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Appendix B Real-Time Analysis

Real-Time Analysis Overview

Real-Time Analysis Workflow

Monitor Run Metrics

Real-Time Analysis Overview

Real-Time Analysis (RTA) runs on the instrument computer, performing base calling and assigning a quality
score to each base call.

The software tracks the status of each tile and determines when to advance it to the next process step.
When advancing a tile, RTA outputs a file for the completed step and then starts the next step. Thus, the
software can determine the status of each tile based on which files exist. If RTA is terminated, it saves run
data and can resume processing.

Input Files

RTA requires the following input files:

u

Cluster intensity files, which contain image analysis results.

u

RunInfo.xml

, which the control software generates automatically at the beginning of the run. From this file,

RTA reads the run name, number of cycles, whether a read is indexed, and the number of tiles on the
flow cell.

u

HiSeq.Configuration.xml

, which is an instrument configuration file in XML format.

u

RTA.exe.config

, which is a software configuration file in XML format.

RTA uses run parameters entered during run setup, and receives commands from the control software that
include information about when to initiate and the location of

RunInfo.xml

.

Output Files

Tiles are small imaging areas on the flow cell defined as 1 field of view by the camera. For each tile that is
analyzed, RTA produces a set of quality-scored base call files and filter files as primary output. Other files
support generation of primary output files.

u

Base call files—For each tile that is analyzed, 1 compressed base call (*.bcl) file is generated for each tile
per cycle. The base call file contains the base call and associated quality score.

u

Filter files—Each tile produces filter information that is included in 1 filter (*.filter) file for each tile over the
whole run. The filter file specifies whether clusters pass filter.

u

Cluster location files—One cluster location (*.locs) file contains the X,Y coordinates for every cluster on the
flow cell.

u

Statistics files—For each cycle, 1 statistics file (*.stats) is produced. The statistics file contains aggregate
statistics for the cycle.

Primary output files are used for subsequent data analysis. Use bcl2fastq for demultiplexing and conversion of
.bcl files into FASTQ files, which can be used as input for alignment. To convert data from the HiSeq, use
bcl2fastq 1.8.4, or later.

RTA provides real-time metrics of run quality stored as InterOp files. InterOp files are binary files containing
tile, cycle, and read-level metrics, and are required for viewing metrics in Sequencing Analysis Viewer (SAV).
For viewing metrics generated by RTA, use SAV v1.8.20 or later.

For details about each output file, see

Sequencing Output Files

.

Document # 15011190 v03

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

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Содержание HiSeq 2000

Страница 1: ...HiSeq 2000 System Guide Document 15011190 v03 May 2019 ILLUMINA PROPRIETARY For Research Use Only Not for use in diagnostic procedures...

Страница 2: ...erly trained personnel in order to ensure the proper and safe use of the product s described herein All of the contents of this document must be fully read and understood prior to using such product s...

Страница 3: ...erform a maintenance wash instead of a water wash after a sequencing run Corrected the run folder naming format to include the flow cell side Updated the maintenance wash procedure Document 15011190 v...

Страница 4: ...rforming the instrument maintenance wash instead of before loading the clustered flow cell Added the following information Overviews of single indexed and dual indexed sequencing runs Description of d...

Страница 5: ...ed description for performing staggered runs before HCS v1 3 Corrected reagent name Part 15011190 Rev E January 2011 Updated software descriptions to HCS v1 3 Updated instructions for resuming a run P...

Страница 6: ...the Run 23 Unload Reagents 23 Perform a Water Wash 24 Chapter 4 Sequencing in TruSeq v3 Mode 26 Introduction 26 Prepare Reagents for Read 1 27 Enter Run Parameters 30 Load and Prime Reagents 33 Load...

Страница 7: ...56 Real Time Analysis Overview 56 Real Time Analysis Workflow 57 Monitor Run Metrics 59 Appendix C Output Files and Folders 61 Sequencing Output Files 61 Output Folder Structure 62 Tile Numbering 62 T...

Страница 8: ...nsities from images and performs quality scored base calling on the instrument computer This method allows monitoring of quality metrics during the run and saves time during subsequent data analysis D...

Страница 9: ...he flow cell imaging A C G and T at the same time using epifluorescence The excitation laser beam passes through the objective and the fluorescence is simultaneously collected through the same objecti...

Страница 10: ...reagent compartment is a high capacity reagent chiller that holds three reagent racks two for SBS reagents and 1 for indexing and paired end reagents Sipper handles lower the sippers into the reagent...

