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Chapter 3 Sequencing

Sequencing Workflow

Prepare Consumables

Prepare Libraries for Sequencing

Set Up a Sequencing Run

Monitor Run Progress

Automatic Post-Run Wash

Remove Used Reservoir from Position #9

Introduction

To perform a sequencing run on the MiniSeq System, prepare run consumables and then follow the software
prompts to set up the sequencing run.

Workflow Overview

Cluster Generation

During cluster generation, single DNA molecules are bound to the surface of the flow cell, and then amplified
to form clusters.

Sequencing

Clusters are imaged using 2-channel sequencing chemistry and filter combinations specific to each of the
fluorescently labeled chain terminators. After imaging of a tile on the flow cell is complete, the next tile is
imaged. The process is repeated for each cycle of sequencing. Following image analysis, the software
performs base calling, filtering, and quality scoring.

Analysis

As the run progresses, the control software automatically transfers base call (BCL) files to the specified output
location for data analysis. Several analysis methods are available depending on your application and the
selected analysis configuration for your system. For more information, see

Configure Analysis Settings

Sequencing Run Duration

Sequencing run duration depends on the number of cycles performed. The maximum run length is a 150-
cycle paired-end run, plus up to 2 index reads of 8 cycles each.

For expected durations and other system specifications, visit the

MiniSeq System specifications page

on the

Illumina website.

Number of Cycles in a Read

In a sequencing run, the number of cycles performed in a read is 1 more cycle than the number of cycles
analyzed. For example, to perform a 150-cycle paired-end run, set up the run to perform 151 cycles per read
(2 × 151) for a total of 302 cycles. At the end of the run, 2 × 150 cycles are analyzed. The extra cycle in each
read is used for phasing and prephasing calculations.

Document # 1000000002695 v02 Material # 20014309

For Research Use Only. Not for use in diagnostic procedures.

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Содержание MiniSeq

Страница 1: ...MiniSeq System Guide Document 1000000002695 v02 Material 20014309 March 2018 ILLUMINA PROPRIETARY For Research Use Only Not for use in diagnostic procedures ...

Страница 2: ...ned 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 AND ...

Страница 3: ...terial 20014309 Document 1000000002695 v01 September 2016 Updated software descriptions to MiniSeq Control Software v1 1 8 which includes Demo Mode Updated the automatic post run wash duration to 60 minutes Added a server configuration step to the instructions for selecting BaseSpace for analysis Noted that mapped drives are not supported by the Local Run Manager software Material 20002370 Documen...

Страница 4: ...p a Sequencing Run 20 Monitor Run Progress 28 Automatic Post Run Wash 30 Remove Used Reservoir from Position 9 30 Chapter 4 Maintenance 32 Introduction 32 Perform a Manual Instrument Wash 32 Software Updates 35 Appendix A Troubleshooting 38 Troubleshooting Files 38 Automatic Check Errors 39 RTA Errors 40 Rehybridization Workflow 41 System Check 42 Network Configuration Settings 45 Custom Genomes 4...

Страница 5: ...t Files 52 Sequencing Output Folder Structure 52 Analysis Input File Requirements 53 Index 54 Technical Assistance 57 Document 1000000002695 v02 Material 20014309 For Research Use Only Not for use in diagnostic procedures v MiniSeq System Guide ...

Страница 6: ...pace Onsite for analysis u Convenient consumable loading A clamping mechanism positions the flow cell as it is loaded onto the instrument A single use prefilled reagent cartridge provides reagents required for a run and subsequent instrument wash The flow cell and reagent cartridge include integrated identification to enable accurate tracking Additional Resources The MiniSeq System support pages o...

Страница 7: ...control software interface B Power button Turns on the integrated instrument computer and operating system C USB ports Convenient connections for peripheral components D Flow cell compartment Holds the flow cell during a sequencing run E Status bar Indicates instrument status as processing blue requires attention orange ready to sequence green or when a wash is due within the next 24 hours yellow ...

