Thermo Scientific Ion Torrent A45727 Скачать руководство пользователя страница 5

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Create and manage run plans .......................... 55

Create a run plan for a sample run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Create a run plan for a library run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

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CHAPTER 6

Dilute the samples and load the sample plate ......... 68

Guidelines for nucleic acid isolation and quantification—sample runs . . . . . . . . . . . . . . . . . 68
Dilute the samples (if needed) and load the sample plate—sample run . . . . . . . . . . . . . . . 69
Guidelines for library quantification—library runs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Dilute and pool libraries, and load the sample plate—library run . . . . . . . . . . . . . . . . . . . . . 71

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CHAPTER 7

Load the sequencer and start a run ..................... 72

Before you begin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Fill Genexus

™

Primer Pool Tubes

(custom assays only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Load the sequencer and start a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Clear the instrument deck and perform a UV Clean . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

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CHAPTER 8

Monitor the run ........................................... 84

View run progress on the instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

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CHAPTER 9

Review data and results .................................. 85

Run Results

screen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Lab Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Download the Lab Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

View sequencing results and run metrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

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TCR Beta

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LR Assay GX run results . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Contents

Genexus

™

Integrated Sequencer User Guide

Содержание Ion Torrent A45727

Страница 1: ...For Research Use Only Not for use in diagnostic procedures Genexus Integrated Sequencer USER GUIDE Catalog Number A45727 Publication Number MAN0017910 Revision A 0 ...

Страница 2: ... ALLOWED BY LAW THERMO FISHER SCIENTIFIC INC AND OR ITS AFFILIATE S WILL NOT BE LIABLE FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING YOUR USE OF IT Revision history Pub No MAN0017910 Revision Date Description A 0 13 November 2019 New user guide for the Genexus Integrated Sequencer Important Licensing Informatio...

Страница 3: ...us GX5 Starter Pack HD 13 Shared reagents and supplies 14 Genexus Templating Strips 3 and 4 GX5 14 Genexus Primer Pool Tubes and Pipette Tips 14 GX5 Chip and Genexus Coupler 15 Genexus Sequencing Kit 15 Genexus Controls 16 Genexus Filter 16 Oncomine GX assays 16 Required materials not supplied 17 Recommended materials for nucleic acid isolation and quantification 18 Genexus Integrated Sequencer co...

Страница 4: ...stem installed assays 30 Manage assays manager administrator 30 Create a new assay manager administrator 32 Copy an assay manager administrator 40 Import an assay manager administrator 41 CHAPTER 4 Enter samples and libraries 42 Add a new sample 42 System installed sample attributes 43 Create a custom sample attribute manager administrator 44 Import samples 45 Manage samples 46 Sort search and fil...

Страница 5: ...4 Load the sequencer and start a run 75 Clear the instrument deck and perform a UV Clean 81 CHAPTER 8 Monitor the run 84 View run progress on the instrument 84 CHAPTER 9 Review data and results 85 Run Results screen 86 Lab Report 88 Download the Lab Report 89 View sequencing results and run metrics 89 Summary of the sample results 91 Run metrics 92 QC results 94 SNV INDEL results 95 Fusion results...

Страница 6: ...with the Qubit Fluorometer 135 Guidelines for using custom assays with the Genexus Integrated Sequencer 136 Planning sequencing runs for efficient use of consumables 138 Configure an Ion Reporter Server account administrator 139 Tag an Ion Reporter Software analysis workflow for use with the IonReporterUploader plugin 140 Configure Thermo Fisher Connect accounts manager administrator 141 APPENDIX ...

Страница 7: ...sposal 156 Safety and electromagnetic compatibility EMC standards 156 Safety standards 157 EMC standards 157 Environmental design standards 158 Radio compliance standards 158 Chemical safety 159 Biological hazard safety 161 APPENDIX F Documentation and support 162 Related documentation 162 Customer and technical support 162 Limited product warranty 162 Contents Genexus Integrated Sequencer User Gu...

Страница 8: ...omated run The instrument supports both sample to results and library to results sequencing runs up to 32 DNA or RNA samples run Additional key features include Sample to results in as little as 14 hours single lane run Flexible and cost effective run planning making use of multi lane multi run sequencing chips Automated library preparation for up to 400 base read libraries using either standard I...

Страница 9: ...ion of the instrument Genexus Installation and Training Kit Contents Part No Quantity Storage Genexus Controls A40267 1 30 C to 10 C Genexus Strip 1 A40254 8 strips 2 C to 8 C Genexus Strip 2 AS A40253 8 strips 30 C to 10 C Genexus Barcodes 1 32 AS A40258 1 plate 15 C to 30 C Genexus Strip 3 GX5 A40265 8 strips 2 C to 8 C Genexus Strip 4 A40264 8 strips 30 C to 10 C Genexus Cartridge A40272 2 cart...

Страница 10: ... AmpliSeq library based chemistry are ordered as kits with eight pairs of strips kit Component Carrier color Part No Quantity per kit Storage Genexus Strip 1 Red A40254 8 strips 2 C to 8 C Genexus Strip 2 AS Blue A40253 8 strips 30 C to 10 C Ion Torrent Genexus Barcodes AS are supplied in plates containing 32 dual barcodes per plate The barcodes can be ordered as a set of three plates Cat No A4025...

Страница 11: ... to 8 C Genexus Strip 2 AS A40253 8 strips 30 C to 10 C Genexus Strip 3 GX5 A40265 8 strips 2 C to 8 C Genexus Strip 4 A40264 8 strips 30 C to 10 C Genexus Barcodes 1 32 AS A40258 1 plate 15 C to 30 C Genexus Pipette Tips A40266 12 racks Genexus Cartridge A40272 2 cartridges 30 C to 10 C Genexus Bottle 2 A40273 4 bottles 15 C to 30 C Genexus Bottles 1 and 3 A40274 2 bottles each Genexus GX5 Starte...

Страница 12: ...eq HD library chemistry have an HD suffix Ion Torrent Genexus Library Strips 1 and 2 HD Cat No A40255 for Ion AmpliSeq HD library based chemistry are ordered as kits with eight pairs of strips kit Component Carrier color Part No Quantity per kit Storage Genexus Strip 1 Red A40254 8 strips 2 C to 8 C Genexus Strip 2 HD Purple A40256 8 strips 30 C to 10 C Ion Torrent Genexus Barcodes 1 32 HD are sup...

Страница 13: ... to 8 C Genexus Strip 2 HD A40256 8 strips 30 C to 10 C Genexus Strip 3 GX5 A40265 8 strips 2 C to 8 C Genexus Strip 4 A40264 8 strips 30 C to 10 C Genexus Barcodes 1 32 HD A40261 1 plate 15 C to 30 C Genexus Pipette Tips A40266 12 racks Genexus Cartridge A40272 2 cartridges 30 C to 10 C Genexus Bottle 2 A40273 4 bottles 15 C to 30 C Genexus Bottles 1 and 3 A40274 2 bottles each Genexus GX5 Starte...

Страница 14: ...olor Part No Quantity per kit Storage Genexus Strip 3 GX5 Grey A40265 8 strips 2 C to 8 C Genexus Strip 4 Yellow A40264 8 strips 30 C to 10 C Genexus Primer Pool Tubes and Pipette Tips can be ordered individually Genexus Primer Pool Tubes are required for custom assays Item Cat No Quantity Storage Genexus Primer Pool Tubes A40262 50 assemblies 2 tubes assembly Bag of 100 caps 15 C to 30 C Genexus ...