Страница 11: ...al Time Analysis software Integrated with the control software Real Time Analysis RTA performs base calling and assigns a quality score to each base for each cycle For more information see Real Time A...

Страница 12: ...rument computer has a storage capacity of over 2 7 TB per flow cell Data from flow cell A is stored on the D drive and data from flow cell B is stored on the E drive At the end of each imaging cycle f...

Страница 13: ...126 275 2 x 100 101 8 7 8 101 225 2 x 75 76 8 7 8 76 175 2 x 50 51 8 7 8 51 125 2 x 35 36 8 7 8 36 95 Dual Indexed Single Read 1 x 125 126 8 8 142 1 x 75 76 8 8 92 1 x 50 51 8 8 67 1 x 35 36 8 8 52 R...

Страница 14: ...ovided in the TruSeq v3 cluster kits are not compatible with Nextera libraries When sequencing Nextera libraries use the Index 1 sequencing primer HP12 and Read 2 sequencing primer HP11 provided in th...

Страница 15: ...oftware operation 7 Open HCS using the shortcut icon on the desktop When the software has initialized the Welcome screen opens and the Initialized icon appears in the bottom right corner of the screen...

Страница 16: ...henticating to Illumina LIMS u LIMS Password The password used when authenticating to Illumina LIMS 3 Select OK to save your work and close the Menu Options window Select Cancel to close without savin...

Страница 17: ...racks positions containing PW1 Instrument wash Collecting and measuring waste volumes Conical tubes 50 ml self standing optional Corning catalog 430921 Storing flow cells Disposable gloves powder free...

Страница 18: ...en a run on the adjacent flow cell is in progress For more information see Stagger Runs on Flow Cell A and Flow Cell B on page 52 Run Types for HiSeq v4 Chemistry The following table shows types of se...

Страница 19: ...haw IRM and USM in a room temperature deionized water bath for about 90 minutes Thaw CRM in a separate water bath NOTE Always replace your gloves after handling CRM 3 Invert each bottle to mix 4 Set I...

Страница 20: ...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 Thaw...

Страница 21: ...pace Sequence Hub the Zip BCL files option is selected by default NOTE The Bin Q Scores setting is enabled by default to reduce required storage space This setting groups quality scores over a wider r...

Страница 22: ...f cycles for Read 1 and Read 2 if applicable The number of cycles performed in a read is 1 more cycle than the number of cycles analyzed For example to perform 125 cycles for Read 1 enter 126 4 For th...

Страница 23: ...e software guides you through these steps in a series of screens on the Pre Run Setup tab Load SBS Reagents 1 Invert each reagent bottle several times to mix CAUTION Handle the bottle of CRM last afte...

Страница 24: ...slot on the top end of the groove Make sure that the handle rests securely in the slot 3 Slide the reagent rack out of the reagent compartment using the rack handle 4 Remove the caps from each reagen...

Страница 25: ...sis Mix 11 FLM2 Fast Linearization Mix 2 13 AMS Fast Amplification Mix 14 FPM Fast Amplification Premix 15 FDR Fast Denaturation Reagent contains formamide 16 HP11 Read 2 Sequencing Primer If you load...

Страница 26: ...om the flow cell to prevent alignment drift 6 Slowly move the flow cell lever to position 1 to engage the vacuum and secure the flow cell When the flow cell lever is blinking green the vacuum is engag...

Страница 27: ...ing pump tubing 2 Place each waste tube into a separate empty 15 ml tube Waste is collected and measured when priming is complete 3 Select Start Prime Monitor priming progress from the Prime screen 4...

Страница 28: ...es Clean the Flow Cell 1 Remove the clustered flow cell from the container using a pair of plastistats 2 Rinse the flow cell with laboratory grade water Dry with a lens cleaning tissue 3 Fold an alcoh...

Страница 29: ...the flow cell and gaskets are properly installed and the manifold is engaged 1 Select Position 5 from the drop down list 2 Confirm the following default values u Volume 250 u Aspirate Rate 250 u Disp...

Страница 30: ...tup the first base confirmation dialog box opens automatically after imaging of the first cycle is complete The run pauses at this step 1 Review the First Base Report from the confirmation dialog box...

Страница 31: ...uctions see Perform a Maintenance Wash on page 45 If the instrument has been idle for one day or more perform a water wash before beginning a new sequencing run 1 From the Welcome screen select Wash W...

Страница 32: ...measure the delivered volume Positions Total Delivered Volume Per Lane Delivered Volume Eight SBS positions 32 ml 4 ml Eight SBS positions and 10 paired end positions 72 ml 9 ml 12 Unwrap the waste t...

