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Part # 15050091 Rev. C

Sequencing Workflow

Prepare the flow cell and reagents for the run.
See

Flow Cell and Reagent Preparation

on page 18

.

Using the prompts on the control software interface, enter run parameters.

Weigh reagents. Load SBS reagents for Read 1 and Read 2, indexing
reagents (if applicable), and paired-end reagents.

With a used flow cell, confirm proper flow.
Prime SBS reagents and measure priming waste.

Load a clustered HiSeq X flow cell, and confirm proper flow.

Start the sequencing run.

[Optional]

After cycle 2, inspect the First Base Report and then continue

Read 1.

When the run is complete, unload and weigh reagents.
Perform an instrument maintenance wash.

Содержание HiSeq X

Страница 1: ...workflow guide with the Custom Protocol Selector support illumina com custom protocol selector html FOR RESEARCH USE ONLY ILLUMINA PROPRIETARY Part 15050091 Rev C October 2014 Catalog SY 412 9001DOC...

Страница 2: ...NeoPrep Nextera NextBio NextSeq Powered by Illumina SeqMonitor SureMDA TruGenome TruSeq TruSight Understand Your Genome UYG VeraCode verifi VeriSeq the pumpkin orange color and the streaming bases de...

Страница 3: ...l research purposes which includes research services provided to third parties and solely in accordance with this Product s Documentation but specifically excluding any use that a would require rights...

Страница 4: ...OING ILLUMINA MAKES NO CLAIM REPRESENTATION OR WARRANTY OF ANY KIND AS TO THE UTILITY OF THIS PRODUCT FOR PURCHASER S INTENDED USES 8 Product Warranty All warranties are personal to the Purchaser and...

Страница 5: ...est Illumina will attempt to pass through any such warranty to Purchaser 9 Indemnification a Infringement Indemnification by Illumina Subject to these terms and conditions including without limitation...

Страница 6: ...g indemnification i promptly notifying the other party in writing of such claim or action ii giving the other party exclusive control and authority over the defense and settlement of such claim or act...

Страница 7: ...ash every 10 days Added option to wash SBS reagent positions only Removed Indexing Reagent Kit ID from the Reagents screen Indexing reagents are packaged with paired end reagents in HiSeq X HD reagent...

Страница 8: ...patterned flow cell Added description of Q score binning Updated available disk space requirements Noted that wait time for instrument devices to initialize is at least 1 minute Added best practice to...

Страница 9: ...Perform a Sequencing Run 17 Introduction 18 Sequencing Workflow 20 Enter Run Parameters 21 Load and Prime Reagents 25 Load the Sequencing Flow Cell 33 Monitor the Run 37 Chapter 3 Post Run Procedures...

Страница 10: ...ing 62 Chapter 5 Troubleshooting 63 Introduction 64 Possible Run Setup Problems 65 Perform a Fluidics Check 66 Pause or End a Run on the HiSeq X 67 Stagger Runs on Flow Cell A and Flow Cell B 69 Possi...

Страница 11: ...tem User Guide 1 Chapter 1 Overview Overview Introduction 2 HiSeq X Components 3 User Supplied Consumables 7 Start the HiSeq X 8 HiSeq X Software 9 Available Disk Space 14 Sample Sheet Overview 15 Add...

Страница 12: ...end runs Integrated paired end fluidics provide reagents from the reagent compartment to the flow cell for Read 2 resynthesis and for indexed sequencing Interface control options The instrument softw...

Страница 13: ...t the same time using epifluorescence The excitation laser beam passes through the objective and the fluorescence is collected through the same objective B Flow cell compartment Contains the vacuum co...

Страница 14: ...positions that correspond to connections on an internal reagent selector valve Indexing and paired end reagent rack The reagent rack is located to the left of racks A and B It has 2 rows of numbered p...

Страница 15: ...e right is flow cell B Each flow cell is seated on the flow cell stage which moves in and out of the optics module as directed by the control software The flow cell stage must be in the forward most p...

Страница 16: ...igned closely together increasing the number of output reads and amount of sequencing data generated The HiSeq X patterned flow cell contains 8 lanes with 2 swaths per lane and has the same general di...

Страница 17: ...ab supplier Cleaning the flow cell and flow cell stage Centrifuge tubes 250 ml Corning catalog 430776 Instrument maintenance wash and water wash Conical tubes 15 ml Corning catalog 430052 Collecting a...

Страница 18: ...iles on it This drive contains hardware configuration files and initializes whenever the instrument is turned on 5 To ensure adequate disk space archive the data on the instrument computer from previo...