Страница 8: ...cess to the reagent compartment to load run consumables and empty the spent reagents bottle Figure 3 Reagent Compartment A Spent reagents bottle Includes a threaded cap to prevent spills when carrying B Reagent cartridge Provides reagents in a single use prefilled consumable C Reagent compartment door Provides access to the reagent compartment The reagent compartment door opens outward from hinges...

Страница 9: ...nnot be reused and sequencing data are not saved System Software The instrument software suite includes integrated applications that perform sequencing runs and on instrument analysis u MiniSeq Control Software The control software guides you through the steps to set up a sequencing run controls instrument operations and shows an overview of run statistics as the run progresses u Real Time Analysi...

Страница 10: ...io frequency identification RFID for accurate consumable tracking and to ensure compatibility with specified run parameters Flow Cell The flow cell is a glass based substrate on which clusters are generated and the sequencing reaction is performed The flow cell is encased in a flow cell cartridge Figure 4 Flow Cell Components A Imaging area B Flow cell gasket C Outlet port D Inlet port Reagents en...

Страница 11: ...inhalation ingestion skin contact and eye contact Wear protective equipment including eye protection gloves and laboratory coat appropriate for risk of exposure Handle used reagents as chemical waste and discard in accordance with applicable regional national and local laws and regulations For additional environmental health and safety information see the SDS at support illumina com sds html Store...

Страница 12: ...lysis methods a reference is required to perform alignment Several reference databases and genomes are preinstalled on the instrument computer Preinstalled Description Databases miRbase for human dbSNP for human RefGene for human Genomes Arabidopsis thaliana cow Bos taurus E coli strain DH10B E coli strain MG1655 fruit fly Drosophila melanogaster human Homo sapiens build hg19 mouse Mus musculus Ph...

Страница 13: ...r button turns on the integrated instrument computer and operating system Figure 8 Power Button Located on Front of Instrument 3 Wait until the operating system has finished loading The MiniSeq Control Software launches and initializes the system automatically The Home screen opens after the initialization step is complete 4 Wait for the system to load and then log on to the operating system If ne...

Страница 14: ... Purging consumables automatically adds additional time to the workflow For example purging reagents after a 300 cycle run 2 151 takes approximately 50 minutes 3 Select Save to save settings and exit the screen Set Automatic Start Option 1 From the Manage Instrument screen select System Customization 2 Select the Skip pre run check confirmation checkbox This setting starts the sequencing run autom...

Страница 15: ...Mode After restart the MiniSeq Control Software banner reads Demonstration Only Exit DemoMode 1 From the Manage Instrument screen select System Customization 2 Clear the Enable Demo Mode checkbox 3 Select Save 4 If a PIN is required enter it when prompted 5 Select OK to restart the software in the standard mode Configure Analysis Settings The control software provides the following options for dat...

Страница 16: ...on on the benefits of this service see Illumina Proactive Technical Note document 1000000052503 This service u Does not send sequencing data u Requires that the instrument be connected to a network with internet access u Is turned on by default To opt out of this service disable the Send instrument health information to Illumina setting 8 Select Save Select BaseSpace Onsite 1 From the Manage Instr...

Страница 17: ...fy default BaseSpace login credentials as follows a Enter a User name and Password b Select the Save credentials as the default checkbox 7 Select Browse and navigate to a preferred output folder location 8 In the Contents pane of the output folder location create a folder named Runs NOTE Local Run Manager does not support mapped drives 9 Optional Select Send instrument health information to Illumi...

Страница 18: ...ximizing instrument uptime For more information on the benefits of this service see Illumina Proactive Technical Note document 1000000052503 This service u Does not send sequencing data u Requires that the instrument be connected to a network with internet access u Is turned on by default To opt out of this service disable the Send instrument health information to Illumina setting 8 Select Save Us...