Страница 33: ...ce specifications Staggering Runs You can 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 A...

Страница 34: ...and SRE and then preparing ICB with LFN and EDP Using the 200 cycle SBS kit requires splitting ICB into 2 portions 1 each for Read 1 and Read 2 during preparation For dual indexing the ICB preparatio...

Страница 35: ...LFN tube with ICB to make sure that all LFN is transferred 5 Add 1 1 ml EDP to the ICB and LFN solution 6 Return the unused portion of EDP to 25 C to 15 C storage 7 Cap the bottle containing EDP ICB a...

Страница 36: ...reagents that are appropriate for the run type and library type Run Type Library Type Index 1 i7 Index 2 i5 Single indexed All libraries except Nextera HP8 Nextera libraries HP12 Dual indexed paired e...

Страница 37: ...nfiguration tab The software guides you through each screen to specify BaseSpace Sequence Hub connectivity enter consumable IDs select indexing options and record other parameters Integration Screen T...

Страница 38: ...Setup screen records information about the flow cell used for the run 1 Scan or enter the flow cell ID barcode number of the flow cell to be sequenced The flow cell ID determines flow cell type and re...

Страница 39: ...lex Sequencing Primer Box or TruSeq Dual Index Sequencing Primer Box c PE turnaround TruSeq PE Cluster Kit v3 6 Optional Select the Use Existing Recipe checkbox to use a custom recipe Sample Sheet Scr...

Страница 40: ...nt compartment door 3 Raise the sippers for the SBS reagent rack as follows a Pull the sipper handle towards you and then raise it b Release the handle into the slot on the top end of the groove Make...

Страница 41: ...matching label color Position Reagent Description 17 HP8 or HP12 Index 1 i7 Sequencing Primer Mix 18 HP3 Denaturation Solution 19 HT2 Wash Buffer Table 3 Single Indexed Run Position Reagent Descriptio...

Страница 42: ...flow cell which indicates flow direction points towards the instrument 4 Gently slide the flow cell towards the top and right guide pins until it stops Figure 12 Flow Cell Positioned Against Top and R...

Страница 43: ...s persist remove the flow cell repeat the cleaning steps and reload the flow cell Position Tubingand Start Prime 1 Remove the 8 waste tubes for the appropriate flow cell from the waste container Figur...

Страница 44: ...urface of the flow cell holder with an alcohol wipe or a lint free tissue moistened with ethanol or isopropanol Do not allow alcohol to drip into the vacuum holes or around the manifolds 4 Dry the sta...

Страница 45: ...ell 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 49 5 Wait about five seconds and then slowly move the flow cell l...

Страница 46: ...ct Start to begin the sequencing run Monitor the Run 1 Monitor run metrics from the Run Overview screen Figure 16 Run Overview Screen A Progress bar Monitor how many cycles have been completed B Fluid...

Страница 47: ...use HP7 Thaw Paired End Reagents 1 Remove the following reagents from 25 C to 15 C storage u For non indexed or single indexed runs AMX2 APM2 AT2 BMX HP3 HP7 or HP11 HT2 LMX2 and RMR u For dual index...

Страница 48: ...every 10 cycles of sequencing to be performed in Read 2 Reagent Volume per 10 Cycles Storage ICB 4 57 ml 2 C to 8 C LFN 0 6 ml 25 C to 15 C EDP 0 11 ml 25 C to 15 C For more information on calculatin...

Страница 49: ...nto the slot on the bottom end of the groove Load ICB for Read 2 1 Raise the sippers for the SBS reagent rack as follows a Pull the sipper handle towards you and then raise it b Release the handle int...

Страница 50: ...ental health and safety information see the SDS at support illumina com sds html Perform a Water Wash A water wash is required after each sequencing run to wash the system and check fluidics A mainten...

Страница 51: ...e water wash Positions Approximate Run Time Eight SBS positions 20 minutes Eight SBS positions and 10 paired end positions 60 minutes 11 When the wash is complete measure the delivered volume Position...

Страница 52: ...after a run when a maintenance wash is an optional alternative to a water wash The Load Gasket screen opens with a maintenance wash every 10 days Replace the 8 port gaskets in the front manifold and...

Страница 53: ...nce wash solution from a previous run load the instrument with solution as follows a Replenish the stored solution and invert to mix Replenish no more than two times after the original use b Load the...