Страница 19: ...se calling and assigns a quality score to each base for each cycle For more information see Real Time Analysis on page 51 Sequencing Analysis Viewer software Sequencing Analysis Viewer SAV provides de...

Страница 20: ...ment has been idle for 1 day or more A water wash is an option following a sequencing run See Perform a Water Wash on page 46 Maintenance Wash The maintenance wash flushes water then NaOH and then wat...

Страница 21: ...s in conditions errors or warnings during run setup steps and during the run Status Icon Status Name Description Status OK No change System is normal Information Information only No action is required...

Страница 22: ...sts Use this log file for troubleshooting purposes Scanner Activates the command to initialize the software manually About Provides information about instrument hardware software versions and technica...

Страница 23: ...The server name for interactions with supported Illumina LIMS LIMS User Name The user name used when authenticating to Illumina LIMS LIMS Password The password used when authenticating to Illumina LIM...

Страница 24: ...flow cell run If disk space drops below the safe threshold during the run the software pauses the run and places the flow cell in a safe state After disk space is made available the run resumes autom...

Страница 25: ...hat stores information about the sequencing run When the run begins the software copies the sample sheet to the run folder where it is later used for analysis Sample sheets are optional unless you are...

Страница 26: ...eagent Kit v2 Reference Guide part 15058861 HiSeq X HD Reagent Kit Reference Guide part 15050092 Provides a description of kit contents and instructions for preparing consumables before a sequencing r...

Страница 27: ...stem User Guide 17 Chapter 2 Perform a Sequencing Run Perform a Sequencing Run Introduction 18 Sequencing Workflow 20 Enter Run Parameters 21 Load and Prime Reagents 25 Load the Sequencing Flow Cell 3...

Страница 28: ...onsumables are packaged in 2 complete sets each set supports 1 flow cell 20 pack kit Each kit includes consumables for sequencing 20 flow cells Consumables are packaged to support 4 flow cells at a ti...

Страница 29: ...ser Guide 19 Stagger Runs It is possible to start a new run on either flow cell A or flow cell B when a run on the adjacent flow cell is in progress For more information see Stagger Runs on Flow Cell...

Страница 30: ...reagents Load SBS reagents for Read 1 and Read 2 indexing reagents if applicable and paired end reagents With a used flow cell confirm proper flow Prime SBS reagents and measure priming waste Load a c...

Страница 31: ...to BaseSpace 2 Select a setting for BaseSpace Storage and Analysis Sends run data to BaseSpace for remote monitoring and data analysis A sample sheet is required with this option NOTE Before selecting...

Страница 32: ...equenced The flow cell ID is used to determine flow cell type and reagent compatibility 2 Confirm that the flow cell type is HiSeq X HD or HiSeq X HD v2 The flow cell type is selected automatically ba...

Страница 33: ...2 and the Index Read if applicable 3 Confirm the following default settings Fields are populated depending on the selected index type a SBS HiSeq X SBS Shows the SBS chemistry used for Read 1 and Read...

Страница 34: ...Remaining field enter the number of SBS cycles that reagents are expected to last from 1 310 cycles NOTE The Cycles Remaining field is populated automatically based on the SBS kit ID If you are using...

Страница 35: ...s not replace the post run instrument wash Load SBS Reagents 1 Invert each bottle several times to make sure that the reagents are mixed thoroughly 2 Remove the cap from each reagent bottle and replac...

Страница 36: ...nt 10 Lower the sippers into the sequencing reagent bottles as follows a Pull the sipper handle towards you and then lower the sipper handle b Visually inspect the sippers to make sure that they do no...

Страница 37: ...Paired End Reagent Positions HP12 is required for indexed runs only If HP12 is not used load a 15 ml conical tube with 10 ml PW1 or laboratory water 6 Slide the reagent rack into the reagent compartm...

Страница 38: ...nt remove it and set aside in a tube of storage buffer or laboratory grade water to keep it from drying out It can be used to confirm proper flow before loading the clustered flow cell 5 Using an alco...

Страница 39: ...er Dry the flow cell with a lens cleaning tissue or lint free tissue 2 Clean the flow cell using alcohol wipes and lens cleaning tissue NOTE Do not remove or replace the flow cell gaskets during this...

Страница 40: ...cuum and secures the flow cell into position When the flow cell lever is blinking green the vacuum is engaged If the lever is not green see Possible Run Setup Problems on page 65 Figure 14 Flow Cell L...

Страница 41: ...the drop down list CAUTION Use water to confirm proper flow on a used flow cell only Never use water to confirm proper flow on a clustered flow cell 2 Confirm the following default values Volume 125 A...

