Signal Processing

1. GS Run Processor : 1.3 Signal Processing

1.3	Signal Processing

1.3.1 Launching the signal processing pipeline

1.3.2 Signal Processing Output

The signal processing step of the GS Run Processor application analyzes the signal data for each flow for all active wells of each loading region of the PicoTiterPlate Device, using the data generated during the image processing step and stored in the .cwf files. The signal processing uses different internal parameters and function calls for signal processing of standard shotgun, Paired End, or cDNA libraries compared to Amplicon libraries and thus the library type must be specified for signal processing jobs.

The signal processing performs a series of normalization, correction, and quality filtering steps and then outputs the remaining (high quality) signals into flowgrams for each well (read). The signal processing step also generates basecalls with associated quality scores for the individual reads, and outputs this data as Standard Flowgram Format (or SFF) files that contain all the sequence trace data for the reads. All the data analysis applications (GS De Novo Assembler, GS Reference Mapper or GS Amplicon Variant Analyzer) use these SFF files as input. In a signal processing job, the GS Run Processor software performs the following operations:

1.	Apply signal filters and corrections (see Section 1.4.1)

2.	Filter (pass or fail) the processed reads based on signal quality (Key Pass, Dot and Mixed filters; see Section 1.4.2)

3.	Trim read ends for low quality and primer sequence (Signal Intensity, Primer and TrimBack Valley filters; see Section 1.4.3)

4.	Generate flowgrams and basecalled sequences with corresponding quality scores for all individual, high quality reads (i.e. those which passed all filters) and output to CWF and SFF files, one per PTP region processed. (Note: For the GS Junior Instrument, the entire PTP device is a single region)

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Version 2.5 or higher of the GS Run Processor contains significant enhancements to the basecaller and CAFIE corrector modules. For any data set that was processed with earlier versions of the GS Run Processor, which will be used for data analysis with V2.5 or higher, signal reprocessing is strongly recommended

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The Amplicons configuration of the signal processing is important because Amplicon libraries tend to generate much stronger signals than other library types. They consist of shorter fragments that amplify to a greater extent during emPCR Amplification. Very strong signals, coupled with the fact that many wells in Runs with Amplicon libraries contain similar sequences, can result in ‘ghost wells’, areas where light is detected but that do not truly correspond to DNA sequencing events. The special signal processing configuration for Amplicons includes two additional well screening steps that help remove these ghost wells. This configuration is triggered by using the runAnalysisPipeAmplicons CLI launch script command or by selecting ‘Full processing for Amplicons’ or ‘Signal processing for Amplicons’ from the Instrument Procedure Wizard or the GS Run Processor Manager tool in the GS Run Browser application.

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With 454 Sequencing Software applications version 2.01 or higher, while a signal processing job is in progress, the cwf files will have a .tmp extension. Users can verify if the pipeline has successfully finished by checking for .cwf files without .tmp suffixes in the regions sub-directory of the processing data (D_...) directory.