Figure 15 Select a Run Screen Please select a run Select Run MyNewRun1234 9JEMIJOS 0J1UO2 XD Y basi Load Flow Cell Screen The Load Flow Cell screen appears when it is time to load the flow cell After loading the flow cell close the flow cell latch and flow cell compartment door Both the latch and compartment door must be closed before beginning the run Figure 16 Load Flow Cell Screen low Cell Barcode x Exit When the flow cell is loaded the software reads and records the RFID A confirmation that the RFID was successfully read appears in the lower
19. For RUO mode Bundle logs Bundle Logs For Illumina Technical Support troubleshooting only ForenSeq manifest genome and recipe files are pre loaded for forensic kits and should not be changed Delete Select the checkbox next to the file or folder listed and then select Delete The Delete feature is available on all tabs Admin user access level is required to delete run files Move Available only for run folders Select the checkbox next to the folder name select Move and then browse to an appropriate location Move copies the run folder to the new location and then deletes the folder from the old location Select All Files Select the checkbox to the left of the Delete button and then select an action Delete or Move The action is applied to all files or folders Upload Files If the MiSeq FGx is not connected to a network use this feature to upload sample sheet or manifest files to the instrument computer from a USB drive Select Upload and browse to the location on a USB drive where the sample sheet resides The file is uploaded to the folder indicated in the Directory field Rename Select the checkbox next to the sample sheet file and then select Rename Use the on screen keyboard to rename the sample sheet Bundle Logs This command combines and zips groups of files for use by Illumina Technical Support for troubleshooting purposes MiSeq FGx Instrument Reference Guide 1 5 9JEMIJOS 0J1UO2 XD basi MiSeq
20. Instrument X Shutdown Table 2 Maintenance During Idle 2 7 days unused Activity Daily Monthly As Needed Standby Wash X Instrument X Shutdown Preventive Maintenance Illumina recommends one preventive maintenance per calendar year If you are not under a service contract please contact your Territory Account Manager or Illumina Technical Support to arrange for a billable preventive maintenance service 5 2 Part 15050524 Rev C Performing a Post Run Wash Always perform an instrument wash after completing a sequencing run Follow the software prompts to load the wash components and perform the wash The post run wash takes approximately 30 minutes NOTE The post run wash contains sodium hypochlorite Do not use sodium hypochlorite for a maintenance wash or a standby wash The software will not proceed to the run setup steps for a subsequent run until a wash has been performed 4 NOTE Leave the used flow cell on the instrument A flow cell must be loaded on the instrument to perform an instrument wash User Supplied Consumables Tween 20 Sigma Aldrich catalog P7949 Sodium hypochlorite Nuclease free water Procedure 1 Prepare fresh wash solution with Tween 20 and nuclease free as follows a Add 5 ml 10096 Tween 20 to 45 ml nuclease free water These volumes result in 1096 Tween 20 b Add 25 ml 10 Tween 20 to 475 ml nuclease free water These volumes result in a 0 5 Tween 20 wash solution c Invert several t
21. On instrument analysis is complete A critical MiSeq Software error occurs a Return 00000 Local SMTP email server address Use the on screen keyboard to enter the local SMTP email server address If necessary contact the facility administrator for this information Sender email address Use the on screen keyboard to enter the sender email address This address can be the email address of an Admin user or a different address specified for sending email notifications The sender email address must have the same domain name as the email server address Recipient addresses Use the on screen keyboard to enter the email addresses of each recipient to receive notifications Separate each email address with a comma Select Test to send a test email to notification recipients Notify via email when Select the checkbox for each of the run events that trigger a notification MiSeq FGx Instrument Reference Guide 1 T 9JEMIJOS 0J1UO2 XD Y SIN MiSeq FGx Software 18 Manage Instrument Screen The Manage Instrument screen contains controls for system settings troubleshooting manual software updates rebooting or shutting down the instrument software v Figure 11 Manage Instrument Screen Please select an activity SYSTEM SYSTEM SETTINGS CHECK MINIMIZE TO WINDOWS System Settings Provides the option to change IP Settings machine name or domain See System Settings Screen on page 19 System Check Pr
22. Sample Sheet Folder D MlluminaMiSeq FGx Control Software SampleSheets Browse Manifest Folder D lllumina MiSeq FGx Control Software Manifests Browse Output Folder D lllumina MiSeqFGxOutput Browse 44 Save and Cancel Return 00000 1 6 Part 15050524 Rev C Recipes Custom recipes are not supported for ForenSeq kits Sample Sheets Sets the default location for sample information to the ForenSeq Universal Analysis Software Sample information is created before library preparation and contains the parameters for the run Manifests ForenSeq kits use manifest files that are pre loaded MiSeqFGxOutput Sets the default location for analysis output files Ilumina recommends changing the default output folder to a network location for sharing and long term storage Email Notifications Tab MiSeq FGx can be configured to send an email notification when RTA analysis is complete when ForenSeq Universal Analysis Software analysis is complete or if a critical MiSeq FGx software error occurs Email notification is only available if the instrument is connected to a network with access to the internet Figure 10 Email Notifications Tab Please make any needed changes to the Run Options Run Settings Folder Settings Email Notifications Local SMTP email server address required Sender address required on SMTP server Recipient addresses separate with commas Notify via email when Primary analysis is complete
23. Software Guide part 15053876 MiSeq FGx Instrument Reference Guide y 3 9JEMIJOS 0J1UO2 XD Y basi Prephasing See Number of Cycles in a Read on page 30 and the Run Metrics Tab section in the ForenSeg Universal Analysis Software Guide part 15053876 Read 1 For information see the Run Metrics Tab section in the ForenSeg Universal Analysis Software Guide part 15053876 Index 1 For information see the Run Metrics Tab section in the ForenSeg Universal Analysis Software Guide part 15053876 Read 2 For information see the Run Metrics Tab section in the ForenSeq Universal Analysis Software Guide part 15053876 Index 2 For information see the Run Metrics Tab section in the ForenSeq Universal Analysis Software Guide part 15053876 Run Setup Screens for an RUO Run After selecting Sequence on the Welcome screen select a run type When you select Research Use Only Run a series of run setup screens open in the following order BaseSpace Option Load Flow Cell Load Reagents Review and Pre Run Check When you select Forensic Genomics a different series of run setup screens opens See Run Setup Screens on page 20 MiSeq FGx Software BaseSpace Option Screen If performing analysis of an RUO run on BaseSpace select Use BaseSpace for storage analysis When you select the BaseSpace option run information transfers to BaseSpace Enter your Mylllumina account information Figure 20 BaseSpace Option Screen
24. a non exclusive non transferable personal non sublicensable right under Illumina s Core IP in existence on the date that this Product ships from Illumina solely to use this Product in Purchaser s facility for Purchaser s internal research purposes which includes research services provided to third parties and solely in accordance with this Products Documentation but specifically excluding any use that a would require rights or a license from Illumina to Application Specific IP b is a re use of a previously used Consumable c is the disassembling reverse engineering reverse compiling or reverse assembling of this Product d is the separation extraction or isolation of components of this Product or other unauthorized analysis of this Product e gains access to or determines the methods of operation of this Product f is the use of non Illumina reagent consumables with Illumina s Hardware does not apply if the Specifications or Documentation state otherwise or g is the transfer to a third party of or sub licensing of Software or any third party software All Software whether provided separately installed on or embedded in a Product is licensed to Purchaser and not sold Except as expressly stated in this Section no right or license under any of Illumina s intellectual property rights is or are granted expressly by implication or by estoppel Part 15050524 Rev C Purchaser is solely responsible for determining whether P
25. bottle and discard the remaining wash solution Refill the wash components with fresh 0 576 Tween 20 wash solution as follows a Add 6 ml wash solution to each reservoir of the wash tray b Add 350 ml wash solution to the 500 ml wash bottle Load the wash tray and wash bottle as follows a Slide the wash tray into the reagent chiller until it stops Close the reagent chiller door b Load the wash bottle and slowly lower the sipper handle making sure that the sippers lower into the wash bottle and waste bottle c Close the reagent compartment door Select Next The final wash begins MiSeq FGx Instrument Reference Guide 5 d USEM 99UEU9IUIEW e Bulvu1oJ19 d Maintenance Procedures After the Wash When the wash is complete leave the used flow cell wash tray and wash bottle containing the remaining wash solution on the instrument E NOTE i The sippers remain in the down position which is normal Leave the unused wash solution in the wash tray and wash bottle to prevent the sippers from drying out and air from entering the system 5 8 Part 15050524 Rev C Performing a Standby Wash If there are no plans to use the instrument within the next seven days prepare the instrument to sit idle by performing a standby wash A standby wash performs two consecutive washes that flush each position of any remaining reagents or salt accumulation Each wash takes approximately 60 minutes Allow approximately 2 hours to complete the s
26. nuclease free water as follows a Add 5 ml 100 Tween 20 to 45 ml nuclease free water These volumes result in 1096 Tween 20 b Add 25 ml 10 Tween 20 to 475 ml nuclease free water These volumes result in a 0 5 Tween 20 wash solution c Invert several times to mix Prepare the wash components with fresh 0 5 Tween 20 wash solution as follows a Add 6 ml wash solution to each reservoir of the wash tray b Add 350 ml wash solution to the 500 ml wash bottle Load the wash tray and wash bottle onto the instrument a Open the reagent compartment door and reagent chiller door and slide the used reagent cartridge or wash tray from the chiller b Slide the wash tray into the reagent chiller until it stops Close the reagent chiller door c Raise the sipper handle in front of the SBS Solution PR2 bottle and waste bottle until it locks into place and replace the SBS Solution PR2 bottle with the wash bottle 4 NOTE Discard the SBS Solution PR2 bottle after each run Do not reuse any remaining SBS Solution PR2 d Remove the waste bottle and discard the contents appropriately Return the waste bottle to the reagent compartment e Slowly lower the sipper handle making sure that the sippers lower into the wash bottle and waste bottle f Close the reagent compartment door Select Next The first wash begins Part 15050524 Rev C Perform Second Wash L 5 NOTE Always use fresh wash solution for each wash step Reusing was
27. on the instrument From the Manage Instrument screen select System Check Select Conduct Volume Test and then select Next Figure 48 System Check Screen Please select the system check options to pertorm O Select All Motion System Optics Other O eser O rose T rone Gh lege mi aal C mea O zs Fill each reservoir of the wash tray with 6 ml nuclease free water Fill the 500 ml wash bottle with 350 ml nuclease free water Load the wash tray and wash bottle onto the instrument a b Open the reagent compartment door and reagent chiller door and slide the wash tray into the reagent chiller until it stops Close the reagent chiller door Raise the sipper handle until it locks into place and load the wash bottle Remove the waste bottle and discard the contents appropriately Return the waste bottle to the reagent compartment Slowly lower the sipper handle making sure that the sippers lower into the wash bottle and waste bottle Following the on screen prompts remove any droplets from the wash bottle sipper as follows a b When prompted slowly raise the sipper handle and check the wash bottle sipper for the presence of a large water droplet When prompted slowly lower the sipper handle far enough into the water to allow the surface tension to remove the droplet When prompted slowly raise the sipper handle and check the wash bottle sipper for the presence of a large water droplet Part
