Sequencing Run Set Up (Instrument Procedure Wizard)

3.3.2

Once the lab materials for the sequencing Run are available and the appropriate Run parameters and processing options have been chosen, the GS FLX+ Instrument is ready to begin a new sequencing Run. The set up is done in the Instrument Procedure Wizard. To launch the Instrument Procedure Wizard, click the ‘Start’ button in the Global Action area of the GS Sequencer application window. (Figure 8).

Figure 8: Start button

The Instrument Procedure Wizard will present a series of questions requesting information for the instrument procedure (a sequencing Run in this instance) that you are setting up. Each screen has a set of navigation buttons at the bottom (‘Back’, ‘Next’, ‘Cancel’, ‘Help’), so the user can modify the Run parameters as needed before starting, or access the Software Manual. There is an additional ‘Door Unlock’ button that can be used in the event you need to open the camera door, on the instrument. After all the parameters have been selected (as shown in the following figures) and there are no validation warning messages (see below), the ‘Start’ button in the last Instrument Procedure Wizard window becomes enabled and can be clicked to launch the sequencing Run.

The succession of questions in the Instrument Procedure Wizard to set up a sequencing Run is described below. For procedures other than sequencing Runs, see Section 3.3.3.

1.	Choose a Procedure (Figure 9) – Choose the procedure to run. The option listed second is the normal sequencing Run. A pre-wash must always be performed before a sequencing Run, and the last option is used to run any special wash or custom instrument script; these are described in Section 3.3.3.

Figure 9: GS Sequencer Instrument Procedure Wizard – ‘Choose a procedure’ window

Enter IDs and barcodes (Figure 10) This form requests you enter the product IDs or barcodes of materials that are associated with this Run. The only required ID is the six-digit PTP ID found as a barcode stamped on the device. The list can only contain one PTP ID so no other six-digit ID can be added to the list.

If a barcode scanner is used, the IDs will automatically be added to the list located below the entry field. If the IDs are entered with the keyboard, press the ‘enter’ key or click the plus button to add each item to the list. Clicking the minus button will remove the selected product ID from the list.

b.	If a PTP ID lookup service (e.g., a LIMS) is available, the Run information associated with that ID will be applied. (See the GS FLX+ System SysAdmin Guide for configuring the PTP ID lookup service.)

c.	The ‘Next’ button is enabled once a valid PTP ID is entered. If the Run parameters were applied using the PTP ID lookup service, the next page will be the Run Comments page (see step 9, below); otherwise the next page will ask for the Run name and Run Group (next step).

Figure 10: GS Sequencer Instrument Procedure Wizard – ‘Enter IDs and barcodes’ window

Enter Run name and Run Group (Figure 11) – Type in a Run name in the Run name entry field, and select a Run Group from the Run Group list. The Run name must contain only 0-9, a-z, underscore, or hyphen characters. This Run name will be part of the directory name used to store the sequencing Run results in the data file system; see step 9 below for more details on Run directory names. The default Run Group is the operator’s default Run Group, but any Run Group in the list can be selected. See section 3.7.2 for more information about Run Groups.

Figure 11: GS Sequencer Instrument Procedure Wizard – ‘Enter Run name and Run Group’ window

4.	Choose sequencing kit [GS FLX+ Instrument only] (Figure 12) – Choose the sequencing kit type that will be used for the sequencing Run.

Figure 12: GS Sequencer Instrument Procedure Wizard – ‘Choose sequencing kit’ window

When using a non-upgraded Genome Sequencer FLX Instrument this screen of the Instrument Procedure Wizard is skipped, and theXLR70 kit is selected automatically.

5.	Choose PicoTiterPlate type (Figure 13) – Specify the bead loading gasket used to load the DNA sample onto the PTP device. The choices of gaskets presented match the sequencing kit choice made in the previous step.

Figure 13: GS Sequencer Instrument Procedure Wizard – ‘Choose PicoTiterPlate type’ window

6.	Choose number of cycles [GS FLX Titanium Sequencing Kit XLR70 only] (Figure 14) – Choose the number of cycles of nucleotide flows for the sequencing Run. The greater the number of cycles chosen, the greater the final read length.

a.	Using the GS FLX Titanium Sequencing Kit XLR70:

i.	100 cycles will produce reads of approximately 250 bases

ii.	150 cycles will produce reads of approximately 375 bases

iii.	200 cycles will produce reads of approximately 500 bases

Figure 14: GS Sequencer Instrument Procedure Wizard – ‘Choose number of cycles’ window (GS FLX Titanium Sequencing Kit XLR70 Only)

When using the GS FLX Titanium Sequencing Kit XL+ this screen of the Instrument Procedure Wizard is skipped, and the number of cycles is automatically set to 400 cycles.