Страница 15: ...The Ion Torrent Genexus Sequencing Kit is ordered as a combo Cat No A40271 and components of the combo are also orderable as listed Component Cat No Quantity Storage Genexus Cartridge A40272 2 cartridges 30 C to 10 C Genexus Bottle 2 A40273 4 bottles 15 C to 30 C Genexus Bottles 1 and 3 A40274 2 bottles each GX5 Chip and Genexus Coupler Genexus Sequencing Kit Chapter 1 Product information Shared r...

Страница 16: ...ns and are supplied in pre measured ready to load Genexus Primer Pool Tubes Assays are provided with Genexus Library Strips 1 and 2 AS Cat No A40252 or Genexus Library Strips 1 and 2 HD Cat No A40255 in the amount listed Contents Carrier color Pool Carriers kit Part No Storage Oncomine Comprehensive Assay v3 GX Cat No A46296 Oncomine Comprehensive Assay v3 DNA GX Purple DNA Pool 1 4 A40281 30 C to...

Страница 17: ...sher com MLS Fisher Scientific fisherscientific com or other major laboratory supplier Item Source MicroAmp EnduraPlate Optical 96 Well Clear Reaction Plates with Barcode 4483352 4483354 Adhesive PCR Plate Foils AB0626 20 200 and 1 000 µL pipettes and appropriate filtered tips MLS Microcentrifuge tubes 1 5 mL or 1 7 mL low retention for nucleic acids MLS Vortex mixer with a rubber platform MLS Glo...

Страница 18: ...e Total Nucleic Acid Isolation Kit A36716 RNaseZap RNase Decontamination Solution AM9780 AM9782 AM9784 Nucleic acid quantification TaqMan RNase P Detection Reagents Kit Recommended for DNA only 4316831 Qubit 4 Fluorometer 1 Q33226 One or more of the following kits for use with the Qubit 4 Fluorometer Qubit dsDNA HS Assay Kit High sensitivity DNA Qubit dsDNA BR Assay Kit Broad range DNA Qubit RNA H...

Страница 19: ...onical Bottles Reusable conical bottles for Genexus Cartridge reagent dilution do not remove or replace unless advised by Thermo Fisher Scientific support representative 5 Genexus Bottle 2 Sequencing Solution 6 Genexus Bottle 3 Cleaning Solution 7 Waste Carboy 8 Waste pipette tip bin 9 Genexus Cartridge 10 Touchscreen 11 Sequencing reagents bay door Doors are locked in the closed position during a...

Страница 20: ...ip install station 7 Zone 1 station Genexus Strip 1 8 Zone 2 station Genexus Strip 2 AS or Genexus Strip 2 HD depending on your assay 9 Zone 3 station Genexus Strip 3 GX5 10 Zone 4 station Genexus Strip 4 11 Enrichment plate station 12 Liquid waste disposal port 13 Waste pipette tip disposal port 14 Sample plate station 15 Genexus Pipette Tips station 16 Robotic pipettor Chapter 1 Product informat...

Страница 21: ...wer switch where the states are on or off O 3 USB ports Connects a USB device to the instrument 4 Ethernet port An RJ45 port that provides Ethernet Gigabit communication between the sequencer and a local area network 5 Ethernet port An RJ45 port that provides Ethernet Gigabit communication between the sequencer and an accessory instrument Chapter 1 Product information Genexus Integrated Sequencer ...

Страница 22: ... 56 Run plans created in Genexus Software contain all of the settings that are used in library preparation templating sequencing and analysis including sample information and plate location assays and barcodes Dilute the samples and load the sample plate page 68 Quantify and dilute your nucleic acid samples then load the sample plate Load the sequencer and start a run page 72 Follow the step by st...

Страница 23: ...ab bench For example touch the deck surface or one of the metal deck stations WARNING Do not use the instrument in close proximity to sources of strong electromagnetic radiation for example unshielded intentional RF sources as these sources can interfere with proper operation WARNING The protection that is provided by the equipment can be impaired if the instrument is operated outside the environm...

Страница 24: ... single pool assay a maximum of 32 samples can be run on a single chip If you are using 2 assays with two primer pools each you can sequence a maximum of 8 samples in a run Similarly for one assay with 4 primer pools you can sequence a maximum of 8 samples in a run if the minimum read count per sample allows it The number of unused lanes on an installed chip A maximum of 4 lanes are available on a...

Страница 25: ...er cannot track barcode usage after the barcode plate has been removed After loading in the sequencer reusable consumables such as barcodes chips and sequencing reagents bay components must be used within 14 days An assay that is selected in a library run cannot include library batches that share one or more samples However two different assays in a run can include shared samples because assays ar...

Страница 26: ...e Genexus Software user interface provides menus to help you add select and manage samples libraries run plans and assays You can also view and manage your sequencing results monitor Genexus Integrated Sequencer runs in progress and manage software settings 1 2 3 4 5 6 7 1 Samples add new samples import samples prepare library batches import library batches and manage attributes 2 Runs plan a run ...

Страница 27: ... metrics for auditable records If the software detects an error at any step for example a scanned barcode is inconsistent with the information given for the run the software alerts the user and does not proceed with the run Only administrator level users can create user accounts After account creation the Genexus Integrated Sequencer automatically sends an email to the new user with the user name ...

Страница 28: ...swords must contain at least one numeric character 0 9 Passwords are case sensitive e Click Change 1 Open the software home page 2 Select your preferred language from the dropdown list in the upper right corner of the page 3 Enter your user name and password then press Enter or click Sign In IMPORTANT Your username and password must be unique and not shared with other users The software opens to t...

Страница 29: ...ssays also define the panels kits and chips that are used in a run and specify the reference files and threshold values for quality control and variant detection Assays are created from system installed templates An assay is a reusable experimental design that contains predefined settings suitable for use with common types of research applications An assay can be used to create many run plans and ...

Страница 30: ...X5 Solid Tumor Fusions Ion AmpliSeq HD RNA Oncomine TCR Beta LR RNA Only Ion AmpliSeq RNA Manage assays manager administrator Manager and administrator level users create and manage assays for use in Genexus Software In the menu bar click Assay 4Manage Assays to open the Assay Manage Assays screen The following tools are available on this screen To Do the following All users Search the assay list ...

Страница 31: ...d A locked assay cannot be edited 1 In the row of a draft assay in the Actions column click Edit The Create Assay workflow reopens 2 Edit the options on each assay step as desired then click Save Copy an assay to create a new assay 1 In the row of an assay in the Actions column click Copy The Create Assay workflow reopens 2 Edit the options on each assay step as desired enter a new name for the as...

Страница 32: ...ew assay manager administrator Manager and administrator level users can create a new assay Assays can be copied from an existing system installed assay or other assay then modified as needed For more information see Copy an assay manager administrator on page 40 To create a new assay from an assay template follow these steps 1 In the menu bar click Assay 4Create Assay The Create Assay screen open...

Страница 33: ...fter selecting the DNA and Fusions template If you are using a custom assay see Guidelines for using custom assays with the Genexus Integrated Sequencer on page 136 for guidelines for setting parameters for Minimum Read Count Per Sample and target amplification in the Panel and Parameters steps Chapter 3 Create and manage assays manager administrator Create a new assay manager administrator 3 Gene...