28. or irregularities X Free Disk Space If disk space is low a message appears indicating how much disk space is reguired Use the Manage Files feature to clear the reguired space from the instrument com puter X Network Connection Make sure that the network cable is plugged into the instrument Active If the network connection is not restored select Reboot on the Manage Instrument screen to reboot the software 10113 dnjas uny BUIAJOSEH If the connection is still not restored select Shut Down on the Manage Instrument screen and then turn off the instrument using the power switch Wait at least 60 seconds and then turn on the instrument and start the software Primary Analysis Ready RTA analysis from the previous run is not complete The default time to allow RTA analysis to complete is 1 hour and a countdown appears on the screen The options are to wait 1 hour or select Terminate Analysis ForenSeq Universal Analysis Software analysis stops for any incomplete cycles X Sample Sheet Present If the sample sheet is not named with the reagent cartridge ID for the run the instrument cannot locate the appropriate sample sheet automatically Browse to the sample sheet for the run If the sample sheet is named with the reagent cartridge ID for the run make sure that the sample sheet is located in the default sample sheet folder Check the default folder location in Run Options on the Welcome screen Make sure that the sample sheet file exte
29. reagent kit barcode number which is located on the kit label directly below the barcode Enter the part number of the reagent cartridge which is located to the right of REF on the reagent kit label Figure 46 Enter Reagent Information Valid bypass code has been entered Please enter the reagent kit barcode and part number Allowed format for barcode is AA1234567 12345 Reagent Kit Barcode Reagent Kit Part Number Select Next to return to the Load Reagents screen Select Next to proceed to the next run setup step MiSeq FGx Instrument Reference Guide 67 eJnjre4 pesy Aldy BulAlos tu Preventing Reboots During a Run If the MiSeq FGx restarts in the middle of a run it might mean the Windows Update software on the network is configured to automatically install software updates This setting should have been turned off during installation Contact the local IT department for help to disable automatic updates to the Windows operating system running in the background on the MiSeq FGx 4 NOTE A restart terminates the run and it cannot be recovered The run has to be started again from the beginning Troubleshooting 6 8 Part 15050524 Rev C Troubleshooting Flow Rate Error The flow rate is the speed in which fluids pass through the fluidics system u1 min It is measured before each run during the pre run check If the system is unable to measure the flow rate pump a volume of reagent SBS Solution PR2 through th
30. right corner of the screen If the RFID cannot be read identifying information can be entered manually However the software allows only one of the three RFID labeled components flow cell reagent cartridge SBS Solution PR2 to fail on a run For more information see Resolving RFID Read Failure on page 66 Select the help icon in the upper right corner of the screen The help file contains a video that illustrates the steps for loading the flow cell MiSeq FGx Instrument Reference Guide 2 1 MiSeq FGx Software Load Reagents Screen The Load Reagents screen has two steps first load the SBS Solution PR2 bottle and empty waste bottle and then load the reagent cartridge When the SBS Solution PR2 bottle and reagent cartridge are loaded the software reads and records the RFID A confirmation that the RFID was successfully read appears in the lower right corner of the screen z NOTE r Use the correct reagent sequencing kit components for the type of run you perform Forensic genomics runs only work with ForenSeq sequencing kits and RUO runs only work with RUO reagent kits Figure 17 Load SBS Solution PR2 Bottle and Waste Bottle Please load the PR2 and waste bottle and walt for the RFID to be read PR2 Botto ID Flow Cell Barcode ASDFG lt Back Figure 18 Load Reagent Cartridge Please load the reagent cartridge If the RFID cannot be read identifying information can be entered manually However the soft
31. software Figure 45 Manage Instrument Screen lumina MiSeq FGx Customer yourlab com Please select an activity SYSTEM SYSTEM SETTINGS CHECK UPDATE MINIMIZE REBOOT HUTDOWN Ee Hw 5 Toggle the power switch to the OFF position Yep NOTE Any time the instrument is turned off wait a minimum of 60 seconds before turning the power switch back to the ON position MiSeq FGx Instrument Reference Guide 61 jueuunJ1su ay umog 6uninus 62 Part 15050524 Rev C Troubleshooting Introduction 2 u ul cnc 64 Resolving Run Setup Errors 65 Resolving RFID Read Failure EE De EG ltd no in S rn osa a 66 Preventing Reboots During a Run 68 Troubleshooting Flow Rate Error 69 Performing aVol me Test da eee Ze Seve US r cdas 70 Measuring Expected Wash Volumes 72 etat er d Mz cus lt ed Pr E so cam TECGGC TEA rogas TC6 ICA D eT o e ar Cac cram rana MiSeq FGx Instrument Reference Guide 6 3 g Je1deuo Troubleshooting Introduction 64 This section describes common troubleshooting steps to take before contacting lumina Technical Support For most errors an on screen message appears with instructions for correcti
32. successfully read E NOTE V If that the RFID cannot be read identifying information can be entered manually However the software allows only one of the three RFID labeled components flow cell reagent cartridge SBS Solution PR2 to fail For more information see Resolving RFID Read Failure on page 66 6 Close the flow cell compartment door 7 Select Next on the Load Flow Cell screen The Load Reagents screen opens Part 15050524 Rev C Loading Reagents There are two steps to loading reagents First load the SBS Solution PR2 bottle and make sure that the waste bottle is empty and then load the reagent cartridge Load SBS Solution PR2 and Check the Waste Bottle 1 Remove the bottle of SBS Solution PR2 from 2 to 8 C storage Gently invert the bottle to mix the SBS Solution PR2 bottle and then remove the lid Open the reagent compartment door Raise the sipper handle until it locks into place Place the SBS Solution PR2 bottle in the indentation to the right of the reagent chiller Figure 41 Load the SBS Solution PR2 Bottle Make sure that the waste bottle is empty If it is not empty empty the contents into the appropriate waste container Slowly lower the sipper handle Make sure that the sippers lower into the SBS Solution PR2 and waste bottles Figure 42 Lower Sipper Handle 7 Check the lower left corner of the screen
33. the flow cell ports are free of obstructions and that the gasket is well seated around the flow cell ports If the gasket appears to be dislodged gently press it back into place until it sits securely around the flow cell ports Part 15050524 Rev C Loading the Flow Cell The Load Flow Cell screen prompts you to load the flow cell 1 4 Raise the flow cell compartment door and then press the release button to the right of the flow cell latch The flow cell latch opens Figure 38 Open Flow Cell Latch Visually inspect the flow cell stage to make sure that it is free of lint If lint or other debris is present clean the flow cell stage using an alcohol wipe or a lint free tissue moistened with ethanol or isopropanol Carefully wipe the surface of the flow cell stage until it is clean and dry Holding the flow cell by the edges of the flow cell cartridge place the flow cell on the flow cell stage Figure 39 Place Flow Cell on Stage Gently press down on the flow cell latch to close it over the flow cell Y NOTE V As the flow cell latch is closed two alignment pins near the hinge of the flow cell latch properly align and position the flow cell An audible click indicates that the flow cell latch is secure MiSeq FGx Instrument Reference Guide A 5 1199 M0 4 ay Buipeo7 Performing a Run 46 Figure 40 Close Flow Cell Latch 5 Check the lower left corner of the screen to confirm that the flow cell RFID was
34. the prompts to load the flow cell and reagents and set up the run described in the following sections Part 15050524 Rev C Cleaning the Flow Cell The flow cell is immersed in storage buffer in a flow cell container 1 2 5 Put on a new pair of powder free gloves Using plastic forceps grip the flow cell by the base of the plastic cartridge and remove it from the flow cell container Figure 34 Remove Flow Cell Lightly rinse the flow cell with nuclease free water making sure that both the glass and plastic cartridge are thoroughly rinsed of excess salts Excess salts can affect flow cell seating on the instrument If salts dry in the imaging area imaging can also be affected Figure 35 Rinse Flow Cell Using care around the black flow cell port gasket outlined in orange in the following illustration thoroughly dry the flow cell and cartridge using a lint free lens cleaning tissue Gently pat dry in the area of the gasket and adjacent glass Figure 36 Flow Cell Ports and Gasket illumina Using an alcohol wipe clean the flow cell glass Make sure that the glass is free of streaks fingerprints and lint or tissue fibers Avoid using the alcohol wipe on the flow cell port gasket MiSeq FGx Instrument Reference Guide A 3 99 Mold ay Buiueej o Performing a Run 44 Figure 37 Dry Flow Cell Dry any excess alcohol with a lint free lens cleaning tissue Visually inspect to make sure that
35. AATTGAGACTAAATATTAAC E ASA CAT GATAACAGTAACACACTTCTG ACCAT IAAGAGCTACCGTGCAACTTAAC ej AATT AGACTAAATAI T GTACCGTAACGAACGTATCAATTGAGCTTCTGT ACITOATOGA GI GATTOAAC GTACCOTAANGATIAC EE AG GATTCAACGTACCGTAACGAACGTATCAAT TGAGACT GTA TAACGAACGTA OA TAAATAT TAACCTACCA MAAGAGCTACCETCTICTGIT TCAATTGA ee ETA ET AAA TATCAATTGAGACTAAGCTACCGTGCAAC AAA ACGTATORA ICAGACTAAATATTAA OCIRCCATIAACAGC TAGE Se EE GGTACCATTAAGAGCTACCGTGCAA GAAAAG TTACHIGATECACT GATTCAAG GACTAAATA ATCCACTGAT T O TTGATOCACTOAT TOAN m CO NE Ee GTTAACCTTAAGAT T A DM NIC UR QA PRACA CAATTGAGACTAM TATY AACC AAATAT TAAC TTAAGAGCTACCG CAACGTACCGTAACGAA CGTACCG TAAI GAACGTATCAATTGAGACTAAATA RE AG GED CTACOGICTICII GTI AACGTACCA a GATTCAACGTACCGTAACGAACGTATCAATTG TGAGACTAAATAI TGCAACGACG I TAACGTACCAI IATCCACTGAT TCAACGTACCGI TTAAGAGCTACC TTACTTGATCCACTGATTCAA TG TTAACG HAAGATIACTI GATCCAGT GATICAAGGIACCGIE TAAGAT EE GE CAGTAACAC AAAAG AROC TAAGATTACI TGATOCACTO TTOAACI AACCTTAAGA AAGAGCTACCGTGCAACGACGAAAAGAATGATAACAG TAACACACT TOT TACT Ee CGTAACGAACGTATCAATTGAGACTAGCAACGAC CAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT TAACG TACCAT TAA CGTACCATTAAGAGCTACCGTCTTCTGTTAACCT TAAGATTACTT GATCCACTGATTCAA BEE IT AG CTAAATA E GE Aler AT TAC ACTAAATATTAACG TACCATTAAG Was UCM ATT AAC AATTG AAC STIAAGATIAGTTGATCCACTGAT CAACGTACCGIAAAGAT IAC
36. ATCAATT e GE WAS TACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGAC TAAATAT TAA STTGATCCACTGATTC GATTACTTGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGCTTCTGTTAAC ARA CAT GATAAGAGTAAGACAGTICTGTIAAGGTTSAGATTAC GATCCACTGATTCAACGTACCGTAAAGATTAC FOR RESEARCH FORENSIC OR PATERNITY USE ONLY ILLUMINA PROPRIETARY Part 15050524 Rev C February 2015 Catalog SY 411 9001DOC GAGAGTA RACGTACCATIAAGAG CTHICTOTIAACOTIAAQATTACY QATA AACCTTAAGATTACTTGATI TATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACCGTCTTCTGTTAACCTTAAGATTACTTGATCCAC SAAAAGAATGATAACAGTAACACACTTCTGTTAACC LTAAGAT TACTTGATCCACTGAT TCAACGTACCGTAAAGATTAC A AAAS ASS ACA TACEAM GTATCAAI MATTA CTACCGTCTTCTGTTAACCT T CTTGAT geg CATTAAGAGCTACCGTGCAACGACGAAAAG CGTG OTTA AA CIMA AA GE AACCTTAAGA GATCCACTGATTCAACG AA ACTGATTCAA CG COACTOATICAACCTACCGTA GH AACAGTAACACA AT CAATTG AGACTAAATATTAAGG IACCAT IAAGAGCIACEGICT ICT GI TAACC TRAGA IAC ER GEAG GAl IGAACC TTACTTGAT This document and its contents are proprietary to Illumina Inc and its affiliates Illumina and are intended solely for the contractual use of its customer in connection with the use of the product s described herein and for no other purpose This document and its contents shall not be used or distributed for any other purpose a