7.	Choose Run Processing Type (Figure 15) – There are up to four different options to launch a data processing job on the GS FLX+ Instrument:

a.	None: only the image capture is done on the GS FLX+ Instrument, and no processing of this data is carried out as part of the sequencing Run. Image and signal processing must be done off-instrument, after the Run.

b.	Image processing only: the image processing step but not the signal processing step of data processing is carried out as part of the sequencing Run. Signal processing must be done off-instrument, after the Run.

c.	Full processing for shotgun or paired end: both the image and signal processing steps are performed on the instrument, as part of the sequencing Run. This option is appropriate for all library types except Amplicon libraries.

d.	Full processing for Amplicons: both the image and signal processing steps are performed on the instrument, as part of the sequencing Run. This option is appropriate for Amplicon libraries sequenced with the GS FLX Titanium Sequencing Kit XLR70 only.

Figure 15: GS Sequencer Instrument Procedure Wizard – ‘Choose Run Processing type’ window

Data Processing Options are discussed in detail in Part B, Section 1 of this Manual.

The Full processing for Amplicons Run processing option is not available for use with the GS FLX Titanium Sequencing Kit XL+.

Request data backup (Figure 16) – If the ‘Backup the Run and Processor data upon completion’ checkbox is checked (which it is by default), the backupScript.sh script will be called at the end of the sequencing Run to archive the data to a data storage location specified in the script. (The backupScript.sh and data management are discussed in more detail in Part B, Section 5 of this manual.)

a.	The backup prompt can be configured in the gssequencer.xml configuration file. The default configuration is:

    <Application id="BackupApp">

        <Command>backupScript.sh</Command>

        <Description>Backup Run upon completion.</Description>

    </Application>

If the <Description></Description> element value is empty, this Instrument Procedure Wizard window will not appear and the backup will always occur at the end of the Run. If the <Command></Command> element value is empty, this Instrument Procedure Wizard window will not appear and the backup script will not run at the end of the Run.

Figure 16: GS Sequencer Instrument Procedure Wizard – ‘Request data backup’ window

In addition to the backupScript.sh script, an optional, user-provided script, postAnalysisScript.sh, can be called at the end of the data processing, and will perform any commands listed (see Part B, Section 5). For example, the postAnalysisScript.sh script can be modified to push the contents of a partially processed D_imageProcessingOnly directory to a DataRig (or cluster) and begin the signal processing on the designated computer.

9.	Run comments (Figure 17) – This window allows operators to supply any comments about the Run. A tab is always present to assign comments to the Run. If the gasket specified contains 2 or more regions, additional tabs are displayed allowing comments to be associated with individual regions.

Figure 17: GS Sequencer Instrument Procedure Wizard – ‘Run comments’ window

10.	Confirmations [GS FLX+ Instrument only] (Figure 18) – This window prompts operators to confirm that the waste container has been emptied (to avoid spillage) and that the Apyrase sleeve has been installed into the chiller for Runs using GS FLX Titanium Sequencing Kit XLR70.

Figure 18: GS Sequencer Instrument Procedure Wizard – ‘Confirmations’ window

When using a non-upgraded Genome Sequencer FLX Instrument this screen of the Instrument Procedure Wizard is skipped.

When using a GS FLX Titanium Sequencing Kit XL+ the “Apyrase sleeve has been installed into the chiller” confirmation does not appear.

11.

Insert new PicoTiterPlate Device (Figure 19) – The final window of the Instrument Procedure Wizard provides a summary of the sequencing Run data entered and displays the name of the Run script (provided with the system) which will be used to carry out the Run. If any of the Run data is incorrect, the back button can be used to navigate to previous windows and correct it. Once all the necessary data has been entered and the PTP device has been inserted (as described in the Sequencing Method Manual), the ‘Start’ button is activated and the GS FLX+ Instrument is ready to begin a new sequencing Run. Clicking on the Start button will initiate the sequencing Run.