Страница 19: ... for Laboratory Grade Water Always use laboratory grade water or deionized water to perform instrument procedures Never use tap water Use only the following grades of water or equivalents u Deionized water u Illumina PW1 u 18 Megohms MΩ water u Milli Q water u Super Q water u Molecular biology grade water User Supplied Equipment Item Source Freezer 25 C to 15 C frost free General lab supplier Ice ...

Страница 20: ...Document 1000000002695 v02 Material 20014309 For Research Use Only Not for use in diagnostic procedures 15 MiniSeq System Guide ...

Страница 21: ...is As the run progresses the control software automatically transfers base call BCL files to the specified output location for data analysis Several analysis methods are available depending on your application and the selected analysis configuration for your system For more information see Configure Analysis Settings on page 10 Sequencing Run Duration Sequencing run duration depends on the number ...

Страница 22: ...epare a new flow cell bring to room temperature and then unwrap Denature and dilute libraries See MiniSeq System Denature and Dilute Libraries Guide document 1000000002697 Load the library dilution onto the reagent cartridge in reservoir 16 From the software interface select Sequence Load the flow cell and reagent cartridge Empty and reload the spent reagents bottle Specify run parameters on the R...

Страница 23: ...egin the run setup steps Select the run name specified on BaseSpace BaseSpace Onsite or Local Run Manager Load the flow cell Load the reagent cartridge Empty and reload the spent reagents bottle Review run parameters on the Run Setup screen After the automated check select Start Monitor the run on the instrument screen from BaseSpace or from a networked computer using Sequencing Analysis Viewer Da...

Страница 24: ...rs 3 Invert the cartridge 5 times to mix reagents 4 Inspect the large reservoirs from the bottom of the cartridge to make sure that reagents have thawed and the reservoirs are free of ice crystals 5 Gently tap on the bench to reduce air bubbles Prepare the Flow Cell 1 Remove a new flow cell package from 2 C to 8 C storage 2 Set the unopened flow cell package aside at room temperature for 30 minute...

Страница 25: ...covering reservoir 16 labeled Load library here using a low lint tissue 2 Pierce the seal with a clean 1 ml pipette tip 3 Add 500 µl prepared 1 8 pM libraries into reservoir 16 Avoid touching the foil seal as you dispense the libraries Figure 10 Load Libraries Set Up a Sequencing Run Run setup steps differ based on system configuration u Standalone configuration You are prompted to define run para...

Страница 26: ...resent clean the flow cell stage with an alcohol wipe 5 Place the flow cell on the flow cell stage over the alignment pins Figure 12 Place Flow Cell on Stage 6 Close the flow cell latch to secure the flow cell Figure 13 Close Flow Cell Latch 7 Close the flow cell compartment door Load the Reagent Cartridge 1 Open the reagent compartment door Document 1000000002695 v02 Material 20014309 For Researc...

Страница 27: ... the reagent compartment until the cartridge stops Figure 14 Load Reagent Cartridge Empty the Spent Reagents Bottle 1 Remove the spent reagents bottle from the compartment Figure 15 Remove the Spent Reagents Bottle 2 To prevent spills when carrying the spent reagents bottle seal the bottle opening with the threaded cap Document 1000000002695 v02 Material 20014309 For Research Use Only Not for use ...

Страница 28: ...t select a recipe Only compatible recipes are listed 4 Select a read type either Single read or Paired end 5 Enter the number of cycles for each read in the sequencing run u Read 1 Enter a value up to 151 cycles u Index 1 Enter the number of cycles required for the Index 1 i7 primer u Index 2 Enter the number of cycles required for the Index 2 i5 primer u Read 2 Enter a value up to 151 cycles This...

Страница 29: ...For any items that do not pass an action is required before you can proceed See Automatic Check Errors on page 39 u To restart the check select Retry The check resumes at the first incomplete or failed check 2 To start the run select from the following options u If the system is not configured to start automatically after a successful check select Start u If the system is configured to start autom...