37. FGx Software Run Options Screen The Run Options screen has three tabs for specifying the default settings for a run Run Settings Folder Settings and Email Notifications Figure 8 Run Settings Tab on Run Options Screen Please make any needed changes to the Run Options Run Settings Folder Settings Email Notifications Recipe Folder C lllumina MiSeq Control SoftwarelRecipe Browse Sample Sheet Folder D lllumina MiSeq FGx Control Software SampleSheets Browse Manifest Folder D lllumina MiSeq FGx Control Software Manifests Browse Output Folder D llluminalMiSegF GxOutput Browse 44 Save and Cancel Return 00000 Run Settings Tab Post Run Wash Option An instrument wash is required after each run The software will not proceed to the run setup steps for a subsequent run until a wash has been performed This setting determines what type of wash will be performed by default after a run is configured here A post run wash takes about 30 minutes and a maintenance wash takes about 1 hour Folder Settings Tab MiSeq FGx requires access to various types of files stored in folders on a local network or on the instrument computer The options on the Folder Settings tab tell the MiSeq FGx where those folders are located Figure 9 Folder Settings Tab Please make any needed changes to the Run Options Run Settings Folder Settings Email Notifications Recipe Folder C llluminalMiSeg Control SoftwarelRecipe Browse
38. I IGATSGACTGAL ICAACOTAG TACC GCTACCGTGCAACGAAAATAACCTIAAG SAAAAGAATGATAACAGTAACACACTICTG GATAACAGTAACACACTICTGTIAACCT I ACCATTAAGAGCTACCGTGCAACAGTAACACACTTGTGITAA Y z O gt O gt gt 53299 O GATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTG GATTCAACGT AACGAACGTATCAATT CTAAATATTAACGTAC GTACCGTAACGAACGTATCATTAAGATTACTIGATCCACTGATTCAACGTACCGTAACGAACGTATCAATTGAGACTAAATATTAACGTACCATTAAGAGCTACCGTGCAA GI ACC GTAACGAACGIATLCAATIGAGCTLTCTOTIAACCAAGAMACIGATCOACTGATTCAACETACCE CTTGATCCACTGATTCAACGTTAAG C TGATCCACTGATT CAACGTACCGTAAAGATTAC 3AAAAGAATGATAACAGTAACACAC lllumina 5200 Illumina Way San Diego California 92122 U S A 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsupport illumina com www illumina com GTACCATTAAGAGCTACCGTCTTCTG GT AACCAACGTATCAATIGAG TAAATAT TAACGTAC CALIAA C AAGATACITGATOCACIG ITOAACGTAC IAACGIACC OTANG GRACO ATA GAGACAAATATTAAUGIACGATIAA AC TGATCCACTGATTCAACGTACCGTAA ZS RE E Kee CTGTTAA ACTGATTCAACGTACCGTAACGAACGTATCAATTGAGAC ATTACTIGATCCAGTGATT CAACCTACCOTAACGAACHA TCAATTG AO CAA AAAS S S GE EI LE AACCT TAAGATTAC AAGAT ACT IGATCCACIG ATTAACGTA E AAG GAGCTTCTGTTAACCT TAAGAT TACT CATTAAGAGCTACCG CGTAA C MAGNO CTACCGTC ATTAAGAGCTACCG EE GEE S GAACGTAI AACGTACCAT T TATTAACGTAC 100 O
39. PhiX v2 6pM Workflow Resequencing 151 151 BaseSpace User am gt D Intensity Q Score All Cycles Flow Cell 1 i gt 030 0 0 T EJ EJ Q Score Value m a SEI IN U c m 2 Ce 00000 75 alues Update After Cycle 5 ters Passing Filter Estimated Yield MB Template Generation Template generation is the process by which cluster positions over the entire flow cell surface are defined according to X and Y coordinate position Real Time Analysis RTA uses the first seven cycles of the sequencing run for template generation for MiSeq FGx and v3 RUO kits and the first four cycles for v1 and v2 RUO kits After the template of cluster positions is generated images produced over every subsequent cycle of imaging are aligned against the template Individual cluster intensities in all four nucleotide color channels are extracted and base calls are produced from the normalized cluster intensities Run Folders Each run on the MiSeq FGx generates three run folders each with a specific purpose DMllumina MiSeqFGxTemp When the run begins a temporary run folder is written to the local drive of the instrument computer and used as a working area for MiSeq FGx Control Software and RTA There is no need to access the Temp folder Contents of this folder are deleted after seven days DMllumina MiSeqFGxOutput RTA copies files from the Temp folder to the Output folder As base call and base call quality scor
40. Q score measurement A Q score is a prediction of the probability of a wrong base call Q scores are calculated after cycle 25 Q Score Probability of Wrong Base Call 040 1 in 10 000 Q30 1 in 1 000 Q20 1 in 100 Q10 1 im 10 Cluster Density K mm Shows the number of clusters per square millimeter for the run Clusters Passing Filter Shows the percentage of clusters passing filter based on the Illumina chastity filter which measures quality This data appears only after cycle 25 NOTE The chastity of a base call is the ratio of the intensity of the greatest signal divided by the sum of the two greatest signals If more than one base call has a chastity value of less than 0 6 in the first 25 cycles reads do not pass the quality filter Estimated Yield Mb Shows the projected number of bases called for the run measured in megabases This data appears only after cycle 25 When the run is complete the Next button appears Review the results on the Sequencing screen before proceeding d NOTE The chastity of a base call is the ratio of the intensity of the greatest signal divided by the sum of the two greatest signals If more than one base call has a chastity value of less than 0 6 in the first 25 cycles reads do not pass the quality filter 3 Select Next to exit the Sequencing screen and proceed to a post run wash Part 15050524 Rev C Figure 26 Sequencing Screen for an RUO Run RG1234567 00CDE 2X101PE
41. R read ngh 30 reagent chiller temperature 12 reagent compartment 3 5 13 Index 14 reagents washes itted 7 benefits of 52 loading 22 25 47 expected volumes 72 Real Time Analysis maintenance 13 56 run folder 29 post run 53 template generation 29 post run wash settings 16 rebooting software 18 prepare to idle 59 pepe managing 14 prepare to shut down 61 RF standby 13 59 flow cell 45 waste bottle 5 reagent cartridge 48 welcome screen 11 SBS Solution 47 Windows minimize to 18 troubleshooting 66 workflow run duration 30 run duration 30 run folders n EE 14 naming 30 temp output analysis 29 run options 16 run setup screens 24 casework 20 database 20 S sample sheet copying to instrument 14 not found 65 SBS Solution loading 47 selecting a run 21 sensor indicators 12 Sequencing Analysis Viewer 27 50 sequencing screen 23 27 50 shutting down the instrument 18 61 sipper handle 5 software disk space checking 30 initialization 6 instrument setup 18 run duration 3 standby wash 13 59 status alerticon 13 stopping a run 31 system settings 19 T technical assistance 75 template generation 29 troubleshooting bundlelogs 15 flow rate 69 fluidics 70 RFID 66 run specific files for 64 run setup errors 65 turning on the instrument 6 U user supplied consumables 7 user name 6 V volume test 70 W wash volumes 72 Part 15050524 Rev C Technical Assistance For technical assis
42. TTCTGTT ARIS Le PME E GOTT Part 15050524 Rev C List Price 50 00 USD T TGAACGIACCGTAACGAACGTATCAAT I AACCTTAAG DAS Ua wes ope et alle LI I TAACGTAC CASAS UNSER TI OS PS SI reer EL ACTGATICAACGTACCG CAI EE Ee GAT EE GCTACCGTGCAACGAAAATAACCTIAAGATTACTTGATCCACTGA TCTGTIAACCTTAAGATTACTTG SANA GAATGATAA CAC AG DI ET CO TAAGATTACI I GATGCACTQATI CANC G TACCGTAAAGATTA e CTGTTAACCT AAS TACT SO GA we CAT T GCTACCGTGCAACAGTAACACACT T GATAACAGTAA AC C I TCTGTTAA OTI A PUn AAGATTACTTGATCCACTGATT TTGATCCACTGAT T ACTTGATCCACTGATTCAA AAAAGAATGATAA AGTAAGAGAGTICTST IAA CCTTAAGATT ACTI TTAAGATTACTTGATCCACTGATT C TGATCCACTGATTCAACGTACCGTAACGAACGTA AAAA AG TAACACACTTCTGTTAACCT TAAGAT TACTTGAT CCACTGATTCAACGTA GCATTAAGAGG DEEG TEER CCTTAAGATT TT AACAGTAACACA G EIE A MIRA A CI ATCAATTG IGATOGACT A AA AATTGAGACTAAAT TATGAAT TGAGACTAAATAT TAACOTACTIAACOTIAAGATTACTI GAC TAAATAT TAACGTACCAT TAAGAGCTACAA EE G TARAS CTIAAGATTACTIGAT CC Ges gt EES g GATTCAACGTACCG CAACGTACCGTAACGAACG TA Oa DOY 3A 9 Pope ESO a BREE p 590 x3 o9 SA 23003300 Beds TTC TAG GTATCAATTGAG AAC CTHAAGATIAC HIGATCCAGTGATICAAG TACCGTAACGAACGTATCAATT GATTCAACGTAC CAATTG AGAHAQIGAYCCAGIQATICAKGOTACCCOTAANQ P DATA CAAT ACTAAATATTAA SO e cic A e P A SN TTGAGACTAAATAT TAACG TATC
43. WISE SHALL IN NO EVENT EXCEED THE AMOUNT PAID TO ILLUMINA FOR THIS PRODUCT 7 Limitations on Illumina Provided Warranties TO THE EXTENT PERMITTED BY LAW AND SUBJECT TO THE EXPRESS PRODUCT WARRANTY MADE HEREIN ILLUMINA MAKES NO AND EXPRESSLY DISCLAIMS ALL WARRANTIES EXPRESS IMPLIED OR STATUTORY WITH RESPECT TO THIS PRODUCT INCLUDING WITHOUT LIMITATION ANY IMPLIED WARRANTY OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE NONINFRINGEMENT OR ARISING FROM COURSE OF PERFORMANCE DEALING USAGE OR TRADE WITHOUT LIMITING THE GENERALITY OF THE FOREGOING ILLUMINA MAKES NO CLAIM REPRESENTATION OR WARRANTY OF ANY KIND AS TO THE UTILITY OF THIS PRODUCT FOR PURCHASER S INTENDED USES 8 Product Warranty All warranties are personal to the Purchaser and may not be transferred or assigned to a third party including an affiliate of Purchaser All warranties are facility specific and do not transfer if the Product is moved to another facility of Purchaser unless Illumina conducts such move a Warranty for Consumables Illumina warrants that Consumables other than custom Consumables will conform to their Specifications until the later of i 3 months from the date of shipment from Illumina and ii any expiration date or the end of the shelf life pre printed on such Consumable by Illumina but in no event later than 12 months from the date of shipment With respect to custom Consumables i e Consumables made to specifications or designs made by Pur
44. ash The standby wash properly prepares the fluidics lines for sitting idle and consists of two consecutive wash cycles Perform a standby wash if you expect the instrument to remain idle for up to seven days See Performing a Standby Wash on page 59 After the instrument has been placed in an idle state a maintenance wash must be performed before a sequencing run can be initiated Post Run Wash The post run wash is the standard instrument wash performed between sequencing runs and consists of a single wash cycle To perform a post run wash at a time other than directly following a run use the command on the Perform Wash screen to initiate the wash The MiSeq FGx can be configured to perform a maintenance wash rather than a post run wash between runs For more information see Run Options Screen on page 16 MiSeq FGx Instrument Reference Guide 1 3 9JEMIJOS 0J1UO2 XD basi MiSeq FGx Software Figure 6 Perform Wash Screen Perform Please select a wash activity The Maintenance Wash should be performed every 30 days for optimal performance Maintenance Wash Perform Standby Wash Perform Post Run Wash Last performed N A The Standby Wash should be performed when the instrument will be idle for more than seven days Note If a Standby Wash is performed a maintenance wash must be conducted before sequencing Last performed N A The Post Run Wash is performed after every sequencing run Last performed N A Raise
45. chaser or provided to Illumina by or on behalf of Purchaser Illumina only warrants that the custom Consumables will be made and tested in accordance with Illumina s standard manufacturing and quality control processes Illumina makes no warranty that custom Consumables will work as intended by Purchaser or for Purchaser s intended uses b Warranty for Hardware Illumina warrants that Hardware other than Upgraded Components will conform to its Specifications for a period of 12 months after its shipment date from Illumina unless the Hardware includes Illumina provided installation in which case the warranty period begins on the date of installation or 30 days after the date it was delivered whichever occurs first Base Hardware Warranty Upgraded Components means Illumina provided components modifications or enhancements to Hardware that was previously acquired by Purchaser Illumina warrants that Upgraded Components will conform to their Specifications for a period of 90 days from the date the Upgraded Components are installed Upgraded Components do not extend the warranty for the Hardware unless the upgrade was conducted by Illumina at Illumina s facilities in which case the upgraded Hardware shipped to Purchaser comes with a Base Hardware Warranty c Exclusions from Warranty Coverage The foregoing warranties do not apply to the extent a non conformance is due to i abuse misuse neglect negligence accident improper storage or use contrar
