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1. Overview of the 454 Sequencing System Software : 1.2 Data Processing Options
1.2
Data Processing Options
1.2.1 GS FLX+ System
1.2.2 GS Junior System
1.2.1
GS FLX+ System
The GS FLX+ System gives the user up to four options for processing data during a sequencing Run (Figure 1).
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None- disable on-instrument processing (image acquisition only). If this is selected, all other steps of image and signal processing are carried out separately on an external DataRig. This maximizes the amount of time the instrument is available for sequencing. Although a backup script can be triggered, no post-analysis script is run.
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Image Processing Only- process images on the instrument concurrently with image acquisition, but handle the time-consuming signal processing step on a DataRig. Image processing completes by the time the instrument is ready for another Run with 200 cycles or fewer, but may extend a few hours beyond the fluidics stage with 400 cycles. A post-analysis script can be triggered, which allows pipeline-specific processing steps to be automated.
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Full Processing for Shotgun or Paired End- include both the image and signal processing steps during the sequencing Run. This is the simplest option from the standpoint of instrument operation, as all data processing up to the generation of read flowgrams and basecalling of the reads is carried out during the Run, without user intervention. However, full processing is computationally intensive.
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GS FLX Titanium Sequencing Kit XLR70: the on-instrument computing resources can take up to 80 hours to process a 200 cycle Run.
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GS FLX Titanium Sequencing Kit XL+: the on-instrument computing resources can take up to 150 hours to process a 400 cycle Run.
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Full processing for Amplicons- include both image and signal processing steps for Amplicon data (currently available only with the GS FLX Titanium Sequencing Kit XLR70). This option uses data processing algorithms that are specially tuned for Amplicon sequencing. It is also used for test Runs that use only Control DNA beads.
Additional post-Run data processing is invoked via the GS Run Processor, either as an option within the GS Run Browser GUI or at the “command line” level. This must be done off-instrument, on a DataRig or a GS Junior Attendant PC. Data Processing will produce comparable results whether it is run on-instrument or off-instrument.
 
The GS De Novo Assembler, GS Reference Mapper and GS Amplicon Variant Analyzer applications, and the SFF Tools commands, are always run separately from the sequencing Run (i.e. off-instrument, on a DataRig or GS Junior Attendant PC). The rationale for this is that these applications either are not usually applied to individual Runs but rather draw on multiple Runs, or require additional information beyond the Run data (such as a reference genome against which to map the sequencing reads). In addition, these applications can take hours to complete, during which time the GS FLX+ or GS Junior Instrument would not be available for another Run. Run troubleshooting may also be time intensive, so the GS Reporter and the GS Run Browser applications are provided both on the GS FLX+ Instrument or GS Junior Attendant PC, and as separate applications that can be run on a DataRig.
For more detail on data processing options, see Section 1 in Part B of this manual. For details on sequencing Run set up, see Section 3 in Part A of this manual (GS FLX+ System version), and the appropriate version of the Sequencing Method Manual.
Figure 1: Data processing options in the GS Junior and GS FLX+ Systems. The blocks identify the various data acquisition, data processing or data analysis applications and their outputs. Raw images are captured as part of the sequencing Run (data acquisition). Depending on the processing type selected during Run set up (top of each column), the image processing and signal processing steps can either be performed as part of the sequencing Run (above the dotted line), or they can be carried out separately following the Run (below the dotted line) using the off-instrument version of the software (on a DataRig or a GS Junior Attendant PC). Note the two options for signal processing: Shotgun or Paired End vs. Amplicons. Image Processing Only (shaded) is not an option on the GS Junior System. Applications launched by direct user input are shown in Bold-Italics; others are run automatically after the previous stage is completed. The GS Run Browser application (not shown) can be invoked on the GS FLX+ Instrument, a DataRig, or a GS Junior Attendant PC to view raw images and other Run data, e.g. for troubleshooting purposes or to reanalyze the data set using different settings.