Страница 42: ...ing step begins Monitor the progress of the priming step from the priming screen 4 When the priming step is complete measure the collected priming volume and confirm that the volume in each tube is 1...

Страница 43: ...er loading the flow cell onto the instrument and confirming proper flow Remove the Used Flow Cell 1 Slowly move the flow cell lever to position 1 to disengage the manifolds Figure 17 Flow Cell Lever i...

Страница 44: ...low lint lab tissue to dry the stage if necessary Figure 19 Inspect Vacuum Holes 3 Visually inspect the flow cell holder to make sure that it is free of lint and the vacuum holes are free of obstruct...

Страница 45: ...me 3 Slowly move the flow cell lever to position 1 which engages the vacuum and secures the flow cell into position When the flow cell lever is blinking green the vacuum is engaged If the lever is not...

Страница 46: ...e 250 Aspirate Rate 250 Dispense Rate 2000 3 Select Pump 4 Visually inspect the flow cell for bubbles passing through the lanes or leaks near the manifolds If bubbles are present check the manifold ga...

Страница 47: ...Review parameters of current run E Analysis graph Monitor quality scores by cycle F Images graph Monitor intensities by cycle Only 1 thumbnail image is shown for each swath scanned All other images do...

Страница 48: ...Sequencing Analysis Viewer opens automatically after run metrics are available Select Refresh at any time during the run to view updated metrics For more information see the Sequencing Analysis Viewe...

Страница 49: ...pter 3 Post Run Procedures Post Run Procedures Introduction 40 Unload and Weigh Reagents 41 Perform a Maintenance Wash 42 Perform a Water Wash 46 Perform a Quick Format of the Output Drive 48 Idle the...

Страница 50: ...nd sippers Prevent salt accumulation and crystallization in the fluidics tubing and sippers Prevent cross contamination from the previous run NOTE A water wash is performed at the end of each run to w...

Страница 51: ...reagent rack out of the reagent compartment using the rack handle 4 Remove each bottle from the reagent rack and record the weight on the lab tracking form Visit the HiSeq X support page on the Illum...

Страница 52: ...aintenance wash replace the 8 port gaskets in the front manifold and back manifold before proceeding to the wash The maintenance wash consists of 3 wash steps First water wash Flushes the system with...

Страница 53: ...cell from the flow cell stage and set it aside Replace the gaskets as follows a Wearing a fresh pair of gloves apply light pressure with your finger to 1 end of the gasket until the other end raises...

Страница 54: ...h is complete measure the delivered volume NOTE The priming pump is washed during the wash steps Keep the waste from the priming pump separate The following tables list the wash volumes collected from...

Страница 55: ...ttles used for the first wash step The water from the first wash step might be contaminated with reagents that were present on the sippers 2 Load the bottles and tubes onto the instrument in the appro...

Страница 56: ...s 8 bottles 250 ml Corning catalog 430776 10 tubes 15 ml Corning catalog 430052 Laboratory grade water Procedure 1 From the Welcome screen select Wash Water 2 Select Yes to wash paired end reagent pos...

Страница 57: ...p all of the ends even Place the bundled tubing ends into a 250 ml bottle 8 Select Next to start the water wash Positions Approximate Run Time 8 SBS positions 20 minutes 8 SBS positions and 10 paired...

Страница 58: ...uick format clears the O drive without removing important system or instrument maintenance files NOTE Instrument maintenance logs are stored on the C drive Therefore it is safe to perform a quick form...

Страница 59: ...to flush the system fully For more information see Perform a Maintenance Wash on page 42 2 Leave the flow cell on the flow cell stage with the flow cell lever in position 2 The manifolds remain in the...

Страница 60: ...2 Remove the flow cell from the flow cell stage 3 Using an alcohol wipe or a lint free tissue moistened with ethanol or isopropanol carefully wipe the surface of the flow cell holder until it is clea...

Страница 61: ...Chapter 4 HiSeq X System User Guide 51 Chapter 4 Real Time Analysis Real Time Analysis Introduction 52 RTA v2 Overview 53 Real Time Analysis Workflow 55 Sequencing Output Files 60 Tile Numbering 62...

Страница 62: ...v2 which includes the following important architecture and feature differences Configuration files output file formats and the processing workflow differ from previous implementations of the software...

Страница 63: ...ware configuration file in XML format RTA v2 receives commands from the control software that include information about the location of RunInfo xml file and if an optional output folder is specified R...