46. cleotides After imaging of one tile of the flow cell is complete the flow cell is moved into place to expose the next tile The process is repeated until all tiles are imaged Following image analysis the software performs base calling filtering and quality scoring MiSeq FGx Instrument Reference Guide 37 MO J440M X94 bD SIIAJ Performing a Run 38 Analysis When the run is complete the ForenSeq Universal Analysis Software automatically performs further analysis Analysis is monitored using an internet connection from another computer See the ForenSeg Universal Analysis Software Guide part 15053876 Part 15050524 Rev C Prepare the Reagent Cartridge The following instructions describe how to thaw the reagent cartridge using a room temperature water bath This method requires approximately one hour NOTE A MiSeg FGx Reagent Cartridge works with ForenSeg seguencing runs and does not work with RUO runs When performing an RUO seguencing run use a standard MiSeg reagent cartridge Remove the reagent cartridge from 25 C to 15 C storage Place the reagent cartridge in a water bath containing enough room temperature deionized water to submerge the base of the reagent cartridge up to the water line printed on the reagent cartridge Do not allow the water to exceed the maximum water line Figure 30 Maximum Water Line koi FA 3
47. ction Part 15050524 Rev C Status Icons In the top right corner of the Welcome screen is a status icon that signals any change in conditions during run setup or during the run Status Icon Status Name Description Status OK No change System is normal Attention Important information Action is recommended Warning Warnings do not stop a run However some warnings require action before proceeding Error Errors usually stop a run and generally require action before proceeding with the run When a change in condition occurs the icon changes to the associated image and blinks to capture attention If this happens select the icon to open the status window which contains a general description of the condition Select any item listed to see a detailed description of the condition and instructions to resolve the condition if applicable Select Acknowledge to accept the message and Close to close the dialog box Messages in the status window can be filtered by selecting the icons along the top margin of the window Selecting an icon toggles the condition to show or hide Perform Wash Screen The Perform Wash screen enables users to perform the following types of washes as well as see the date of the most recent wash performed Maintenance Wash The maintenance wash consists of three consecutive wash cycles that thoroughly flush the system Perform a maintenance wash at least every 30 days See Procedure on page 56 Standby W
48. d loading The MiSeq FGx Control Software launches and initializes the instrument automatically After the initialization step is complete log in using your ForenSeq Universal Analysis Software user name and password Figure 2 Log In Screen Enter your ForenSeq UAS username and password Password If you do not have an account please contact your facility administrator 5 Click Next The Welcome screen opens See Welcome Screen on page 11 Part 15050524 Rev C Required Consumables Sequencing Consumables The sequencing consumables required to run the MiSeq FGx are provided separately User Supplied Consumables Make sure that the following user supplied consumables are available before beginning a run Consumable Alcohol wipes 70 Isopropyl or Ethanol 70 Disposable gloves powder free Lab tissue low lint Lens paper 4 x 6 in Microcentrifuge tubes Sodium hypochlorite Tween 20 Tweezers square tip plastic optional Water nuclease free or equivalent Supplier VWR catalog 95041 714 General lab supplier General lab supplier VWR catalog 21905 026 VWR catalog 52846 001 General lab supplier General lab supplier Sigma Aldrich catalog P7949 McMaster Carr catalog 7003A22 General lab supplier Guidelines for Nuclease free Water Always use nuclease free water to perform instrument procedures Never use tap water or deionized water Any of the followi
49. e Guide 3 Getting Started Flow Cell Compartment Flow Cell Stage Flow Cell Compartment Door Flow Cell Latch Flow Cell Flow Cell Latch Release Button moou o The flow cell compartment houses the flow cell stage thermal station and fluidics connections to the flow cell The flow cell stage holds the flow cell and the flow cell latch secures and positions the flow cell When the flow cell latch closes two pins near the latch hinge auto position the flow cell The thermal station located beneath the flow cell stage controls changes in flow cell temperature required for cluster generation and sequencing Flow Cell A Outlet Port B Imaging Area C Inlet Port The MiSeq FGx flow cell is a single use glass based substrate on which clusters are generated and the sequencing reaction is performed Reagents enter the flow cell through the inlet port pass through the single lane imaging area and then exit the flow cell through the outlet port Waste exiting the flow cell is delivered to the waste bottle During the sequencing run the single lane is imaged in small imaging areas called tiles Part 15050524 Rev C Reagent Compartment Reagent Chiller Sipper Handle shown in raised position SBS Solution PR2 Bottle Waste Bottle MiSeq FGx Reagent Cartridge muou gt The reagent compartment contains the reagent chiller and positions for the SBS Solution PR2 bottle and the waste bottle During t
50. e files are generated RTA copies files back to the Temp folder and populates the Analysis folder Focus images and thumbnail images are not copied to the Analysis folder The location of the output folder can be changed in the Output Folder field on the Run Options screen For more information see Run Options Screen on page 16 DMllumina MiSeqFGxAnalysis When RTA analysis is complete ForenSeq Universal Analysis Software accesses the Analysis folder on the instrument local drive to begin further analysis All files written to the Analysis folder are copied to the Output folder For more information see the ForenSeg Universal Analysis Software Guide part 15053876 Root Folder Naming The root run folder name identifies the date of the run the instrument number and the flow cell used for the run For any one run each run folder has the same root folder name MiSeq FGx Instrument Reference Guide 2 9 9JEMIJOS 0J1UO2 XD Y basi MiSeq FGx Software By default the folder name uses the following format YYMMDD lt lnstrumentNumber gt lt Run Number gt A lt FlowCellBarcode gt The run number increments by one each time a run is performed on a given instrument Run Duration Run duration depends on the number of cycles performed MiSeg FGx can perform 8 8 indexed seguencing run of 351 31 cycles for ForenSeg kits Number of Cycles in a Read The number of cycles performed in a read is one more cycle than the number of cycle
51. e system before checking the flow rate again 1 Use the drop down list or on screen keyboard to enter the following information Solution PR2 Volume 250 ul Aspirate Rate 2500 ul min Dispense Rate 2500 ul min 2 Select Pump Figure 47 Measure Flow Rate wa Load Review un Unable to Measure Flow Rate Please check the fluidics and press Restart Check to continue Solution PR2 3 Y Volume 250 uL Eg Aspirate Rate 2500 uL min pug Dispense Rate 2500 y L min Eug ET ES sa m D Z y 3 When the pump step is complete select Restart Check 4 If the error persists set the volume to pump 500 ul SBS Solution PR2 and repeat the process one more time Contact Illumina Technical Support if the second attempt does not resolve the error MiSeq FGx Instrument Reference Guide 6 9 10413 e1eH MO 4 6unoouse qnoaJ Troubleshooting Performing a Volume Test ro An obstruction in the fluidics lines can cause poor reagent delivery and affect seguencing results If an obstruction in the fluidics lines is suspected perform a volume test A volume test checks the health of the fluidics system by estimating the volume between two bubbles as they pass by the sensors To perform a volume test the wash tray and wash bottle must be loaded with nuclease free water and a used flow cell must be in place Follow the onscreen prompts to perform the test 1 2 3 Make sure that a used flow cell is loaded
52. each by Purchaser of these terms and conditions iii the use of this Product in combination with any other products materials or services not supplied by Illumina iv the use of this Product to perform any assay or other process not supplied by Illumina or v Illumina s compliance with specifications or instructions for this Product furnished by or on behalf of Purchaser each of i v is referred to as an Excluded Claim Indemnification by Purchaser Purchaser shall defend indemnify and hold harmless Illumina its affiliates their non affiliate collaborators and development partners that contributed to the development of this Product and their respective officers directors representatives and employees against any claims liabilities damages fines penalties causes of action and losses of any and every kind including without limitation personal injury or death claims and infringement of a third party s intellectual property rights resulting from relating to or arising out of i Purchaser s breach of any of these terms and conditions ii Purchaser s use of this Product outside of the scope of research use purposes iii any use of this Product not in accordance with this Products Specifications or Documentation or iv any Excluded Claim Conditions to Indemnification Obligations The parties indemnification obligations are conditioned upon the party seeking indemnification i promptly notifying the other party in writin
53. empty the waste bottle or check the volume remaining in the SBS Solution PR2 bottle and then resumed to continue sequencing Y y CAUTION Do not pause a run during cluster generation or within the first eight cycles of sequencing It is not possible to resume a run that was paused during this time If you use the MiSeq FGx in RUO mode consult Run Metrics on page 50 to see cycle information for reagent cartridge kits To pause a run from the Sequencing screen select Pause The current command is completed after which the run is paused the flow cell is placed in a safe state and the button changes to Resume At this point the waste bottle can be emptied for example Select Resume to continue with the run Part 15050524 Rev C Stopping a Run A run can be stopped during sequencing if necessary for example if the run was set up incorrectly if the data quality is bad or if there is a hardware error To stop a run from the Sequencing screen select Stop When a run is stopped the current command is not completed and the flow cell stage moves to the forward position RTA analysis continues for the last completed cycle Figure 27 Stopping a Run z Confirm Stopping a run is final You can not resume the run Are you sure you want to stop the run 1 Stopping a run is final A stopped run cannot be resumed The only option is to proceed to an instrument wash If you stop a run you cannot use reagents remaining in
54. ent cartridge loaded on the instrument If no sample sheet is found a window opens so you can browse to the sample sheet Select the Change Sample Sheet button on the Load Reagents screen and browse to the appropriate sample sheet Part 15050524 Rev C Review Screen The Review screen confirms that the software has located the sample sheet for the run and lists the sample sheet name and parameters provided in the sample sheet Figure 24 Review Screen ilumina Miseq FGx mu customer yo m Load Reagents Please review the run setup Run Name SampleSheet Workflow Type None Read 1 Index 1 Index 2 Read2 301 8 8 31 User customer yourlab com Read Length Pre Run Check Screen The software automatically performs a pre run check of required run components and conditions before allowing the run to begin If any errors occur during the pre run check a message appears on the screen that describes the error and the action to correct it For more information see Resolving Run Setup Errors on page 65 Figure 25 Pre Run Check Screen Checking flow Network Connection Active Sample Validated Flow Cell Cartridge Loaded Free Disk Space 86 Waste Bottle Loaded PR2 Bottle Loaded Reagent Cartridge Loaded Flow Cell Door Closed Chiller Door Closed Flow Rate Measured Primary Analysis Ready 00 56 29 The system is waiting 1 hour for primary analysis to complete for the previous run Reagent ID RG1234567 00CDE PR2 Bo
