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1. GS Run Processor : 1.2 Image Processing
1.2
Image Processing
1.2.1 Launching the image processing pipeline
1.2.2 Image Processing Output
In the image processing step, the initial PPi or ATP flow is used to define the PTP regions that are loaded and the shape of the background across the plate. The specific background for each nucleotide type is also computed from the key flows and these are combined to subtract the background for each signal producing location on the plate for the key flow images. The sum of all signal locations across the initial key flow images defines the total set of initial raw wells. Well centers are thus defined at consistent locations across the remaining series of nucleotide flows (distinct image files) comprising the sequencing Run. Flow signal information is extracted for each raw well and organized into composite well format (cwf) for further processing.
There is one CWF file generated for each sequencing run / PTP Device on a GS Junior Instrument, while for the GS FLX+ Instrument, there is one CWF file generated for each region of the PTP Device. The total size of the raw images for a 2-region, 200 cycle GS FLX Titanium Sequencing Kit XLR70 Run is about 17GB, and for a 2-region, 400 cycle GS FLX Titanium Sequencing Kit XL+ Run is about 35GB. For a GS Junior Titanium Run, the total size of the raw images is about 2.5GB.
The image processing computation is fast enough to be run concurrently with a 200 cycle sequencing Run, but continues a few hours beyond the fluidics stage of a 400 cycle Run.
Image processing filter parameters are described in Table 4.
Image Processing Parameter
Description
Values
Shotgun/PE
Amplicon
minConsensusSignal
Minimum average intensity of the positive key flows to be considered as a potential well.
integer
Junior Kit: 20*
XLR70 Kit: 20
XL+Kit: 20
Junior Kit: 60
XLR70 Kit: 20
XL+Kit: n/a
useBicubic
Use bicubic upsampling of images (‘false’ = bilinear).
true/false
true
true
Table 4: Image Processing Parameters. Stringency can be increased by using larger green values or lower red values.
* The imageProcessingOnly pipeline for the GS Junior System uses an alternative value of ‘60’.
The following files are generated by the sequencing Run image capture and are used as input to the image processing step;
•
dataRunParams.parse
•
imageLog.parse
•
ptpImage.png
•
runLog.parse
•
aaLog.txt
•
rawImages (subdirectory)- containing the image data in .png formatted files
◦
00000.png (initial PPi or ATP flow image)
◦
00001.png. 00002.png, 00003.png, … (sequential nucleotide flow images)
Image data from the GS FLX+ Instrument are stored as standard .png image files. The legacy .pif image format is still supported, so older Run image data (and data from GS Junior Instrument Runs) can be used directly, without re-processing.
 
For the 454 Sequencing System Software Applications version 2.3 and higher, all the image files are used by data processing applications, thus it is strictly required that these files not be altered.