AlanFung:Protocol/Genome Analyzer: Difference between revisions

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==Preparing Reagents for the Genome Analyzer==
==Genome Analyzer User Guide==
==Genome analyzer Reagent Compartment & Reagent Position==
==SCS v2.4==
[[Image:Reagent Position.jpg]]
*start up scsv2.4
*Position of each reagent in the Genome Analyzer
===setting the run parameters===
  NOTE:LABEL ALL REAGENT CONTAINERS WITH THE APPROPRIATE NUMBER
*click <view>
'''Position    Reagent                Volume              Container'''
*clear box next to show on start up
1          Incorporation Mix      Varies w/ cycle #  50ml amber tube
*from the data collection screen
2          DI H2O                                    250ml bottle
*edit|run parameters|GA Settings / RunBrowser Settings
3          Scanning Mix          50ml                50ml tube
===GA Setting===
4          High Salt Buffer                          250ml bottle
*specify the network location for run results
  5          Incorporation buffer  150ml              250ml bottle
*set perferences for real time analysis results
6          Cleavage Mix          50ml                50ml tube
*set settings for auto calibration
7          Cleavage Buffer        70ml                250ml bottle
  *delete images (select this option to delete images from the genome analyzer computer after analysis
====Validation strings====
*edit if you are using a customized identification system
====Use auto calibration====
*select this option to enable the auto calibration feature
*refer to page 29 for a description of the auto calibration feature
*note: if this option is cleared, focus calibration must be performed manually as described in appendix B
*
===Run Browser Settings===
*runbrowzer reports, specify which runbrowser reports you want to show and export
  if selected the first cycle is generated by the software
*enable runbrowser report emails to sens reports to the email addresses specified
*after you have finished selecting the settings in the run parameters window, click <save & continue>/<cancel & continue>/<reset default configs>


==Scanning Mix==
===initialize the software===
'''Required Materials'''
*click the manual control/setup tab
*Scanning Reagent
*perform any operation to trigger the genome analyzer initialization
*Scanning Buffer
*Filter Unit
*Scanning Mix Tube
*Add the Scanning Reagent vial to the Scanning Buffer on ice
*Rinse the empty vial into the Scanning Buffer three times with 1 ml Scanning Buffer
*Invert the Scanning Buffer several times to mix
*Leave on ice for 10 minutes
*Invert several times to mix. Check to ensure that the solids are completely dissolved
*Filter the Scanning Mix with the provided Scanning Mix Filter Unit
*Pour 50 ml of the Scanning Mix into the 50 ml Scanning Mix Tube
'''*If you are running more than 26 cycles on the Genome Analyzer, you must top off the Scanning Mix at the end of the first day of the sequencing run. The tube should be approximately one-third empty after running for six or seven hours'''
*Record the weight of the reagent in the lab tracking worksheet
*Leave the remaining solution in the Filter Unit at 4C for later cycles
*Keep the Scanning Mix on ice until ready to load