Страница 34: ... baseline click Assay 4Manage Presets4Copy Number Baselines then click Create New or Import d Optional If you selected a CNV baseline select a value to copy number based on tissue content if you want the CNV analysis to adjust for heterogenous tumor content in your samples Samples with cellularity below this value when created are rejected If you do not want to make this adjustment select Do Not A...

Страница 35: ...mplate sequencing and control kits then select or enter values for template size and sequencing flows if different from the prepopulated values Click Next Note If you select Other AmpliSeq from the Library Kit dropdown list the Barcode Kit dropdown list includes barcodes sets such as IonCode and Ion Xpress barcodes that are compatible with manually or Ion Chef prepared libraries Chapter 3 Create a...

Страница 36: ... to the CF 1 reference Mean AQ20 Read Length The mean length of CF 1 reads having 99 accuracy at each position Run QC Key Signal The average signal after software processing for all ISPs that identically match the library key TCAG A measure of the efficiency of template amplification Percent Loading The percentage of addressable wells on a chip lane that are loaded with an ISP Raw Read Accuracy Th...

Страница 37: ... of sample reads aligned to a reference sequence that have 99 accuracy at each position Mean Read Length bp The mean length of all sample reads Uniformity of Amplicon Coverage The percentage of reads showing a depth of coverage 20 of the mean base coverage Sample QC RNA Mapped Reads The total number of reads mapping to a fusion reference sequence Mean AQ20 Read Length bp The mean length of called ...

Страница 38: ... settings for the cycling and input parameters used in library and template preparation Primary Analysis Parameters Annotation CNV Finding Fusions Read Mapping Variant Finding A parameter category can be quickly brought to the top of the screen by selecting it in the Filter Parameters by list at the upper left Chapter 3 Create and manage assays manager administrator Create a new assay manager admi...

Страница 39: ...n your drive and upload Click Download Parameters to download parameters settings as a JSON file to your hard drive 7 In the Plugins step select plugins that you want to include in the sequencing data analysis from the plugin list then click Next Chapter 3 Create and manage assays manager administrator Create a new assay manager administrator 3 Genexus Integrated Sequencer User Guide 39 ...

Страница 40: ...n create a new assay by copying an existing system installed assay or other custom assay and modifying it as needed 1 In the menu bar click Assay 4Manage Assays 2 In the Assay Manage Assays screen in the Actions column for the assay that you want to copy click Copy The Copy command may be located in the Expand menu The Assay Create Assay screen opens to the Panel step The assay settings can be mod...

Страница 41: ... administrator level user can import an assay from another Genexus Integrated Sequencer if the assay has been first exported to a local drive 1 In the menu bar click Assay 4Manage Assays 2 In the Assay Manage Assays screen click Import Assay 3 In the Import Assay dialog box click Select file navigate to the exported assay ZIP folder on your drive then select it 4 Click Import The assay appears in ...

Страница 42: ...nformation From the Samples menu samples can either be added directly to the software or be imported into the software by uploading a data file Add a new sample If Genexus Software does not display the sample that you need for your run you can add a new sample manually on the Samples Manage Samples screen The new sample will be available to use in your run plan 1 In the menu bar click Samples4Mana...

Страница 43: ...escription Sample Name A unique identifier representing the sample The sample name can contain only alphanumeric characters 0 9 Aa Zz full stops periods underscores _ or hyphens cannot contain spaces and is limited to a maximum of 20 characters IMPORTANT To prevent erroneous sample selection during run plan creation make sure that you assign a unique and distinguishable sample name for each sample...

Страница 44: ...e been added to a library To remove a custom sample attribute from use see Remove a sample attribute administrator on page 45 1 In the menu bar click Samples4Manage Attributes 2 In the Samples Manage Attributes screen click Add New Attribute 3 Complete the fields in the Add New Attribute dialog box a In Attribute Name enter the name of the attribute Attribute names are limited to 20 alphanumeric c...

Страница 45: ...eactivate replaces Obsolete in the Actions column To reactivate an attribute click Reactivate in the Actions column Active sample attributes are listed in the Add New Sample dialog box Import samples You can import sample data with a file in one of the following formats TXT XLS XLSX or CSV The import file includes the same sample attributes that are listed in the Add New Sample dialog box For ease...

Страница 46: ...the reason for failure for example an invalid character was used For additional troubleshooting see Batch sample import fails on page 117 Successfully imported samples are listed in the Samples Manage Samples screen Manage samples You can find tools for creating searching sorting editing deleting and exporting samples and for viewing the sample history in the Samples Manage Samples screen Use the ...

Страница 47: ...e sample or samples that match the sample name search entry are listed 3 Click Remove to return to the complete list of samples The Export function generates an XLS file that contains details about the selected sample 1 In the menu bar click Samples4Manage Samples 2 In the Samples Manage Samples screen select the checkbox in the row of each sample that you want to export To select all samples sele...

Страница 48: ...efore execution of a run plan that uses that sample After the sample is used in a run the Edit link is no longer available You can edit a sample after executing a run by using the Edit Amend Report command For more information see Edit a sample and amend a report after a run manager administrator on page 49 To add a custom sample attribute to a sample see Create a custom sample attribute manager a...

Страница 49: ...e updated The entire history of defining a sample is available to review export or print using the audit feature The history shows the original sample values and any new values or activities This feature allows you to meet Title 21 CFR Part 11 of Federal Regulations that establishes the United States Food and Drug Administration regulations on electronic records and signatures password policies an...

Страница 50: ...you have already prepared manually you must first create a library batch in Genexus Software from samples that you have added or imported For more information see Add a new sample on page 42 or Import samples on page 45 Select the library batch when you create the run plan If you are creating a run plan starting from nucleic acid samples skip this step and proceed to Create a run plan for a sample...

Страница 51: ...run including any required controls and post run data analysis settings 5 In the expanded screen in Library Batch ID enter a unique identifier for the library batch Note Library Prep Type autofills to the nucleic acid type specified by the assay you selected DNA RNA DNA RNA or TNA Chapter 4 Enter samples and libraries Prepare or import a library batch 4 Genexus Integrated Sequencer User Guide 51 ...

Страница 52: ...og box between DNA and RNA by clicking Each library in a library batch must have a different barcode ID When preparing the physical libraries best practice is to swap barcodes between DNA and RNA libraries in consecutive sequencing runs to prevent carryover contamination The barcodes that are listed in the DNA Barcode or RNA Barcode dropdown list belong to the barcode set that was selected when th...

Страница 53: ...brary batch information Assay name auto populated Reagent kit barcodes Library kit barcode for example Genexus Library Strips 1 and 2 AS optional Panel kit barcode for example Oncomine Comprehensive Assay v3 DNA GX optional Unique Library Batch ID must be alphanumeric 0 9 Aa Zz full stop period underscore _ and hyphen Sample Names same character requirement as Library Batch ID Library names same c...

Страница 54: ...ils an error message indicates the reason for failure for example an invalid character was used For additional troubleshooting support see Library batch import fails on page 117 Chapter 4 Enter samples and libraries Prepare or import a library batch 4 54 Genexus Integrated Sequencer User Guide ...