Страница 30: ...resent clean the flow cell stage with an alcohol wipe 5 Place the flow cell on the flow cell stage over the alignment pins Figure 18 Place Flow Cell on Stage 6 Close the flow cell latch to secure the flow cell Figure 19 Close Flow Cell Latch 7 Close the flow cell compartment door Load the Reagent Cartridge 1 Open the reagent compartment door Document 1000000002695 v02 Material 20014309 For Researc...

Страница 31: ... the reagent compartment until the cartridge stops Figure 20 Load Reagent Cartridge Empty the Spent Reagents Bottle 1 Remove the spent reagents bottle from the compartment Figure 21 Remove the Spent Reagents Bottle 2 To prevent spills when carrying the spent reagents bottle seal the bottle opening with the threaded cap Document 1000000002695 v02 Material 20014309 For Research Use Only Not for use ...

Страница 32: ...onfirm run parameters The control software confirms the number of specified cycles using the following criteria u Total cycles do not exceed the maximum cycles allowed based on the reagent cartridge loaded for the run u Cycles for Read 1 are greater than the 5 cycles required for template generation u Index Read cycles do not exceed Read 1 and Read 2 cycles NOTE Make sure that you specify the appr...

Страница 33: ...gress intensities and quality scores as metrics appear on the screen Figure 23 Sequencing Run Progress and Metrics A Run progress Shows the current step and number of cycles completed for each read The progress bar is not proportional to the run rate of each step B Q Score Shows the distribution of quality scores Q scores See Quality Scoring on page 51 C Intensity Shows the value of cluster intens...

Страница 34: ...w updated metrics For more information see the Sequencing Analysis Viewer User Guide part 15020619 The Sequencing Analysis Viewer is included in the software installed on the instrument computer You can also install Sequencing Analysis Viewer on another computer linked to the same network as the instrument to monitor run metrics remotely Data Transfer Status Depending on the analysis configuration...

Страница 35: ...The automatic post run wash takes approximately 60 minutes When the wash is complete the Home button becomes active Sequencing results remain visible on the screen during the wash After the Wash After the wash the sippers remain in the down position to prevent air from entering the system Leave the cartridge in place until the next run Remove Used Reservoir from Position 9 The reservoir in positio...

Страница 36: ...rdous chemicals Personal injury can occur through inhalation ingestion skin contact and eye contact Wear protective equipment including eye protection gloves and laboratory coat appropriate for risk of exposure Handle used reagents as chemical waste and discard in accordance with applicable regional national and local laws and regulations For additional environmental health and safety information ...

Страница 37: ...a Manual Instrument Wash Manual instrument wash options include the Quick Wash and the Manual Post Run Wash Wash Types Description Quick Wash Duration 20 minutes A quick wash is required every 7 days that the instrument is idle or after an instrument shutdown The wash flushes the system with a user supplied wash solution of laboratory grade water and Tween 20 Manual Post Run Wash Duration 90 minut...

Страница 38: ...r NaOCl and Wash Solution A 0 12 NaOCl B Wash solution 3 Combine the following volumes to result in a 0 05 Tween 20 wash solution u 100 Tween 20 40 µl u Laboratory grade water 80 ml 4 Add 80 ml wash solution to the wash cartridge The correct reservoir is equivalent to position 40 on the prefilled reagent cartridge 5 From the Home screen select Perform Wash and then select Manual post run wash Docu...

Страница 39: ...red wash cartridge 4 Remove the spent reagents bottle and discard the contents in accordance with applicable standards WARNING This set of reagents contains potentially hazardous chemicals Personal injury can occur through inhalation ingestion skin contact and eye contact Wear protective equipment including eye protection gloves and laboratory coat appropriate for risk of exposure Handle used reag...

Страница 40: ...tes For instruments connected to a network with internet access an alert icon appears on the Manage Instrument button on the Home screen when an update is available u Manual updates Download the System Suite installer from the MiniSeq System support page on the Illumina website NOTE Cancelling an update before installation is complete stops the update at the current point in the installation Any c...