Страница 64: ...o the final output destination at the end of processing The following log files are generated when errors occur GlobalLog tsv summarizes important run events LaneNLog tsv logs processing events for ea...

Страница 65: ...on the patterned flow cell cluster positions are predetermined according to the number of rows number of columns and distance between the nano wells on the flow cell For more information see HiSeq X...

Страница 66: ...action each DNA strand in a cluster extends by 1 base per cycle Phasing and prephasing occurs when a strand becomes out of phase with the current incorporation cycle Phasing occurs when a base falls b...

Страница 67: ...chastity value below 0 6 in the first 25 cycles Chastity is defined as the ratio of the brightest base intensity divided by the sum of the brightest and the second brightest base intensities The perce...

Страница 68: ...eliable and more likely to be correct The Q score serves as a compact way to communicate small error probabilities Quality scores are represented as Q X where X is the score The following table shows...

Страница 69: ...ion see Sequencing Output Files on page 60 Q Score Binning RTA v2 groups quality scores into specific ranges or bins and assigns a value to each range Q score binning significantly reduces storage spa...

Страница 70: ...cation files are the result of template generation Data Intensities One file for the run is stored in the Intensities folder s locs Filter files The filter file specifies whether a cluster passed filt...

Страница 71: ...the Real Time Analysis configuration file lists settings for the run Root folder RTAConfiguration xml Run information file Lists the run name number of cycles in each read whether the read is an index...

Страница 72: ...he flow cell The first digit represents the surface 1 is for top 2 is for bottom The second digit represents the swath 1 is for the first swath 2 is for the second swath NOTE A swath is a column of ti...

Страница 73: ...63 Chapter 5 Troubleshooting Troubleshooting Introduction 64 Possible Run Setup Problems 65 Perform a Fluidics Check 66 Pause or End a Run on the HiSeq X 67 Stagger Runs on Flow Cell A and Flow Cell B...

Страница 74: ...Troubleshooting 64 Part 15050091 Rev C Introduction This section describes what to do if a problem occurs during a sequencing run and how to perform a fluidics check from the Welcome screen...

Страница 75: ...eaning steps Make sure that the gaskets are present and well seated Reload the flow cell If the preceding steps do not work try replacing the gaskets and then reload the flow cell Flow cell lever is b...

Страница 76: ...the instrument 3 Select Check on the Welcome screen 4 Select solution 5 PB2 from the drop down list If you are performing a fluidics check with a used flow cell select solution 2 which is water 5 Ent...

Страница 77: ...m the Run Overview screen select Pause The pause menu opens Figure 29 Pause Options 2 Select Normal Pause 3 Select Yes to confirm the pause command The software completes the current chemistry or imag...

Страница 78: ...d the software does not resume processing and run data are not saved Therefore a run cannot resume after it has been stopped 1 To end the run select Abort Confirm or cancel the command Confirming the...

Страница 79: ...to an imaging step 2 Select Normal Pause 3 Wait for the software to complete the current chemistry step The system is placed in a safe state automatically 4 After the adjacent flow cell run is paused...

Страница 80: ...the flow cell Read 1 primer rehybridization is performed on the cBot and does not damage clusters on the flow cell Read 1 primer hybridization on a HiSeq X patterned flow cell requires the following I...

Страница 81: ...nt 3 optics module 3 reagent compartment 3 4 config file Real Time Analysis 53 config file RTA v2 61 confirm first base 22 confirm proper flow default values 31 36 consumables Illumina sequencing kits...

Страница 82: ...ges monitoring 37 saving images samples 21 thumbnails 21 61 indexing reagents loading 26 indexing scheme 23 instrument health 12 instrument shutdown 50 instrument wash 40 intensity extraction 55 Inter...

Страница 83: ...0 Q score binning 59 SAV index tab 52 stopped run no resume 53 stopping a run no resume 68 RTA exe config file 53 run monitoring 37 run setup chemistry selections 23 confirm proper flow 31 36 cycles r...

Страница 84: ...d consumables 7 W washes maintenance wash 40 recommendations 40 water wash 40 46 water wash 10 40 consumables 46 duration 46 expected volumes 46 weighing reagents after run 41 before run 25 Welcome sc...

Страница 85: ...223859 Belgium 0800 81102 Norway 800 16836 Denmark 80882346 Spain 900 812168 Finland 0800 918363 Sweden 020790181 France 0800 911850 Switzerland 0800 563118 Germany 0800 180 8994 United Kingdom 0800 9...

Страница 86: ...Illumina San Diego California 92122 U S A 1 800 809 ILMN 4566 1 858 202 4566 outside North America techsupport illumina com www illumina com...

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