==Incorporation Mix==
===run window===
'''Required Materials'''
====recipe tab====
'''*The volumes of the dNTPs in the various kits are specific to that kit'''
*hover over a stop to show the parameters specific to that step
'''*Do not mix and match dNTP vials form different cycle number kits'''
====image cycletab====
  *dNTPs: ff-dATP, ff-dCTP, ff-dGTP, ff-dTTP
  white tile are queued for imaging
  2 each for the 36-cycle kit
  blue tiles haven been imaged
  1 each for the 18-cycle and 26-cycle kits
  gray tiles are not defined in the current run
*10X Incorporation Buffer v2.0 (5 ml)
right side of the window shows the photographs being taken of the current tile
*dNTP Mix Diluent
*SBS Polymerase
  2 for the 36-cycle kit
  1 for the 18-cycle and 26-cycle kits
*Magnesium
*Plastic Syringe
*0.22 μm Cellulose Acetate Syringe Filter
*50 ml Amber Tube
*The volumes of dNTP Diluent, 10X Incorporation Buffer, and Magnesium vary depending on the sequencing kit you are using
*Refer to the table below for correct volumes
'''Reagents                      18-Cycle          26-Cycle          36-Cycle'''
  dNTP Diluent                  21ml              29ml              43ml
  10X Incorporation Bufferv2.0  2.5ml            3.5ml            5ml
Magnesium                      150ul            210ul            300ul
*Transfer the correct volume of dNTP Diluent to a 50 ml tube
*Refer to the table of volumes for the sequencing kit you are using
*Add the correct volume of 10X Incorporation Buffer v2.0
*Refer to the table of volumes for the sequencing kit you are using.
*Vortex for 10 seconds.
'''*NOTE Save the remaining 10X Buffer for the Incorporation Buffer'''
*Pipette the contents of every ff-dNTP vial into the Incorporation Mix
*Rinse each of the ff-dNTP tubes into the dNTP mix with 1 ml of Incorporation Mix
*Invert several times to mix
*Filter the dNTP mix through a 0.22 μm membrane filter
*Transfer the mix into the supplied 50 ml amber tube labelled “dNTP Mix Tube.”
*Place the dNTP mix on ice.
*Remove the Magnesium solution from ice and allow it to thaw at room temperature for 10 minutes.
*Vortex to mix.
*Centrifuge to 10,000 xg for 2 minutes at 22°C.
*Ensure that the vial contains no undissolved material or ice.
*Add the correct volume of Magnesium solution to the dNTP mix on ice.
*Refer to the table of volumes for the sequencing kit you are using.
*Invert several times to mix and return to ice.
*Centrifuge the SBS Polymerase to 10,000 xg for 1 minute at 22°C.
'''*NOTE Two vials of SBS Polymerase are used in the 36-Cycle Kit. Only one vial of SBS Polymerase is used in the 18-Cycle and 26-Cycle Kits'''
*Add the contents of each polymerase vial into the dNTP mix on ice.
*Rinse each empty vial into the dNTPs mix three times with 1 ml dNTP mix
*Record the weight of the reagent in the lab tracking worksheet.
*Invert the dNTP mix several times to mix and immediately return to ice.
*Keep on ice until ready to load.


==Incorporation Buffer==
====temperature tab====
Required Materials
*for monitor various run temperatures
*10X Incorporation Buffer v2.0
*Incorporation Buffer Diluent
*Add 15ml of 10X Incorporation Buffer v2.0 to the 150ml bottle of Incorporation Buffer Diluent
*Record the weight of the reagent in the lab tracking worksheet


==High Salt Buffer==
===manual control/setup window===
*Invert the High Salt Buffer several times to mix it before loading it on the Genome Analyzer
====manual control====
*Record the weight of the reagent in the lab tracking worksheet
*settings used for manual focus calibration
====pump tab====
command
to
solution
volume(ml)
aspirate rate(ml/min)
dispense rate(ml/min)
====recipe viewer====
*genome analyzer recipes found can be found in
<install directory>\datacollection_v<#>\bin\recipes
*to access the recipe view click file|open recipe from the toolbar
*the following information is listed for each recipe
recipe
subfolder
cycle
read end
multiplexing
service
version
comment
file status
====editing cycle number====
*click edit on the recipe viewer toolbar
===reagent tracking===
*provides email notifications and warnings when reagents are getting low and need replacing
*beneficial when performing runs longer than 36 cycles
*click reagents from the toolbar at the top of the data collection software screen
====current volumes====


==1X Cleavage Buffer==
====reset volumes====
  Required Materials
====reagent barcodes====
*to track reagents for any given run
barcodes are stored in the Reagentsld.xml file in the runfolder
*if the remaining reagents are not to be used click clear
====configure tracking====
*to enable or disable reagent tracking features select reagents|configure tacking
enable volume tracking
autostop when empty
display and email warnings
===disk space checking===
*during the run at the end of each cycle  the software checks available disk space
*if there is insufficient disk space to accommodate one cycle the software pauses the run and a dialog box appears to tell you how much dish space remains. when sufficient disk space is available the dialog box closes and the run automatically resumes
===image control===
zoom in
move the zoomed image
zoom out
modify the color display
view or hide the center mark
see intensity values
see focus quality and uniformity
show region of interest
  save the image
==run folders==
*each run on the genome analyzer generates a run folder that contains data files and log files specific for that run
*when you start a run the software prompts you to enter the folder name for it
by default the folder is named YYMMDD_<Workstation Name>_<Run #>
*the run folder name may not contain any spaces
====run folder path====
*the name and location of this folder are set in <install location>\bin\config\rcmconfig in this line
<Run Path="D:\Runs" />
* to change the run folder path wither change this line or select run|select run folder root in the software
 