55. ent or the remaining period on the original Hardware warranty whichever is shorter If only a component is being repaired or replaced the warranty period for such component is 90 days from the date of shipment or the remaining period on the original Hardware warranty whichever ends later The preceding states Purchaser s sole remedy and Illumina s sole obligations under the warranty provided hereunder Third Party Goods and Warranty Illumina has no warranty obligations with respect to any goods originating from a third party and supplied to Purchaser hereunder Third party goods are those that are labeled or branded with a third party s name The warranty for third party goods if any is provided by the original manufacturer Upon written request Illumina will attempt to pass through any such warranty to Purchaser Indemnification a Infringement Indemnification by Illumina Subject to these terms and conditions including without limitation the Exclusions to Illumina s Indemnification Obligations Section 9 b below the Conditions to Indemnification Obligations Section 9 d below Illumina shall i defend indemnify and hold harmless Purchaser against any third party claim or action alleging that this Product when used for research use purposes in accordance with these terms and conditions and in accordance with this Product s Documentation and Specifications infringes the valid and enforceable intellectual property rights of a third party
56. f a new sequencing run is started on the MiSeq FGx before secondary analysis of an earlier run is complete a confirmation dialog box appears After confirming the sequencing run secondary analysis stops MiSeq FGx Instrument Reference Guide 3 3 SUNY ONY JO suondo slemijos sisAleuy MiSeq FGx Software 34 To restart analysis use the Requeue feature on the MiSeq Reporter interface after the new sequencing run is complete At that point analysis starts from the beginning Part 15050524 Rev C Performing a Run cn EE 36 MiSeq FGx Workflow 37 Prepare the Reagent Cartridge 39 Log In and Select Run Type 42 Cleaning the Flow Cell 43 Loading the Flow Gell 225 2 inches ss athena o vn 45 Loading CAG SINS ae uuu etd te Seis sng inet inne ate E 47 Starting the RUN uoce rio de Ee Eb ee dl 49 Monitoring eum 50 H lt as ES e NE 3 E A axe WAR na Sa E fi sa Se Ee 7 NE kas gt SCT Fi A a gt 1 T SSC LAM SN 2 ATI E s lt mu LE se I GEGGGA TRA rogaSTEGTO te prpTacoTa a a eg ar pene E MiSeq FGx Instrument Reference Guide 3 5 Je1deuo Performing a Run Introduction
57. g an RUO run follow sample loading instructions in Load Sample Libraries onto Cartridge for RUO on page 41 After you have prepared the MiSeg FGx reagent cartridge for use you are ready to load your samples 1 4 Use a low lint lab tissue to clean the foil seal covering the reservoir labeled Load Samples Use a clean 1 ml pipette tip to pierce the foil seal covering the reservoir labeled Load Samples Y NOTE Do not pierce any other reagent positions Other reagent positions are pierced automatically during the run Pipette 600 ul of the DNL sample libraries into the Load Samples reservoir Avoid touching the foil seal as you dispense the sample Check for air bubbles in the reservoir after loading sample If air bubbles are present gently tap the cartridge on the bench to release the bubbles Figure 31 Load Libraries Proceed directly to the run setup steps using the MiSeq FGx Control Software interface Part 15050524 Rev C Load Sample Libraries onto Cartridge for RUO NOTE If necessary for your library type denature and dilute libraries and add optional PhiX control See Preparing Libraries for Sequencing on the MiSeg part 15039740 This step does not apply to all library types Some Illumina sample preparation methods result in a ready to use normalized concentration of pooled libraries Refer to the sample preparation guide for the kit used to prepare sample libraries NOTE If you are using cus
58. g of such claim or action ii giving the other party exclusive control and authority over the defense and settlement of such claim or action iii not admitting infringement of any intellectual property right without prior written consent of the other party iv not entering into any settlement or compromise of any such claim or action without the other party s prior written consent and v providing reasonable assistance to the other party in the defense of the claim or action provided that the party reimburses the indemnified party for its reasonable out of pocket expenses incurred in providing such assistance Third Party Goods and Indemnification Illumina has no indemnification obligations with respect to any goods originating from a third party and supplied to Purchaser Third party goods are those that are labeled or branded with a third party s name Purchaser s indemnification rights if any with respect to third party goods shall be pursuant to the original manufacturer s or licensor s indemnity Upon written request Illumina will attempt to pass through such indemnity if any to Purchaser Part 15050524 Rev C Revision History Part 4 Revision Date Description of Change 15050524 C February Corrected formatting errors 2015 15050524 January Updated the name of the reagent kit from MiSeq ForenSeq 2015 Sequencing Kit to MiSeq FGx Reagent Kit 15050524 A October Initial release 2014 MiSeq FGx Instrument Reference Guide
59. h solution from the previous wash can return waste to the fluidics lines Prepare fresh wash solution with Tween 20 and nuclease free water as follows a Add 5 ml 100 Tween 20 to 45 ml nuclease free water These volumes result in 10 Tween 20 b Add 25 ml 10 Tween 20 to 475 ml nuclease free water These volumes result in a 0 5 Tween 20 wash solution c Invert several times to mix When the first wash is complete remove the wash tray and wash bottle and discard the remaining wash solution Refill the wash components with fresh 0 576 Tween 20 wash solution as follows a Add 6 ml wash solution to each reservoir of the wash tray b Add 350 ml wash solution to the 500 ml wash bottle Load the wash tray and wash bottle as follows a Slide the wash tray into the reagent chiller until it stops Close the reagent chiller door b Load the wash bottle and slowly lower the sipper handle making sure that the sippers lower into the wash bottle and waste bottle c Close the reagent compartment door Select Next The second wash begins Perform Final Wash 1 Prepare fresh wash solution with Tween 20 and nuclease free water as follows a Add 5 ml 10096 Tween 20 to 45 ml nuclease free water These volumes result in 1096 Tween 20 b Add 25 ml 10 Tween 20 to 475 ml nuclease free water These volumes result in a 0 5 Tween 20 wash solution c Invert several times to mix When the second wash is complete remove the wash tray and wash
60. he run the reagent chiller holds a single use reagent cartridge During the instrument wash the reagent chiller holds the wash tray The software automatically lowers sippers into each reservoir of the reagent cartridge at the appropriate time during a run depending on the process being performed To the right of the reagent chiller are two form fitted slots one for the SBS Solution PR2 bottle and one for the waste bottle The sipper handle locks the bottles in place and lowers the appropriate sipper into each bottle Reagents are pumped through the sippers and fluidics lines and then to the flow cell Reagent waste is delivered to the waste bottle throughout the process MiSeq FGx Instrument Reference Guide 5 sjuauoduo Getting Started Starting the MiSeq FGx NOTE Illumina recommends that you leave the instrument on continuously However if the instrument must be turned off follow the shutdown procedure described in Shutting Down the Instrument on page 61 Wait a minimum of 60 seconds before turning the power switch back to the ON position If the MiSeq FGx is not already on reach around the right side of the instrument to locate the power switch on the back panel It is in the lower corner directly above the power cord Figure 1 Power Switch Location Turn the power switch to the ON position The integrated instrument computer starts Log in to the operating system Wait until the operating system has finishe
61. illumina MiSeg FGx Instrument Reference Guide 3AAAAGAATGATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTACT GATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGT T STE LTS AAA CAATTCAGAC TAANI SIG T AAA S K A TINA SR EI IA NAS ENS SN ZACTGATTCAACGTACCAAGATTACTTGATCCACTGAT I CAACG GTAACGAACGTATCAATTGAGACTAAATAT TAACG AGCTACCGICTICT 3AAAAGAATGATAACAG TAACACACTTCTGTTAACCTTAAGAT TACTTGATCCACTGAT TCAACG TACCGTAAAGAT TAC T GATAACAGTAA eT CTGTTAACCTTAAGAT TACI TGATCCACTGAI TCAACGTACCGTAACGAACGTATCAATTGA TGAGACTAAATAT TAACGTTGTTAACCTTAAGATTACT TGATCCACTGATTCAACGTACCGTAACGAACGTATCAAT TGAGAC TAAATAT GACTAAATAT TAACGTACCA I TGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT 1 TTGATCCACTGAT TCAACGTACCGTAACGAACGTATCAAT TGAGACTAAATAT I GAGCTACCGTCTTCTGTTAACC GAT TACT TGATCCACTG EE IAACGTACCATTAAGAGCTACC CCGTAACG IAACGTACCAT TAAGAGCTACC AAGA IAACCTTAAGATTACTTGATCCACTGATTCAACC AACGTACCATTAAGAGCT TCTGTTAACCT TAAGAT TACTTGATCCACTGATTCAACGTACCGTA AT CAATTGAGAC TAAATALTAACGTACTTAR SC TAAGATACI TG TOCACI GATTCAA IA COGTAACGAACG ICITGIGTTA CC TAAGATIACI TOATCCACITGATT CAACG TA COTAACGAA CI TATCAATTGAGACTAACGACGA GACTAAATAT TAACGTACCAT TAAGAGCTACAACCTTAAGAT TACTTGATCCACTGATTCAACGTACCGTAACGAACG TATCAAT TGAGACTAAATAT TAACG TACCAT TAAGAGC TAC GATAACAGTAACACACTTCTGTTAACCTTAAGATTACTTGATCCACTGAT ICAACG TACCGTAACGAACG TATCAAT TGAGACTAAATAT TAACG TACCAT TAAGAGC C ACCATTAAGAG EH ACCGTGCAACTTAAC E
62. imes to mix 2 Prepare fresh sodium hypochlorite wash solution with nuclease free water as follows a Add 30 ul of 6 sodium hypochlorite to 870 ul nuclease free water These volumes result in a 1 30 sodium hypochlorite dilution b Add 50 ul of the 1 30 sodium hypochlorite dilution to 950 ul of nuclease free water in an Illumina supplied MiSeq tube part MS 102 9999 d NOTE i Make sure to check the percentage of sodium hypochlorite on the product label Using the correct concentration of sodium hypochlorite is important If the concentration is too high it can make cluster generation fail in subsequent runs If 6 sodium hypochlorite is not available make a 1 ml solution of 0 01 sodium hypochlorite in nuclease free water c Mix the sodium hypochlorite dilution and water mixture well pipetting up and down with a P1000 micropipette 3 Prepare the wash components with fresh 0 5 Tween 20 wash solution as follows a Add 6 ml wash solution to each reservoir of the wash tray b Add 350 ml wash solution to the 500 ml wash bottle 4 Insert the MiSeq tube containing 01 sodium hypochlorite wash solution into position 17 of the wash tray until the neck of the tube is flush with the tray The tube displaces the Tween 20 and nuclease free wash solution from position 17 MiSeq FGx Instrument Reference Guide 5 3 USEM uny 1s0 y e BUIUUOMSd Maintenance Procedures 94 5 7 10 Figure 44 MiSeq Tube in Position 17 of the Wash Tray
63. inations of biomarkers or sequences are examples of Application Specific IP Consumable s means Illumina branded reagents and consumable items that are intended by Illumina for use with and are to be consumed through the use of Hardware Documentation means Illumina s user manual for this Product including without limitation package inserts and any other documentation that accompany this Product or that are referenced by the Product or in the packaging for the Product in effect on the date of shipment from Illumina Documentation includes this document Hardware means Illumina branded instruments accessories or peripherals Illumina means Illumina Inc or an Illumina affiliate as applicable Product means the product that this document accompanies e g Hardware Consumables or Software Purchaser is the person or entity that rightfully and legally acquires this Product from Illumina or an Illumina authorized dealer Software means Illumina branded software e g Hardware operating software data analysis software All Software is licensed and not sold and may be subject to additional terms found in the Software s end user license agreement Specifications means Illumina s written specifications for this Product in effect on the date that the Product ships from Illumina 2 Research Use Only Rights Subject to these terms and conditions and unless otherwise agreed upon in writing by an officer of Illumina Purchaser is granted only