Страница 55: ...les and chips throughout the library preparation templating sequencing and data analysis workflow Using the Runs menu you can create run plans for sequencing runs that use either nucleic acid samples sample run or libraries that you have previously prepared manually library run as input Genexus Software guides you step by step to set up a run plan that tells you what consumables are needed and pro...

Страница 56: ... Runs Run Plan screen Before creating a run plan for a sample run sample to result workflow you must enter sample information into Genexus Software For more information see Add a new sample on page 42 or Import samples on page 45 1 In the menu bar click Runs4Plan Sample Run You can also click Plan Sample Run in the Runs Manage Runs screen Chapter 5 Create and manage run plans Create a run plan for...

Страница 57: ... files to Ion Reporter Software Select this option to upload data for further analysis with Ion Reporter Software Select your Ion Reporter Software account and release version To configure an Ion Reporter Server account see Configure an Ion Reporter Server account administrator on page 139 To configure an Ion Reporter Software on Connect account see Configure Thermo Fisher Connect accounts manager...

Страница 58: ...istrator IMPORTANT Ensure that you select the assay that corresponds with the sample type that you will use in the run If you select a wrong assay when you create a run plan the instrument will use incorrect settings during the run resulting in invalid sequencing results To determine which system installed assay to use for your sample type see System installed assays on page 30 Chapter 5 Create an...

Страница 59: ...ata and launch an analysis in Ion Reporter Software automatically upon run completion Note In order for the Ion Reporter analysis workflow to appear in the list you must tag the analysis workflow for use with the IonReporterUploader plugin For more information see Tag an Ion Reporter Software analysis workflow for use with the IonReporterUploader plugin on page 140 4 In the Samples step select the...

Страница 60: ...e Chip View updates and shows the lane color as red instead of green In the example shown at right seven samples were selected instead of four The run is allowed but you may not achieve the required reads per sample to pass QC metrics 5 If you selected more than one assay repeat step 4 for each additional assay 6 If needed edit samples in one of the following ways then click Next Click View and Re...

Страница 61: ...centration is greater than this value you must manually dilute the sample to the target concentration or to a value within range for automated dilution before loading on the sample plate For more information see Dilute the samples if needed and load the sample plate sample run on page 69 Note The sample volume that is required for library preparation is not adjustable The volume depends on the num...

Страница 62: ... consumables that are installed on the sequencer and available for the run 4 Positions to load primer pool tubes 5 Chip view showing the lanes to be used in the run 6 Positions in the sample plate to load the samples 7 Table listing the sample plate position sample type volume to load concentration dilution factor and assay for each sample 8 Description of each primer pool and its position Note If...

Страница 63: ...information and add a library batch in Genexus Software For more information see Prepare a library batch on page 50 Library run planning is organized into four steps Setup Assays Library Batches and Review Progress through the steps is tracked in the upper left corner of the Runs Run Plan screen 1 On the menu bar click Runs4Plan Library Run Note You can also click Plan Library Run in the Runs Mana...

Страница 64: ...d BAM files to Ion Reporter Software Select this option to upload data for further analysis with Ion Reporter Software Select your Ion Reporter Software account and release version To configure an Ion Reporter Server account see Configure an Ion Reporter Server account administrator on page 139 To configure an Ion Reporter Software on Connect account see Configure Thermo Fisher Connect accounts ma...

Страница 65: ...ng option in substep 2b make the following selections from the dropdown list in the Ion Reporter Workflow column in the row of each assay that you selected Select Upload Only to upload sample data to Ion Reporter Software automatically upon run completion Select the desired Ion Reporter analysis workflow to upload sample data and launch an analysis in Ion Reporter Software automatically upon run c...

Страница 66: ...per sample the number of samples exceeds the chip or lane well capacity a dialog box appears after you click Next asking you to confirm that you want to continue After clicking Yes the Chip View updates and shows the lane color as red instead of green In the example shown at right seven samples were included in a library batch instead of six The run is allowed but you may not achieve the required ...

Страница 67: ...stalled on the sequencer and available for the run 4 Position s in the sample plate to load the library batch 5 Chip view showing the lanes to be used in the run 6 Table listing assay and run information 7 Table listing the well position library batch ID volume to load and assay for each library batch After saving the run plan appears in the run plan list on the Manage Runs screen with the name yo...

Страница 68: ...ng the TaqMan RNAse P Detection Reagents Kit Pub No MAN0007732 available at thermofisher com The Qubit dsDNA HS Assay Kit Cat No Q32851 or Q32854 can also be used for quantification particularly for FFPE DNA and highly degraded DNA samples See Quantify FFPE DNA with the Qubit Fluorometer on page 135 for a detailed procedure for quantifying FFPE DNA We recommend the Qubit RNA HS Assay Kit Cat No Q3...

Страница 69: ... target concentration Run is allowed but sample concentration may not be optimal for library preparation Concentrate the sample to greater than or equal to the target concentration 1 024X of the target concentration No manual dilution is necessary unless the concentration is 1 000 ng µL The sequencer dilutes the sample to the target concentration automatically during the run 1 024X of the target c...

Страница 70: ...me Ion AmpliSeq chemistry DNA 1 15 µL DNA 2 25 µL RNA 1 20 µL RNA 2 20 µL Ion AmpliSeq HD chemistry DNA 1 20 µL RNA 1 20 µL TNA 1 20 µL 3 Seal the plate with a sheet of Adhesive PCR Plate Foils Thermo Fisher Scientific Cat No AB0626 Note The use of other plate seals may affect performance 4 Keep the plate on ice until ready to load it in the sequencer Chapter 6 Dilute the samples and load the samp...

Страница 71: ...sample library to 125 pM with nuclease free water Note Each library must be barcoded with a unique barcode or barcode pair Use this concentration as a starting point then titrate up or down based on sequencing results if needed 2 Add equal volumes of each library to a new 1 5 mL low DNA retention tube so that the total volume is greater than the volume specified in the run setup guide provided by ...

Страница 72: ...HD depending on your assay and Genexus Strip 4 thaw on ice for at least 30 minutes Keep the strips on ice until you load them in the sequencer IMPORTANT Confirm that the strip contents are completely thawed before installing in the sequencer 2 Remove primer pool tubes in tube carriers that are needed for the run from the freezer then thaw for at least 30 minutes on ice After thawing gently tap the...

Страница 73: ...apped in keyholes see the following figure then repeat the centrifugal motion if needed It is acceptable if a few beads remain in the keyhole or on the tube wall but most should be either in suspension or in a pellet at the bottom of the tube 1 2 Example Genexus Strip 3 GX5 before upper and after lower inversion The carrier has been removed to show tube contents more easily 1 Magnetic beads trappe...

Страница 74: ...Tubes must be manually filled with the custom Ion AmpliSeq or Ion AmpliSeq HD panels at the appropriate volume and in the correct primer pool tube positions For Ion AmpliSeq library panels use one carrier per DNA or RNA primer pool The two positions in the primer pool tube carrier are designated as shown in the following figure 1 2 1 Position 1 tube Contains Ion AmpliSeq DNA Ion AmpliSeq RNA or Io...

Страница 75: ... tubes in the same carrier Ensure that no bubbles are introduced at the bottom of the tube when adding the primer pool 2 If you do not install the primer pool tube carriers in the sequencer immediately cap the tubes that contain primer pools then store the tube carriers on ice Remember to uncap all tubes before installing Load the sequencer and start a run 1 Tap Run on the sequencer home screen to...