Страница 41: ...are Update 4 Select Manually install the update from the following location 5 Select Browse to navigate to the location of the software installation file and then select Update 6 Follow the prompts in the installation wizard a Accept the licensing agreement b Review the list of software included in the update When the update is complete the control software restarts automatically NOTE If a firmwar...

Страница 42: ...Document 1000000002695 v02 Material 20014309 For Research Use Only Not for use in diagnostic procedures 37 MiniSeq System Guide ...

Страница 43: ...he run Log files Logs Log files describe each step performed by the instrument for each cycle and list software and firmware versions used with the run The file named InstrumentName _CurrentHardware csv lists the serial numbers of instrument components Error log files ErrorLog txt RTA Logs Log of RTA errors Error log files are updated whenever an error occurs Global log files GlobalLog tsv RTA Log...

Страница 44: ... RFID is not locked and can be used for a subsequent run However the RFID is locked after the foil seals have been pierced System Checks Recommended Action Doors closed Make sure that the compartment doors are closed Consumables loaded Consumable sensors do not register Make sure that each consumable is properly loaded On the run setup screens select Back to return to the loading step and repeat r...

Страница 45: ...lder the files are copied to the output folder Therefore if you specify an output folder location on the instrument hard drive 2 copies of that run are written to the hard drive The software saves the most recent 3 temporary run folders When using the Local Run Manager software for analysis temporary files are not deleted by default The retention policy is set manually from the Local Run Manager S...

Страница 46: ...rmal u Skips the clustering step u Removes the previous Read 1 primer u Hybridizes a fresh Read 1 primer u Continues through Read 1 and the remainder of the run based on specified run parameters Points to End a Run for Rehybridization Later rehybridization is possible only if you end the run at the following points u After cycle 5 Intensities appear after template registration which requires the f...

Страница 47: ...e saved flow cell was stored allow it to reach room temperature 15 30 minutes 3 Clean and load the saved flow cell The system reads the flow cell RFID as a saved flow cell and confirms a valid rehybridization date 4 Remove the spent reagents bottle and discard the contents appropriately and then reload the empty container 5 Load the new reagent cartridge 6 From the Run Setup screen select from the...

Страница 48: ...lect screen indicate tests that require assistance from an Illumina field representative Perform a System Check 1 From the Manage Instrument screen select System Check When prompted to close the control software select Yes 2 Add 40 ml deionized water to the wash cartridge The correct reservoir is equivalent to position 40 on the prefilled reagent cartridge Figure 30 Position for Wash Solution A Wa...

Страница 49: ...stage and Z stage Optics Check System Check Description Flow Cell Registration Measures flow cell tilt on an optical plane tests camera functionality tests the imaging module and verifies registration of the flow cell in the correct imaging position Fluidics Checks System Check Description Valve Response Checks the accuracy of the valve and pump movements and tests the pump syringe range of moveme...

Страница 50: ...t Obtain a DNS server address automatically to connect the instrument to the domain name server associated with IP address Alternatively select Use the following DNS server addresses to connect the instrument to the domain name server manually as follows u Enter the preferred DNS address The DNS address is the server name used to translate domain names into IP addresses u Enter the alternate DNS a...

Страница 51: ...c characters as chromosome names Shut Down the Instrument Under normal conditions there is no reason to shut down the instrument 1 Select Manage Instrument 2 Select Shutdown options 3 Select Shut down The command safely shuts down the software and turns off instrument power Wait at least 60 seconds before turning on the instrument again A wash is required before the next sequencing run CAUTION Do ...

Страница 52: ...Document 1000000002695 v02 Material 20014309 For Research Use Only Not for use in diagnostic procedures 47 MiniSeq System Guide ...