Figure 19: GS Sequencer Instrument Procedure Wizard – ‘Insert new PicoTiterPlate device’ window

If the ‘Start’ or ‘Next’ button is not enabled in the Instrument Procedure Wizard, look for one or more validation messages above the buttons specifying the nature of the problem. Below is a list of the problems that may occur and how to resolve them:

Validation message	Cause	Resolution
Camera door must be closed.	The camera door sensor detected the camera door was not closed.	Close the camera door. If the door is already closed, contact your Roche Representative.
Insert a Cassette for this procedure. Or Insert a Pre-wash Cassette for this procedure. Or Insert a Reagents Cassette for this procedure.	There is a sensor that detects the presence of a cassette and its type. The sensor did not detect a correctly seated cassette.	For a sequencing Run or Maintenance Wash, insert a Reagent Cassette. For a Pre-wash procedure, ensure the Pre-wash cassette is inserted. If the correct cassette is already inserted contact your Roche Representative.
Sipper must be lowered.	There is a sensor that detects the position of the sipper. Before a procedure can start, the sipper must be lowered into the cassette.	Lower the sipper. If the sipper is already lowered contact your Roche Representative.
Run script is not defined.	In choosing a custom Run script, a path was supplied that did not point to a valid script. This can happened if you manually type in the path to the script instead of selecting it from the file chooser dialog.	Go to the custom script selection page (see Section 3.3.3) and verify that the script path is referring to a valid Run script .icl file at that location. Use the script file chooser button to ensure that the correct path is supplied.
A valid instrument icl script file must be chosen. Or The instrument icl script file cannot be found. Make sure the file % exists.	In choosing a Run script a valid script must be entered in that wizard page. If the PTP ID lookup feature is used to obtain the script the script reference must be valid.	Ensure the correct script name has been provided in the Run script dialog or in the information returned from the PTP ID lookup service.
A Run Group must be selected.	The Configuration panel ensures the operator default run group is correct but it can be made invalid if the sequencer configuration file is edited or the PTP ID lookup feature returned an invalid Run group id.	Ensure the sequencer configuration has a default run group associated with the operator or the information returned from the PTP ID lookup service is correct.
Unlocking camera door...	The camera door unlocking procedure takes a few moments to run and complete. Another procedure cannot be started until this completes.	Wait for the unlock procedure to complete. Once the next procedure is started, the door will relock.
Connection to the instrument services has failed. Or The instrument services failed to start. Or The instrument services have failed.	Communication to the backend services has failed or failed to start. These conditions are unusual since the Instrument Procedure Wizard cannot be accessed until the instrument is in an available state.	In rare conditions, the instrument backend service may fail to respond. To resolve the problem, exit the GS Sequencer application and restart the backend services with ‘systemStart’. If the problem persists contact your Roche Representative.
The instrument services are starting...	The backend services have been restarted. This condition is unusual since the Instrument Procedure Wizard cannot be accessed until the instrument is in an available state.	This startup process takes a few minutes; wait for the backend to restart and return to the available state.
An unexpected instrument state is preventing the start of this procedure. Or A maintenance wash is unexpectedly running. Or A pre-wash is unexpectedly running. Or A sequencing Run is unexpectedly running.	The instrument is in an unexpected state. These conditions are unusual since the Instrument Procedure Wizard cannot be accessed until the instrument is in an available state.	Let any running process complete and perform a system restart. To restart, exit the GS Sequencer application and restart the backend services with ‘systemStart’. If the problem persists contact your Roche Representative.
Connection to the data processor has failed	The data processing service is not running. This is a warning so a procedure can be started, but no data processing of a Run will occur.	Exit the GS Sequencer application. Run systemStart then start the GS Sequencer again. If the problem persists contact your Roche Representative.
There is not enough space available to complete this sequencing Run	The drive where $RIGDATA is located does not have enough free space to store the Run.	Free up some disk space on the drive by removing data sets that are no longer needed.
Could not get Run processing types.	The GS Run Processor is not running.	A Run can be started, but no Run processing types can be selected. Install and start the GS Run Processor. Ensure the GS Run Processor is started
A PicoTiterPlate ID must be added to the list. The PicoTiterPlate ID consists of only digits 0-9 and must be six (6) digits in length.	An invalid product id was entered on the products id view. The id can only include the numeric digits 0-9 and must be 6 digits in length.	Before you are allowed to continue you must enter a valid product id. At minimum, a valid PicoTiterPlate ID must be entered.
The 454 Life Sciences product ids have 11 digits for the material, 8 digits for the lot number, 2 digits for the month and 4 digits for the year. The string length is 25 digits and the 1st character in the year is always 2.	An invalid product id was entered on the products id view. The id can only include the numeric digits 0-9. The length of the id must be 25 digits in length with the following additional requirements: it must start with 11 digits for the material, followed by 8 digits for the lot number, then followed by 2 digits for the month & 4 digits for the year. The year portion must start with the digit ‘2’.	Before you are allowed to continue you must enter a valid product id. At minimum a valid PicoTiterPlate ID must be entered.