Страница 76: ...med then the sequencer prompts you to perform the Clear Deck UV Clean Load Deck Clear Sequencing Reagents and Load Sequencing Reagents steps 3 In the Review Run screen confirm the selected run plan then tap Next The deck door opens automatically Note If the instrument vision system detects consumables loaded on the deck the sequencer prompts you to remove the consumables then starts a UV Clean Cha...

Страница 77: ...r pool tube cap or caps before installing the tube carrier on the deck Note A primer pool tube carrier can only be installed with the position 1 tube in the back row of the Primer Pool Tube Station Follow the guidance in the run setup guide for loading the primer pool tube carrier or carriers in the correct position and order in the station 1 2 1 Position 1 2 Position 2 Chapter 7 Load the sequence...

Страница 78: ... moves the installed chip to loading and sequencing positions during the run 6 When the deck consumables have been loaded lock the library and templating strips in place by sliding the latches toward the rear of the deck If a chip is detected and the strip latches are closed the Close Deck Door screen appears Chapter 7 Load the sequencer and start a run Load the sequencer and start a run 7 78 Gene...

Страница 79: ... in the sequencing reagents bay must be replaced every time that a new chip is installed regardless of how many lanes were used in the previous chip 8 Follow on screen instructions to empty the waste in the Waste Carboy remove waste pipette tips remove the used Genexus Bottle 1 Genexus Bottle 2 Genexus Bottle 3 and Genexus Cartridge then tap Next IMPORTANT Follow all applicable local state provinc...

Страница 80: ...reen A sequencing run encompasses six stages Library preparation Priming Reagent Check Pre sequencing Sequencing Cleaning When the run finishes the sequencer displays the Run Complete screen Note If all the lanes of a chip are used the chip shuttles to the install position You are asked to remove the chip and coupler and clear the sequencing reagents Chapter 7 Load the sequencer and start a run Lo...

Страница 81: ...k door opens 2 In the Clear Deck screen the sequencer provides step by step instructions by highlighting the components to be removed Unlock the library and templating strips by sliding the latches toward the front of the deck then remove the used strips Remove the remaining deck components specified by the sequencer Chapter 7 Load the sequencer and start a run Clear the instrument deck and perfor...

Страница 82: ... replace it with a new filter For more information see Replace the Genexus Filter on page 135 If the Genexus Filter does not appear to be clogged contact Technical Support and report a possible deck liquid waste line clog 4 When finished close the deck door then tap Next A two minute UV Clean starts Chapter 7 Load the sequencer and start a run Clear the instrument deck and perform a UV Clean 7 82 ...

Страница 83: ...icable local state provincial and or national regulations when recycling or disposing of Genexus Integrated Sequencer consumables and liquid waste CAUTION The Genexus Bottle 1 small waste bottle contains small amounts of formamide Dispose of this waste appropriately 6 After removal of used components close the sequencing reagents bay doors then tap Next The sequencer returns to the home screen Cha...

Страница 84: ...w 2 In the Monitor Instrument View screen view the status of a run in progress or the status of an instrument The following information is provided Sequencer operator and run names PQ status of the instrument Instrument status at various stages of the run and post run Status of the results analysis Run start and completion times 3 Click Refresh to update the information 8 84 Genexus Integrated Seq...

Страница 85: ...ts files to Ion Reporter Software 111 Verification runs 113 Use the Results menu to review run results and data analysis and perform data management tasks In the menu bar click Results4Run Results to review completed sample run results and reports In the menu bar click Results4Verification Results to review data from completed verification runs that were performed during sequencer installation or ...

Страница 86: ...sample name Enter a search term then click Search The sample list associated with a run plan can be collapsed and hidden by clicking the triangle to the left of the run plan name Click the triangle again to view the list The following information appears in the Results Run Results screen Chapter 9 Review data and results Run Results screen 9 86 Genexus Integrated Sequencer User Guide ...

Страница 87: ...con in this column alerts you that a block failure has occurred on the chip Hover over the icon to identify the affected block or blocks QC Status The QC status of a completed run for example Passed Failed Not Set Started On The date and time when the run analysis was started Last Updated On The date and time when the last action was completed on the run Actions Click the appropriate link To see m...

Страница 88: ...on depending on the assay used Section Description Sample Details The sample information that is entered into the software You can customize the format of the Sample Details section when you create a new report template To create a new report template click Assay4Presets then in the Report Templates tab click Add New Sequence Variations Detected Variants and fusions detected in the sample based on...

Страница 89: ... specific information To view sequencing results for a particular sample including variant and fusion calls in the Sample Name column click the sample name The View Results screen opens showing the QC results for that sample To view metrics and other quality information for the run in the Run Plan column click the run name For runs with multiple assays only the metrics for the first assay are show...

Страница 90: ...se the double arrows to navigate between assay results if more than one assay was used in the run Select the sample or assay you want to view from the dropdown list All of the samples that were run with the same assay share the same run metrics All other results are sample specific Chapter 9 Review data and results View sequencing results and run metrics 9 90 Genexus Integrated Sequencer User Guid...

Страница 91: ...number of base reads on target divided by the number of targeted bases and therefore includes any bases that had no coverage Uniformity Of Base Coverage The percentage of bases in all targeted regions or whole genome that are covered by at least 20 of the average base coverage depth reads Cumulative coverage is linearly interpolated between the nearest integer base read depths Base Reads On Target...

Страница 92: ...ry The number and percentage of wells with live ISPs that contain Library ISPs Final Reads Library reads passing all filters that are recorded in the output BAM files This value can be different from the total number of reads due to technicalities associated with read trimming beyond a minimal requirement Total Bases The number of filtered and trimmed base pairs that are reported in the output BAM...

Страница 93: ... pair assigned to the DNA and or RNA library for a sample Total Reads The total number of filtered and trimmed reads with the listed dual barcodes assigned to the sample The reads are independent of length reported in the output BAM file Mean Read Length The average length in base pairs of usable library reads for each sample Q20 Bases The total number of called bases that have 99 accuracy or less...

Страница 94: ...Average length in base pairs at which the accuracy rate is 99 for CF 1 reads Sample QC DNA Sequencing quality metrics of the sample DNA library MAPD MAPD Median of the Absolute values of all Pairwise Differences is a quality metric that estimates coverage variability between adjacent amplicons in CNV analyses A MAPD value of 0 5 indicates an acceptable level of coverage variability High MAPD value...

Страница 95: ...axis The shape of the histogram should closely resemble the library size distribution trace without the adapter sequences The SNVs Indels table lists the calls and other information for the SNV and INDEL variants that are analyzed in each sample in a run To view the SNVs Indels table in the Results Run Results screen click the sample name in the Sample Name column then click SNVs Indels in the lef...

Страница 96: ... or the variant hypothesis interval cutoff 1 Phred scaled 10 log10 A higher score means more evidence for the variant call Coverage The number of reads covering the position Allele Ratio The total read count of variants that are the alternative alleles of the current variant Variant ID The name of the hotspot as defined in the BED file Filter Indicates the presence PASS or absence ABSENT of an SNV...

Страница 97: ...was used in the run Filter the results list in the table using the filtering tools For more information see Filter results on page 102 Click Export in the upper right corner of the screen to export the results data in tabular format Column Description User Classifications A user defined classification selected from the list For more information see Create and assign variant classifications on page...