64. indicators 12 analysis during SE 33 options 3 B base calls 23 BaseSpace 24 connection 12 bundle logs 15 C chastity filter 23 cluster density 23 components ow cell 4 flow cell compartment 3 4 optics module 3 reagent compartment 3 5 consumables S 7 user supplied 7 copying files and folders 14 customer support 75 cycles in a read 30 D deleting files and folders 14 disk space checking 30 low disk space 65 documentation 75 domain name 19 E email alerts 16 flow cell cleaning 43 loading 21 25 45 overview 4 flow cell clamp 4 flow cell compartment 3 4 flow cell door sensor 12 flow rate troubleshooting 69 fluidics troubleshooting 69 70 washing 56 59 folder locations default settings 16 G genome references 14 MiSeq FGx Instrument Reference Guide x pu H help Live Help 31 help technical 75 icons activity indicators 12 errors and warnings 13 sensors 12 status alert 13 idling the instrument 59 initialization 65 instrument setup 18 intensities 23 IP address 19 L Live Help 31 loading flow cell 21 25 loading reagents 22 25 cartridge 48 SBS Solution 47 M maintenance wash 13 56 manifest file AS to instrument 14 MiSeq Reporter overview 33 monitoring the run 23 27 50 moving files and folders 14 N network connection 65 network settings 19 O optics module 3 p password 6 pausing a run 30 postrun wash 53 power switch 6 Q Q scores 23
65. intended for targeted sequencing of DNA for forensic casework database and other human identification applications The MiSeq FGx also features a Research Use Only RUO mode RUO mode works with all Illumina RUO library preparation kits compatible with the MiSeq 2 Part 15050524 Rev C Components The MiSeq FGx has the following exterior components sjuauoduo A Flow cell compartment Contains the flow cell stage that houses the flow cell throughout the run Flow cell stage motors move the stage out of the enclosed optical module for flow cell loading and returns the stage when the run begins B Enclosed optics module Contains optical components that enable imaging of the flow cell C Status bar Uses three colors to indicate instrument status Blue indicates that the instrument is processing orange indicates the instrument needs attention and green indicates that the instrument is ready to begin the next run D Touch screen monitor Enables on instrument configuration and run setup using the software interface E External USB port Facilitates the transfer of files and data to the instrument computer from the touch screen monitor F Reagent compartment Holds reagents at proper temperatures wash solutions and the waste bottle A magnetic latch secures the reagent compartment door The MiSeq FGx interface guides users through the run setup steps using the touch screen monitor MiSeq FGx Instrument Referenc
66. kit at support illumina com sds html Slowly lower the sipper handle making sure that the sippers lower into the wash bottle and waste bottle Close the reagent compartment door Select Next The post run wash begins When the wash is complete leave the used flow cell wash tray and wash bottle containing the remaining wash solution on the instrument A small amount of wash solution remains in the MiSeq tube in position 17 Part 15050524 Rev C NOTE i The sippers remain in the down position which is normal Leave the unused wash solution in the wash tray and wash bottle to prevent the sippers from drying out and air from entering the system USEM uny 1s0 y e BUIUUOMSd MiSeq FGx Instrument Reference Guide 5 5 Maintenance Procedures Performing a Maintenance Wash Procedure o6 Perform a maintenance wash every 7 days to ensure optimal performance The maintenance wash includes a series of three wash steps using a wash solution of nuclease free water mixed with Tween 20 Allow approximately 90 minutes to complete the wash User Supplied Consumables Tween 20 Sigma Aldrich catalog P7949 Nuclease free water Make sure that a used flow cell is loaded on the instrument From the Welcome screen select Perform Wash From the Perform Wash screen select Maintenance Wash The software automatically raises the sippers in the reagent chiller Perform First Wash 1 Prepare fresh wash solution with Tween 20 and
67. nalysis files will continue uploading after the connection is restored from the point when the interruption occurred As soon as the last base call file is uploaded to BaseSpace additional analysis of your data begins The same analysis workflows are supported on BaseSpace as with on instrument analysis using MiSeq Reporter BaseSpace only supports the same genomes that are provided with MiSeq Reporter You can connect to BaseSpace at basespace com Log in using your Mylllumina account login For more information about using BaseSpace see the BaseSpace online help on the Illumina website www illumina com help BaseSpaceHelp BaseSpaceHelp htm MiSeq Reporter Overview MiSeq Reporter is a Windows Service application that processes base calls generated by initial analysis MiSeq Reporter begins additional analysis immediately after the completion of RTA analysis of the sequencing run MiSeq Reporter runs on the MiSeq FGx instrument computer However the software interface must be viewed through a web browser on another computer that is connected to the same network as MiSeq Reporter When secondary analysis is complete a file named CompletedJobInfo xml is written to the run folder For more information see the MiSeg Reporter User Guide part 15042295 Sequencing During Analysis If you choose MiSeq Reporter for additional analysis of an RUO run the MiSeq FGx instrument computing resources are dedicated to either sequencing or analysis I
68. nd or otherwise communicated disclosed or reproduced in any way whatsoever without the prior written consent of Illumina Illumina does not convey any license under its patent trademark copyright or common law rights nor similar rights of any third parties by this document The instructions in this document must be strictly and explicitly followed by qualified and properly trained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product s FAILURE TO COMPLETELY READ AND EXPLICITLY FOLLOW ALL OF THE INSTRUCTIONS CONTAINED HEREIN MAY RESULT IN DAMAGE TO THE PRODUCT S INJURY TO PERSONS INCLUDING TO USERS OR OTHERS AND DAMAGE TO OTHER PROPERTY ILLUMINA DOES NOT ASSUME ANY LIABILITY ARISING OUT OF THE IMPROPER USE OF THE PRODUCT S DESCRIBED HEREIN INCLUDING PARTS THEREOF OR SOFTWARE OR ANY USE OF SUCH PRODUCT S OUTSIDE THE SCOPE OF THE EXPRESS WRITTEN LICENSES OR PERMISSIONS GRANTED BY ILLUMINA IN CONNECTION WITH CUSTOMER S ACQUISITION OF SUCH PRODUCT S FOR RESEARCH FORENSIC OR PATERNITY USE ONLY 2015 Illumina Inc All rights reserved Illumina 24sure BaseSpace BeadArray BlueFish BlueFuse BlueGnome cBot CSPro CytoChip DesignStudio Epicentre GAIIx Genetic Energy Genome Analyzer GenomeStudio GoldenGate HiScan HiSeq HiSeq X Infinium iScan iSelect ForenSeq MiSeq FGx NeoPrep Nextera
69. ng are acceptable examples Illumina PW1 18 Megaohm MQ water Milli O water Super Q water Molecular biology grade water MiSeq FGx Instrument Reference Guide Purpose Cleaning the flow cell holder General use Cleaning the flow cell stage and the foil seal covering the load samples reservoir Cleaning the flow cell Denaturing and diluting sample libraries and PhiX control DNA Washing the instrument Washing the instrument Removing flow cell from flow cell shipping container Washing the instrument se qeuunsuo parmbay Part 15050524 Rev C MiSeg FGx Software Introduction MiSeq FGx Control Software Analysis Software Options for RUO Runs 3 a ad NE if A t I MiSeq FGx Instrument Reference Guide c Je1deuo MiSeq FGx Software Introduction 10 The software described in this chapter is used to configure run and analyze data from the MiSeq FGx MiSeq FGx Control Software Controls the instrument operation including various configuration settings This software is installed and runs on the instrument See MiSeq FGx Control Software on page 11 Real Time Analysis RTA Performs image processing and base calling and assigns a quality score to each base for each cycle Images are temporarily stored in the run folder for processing by RTA and then automatically deleted when RTA analysis is complete This
70. ng the error For technical questions visit the MiSeq FGx support pages on the Illumina website for access to frequently asked questions or log in to your Mylllumina account for access to support bulletins For problems with run quality or performance contact Illumina Technical Support For more information see Technical Assistance on page 75 The Illumina Technical Support representative might ask for copies of run specific files for troubleshooting purposes The following files are located at the root level of the output run folder SampleSheet csv if using the instrument in RUO mode RunParameters xml RunInfo xml Interop folder Part 15050524 Rev C Resolving Run Setup Errors If any checks in the pre run check fail a red icon X appears next to the item A message appears on the screen that describes the error and how to correct it Error Action X Flow Rate Measured The flow rate check screen opens Use the drop down list or on screen keyboard to enter the following Solution PR2 Volume 250 Aspirate Rate 2500 Dispense Rate 2500 Select Pump If the error persists set the volume to pump 500 ul SBS Solution PR2 and repeat the process When fluids have been pumped select Restart Check When the pre run check is successful the Start Run button becomes active If the flow check fails again reseat the flow cell to make sure that flow is not interrupted due to misalignment Inspect the flow cell gasket for lint
71. nitor run progress intensities and quality scores that appear on the Sequencing screen The Sequencing screen is view only For more information see Sequencing Screen on page 23 For an RUO run see Sequencing Screen for RUO on page 27 2 When the run is complete the Next button appears Review the results on the Sequencing screen before proceeding y NOTE The Sequencing screen remains viewable until Next is selected After you select Next it is not possible to return to the Sequencing screen 3 Select Next to exit the Sequencing screen and proceed to a post run wash Run Metrics Run metrics appear on the Sequencing screen at different points in a run During cluster generation steps no metrics appear After sequencing begins the following metrics appear o0 at the indicated cycles Metric Intensity Intensity and Cluster Density Intensity Cluster Density PF Yield and Q scores Kit MiSeq FG MiSeq RU MiSeq RU MiSeq RU MiSeq RU MiSeq RU MiSeq RU MiSeq FG MiSeq RU MiSeq RU MiSeq RU x Reagent Kits O Reagent Kits v3 O Reagent Kits v2 O Reagent Kits v1 MiSeq FGx Reagent Kits O Reagent Kits v3 O Reagent Kits v2 O Reagent Kits v1 x Reagent Kits O Reagent Kits v3 O Reagent Kits v2 O Reagent Kits v1 Cyde Cycle 1 7 Cycle 1 7 Cycle 14 Cycle 14 Cycle 8 25 Cycle 8 25 Cycle 5 25 Cycle 5 25 Cycle 26 through run completion Part 15050524 Rev C Maintenance Procedures
72. nsion is csv If the sample sheet is missing create one and copy it to the sample sheet locations specified in Run Options After locating a sample sheet select Restart Check MiSeq FGx Instrument Reference Guide 6 5 Troubleshooting Resolving RFID Read Failure 66 RFID failures are triggered if The component loaded is not part of a forensics kit For an RUO run the component loaded is not part of the kit identified by the sample sheet There is a technical failure with the REID tag in the component The following steps can be used to resolve REID failures resulting from a technical failure r NOTE A run is allowed one RFID read failure If the RFID of two consumables cannot be read the software cannot proceed to the next run setup step If this occurs contact Ilumina Technical Support Flow Cell 1 Always retry the RFID read before proceeding To do so open and then close the flow cell compartment door 2 If the RFID fails a second time open the flow cell compartment door and remove the flow cell carefully 3 Check the flow cell and the flow cell stage for debris or salts that can stop the RFID read 4 Reseat the flow cell Make sure that the latch brings the flow cell into proper alignment 5 Close the flow cell compartment door 6 If the RFID continues to fail select the keyboard icon to open the on screen keyboard 7 Enter the barcode number of the flow cell which is located on the flow cell container