The Run name is used to create the file storage name, so it must contain only 0-9, a-z, underscores or hyphens.	An invalid run name was entered or is missing. The run name can only include the characters A-Z, a-z, 0-9, underscores or hyphens.	Before you are allowed to continue you must enter a valid Run name.
The Operator ID is used to create the file storage name, so it must contain only 0-9, a-z, underscores or hyphens. The ID must also be unique.	An invalid operator id was provided. The Configuration panel ensures the operator ID is correct but it can be made invalid if the sequencer configuration file is edited or the PTP ID lookup feature returned an invalid Operator ID. The ID can only include the characters A-Z, a-z, 0-9, underscores or hyphens.	Before you are allowed to continue you must provide a valid Run name. Ensure the sequencer configuration file contains a valid Operator ID or, if performing the PTP ID lookup, ensure the service is returning a valid operator ID.
The email must be a valid email address.	An invalid email address was provided. The Configuration panel ensures the operator email is correct but it can be made invalid if the sequencer configuration file is edited or the PTP ID lookup feature returned an invalid email address. The email address can only include the characters A-Z, a-z, 0-9, underscores, hyphens and the ‘@’ symbol. There must be at least 1 valid character before and after the ‘@’ symbol.	Before you are allowed to continue you must provide a valid email address name. Ensure the sequencer configuration file contains a valid email address for the signed in Operator, or if performing the PTP ID lookup, ensure the service is returning a valid email address.
Calculating space requirements for this procedure...	This is an information message that appears briefly when the space calculation feature is searching for the Run information.	Wait for the calculation to complete. Should be less than 2 seconds.
Available space could not be calculated.	A system error occurred when calculating the available space.	Contact your Roche Representative.
There is not enough space available to complete the %. Please ensure there is at least % kilobytes available.	The data set storage location is full.	Using the Data tab, remove any data sets that have been externally backed up and copied.
Referencing Run information...	This is an information message that appears briefly while the PTP ID lookup feature is searching for the Run information.	Wait for the process to complete. This should take less than 2 seconds. If it takes longer, the Run Processor service may not be installed or running.
Could not find Run information associated with the PicoTiterPlate ID.	This is a warning which only appears if the PTP ID lookup feature is enabled and the PTP ID entered could not be found. This is only a warning and the Run can still be started after providing the information manually.	Ensure you entered the correct PTP ID. The wizard will prompt you through the normal series of questions if the Run information could not be found.
There are instrument checks that failed.	This is a warning. The run can still be started, but keep in mind the Run may be compromised if the instrument is not performing correctly.	Exit the wizard and click on the instrument name on the Status bar, to view the list of instrument validation messages.
Connection to the instrument services has failed.	The GS Junior Sequencer Application can not communicate with the instrument services.	Shutdown and reboot. If this fails to resolve the problem, contact your Roche Representative.
The instrument services failed to start.	The instrument services are in an unexpected state.	Shutdown and reboot. If this fails to resolve the problem, contact your Roche Representative.
The instrument services have failed.	The instrument services are in an unexpected state.	Shutdown and reboot. If this fails to resolve the problem, contact your Roche Representative.
A sequencing Run is unexpectedly running.	The Instrument Procedure Wizard was started while a procedure was running.	Shutdown and reboot. If this fails to resolve the problem, contact your Roche Representative.
The instrument services failed to start.	The instrument services are in an unexpected state.	Shutdown and reboot. If this fails to resolve the problem, contact your Roche Representative.
The instrument services are starting...	This message will appear if the connection to the instrument is lost while in the Instrument Procedure Wizard.	Shutdown and reboot. If this fails to resolve the problem, contact your Roche Representative.
Verifying instrument...	This message will appear if the instrument services are restarted while in the Instrument Procedure Wizard.	Shutdown and reboot. If this fails to resolve the problem, contact your Roche Representative.
Data is synchronizing...	This message will appear if the instrument services are restarted while in the Instrument Procedure Wizard.	Shutdown and reboot. If this fails to resolve the problem, contact your Roche Representative.
An unexpected instrument state is preventing the start of this procedure.	The instrument services are in an unexpected state.	Shutdown and reboot. If this fails to resolve the problem, contact your Roche Representative.
Connection to the data processor has failed.	The data processing service is not running. This is a warning so a procedure can be started, but no data processing of a Run will occur.	Shutdown and reboot. If this fails to resolve the problem, contact your Roche Representative.
Insufficient space on drive data to complete job. Approximately % kilobytes required for results.	The data set storage location is full.	Using the Data tab, remove any data sets that have been externally backed up and copied.