Страница 98: ...ng assays Ratio To Wild Type The molecular ratio for exon skipping assays relative to wild type control amplicons Norm Count Within Gene 1 Lung panel only Exon skipping assay coverage normalized to molecular coverage of wild type WT MET control amplicons 1 Column appears in analyses of Ion AmpliSeq HD sequencing data only 2 Column appears in analyses that use the exon tiling fusion detection metho...

Страница 99: ...calls and other information for the copy number variants CNVs analyzed in each sample in a run To view the CNVs table in the Results Run Results screen click the sample name in the Sample Name column then click CNVs in the left navigation pane of the View Results screen The data displayed in the table depend on the assay that was used in the run Filter the results list in the table using the filte...

Страница 100: ...ive call is made No Call Reason Details about the No Call result in the Filter column CNV Confidence The CNV confidence interval associated with the call The 5 lower confidence bound value is the ploidy estimate where there is a 5 chance that the true ploidy is below that value The 95 upper confidence bound is the ploidy estimate where it is 95 certain that the true ploidy is below that value Vari...

Страница 101: ...ariant 2 Review annotation information in the View Annotation Sources window a Click View IGV to download a JNPL file that can be opened in the Integrative Genomics Viewer IGV from the Broad Institute see software broadinstitute org software igv b If available click a link in the row of an annotation source to navigate to the online annotation information View annotation sources Chapter 9 Review d...

Страница 102: ...classification from a variant click Remove To delete a classification from the list click Delete next to the classification name The classification will be removed from all variants in all results You can filter results on the View Results screen in two ways You can apply filters to columns of information that appear on the screen The filters available at the top of each column immediately narrow ...

Страница 103: ...er data in the Type column to show one specific variant type The column or columns to which you applied a filter change to reflect the filter and selected options Filter results using a filter chain You can filter the results that are listed in the View Results screens with a system installed or custom filter chain A filter chain is a set of filters that Genexus Software uses to narrow the list of...

Страница 104: ...o reflect the selected filter chain b Optional Click Save Filter Chain to associate the filter chain with the assay The filter chain is applied by default to all results that use the assay 7 Create a new custom filter chain a In Filter Chains click New to open the Create Filter Chain dialog box b In Name enter a name for the new filter chain c Optional In Description enter a description for the fi...

Страница 105: ...4 Oncomine TCR Beta LR Assay GX results do not include a Summary screen To view the analysis of Oncomine TCR Beta LR Assay GX expression data in the Results Run Results screen click the sample name in the Sample Name column then click Results in the left navigation pane in the View Results screen Results are viewed in two tabs Sample Results and Sample QC In the Sample Results tab select from the ...

Страница 106: ...assification Base composition of overcalled and undercalled homopolymers Downsampling analysis QC metrics For detailed information on viewing and analyzing Oncomine TCR Beta LR Assay GX results see the Oncomine TCR Beta LR Assay GX User Guide Pub No MAN0018513 Chapter 9 Review data and results View sequencing results and run metrics 9 106 Genexus Integrated Sequencer User Guide ...

Страница 107: ...and Drug Administration regulations on electronic records and signatures password policies and user activity auditing 1 In the menu bar click Results4Run Results 2 In the Results Run Results screen click the sample of interest in the Sample Name column 3 In the View Result screen click Expand 4 In the Electronic Signature dialog box enter your user name password and comments Items identified with ...

Страница 108: ... In the View Result screen click Expand 4 Generate Report 4 In the Generate Report dialog box select the desired language report template and variants to include in the report 5 Click Generate A message displays when the report has been generated The new report overwrites the existing report 6 Click Download Files then in the Download Files dialog box select the new report and any other run relate...

Страница 109: ...ber variant results in tab separated value format TSV Variants vcf Oncomine_ LibPrepID _ analysisID vcf Summary of variant results in variant call format VCF Variant tsv Summary tsv File that lists SNV INDEL copy number and fusion results in tab separated value format TSV Fusion tsv Fusion tsv File that lists fusion results in tab separated value format VCF Sequencing results DNA mapped bam file b...

Страница 110: ...d bam ptrim bam Mapped BAM file of combined barcode reads DNA Barcode ID barcode unmapped bam file bam barcode _rawlib basecaller bam Unmapped DNA barcode BAM file output after mapping reads to reference DNA Barcode ID barcode basecaller FASTAQ file fastq barcode _rawlib basecaller fastq FASTQ file of the DNA barcodes used RNA Mapped bam file bam barcode _rawlib basecaller_align ments bam Mapped B...

Страница 111: ...eport summary For more information on how to review and interpret read statistics and output files see Appendix D coverageAnalysis plugin in Genexus Software 1 View coverageAnalysis plugin summary In the View Results screen for a sample of interest in the left navigation pane click Plugins then scroll to the coverageAnalysis plugin results In the View Results screen for a sample of interest in the...

Страница 112: ...he following selections a From the Ion Reporter Software Account list select your Ion Reporter Server account b In the Assay column select the appropriate assay from the list c In the Ion Reporter Workflow column do one of the following Select Upload Only to upload the sample results BAM files Ion Reporter Software You will need to sign in to Ion Reporter Software to launch an analysis Select an a...

Страница 113: ...e of the sequencer that was verified Run Status The current status of the full sequencing run including analysis QC Status Indicates whether a sequencing run passed or failed based on the sequencing QC metrics selected for the assay Last Updated on The date and time that the sequencing run was last updated Actions View Results Click this link to view results for completed runs that have not yet be...

Страница 114: ...rate is 1 for all aligned reads Raw Read Accuracy The percentage of raw reads mapped to the reference genome Run Balance Factor The minimum Sample Balance Factor in a set of four tested samples A D Total Reads The total number of filtered and trimmed reads with the listed barcodes assigned to the samples Templating Control QC CF 1 Average Reads Per Lane Average number of CF 1 reads per GX5 Chip la...

Страница 115: ...hreshold Run QC AQ20 Mean Read Length bp Average read length in base pairs at which the error rate is 1 for all aligned reads 95 Raw Read Accuracy The percentage of raw reads mapped to the reference genome 98 5 Run Balance Factor The minimum Sample Balance Factor in a set of four tested samples A D 0 5 Total Reads The total number of filtered and trimmed reads with the listed barcodes assigned to ...

Страница 116: ... reads mapped to the reference sequence 1 100 000 Mean AQ20 read length bp Average length in base pairs at which the error rate is 1 for all aligned reads of the sample DNA library 95 Percent Reads On Target The percentage of reads mapped on target over total reads 90 Sample Balance Factor For a given sample in a PQ run Sample Balance Factor Number of mapped reads Mean number of mapped reads for f...

Страница 117: ...name Every sample name in the software must be unique Ensure that the spreadsheet does not contain any duplicate sample names and repeat the import Note that the system check is not case sensitive so a sample name of ABC1 conflicts with abc1 The headings in the sample import spreadsheet do not match the sample attributes in the software The headings must match the sample attributes in the software...

Страница 118: ...your assay Go to Assay tab4Manage Assays and make sure that the assay is locked Cannot upload my panel or hotspots Issues with BED file format or files do not end in bed Ensure your file is in the correct BED format and has a bed extension Allele coverage does not match hotspot coverage The coverage value reported under the Variants tab and Allele Coverage tab can be different No action required T...