73. ovides troubleshooting options for checking the operational status of instrument components See System Check Screen on page 19 Manual Update Provides the option to update software on the instrument computer manually See Manual Update Screen on page 20 Reboot Use the Reboot command to reboot the system software There is no requirement to reboot the software as part of regular maintenance Shut Down Use the Shut Down command to shut down the control software and Windows on the instrument computer See Shutting Down the Instrument on page 61 Minimize to Windows Provides quick access to the instrument operating system and any folders on the instrument computer when MiSeq FGx is running in kiosk mode as opposed to Windows mode Admin or SuperUser user access level is required to use this feature Part 15050524 Rev C System Settings Screen System Settings are normally configured when the instrument is initially installed and started for the first time If any settings changes are required due to a network or facility change use the System Settings feature Figure 12 System Settings Please select from the following options to set up your IP settings Obtain an IP address automatically O Obtain DNS server address automatically O Use the following IP address O Use the following DNS server addresses IP address Preferred DNS server Subnet mask Alternate DNS server Default gateway x Save and Cancel Continue
74. presentation of the run in progress including intensities and quality scores Q scores Run Progress Shows run progress in a status bar and lists the number of cycles completed Intensity Shows the value of cluster intensities of the 90 percentile for each tile The graphic in the Intensity area represents the number of tiles and number of surfaces being imaged The flow cell is imaged on the top surface only A single column graphic appears Flow Cell Shows the current temperature of the flow cell indicated by color Blue indicates cooler temperatures while orange and red indicate warmer temperatures During imaging imaged tiles appear dark gray During chemistry wavy lines indicate that reagents are pumping through the flow cell Cluster Density K mm Shows the number of clusters per square millimeter for the run Optimally expect a cluster density range of 400 1650 K mm Clusters Passing Filter Y Shows the percentage of clusters passing filter based on the Illumina chastity filter which measures quality This data appears only after cycle 25 1 NOTE The chastity of a base call is the ratio of the intensity of the greatest signal divided by the sum of the two greatest signals If more than one base call has a chastity value of less than 0 6 in the first 25 cycles reads do not pass the quality filter Phasing See Number of Cycles in a Read on page 30 and the Run Metrics Tab section in the ForenSeg Universal Analysis
75. r and reagent chiller door and slide the used reagent cartridge or wash tray from the chiller b Slide the wash tray into the reagent chiller until it stops Close the reagent chiller door c Raise the sipper handle in front of the SBS Solution PR2 bottle and waste bottle until it locks into place and replace the SBS Solution PR2 bottle with the wash bottle MiSeq FGx Instrument Reference Guide 5 9 Maintenance Procedures f NOTE Discard the SBS Solution PR2 bottle after each run Do not reuse any remaining SBS Solution PR2 Remove the waste bottle and discard the contents appropriately Return the waste bottle to the reagent compartment Slowly lower the sipper handle making sure that the sippers lower into the wash bottle and waste bottle Close the reagent compartment door Select Next The first wash begins Perform Second Wash L NOTE Always use fresh wash solution for each wash step Reusing wash solution from the previous wash can return waste to the fluidics lines 1 Prepare fresh wash solution with Tween 20 and nuclease free water as follows a Add 5 ml 100 Tween 20 to 45 ml nuclease free water These volumes result in 10 Tween 20 b Add 25 ml 10 Tween 20 to 475 ml nuclease free water These volumes result in a 0 5 Tween 20 wash solution c Invert several times to mix 2 When the first wash is complete remove the wash tray and wash bottle and discard the remaining wash solution 3 Refill
76. rform Wash Screen on page 13 Manage Files Provides controls for moving deleting and uploading files on the instrument computer See Manage Files Screen on page 14 Run Options Provides options for the post run wash changing default locations of data folders and specifying email notification preferences See Run Options Screen on page 16 Manage Instrument Provides options to go to system settings perform a systems check update software manually and reboot or shut down the instrument See Manage Instrument Screen on page 18 MiSeq FGx Instrument Reference Guide 1 1 MiSeq FGx Software 12 Activity Indicators A series of icons are located in the lower right corner of each interface screen Each icon is an activity indicator that shows which activity the instrument is performing Figure 4 Activity Indicators From left to right the activity indicators represent the following activities Moving the Y stage Moving the Z stage Activating electronics functionality Using the camera Pumping through the fluidics system Sensor Indicators Four sensor indicators at the base of each interface screen represent the status of an instrument component Figure 5 Sensor Indicators From left to right the sensor indicators represent the following components Flow cell compartment door in the closed or open positions Temperature of the reagent chiller in C Temperature of the flow cell in C Status of server conne
77. s analyzed The one extra cycle is required for phasing and prephasing calculations For example a paired end 350 30 cycle run performs a 351 cycle read and a 31 cycle read for a total of 382 cycles At the end of the run 380 cycles are analyzed For information on the number of cycles that can be performed with one kit see the MiSeg FGx Reagent Kit Reference Guide part 15055483 If performing an RUO run see the appropriate MiSeq Reagent Kit reagent preparation guide for RUO Required Disk Space The integrated instrument computer has approximately 550 GB of storage capacity Before starting a run the software checks available disk space If there is not enough disk space for the run a software prompt appears The message indicates how much disk space is required for the run and how much disk space must be cleared before the run can proceed If prompted to make disk space available go to the Welcome screen and select Manage Files From the Manage Files screen select the Runs tab Move or delete older run folders as appropriate For more information see Manage Files Screen on page 14 After clearing adequate disk space select Restart Check Pausing or Stopping a Run 30 The MiSeq FGx is designed to complete a run from beginning to end without user intervention However it is possible to pause a run or stop a run from the Sequencing screen Pausing a Run A run can be paused during sequencing if necessary for example to
78. software is installed and runs on the instrument ForenSeq Universal Analysis Software Performs user authentication and analysis such as demultiplexing alignment variant calling and report generation The specific functions that are supported vary by application This software is installed off instrument on a stand alone server The output is viewed through a web browser See the ForenSeg Universal Analysis Software Guide part 15053876 for information on analyzing data For RUO runs BaseSpace and MiSeq Reporter are available to perform additional analyses See Analysis Software Options for RUO Runs on page 33 Part 15050524 Rev C MiSeq FGx Control Software During the run MiSeq FGx Control Software operates the flow cell stage dispenses reagents controls flow cell temperatures and captures images of clusters on the flow cell The control software performs the run according to parameters specified in the ForenSeq Universal Analysis Software Welcome Screen The MiSeq FGx Control Software interface opens to the Welcome screen when the software launches Figure 3 Welcome Screen 9JEMILOS 01JU0 XD basi SEQUENCE PERFORM WASH Sequence This option opens a series of run setup screens that guide users through the run setup steps See Run Setup Screens on page 20 Perform Wash Provides options to start the following types of instrument washes a maintenance wash standby wash or a post run wash See Pe
79. tance contact Illumina Technical Support Table 3 Illumina General Contact Information Website www illumina com Email techsupport illumina com Table4 Ilumina Customer Support Telephone Numbers Region Contact Number Region Contact Number North America 1 800 809 4566 Italy 800 874909 Australia 1 800 775 688 Netherlands 0800 0223859 Austria 0800 296575 New Zealand 0800 451 650 Belgium 0800 81102 Norway 800 16836 Denmark 80882346 Spain 900 812168 Finland 0800 918363 Sweden 020790181 France 0800 911850 Switzerland 0800 563118 Germany 0800 180 8994 United Kingdom 0800 917 0041 Ireland 1 800 812949 Other countries 44 1799 534000 Safety Data Sheets Safety data sheets SDSs are available on the Illumina website at support illumina com sds html Product Documentation Product documentation in PDF is available for download from the Illumina website Go to support illumina com select a product then click Documentation amp Literature MiSeq FGx Instrument Reference Guide T D 9UB SISSY EDIUYD9 TAACC AC AAGAT CC HGALGE GIE AAGATTACTTGATCCACTGATTCAACGTACCGTAAAGAT SE ATTCAACGTACCGTAACGAACGTATCAATTGAGAC TAAATAT T ACTTGI OE TA ACH GT AAGATTACTTGATCCACTGATTCAACGTACCGTAAAGATTAC HET es AGCTTGATCCACTGATTCAACGTACGGTAACGAAGGTATGAAT T G GAAAAGAAT GATAACAGTAACACAC TTGAGACTAAATATTAACG TGATCCACTGAT IAACGTACCAT TAAGAGCTACCGTCTTCTGT T IACCATTAAGAGCTACCGTC
80. tandby wash When the standby wash is complete the instrument is in standby mode and a message appears on the Welcome screen stating the status of the instrument When the instrument is in standby mode a maintenance wash must be performed before a sequencing run can be initiated See Maintenance Frequency on page 52 for information on when to perform washes dy NOTE Illumina recommends repeating the standby wash every 30 days that the instrument remains idle User Supplied Consumables Tween 20 Sigma Aldrich catalog P7949 Nuclease free water useA qpuezs e Buiuuoje d Procedure 1 Make sure that a used flow cell is loaded on the instrument 2 From the Welcome screen select Perform Wash 3 From the Wash Options screen select Standby Wash The software automatically raises the sippers in the reagent chiller Perform First Wash 1 Prepare fresh wash solution with Tween 20 and nuclease free water as follows a Add 5 ml 100 Tween 20 to 45 ml nuclease free water These volumes result in 1096 Tween 20 b Add 25 ml 10 Tween 20 to 475 ml nuclease free water These volumes result in a 0 5 Tween 20 wash solution c Invert several times to mix 2 Prepare the wash components with fresh 0 5 Tween 20 wash solution as follows a Add 6 ml wash solution to each reservoir of the wash tray b Add 350 ml wash solution to the 500 ml wash bottle 3 Load the wash tray and wash bottle onto the instrument a Open the reagent compartment doo
81. tempt to determine the methods of operation of this Product or iv transfer to a third party or grant a sublicense to any Software or any third party software Purchaser further agrees that the contents of and methods of operation of this Product are proprietary to Illumina and this Product contains or embodies trade secrets of Illumina The conditions and restrictions found in these terms and conditions are bargained for conditions of sale and therefore control the sale of and use of this Product by Purchaser 5 Limited Liability TO THE EXTENT PERMITTED BY LAW IN NO EVENT SHALL ILLUMINA OR ITS SUPPLIERS BE LIABLE TO PURCHASER OR ANY THIRD PARTY FOR COSTS OF PROCUREMENT OF SUBSTITUTE PRODUCTS OR SERVICES LOST PROFITS DATA OR BUSINESS OR FOR ANY INDIRECT SPECIAL INCIDENTAL EXEMPLARY CONSEQUENTIAL OR PUNITIVE DAMAGES OF ANY KIND ARISING OUT OF OR IN CONNECTION WITH WITHOUT LIMITATION THE SALE OF THIS PRODUCT ITS USE ILLUMINA S PERFORMANCE HEREUNDER OR ANY OF THESE TERMS AND CONDITIONS HOWEVER ARISING OR CAUSED AND ON ANY THEORY OF LIABILITY WHETHER IN CONTRACT TORT INCLUDING NEGLIGENCE STRICT LIABILITY OR OTHERWISE 6 ILLUMINA S TOTAL AND CUMULATIVE LIABILITY TO PURCHASER OR ANY THIRD PARTY ARISING OUT OF OR IN CONNECTION WITH THESE TERMS AND CONDITIONS INCLUDING WITHOUT LIMITATION THIS PRODUCT INCLUDING USE THEREOF AND ILLUMINA S PERFORMANCE HEREUNDER WHETHER IN CONTRACT TORT INCLUDING NEGLIGENCE STRICT LIABILITY OR OTHER