Страница 53: ... view by the camera From these images the software produces output as a set of quality scored base call files and filter files Output files are used for downstream analysis on BaseSpace or by Local Run Manager analysis modules File Type Description Base call files Each tile that is analyzed is included in an aggregated base call bcl file for each lane and for each cycle The aggregated base call fi...

Страница 54: ...ow cell are written to cluster location locs files 1 file for each lane Registration and Intensity Extraction Registration and intensity extraction begin after template generation u Registration aligns images produced over every subsequent cycle of imaging against the template u Intensity extraction determines an intensity value for each cluster in the template for a given image If registration fa...

Страница 55: ... populations each corresponding to a nucleotide The base calling process determines to which population each cluster belongs Figure 32 Visualization of Cluster Intensities Base Red Channel Green Channel Result A 1 on 1 on Clusters that show intensity in both the red and green channels C 1 on 0 off Clusters that show intensity in the red channel only G 0 off 0 off Clusters that show no intensity at...

Страница 56: ...s ensures accuracy when analyzing low plex samples Quality Scoring A quality score or Q score is a prediction of the probability of an incorrect base call A higher Q score implies that a base call is higher quality and more likely to be correct The Q score is a compact way to communicate small error probabilities Q X represents quality scores where X is the score The following table shows the rela...

Страница 57: ...ation files are the result of template generation Data Intensities L001 s_ lane locs Filter files The filter file specifies whether a cluster passed filters Filter information is aggregated into a filter file for each read Filter files are generated at cycle 26 using 25 cycles of data Data Intensities BaseCalls L001 s_ lane filter InterOp files Binary reporting files used for Sequencing Analysis V...

Страница 58: ...e analysis module you are using File Name Type Description RTAComplete txt A marker file that indicates RTA processing is complete The presence of this file triggers Local Run Manager to queue analysis RunInfo xml Contains high level run information such as the number of reads and cycles in the sequencing run and whether a read is indexed Base call files bcl Contains the base call and encoded qual...

Страница 59: ...instrument maintenance 14 laboratory grade water 14 reagent cartridge 6 sequencing runs 13 user supplied 13 wash consumables 32 33 control software 4 customer support 57 cycles in a read 16 D data transfer activity icons 29 BaseSpace Broker 30 run copy service 29 databases preinstalled 7 dbSNP database 7 Demo Mode 9 documentation 1 57 E empirical phasing 49 errors probability 51 errors and warning...

Страница 60: ...eventive maintenance 32 primer rehybridization 41 purge consumables 9 Q Q scores 51 quality tables 51 R read length 16 reagent cartridge overview 6 preparation 19 reservoir 28 33 reagent compartment 2 reagents kitted 5 proper disposal 22 26 Real Time Analysis software 1 4 results 52 reference genomes custom genomes 46 file format 7 preinstalled 7 RefGene database 7 rehybridization Read 1 41 RFID t...

Страница 61: ...etrics 41 pre run check 39 run specific files 38 system check 42 U updating software 35 user supplied consumables 13 14 user name and password 8 W wash automatic 30 manual wash 32 user supplied consumables 32 wash components 32 workflow advanced loading option 9 analysis configuration 20 BaseSpace login 24 BaseSpace mode 24 indexing considerations 51 overview 17 18 pre run check 24 28 reagent cart...

Страница 62: ...50506 Italy 39 800985513 39 236003759 Japan 0800 111 5011 Netherlands 31 8000222493 31 207132960 New Zealand 0800 451 650 Norway 47 800 16836 47 21939693 Singapore 1 800 579 2745 Spain 34 911899417 34 800300143 Sweden 46 850619671 46 200883979 Switzerland 41 565800000 41 800200442 Taiwan 00806651752 United Kingdom 44 8000126019 44 2073057197 Other countries 44 1799 534000 Safety data sheets SDSs A...

Страница 63: ...2122 U S A 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsupport illumina com www illumina com For Research Use Only Not for use in diagnostic procedures 2018 Illumina Inc All rights reserved Document 1000000002695 v02 Material 20014309 ...

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