Страница 119: ...om temperature Genexus Strip 1 or incompletely thawed Genexus Strip 2 AS or Genexus Strip 2 HD before loading in the sequencer Ensure that Genexus Strip 1 strips are fully equilibrated to room temperature and Genexus Strip 2 AS or Genexus Strip 2 HD strips are completely thawed before loading in the sequencer The number of sample reads is low and CF 1 metrics fail QC If CF 1 metrics failed QC a pr...

Страница 120: ...2 5 6 Number Icon Description 1 Network connectivity connected Network connectivity not connected 2 Instrument idle Sequencing in progress Instrument ready Error 3 Chip status Absent Chip status Standby B 120 Genexus Integrated Sequencer User Guide ...

Страница 121: ...for sufficient disk space before each run and notifies the user if there is not enough 6 Instrument Server File System Space the percent of file space used is indicated Note If the indicator turns red archive data from the server to free up disk space Refer to the Genexus Software Help for more information on archiving data 1 Indicator turns yellow when disk space is 67 full indicator turns red wh...

Страница 122: ...to use IP Address Configuration Allows users to set or change the IP configuration DHCP or Static IP 1 Select either the DHCP or Static IP radio button 2 Touch the screen in the field you want to edit to activate the field A virtual keyboard appears 3 Enter the new information then press Done Network Settings Appendix B Touchscreen reference Settings B 122 Genexus Integrated Sequencer User Guide ...

Страница 123: ...e address unless directed to do so by a Thermo Fisher Scientific field service engineer FTP Server Configuration Allows users to change the FTP Server IP address and user information Enter the following IP address in the FTPserver field for your instrument 192 168 201 1 When a change to the FTP Server IP address or user information is required Note Do not change address unless directed to do so by...

Страница 124: ...he cleaning procedure starts automatically 2 Follow the on screen instructions then tap Next For more information see Clear the instrument deck and perform a UV Clean on page 81 The user interface returns to the Home screen when the cleaning is complete The System Tools menu enables you to upload instrument diagnostics manage data and shut down or reboot the instrument Perform a Clean instrument o...

Страница 125: ... a period of instrument shutdown have expired 1 Tap the screen within the row of the product to view an RFID Tag Detail page 2 Tap Done to return Noise Test Provides real time measurement of electrical noise readings on the chip For troubleshooting if directed to do so by Technical Support Appendix B Touchscreen reference Settings B Genexus Integrated Sequencer User Guide 125 ...

Страница 126: ...into the chip clamp 2 Tap Chip Calibration to start the chip calibration 3 In the dropdown lists select the Image to display and the type of Calibration to perform Note Tap Cancel to quit the chip calibration Appendix B Touchscreen reference Settings B 126 Genexus Integrated Sequencer User Guide ...

Страница 127: ...ystems and disks The Filesystems screen opens 2 Tap the screen within the row of the Filesystem you want to view then press Details The Disk Drives in Filesystem screen opens 3 Tap the screen within the row of the Drive to view details of the disk drive The Details for Drive screen opens 4 Tap Done to return to the previous screen Appendix B Touchscreen reference Settings B Genexus Integrated Sequ...

Страница 128: ...out click Instrument Diagnostics then follow the on screen instructions Shut Down Access to Shut Down and Reboot commands If directed to do so by Technical Support as part of a troubleshooting procedure or if the instrument will not be used for more than three days Note It is not necessary recommended to shut down the instrument overnight or over the weekend To power on the instrument after a shut...

Страница 129: ...om the instrument hard drive to the Ion Torrent Server 1 Tap the screen in the Experiment Name row of the individual experiment to be transferred 2 Tap Transfer 3 Select the radio button of the action to be performed 4 Select the radio button of the files to be transferred 5 Tap OK Analysis Controls Allows the user to stop analysis the data of individual runs 1 Tap the screen in the Experiment Nam...

Страница 130: ...xternal server 1 In the Settings menu tap Data Management to access the Data Management screen then tap Manage 2 Tap Select All to select all the available experiments or tap the screen in the Experiment Name row of the individual experiment to be managed 3 Tap Delete Selected Appendix B Touchscreen reference Settings B 130 Genexus Integrated Sequencer User Guide ...

Страница 131: ...ibrate the touchscreen Item Description When How to use About Provides instrument details To view instrument reference information or access regulatory information Click Regulatory info to view the following screen Instrument settings Appendix B Touchscreen reference Settings B Genexus Integrated Sequencer User Guide 131 ...

Страница 132: ...c regulatory information To view instrument RFID regulatory information See Radio compliance standards on page 158 for compliance information Instrument Name To change the instrument name Appendix B Touchscreen reference Settings B 132 Genexus Integrated Sequencer User Guide ...

Страница 133: ...with your finger or a stylus each time it appears In total you touch the screen 4 times one time in each corner Smart Monitor Enabled Select the checkbox to enable remote monitoring of instrument runs by Thermo Fisher Scientific for troubleshooting Appendix B Touchscreen reference Settings B Genexus Integrated Sequencer User Guide 133 ...

Страница 134: ...stallation and Training Kit If a spill or leak occurs inside the instrument perform the following steps to clean or decontaminate the sequencer Note Dispose of all waste in appropriate liquid or solid waste containers 1 Remove each Genexus Bottle 2 then remove and empty the Genexus Bottle 1 and Waste Carboy 2 Remove the Genexus Cartridge 3 Inspect the floor of the sequencing reagents bay and Genex...

Страница 135: ...ntration can be estimated using a Qubit Fluorometer and the Qubit dsDNA HS Assay Kit Cat No Q32851 See the Qubit dsDNA HS Assay Kits User Guide Pub No MAN0002326 for more information 1 Set up the required number of 0 5 mL Qubit Assay tubes for standards and samples The Qubit dsDNA HS Assay requires 2 standards 2 Prepare sufficient Qubit working solution for all samples and standards by diluting Qu...

Страница 136: ... up your assay Example Your panel has 500 amplicons in each of two primer pools Minimum Read Counts Per Sample 500 amplicons 2 pools 2 500 reads amplicon sample 2 500 000 Refer to the information in the following table for entering the number of target amplification cycles and anneal extend time parameters for a custom Ion AmpliSeq panel Primer pairs per pool Recommended number of amplification cy...

Страница 137: ...mer pools and you require 0 1 LOD and 50 000 reads amplicon for a cfDNA sample Minimum Read Counts Per Sample 100 amplicons 2 pools 50 000 reads amplicon sample 10 000 000 Note These calculations are based on 100 uniformity and 100 on target reads Depending on the actual uniformity of the panel the quality of input sample and panel on target percentage minimum read counts per sample may need adjus...

Страница 138: ...ical use of reagents therefore you should try to group samples in your runs in multiples of four if possible The following figures illustrate the relationship between sample number the number of pools in an assay the required number of reads per sample and the amount of deck consumables required for a sequencing run 1 2 1 1 1 1 2 1 One pool assay requiring 500K reads per sample 2 2 4 4 1 1 1 1 2 2...

Страница 139: ...exus Software directly to Ion Reporter Software 1 Click Settings 4Thermo Fisher Account 2 In the Thermo Fisher Account Settings screen click Add New 3 In the Add Thermo Fisher Account dialog box in Account Type select Ion Reporter Local The fields that are required for configuring the account are populated in the dialog box 4 Complete the information in the required fields click Get Ion Reporter S...