82. the reagent cartridge for a subseguent run A new run reguires a new reagent cartridge Live Help The MiSeg FGx must be connected to a network with internet access to enable Live Help The Live Help feature is an online assistance tool that enables a representative from Illumina Technical Support to view the MiSeq FGx screen with your permission and share control of the instrument You have overriding control and can end the screen sharing session at any time Access Live Help from the help icon on the Welcome screen Figure 28 Help Menu LIVE HELP ABOUT To enable a connection obtain a unique access code from Illumina Technical Support enter that code on the Live Help screen and then select Next MiSeg FGx Instrument Reference Guide 31 9JEMIJOS 0J1UO2 XJ basi Figure 29 Live Help Screen NT MiSeq FGx customer yourlab com Please follow below instructions to log into MiSeq Live Help EA Enter the access code provided to you by your Illumina Representative in the Access Code field below using the keyboard icon and select Next to start the screen sharing session You might be prompted to download a small virus free plug in This plug in will not stay on the instrument and will be removed once the support session is closed When your session has started your Illumina support representative will ask to take control of your instrument You have overriding control and can end the screen sharing session at any time
83. the wash components with fresh 0 5 Tween 20 wash solution as follows a Add 6 ml wash solution to each reservoir of the wash tray b Add 350 ml wash solution to the 500 ml wash bottle 4 Load the wash tray and wash bottle as follows a Slide the wash tray into the reagent chiller until it stops Close the reagent chiller door b Load the wash bottle and slowly lower the sipper handle making sure that the sippers lower into the wash bottle and waste bottle c Close the reagent compartment door 5 Select Next The second wash begins After the Wash When the wash is complete leave the used flow cell wash tray and wash bottle containing the remaining wash solution on the instrument 60 NOTE The sippers remain in the down position which is normal Leave the unused wash solution in the wash tray and wash bottle to prevent the sippers from drying out and air from entering the system Part 15050524 Rev C Shutting Down the Instrument It is best to leave the instrument on at all times However if the instrument must be turned off use the following procedure to shut down Windows and prepare the fluidics lines 1 Perform a maintenance wash For more information see Procedure on page 56 2 Remove the waste bottle and discard the contents appropriately Return the waste bottle to the reagent compartment 3 Close the reagent compartment door 4 From the Manage Instrument screen select Shut Down This command shuts down the
84. to confirm that the RFID of the SBS Solution PR2 bottle was read successfully MiSeq FGx Instrument Reference Guide 47 sjuebesy Bulpeo7 Performing a Run NOTE e If the REID cannot be read identifying information can be entered manually However the software allows only one of the three RFID labeled components flow cell reagent cartridge SBS Solution PR2 to fail on a forensic genomics run For more information see Resolving RFID Read Failure on page 66 8 Select Next on the Load Reagents screen Load the Reagent Cartridge t NOTE Do not leave the reagent chiller door open for extended periods of time 1 Open the reagent chiller door 2 Hold the reagent cartridge on the end with the Illumina label and slide the reagent cartridge into the reagent chiller until the cartridge stops Figure 43 Load Reagent Cartridge 3 Close the reagent chiller door 4 Check the lower left corner of the screen to confirm that the RFID of the reagent cartridge was read successfully dy NOTE If that the RFID cannot be read identifying information can be entered manually However the software allows only one of the three RFID labeled components flow cell reagent cartridge SBS Solution PR2 to fail For more information see Resolving RFID Read Failure on page 66 5 Close the reagent compartment door 6 Select Next on the Load Reagents screen The Review screen opens A
85. tom primers prepare primers and set up the sample sheet as described in Using Custom Primers on the MiSeg part 15041638 After you have prepared the MiSeq FGx reagent cartridge for use you are ready to load your samples 1 E Use a low lint lab tissue to clean the foil seal covering the reservoir labeled Load Samples Use a clean 1 ml pipette tip to pierce the foil seal covering the reservoir labeled Load Samples Y NOTE Do not pierce any other reagent positions Other reagent positions are pierced automatically during the run Pipette 600 ul of prepared libraries into the Load Samples reservoir Avoid touching the foil seal as you dispense the sample Check for air bubbles in the reservoir after loading sample If air bubbles are present gently tap the cartridge on the bench to release the bubbles Figure 32 Load Libraries Proceed directly to the run setup steps using the MiSeq FGx Control Software interface MiSeq FGx Instrument Reference Guide 41 oDpUUED juebesy ou eyedojg Performing a Run Login and Select Run Type 42 From the log in screen enter your ForenSeq Universal Analysis Software user name and password and then select Next From the Welcome screen select Sequence When the Select Run Type screen appears select Forensic Genomics or Research Use Only Run Figure 33 Select Run Type Please select a run type F ORENSIC RESEARCH USE GENOMICS ONLY RUN m Z 00000 4 Follow
86. ttle ID PR1234567 ABCDE Flow Cell Barcode FC1234567 ABCDE ET ZS m Z E 00000 400 C ECK When the pre run check is successful the Start Run button becomes active Sequencing Screen for RUO 1 During the RUO run monitor run progress intensities and quality scores that appear on the Sequencing screen The Sequencing screen is view only To monitor the run in greater detail use the Sequencing Analysis Viewer SAV MiSeq FGx Instrument Reference Guide 2 7 9JEMILOS 01JU0 XD basi MiSeq FGx Software 28 installed on another computer independent of the instrument computer A network connection is required See Sequencing Analysis Viewer User Guide part 15020619 Alternatively if you are connected to BaseSpace you can monitor your run using SAV in BaseSpace Run Progress Shows run progress in a status bar and lists the number of cycles completed Intensity Shows the value of cluster intensities of the 90 percentile for each tile The graphic in the Intensity area represents the number of tiles and number of surfaces being imaged If the flow cell is imaged on the top surface only a single column graphic appears If the flow cell is image on the top surface and bottom surface a two column graphic appears A Indicates two tiles top surface only B Indicates four tiles top and bottom surface Q Score All Cycles Shows the average percentage of bases greater than Q30 which is a quality score
87. urchaser has all intellectual property rights that are necessary for Purchaser s intended uses of this Product including without limitation any rights from third parties or rights to Application Specific IP Illumina makes no guarantee or warranty that purchaser s specific intended uses will not infringe the intellectual property rights of a third party or Application Specific IP 3 Regulatory This Product has not been approved cleared or licensed by the United States Food and Drug Administration or any other regulatory entity whether foreign or domestic for any specific intended use whether research commercial diagnostic or otherwise This Product is labeled For Research Use Only Purchaser must ensure it has any regulatory approvals that are necessary for Purchaser s intended uses of this Product 4 Unauthorized Uses Purchaser agrees a to use each Consumable only one time and b to use only Illumina consumables reagents with Illumina Hardware The limitations in a b do not apply if the Documentation or Specifications for this Product state otherwise Purchaser agrees not to nor authorize any third party to engage in any of the following activities i disassemble reverse engineer reverse compile or reverse assemble the Product ii separate extract or isolate components of this Product or subject this Product or components thereof to any analysis not expressly authorized in this Product s Documentation iii gain access to or at
88. ution PR2 bottle and reagent cartridge are loaded the software reads and records the REID A confirmation that the RFID was successfully read appears in the lower right corner of the screen i NOTE r Use the correct reagent sequencing kit components for the type of run you perform Forensic genomics runs only work with ForenSeq sequencing kits and RUO runs only work with RUO reagent kits MiSeq FGx Instrument Reference Guide 2 5 9JEMIJOS 0J1UO2 XD Y basi MiSeq FGx Software Figure 22 Load SBS Solution PR2 Bottle and Waste Bottle Please load the PR2 and waste bottle and walt for the RFID to be read FR Botto ID Flow Cell Barcode ASDFG 4 Back Figure 23 Load Reagent Cartridge Please load the reagent cartridge Reagent D MS2345688 0FSV1 TEST If the RFID cannot be read identifying information can be entered manually However the software allows only one of the three RFID labeled components flow cell reagent cartridge SBS Solution PR2 to fail on a forensics run For more information see Resolving RFID Read Failure on page 66 Select the help icon in the upper right corner of the screen The help file contains a video that illustrates the steps for loading the reagent cartridge and for loading the SBS Solution PR2 bottle Change Sample Sheet A sample sheet is required for an RUO run By default the software searches for a sample sheet file with a name that matches the barcode number of the reag
89. ware allows only one of the three RFID labeled components flow cell reagent cartridge SBS Solution PR2 to fail on a forensics run For more information see Resolving RFID Read Failure on page 66 Select the help icon in the upper right corner of the screen The help file contains a video that illustrates the steps for loading the reagent cartridge and for loading the SBS Solution PR2 bottle Parts 15050524 Rev C Pre Run Check Screen The software automatically performs a pre run check of required run components and conditions before allowing the run to begin If any errors occur during the pre run check a message appears on the screen that describes the error and the action to correct it For more information see Resolving Run Setup Errors on page 65 Figure 19 Pre Run Check Screen Checking flow Network Connection Active Sample Validated Flow Cell Cartridge Loaded Free Disk Space 86 Waste Bottle Loaded PR2 Bottle Loaded Reagent Cartridge Loaded Flow Cell Door Closed Chiller Door Closed Flow Rate Measured Primary Analysis Ready 00 56 29 The system is waiting 1 hour for primary analysis to complete for the previous run Reagent ID RG1234567 00CDE PR2 Bottle ID PR1234567 ABCDE Flow Cell Barcode FC1234567 ABCDE m 2 00000 When the pre run check is successful the Start Run button becomes active Sequencing Screen The Sequencing screen opens when the run begins This screen provides a visual re
90. y to the Documentation or Specifications ii improper handling installation maintenance or repair other than if performed by Illumina s personnel iii unauthorized alterations iv Force Majeure events or v use with a third party s good not provided MiSeq FGx Instrument Reference Guide by Illumina unless the Product s Documentation or Specifications expressly state such third party s good is for use with the Product Procedure for Warranty Coverage In order to be eligible for repair or replacement under this warranty Purchaser must i promptly contact Illumina s support department to report the non conformance ii cooperate with Illumina in confirming or diagnosing the non conformance and iii return this Product transportation charges prepaid to Illumina following Illumina s instructions or if agreed by Illumina and Purchaser grant Illumina s authorized repair personnel access to this Product in order to confirm the non conformance and make repairs Sole Remedy under Warranty Illumina will at its option repair or replace non conforming Product that it confirms is covered by this warranty Repaired or replaced Consumables come with a 30 day warranty Hardware may be repaired or replaced with functionally equivalent reconditioned or new Hardware or components if only a component of Hardware is non conforming If the Hardware is replaced in its entirety the warranty period for the replacement is 90 days from the date of shipm

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