Страница 54: ...However the delivered volume for each position varies so the bottles and tubes contain different volumes when the wash is complete 22 Unwrap the waste tubes and return them to the waste container Idle...

Страница 55: ...s Use a low lint lab tissue to dry the stage if necessary 4 Load 10 ml laboratory grade water in each position in the reagent racks and then lower the sippers 5 Turn off the instrument 6 To restart th...

Страница 56: ...reload the flow cell Flow cell lever is blinking orange Vacuum is being provided but is inadequate Remove the flow cell and repeat the cleaning steps Make sure that the gaskets are present and well s...

Страница 57: ...resuming a stopped run select the appropriate normal stop option that allows the run to resume Stop Option Real Time Analysis RTA Option Able to Resume Normal Stop End of Lane Chemistry Keep As Is Ye...

Страница 58: ...wing steps to resume a run that was stopped using a normal stop option with an RTA option that allows the run to be resumed NOTE If the adjacent side is performing cluster generation or paired end che...

Страница 59: ...s use the Change Reagents feature to pause the run and replenish reagents NOTE Priming is not required 1 From the Run Overview screen select Pause to open the pause menu 2 Select Change Reagents 3 Sel...

Страница 60: ...ycles See Thaw SBS Reagents on page 12 2 Label 7 250 ml bottles with the position number and reagent name as follows u 1 IRM u 3 USM u 4 SB1 u 5 SB2 u 6 SB2 u 7 CRM u 8 SB3 3 Add the following volume...

Страница 61: ...o the ICB and LFN solution 4 Return the unused portion of EDP to 25 C to 15 C storage 5 Cap the bottle containing EDP ICB and LFN and invert to mix 6 Set aside on ice Primer Rehybridization A rehybrid...

Страница 62: ...or Nextera libraries use HP10 from the TruSeq Dual Index Sequencing Primer Box Rehyb Kit Name Workflow Instructions TruSeq v2 cBot Multi Primer Rehyb Kit Catalog GD 304 2001 Read 1 Primer Rehyb on a T...

Страница 63: ...low cell defined as 1 field of view by the camera For each tile that is analyzed RTA produces a set of quality scored base call files and filter files as primary output Other files support generation...

Страница 64: ...cluster Quality scoring Assigns a quality score to every base call Template Generation Template generation locates and defines the position of each cluster in a tile using X and Y coordinates The tem...

Страница 65: ...he call for that cluster in that cycle Base calling on the HiSeq 2000 using RTA begins after cycle 12 Base calling determines a base A C G or T for every cluster of a given tile at a specific cycle Ba...

Страница 66: ...ace requirements without affecting accuracy or performance of downstream applications Q score binning contributes to the efficiency of analysis processes and data transfer requirements associated with...

Страница 67: ...Inline controls are generated only for TruSeq library preparation methods Index count After the index reads are complete The index count per lane is generated when a sample sheet is provided Sequenci...

Страница 68: ...relative to the template SubTileOffsets txt Contains the measured shift for each quadrant of each image relative to the frame of reference Data Intensities Offsets Phasing files Contains empirical pha...

Страница 69: ...e prompts you to enter the path for the run folder By default the folder is named using the following format YYMMDD_ Computer Name _ Run Number _ Flow Cell Side Flow Cell ID Example 110114_SN106_0716_...

Страница 70: ...the top surface of the flow cell the second swath and the seventh tile Thumbnail Images You can configure the control software to generate thumbnail images in jpg file format Thumbnail images are gene...

Страница 71: ...rs status bar 2 compartments 2 configuration file 61 connecting USB cables 8 consumables Illumina 5 user supplied 10 control lanes 15 31 cross contamination preventing 46 cross talk 58 custom recipes...

Страница 72: ...31 indexing number of cycles 11 26 required reagents 12 29 indexing reagent positions 17 34 indexing reagents HiSeq v4 12 TruSeq v3 29 indexing scheme 15 32 initializing software 8 initializing softw...

Страница 73: ...k 16 33 reagent racks 3 reagent volumes split SBS kits 53 54 reagents changing midrun 52 handling post run 23 43 shorter runs 53 reagents SBS creating 2 sets 53 TruSeq v3 workflow 27 reagents storing...

Страница 74: ...nical assistance 68 temperature reagent chiller 3 temporary folders 62 thumbnails 61 63 saving 14 31 tiles 56 62 TruSeq Controls tab 60 TruSeq Dual Index Sequencing Primer Box purpose 7 required reage...

Страница 75: ...06 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 South Korea 82 80 234 53...

Страница 76: ...lifornia 92122 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...

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