==Cluster Station==
==GA Pipeline 1.4==
*[[Media: Pipeline_CASAVA_User_Guide_15003807_A.pdf|Manual]]

Latest revision as of 02:48, 24 July 2009

Genome Analyzer User Guide[edit]

SCS v2.4[edit]

  • start up scsv2.4

setting the run parameters[edit]

  • click <view>
  • clear box next to show on start up
  • from the data collection screen
  • edit|run parameters|GA Settings / RunBrowser Settings

GA Setting[edit]

  • specify the network location for run results
  • set perferences for real time analysis results
  • set settings for auto calibration
*delete images (select this option to delete images from the genome analyzer computer after analysis

Validation strings[edit]

  • edit if you are using a customized identification system

Use auto calibration[edit]

  • select this option to enable the auto calibration feature
  • refer to page 29 for a description of the auto calibration feature
  • note: if this option is cleared, focus calibration must be performed manually as described in appendix B

Run Browser Settings[edit]

  • runbrowzer reports, specify which runbrowser reports you want to show and export
if selected the first cycle is generated by the software
  • enable runbrowser report emails to sens reports to the email addresses specified
  • after you have finished selecting the settings in the run parameters window, click <save & continue>/<cancel & continue>/<reset default configs>

initialize the software[edit]

  • click the manual control/setup tab
  • perform any operation to trigger the genome analyzer initialization

run window[edit]

recipe tab[edit]

  • hover over a stop to show the parameters specific to that step

image cycletab[edit]

white tile are queued for imaging
blue tiles haven been imaged
gray tiles are not defined in the current run
right side of the window shows the photographs being taken of the current tile

temperature tab[edit]

  • for monitor various run temperatures

manual control/setup window[edit]

manual control[edit]

  • settings used for manual focus calibration

pump tab[edit]

command
to
solution
volume(ml)
aspirate rate(ml/min)
dispense rate(ml/min)

recipe viewer[edit]

  • genome analyzer recipes found can be found in
<install directory>\datacollection_v<#>\bin\recipes
  • to access the recipe view click file|open recipe from the toolbar
  • the following information is listed for each recipe
recipe
subfolder
cycle
read end
multiplexing
service
version
comment 
file status

editing cycle number[edit]

  • click edit on the recipe viewer toolbar

reagent tracking[edit]

  • provides email notifications and warnings when reagents are getting low and need replacing
  • beneficial when performing runs longer than 36 cycles
  • click reagents from the toolbar at the top of the data collection software screen

current volumes[edit]

reset volumes[edit]

reagent barcodes[edit]

  • to track reagents for any given run
barcodes are stored in the Reagentsld.xml file in the runfolder
  • if the remaining reagents are not to be used click clear

configure tracking[edit]

  • to enable or disable reagent tracking features select reagents|configure tacking
enable volume tracking
autostop when empty
display and email warnings

disk space checking[edit]

  • during the run at the end of each cycle the software checks available disk space
  • if there is insufficient disk space to accommodate one cycle the software pauses the run and a dialog box appears to tell you how much dish space remains. when sufficient disk space is available the dialog box closes and the run automatically resumes

image control[edit]

zoom in
move the zoomed image
zoom out
modify the color display
view or hide the center mark
see intensity values
see focus quality and uniformity 
show region of interest
save the image

run folders[edit]

  • each run on the genome analyzer generates a run folder that contains data files and log files specific for that run
  • when you start a run the software prompts you to enter the folder name for it
by default the folder is named YYMMDD_<Workstation Name>_<Run #>
  • the run folder name may not contain any spaces

run folder path[edit]

  • the name and location of this folder are set in <install location>\bin\config\rcmconfig in this line
<Run Path="D:\Runs" />
  • to change the run folder path wither change this line or select run|select run folder root in the software

Cluster Station[edit]

GA Pipeline 1.4[edit]