Страница 140: ...sly tagged workflow from the list of workflows that are available for selection in the Genexus Software click Actions4Untag from IRU The analysis workflows are available for selection when you perform one of the following actions Action Description Create a run plan 1 In the Setup step of the run plan creation select Upload BAM files to Ion Reporter Software then select an Ion Reporter Software ac...

Страница 141: ...a new account c Click Create an account at the bottom of the screen to complete the registration 4 In Genexus Software click Add new 5 In the Add Thermo Fisher Account dialog box enter the information that is required to create a new account then click Save Item Description Account Type Select Thermo Fisher Cloud from the list Name Enter a unique account name The name can contain only alphanumeric...

Страница 142: ...mple data to Ion Reporter Software on Connect and launch analyses When updated software packages are available for download from Connect cloud based platform appears as an alert in the Genexus Software banner on the top of the screen When you see this alert navigate to the Settings Software Updates screen then click Software Updates to see what updated software packages are available Appendix C Su...

Страница 143: ...rovides tooltips with definitions Hover over a heading or description in the report to view the tooltip General statistics Statistic Description Number of mapped reads The total number of reads mapped to the reference genome Percent reads on target The percentage of filtered reads mapped to any targeted region relative to all reads mapped to the reference If no target regions file is specified thi...

Страница 144: ...al ends of the region Amplicon base composition bias A number that represents the proportion of amplicons showing low representation 0 2x mean reads in the lower and or upper quartiles of amplicons ordered by increasing G C base pair content of their insert sequences The value is relative to that in the center 50th percentile of amplicons and weighted by the standard deviation of representation ov...

Страница 145: ...total reads is greater than 70 Target bases with 10 reads are considered to have no strand bias Percent end to end reads The percentage of on target reads that fully cover their assigned amplicon insert from end to end To allow for error the effective ends of the amplicon region for read alignment are within 2 bases of the actual ends of the region Example Coverage Analysis Report The following is...

Страница 146: ...Appendix D coverageAnalysis plugin in Genexus Software Example Coverage Analysis Report D 146 Genexus Integrated Sequencer User Guide ...

Страница 147: ...of the number of reads at a given read depth or greater as a percentage of the total number of reads The individual orange bars represent the percentage of reads in the specific range of base read depths The blue curve measures the cumulative reads at a given base read depth or greater If your analysis includes a regions of interest file this chart reflects only target regions reads that fall with...

Страница 148: ...d in the tables at the top of the plugin report The first line is the title Each subsequent line is either blank or a particular statistic title followed by a colon and its value Base depth of coverage Coverage summary data used to create the Depth of Coverage Chart This file contains the following fields read_depth the depth at which a targeted reference base has been read base_cov the number of ...

Страница 149: ...n rev_reads the number of reverse strand reads assigned to this amplicon cov20x the number of bases of the amplicon target that had at least 20 reads cov100x the number of bases of the amplicon target that had at least 100 reads cov500x the number of bases of the amplicon target that had at least 500 reads Chromosome base coverage summary Base reads per chromosome summary data used to create the d...

Страница 150: ... the Documentation and Support section in this document Symbols on this instrument Symbols may be found on the instrument to warn against potential hazards or convey important safety information In this document the hazard symbol is used along with one of the following user attention words CAUTION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injur...

Страница 151: ...isque de danger Consulter le manuel pour d autres renseignements de sécurité MISE EN GARDE Risque de choc électrique MISE EN GARDE Surface chaude MISE EN GARDE Danger biologique potentiel MISE EN GARDE Rayonnement ultraviolet Appendix E Safety Symbols on this instrument E Genexus Integrated Sequencer User Guide 151 ...

Страница 152: ...et description MISE EN GARDE Parties mobiles MISE EN GARDE Danger de perforation Label and location A44096 6191 A44096 6191 Rear panel Additional safety symbols Location of safety labels Appendix E Safety Symbols on this instrument E 152 Genexus Integrated Sequencer User Guide ...

Страница 153: ... standards for electromagnetic compatibility Indicates conformity with the WEEE Directive 2012 19 EU CAUTION To minimize negative environmental impact from disposal of electronic waste do not dispose of electronic waste in unsorted municipal waste Follow local municipal waste ordinances for proper disposal provision and contact customer service for information about responsible disposal options Co...

Страница 154: ...rmanent back injury Things to consider before moving the instrument Depending on the weight moving may require two or more persons If you decide to move the instrument after it has been installed do not attempt to do so without the assistance of others the use of appropriate moving equipment and proper lifting techniques Make sure that the path from where the object is to where it is being moved i...

Страница 155: ... mise à la terre est déconnecté La continuité de la mise à la terre est impérative pour le fonctionnement de l instrument en toute sécurité WARNING Power Supply Line Cords Use properly configured and approved line cords for the power supply in your facility AVERTISSEMENT Cordons d alimentation électrique Utiliser des cordons d alimentation adaptés et approuvés pour raccorder l instrument au circui...

Страница 156: ... de nettoyage ou de décontamination susceptibles de réagir avec certaines parties de l appareil ou avec les matières qu il contient et de constituer de ce fait un DANGER L instrument doit être correctement décontaminé a si des substances dangereuses sont renversées sur ou à l intérieur de l équipement et ou b avant de le faire réviser sur site ou de l envoyer à des fins de réparation de maintenanc...

Страница 157: ... medical equipment EMC standards Reference Description EU Directive 2014 30 EU European Union EMC Directive EN 61326 1 IEC 61326 1 Electrical Equipment for Measurement Control and Laboratory Use EMC Requirements Part 1 General Requirements EN 61326 2 6 IEC 61326 2 6 Electrical equipment for measurement control and laboratory use EMC requirements Particular requirements In vitro diagnostic IVD medi...

Страница 158: ...ncluding interference that may cause undesired operation Changes and modifications not expressly approved by Thermo Fisher Scientific can void your authority to operate this equipment under Federal Communications Commissions rules RFID Canada English This device complies with Industry Canada licence exempt RSS standard s Operation is subject to the following two conditions 1 this device may not ca...

Страница 159: ...f needed the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply AVERTISSEMENT PRÉCAUTIONS GÉNÉRALES EN C...

Страница 160: ...re les déchets générés par les applications les réactifs et les substrats particuliers utilisés dans le laboratoire Vérifier que les déchets sont convenablement stockés transférés transportés et éliminés en respectant toutes les réglementations locales nationales et ou communautaires en vigueur IMPORTANT Les matériaux représentant un danger biologique ou radioactif exigent parfois une manipulation...

Страница 161: ...oggles Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially biohazardous materials Follow all applicable local state provincial and or national regulations The following references provide general guidelines when handling biological samples in laboratory environment U S Department of Health and Human Services Biosafet...

Страница 162: ... Worldwide contact telephone numbers Product support information Product FAQs Software patches and updates Training for many applications and instruments Order and web support Product documentation User guides manuals and protocols Certificates of Analysis Safety Data Sheets SDSs also known as MSDSs Note For SDSs for reagents and chemicals from other manufacturers contact the manufacturer Limited ...

Страница 163: ...thermofisher com support thermofisher com askaquestion thermofisher com 13 November 2019 ...

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