Sanger Sequencing Data Analysis
After electrophoresis, Data Collection software creates a sample file of the raw Sanger sequencing data. Using downstream software applications, further data analysis is required to translate the collected color-data images into the corresponding nucleotide bases.
Sanger Sequencing Primary Analysis
The primary data analysis tools convert the images gathered during Data Collection into all four colors, representing the four corresponding nucleotide bases (Figure 1). For example, our sequencing analysis software is a primary analysis tool that must be used after collection is completed. The sequencing analysis software application allows users to basecall and re-basecall, trim data ends, display, edit and print sample files. Primary data analysis software processes your raw sequence data in an *.ab1 file using algorithms and applies the following analysis settings to the results:
The selected basecaller processes the fluorescence signals, then assigns a base to each peak (A, C, G, T, or N). If the KB™ basecaller is used, it also provides per-base quality value predictions, optional mixed base calling, and automatic identification of failed samples (Figure 1).
- Mobility Shift Correction
The mobility file compensates for the change in DNA fragment mobility caused by the dye molecule attached to the DNA fragment and changes the color designation of bases depending on the type of chemistry used to label the DNA.
- Quality Value (QV)
If the KB basecaller is used for sequencing data analysis, the software assigns a QV for each base. The QV predicts the probability of a basecall error. For example, a QV of 20 predicts an error rate of 1%. The quality prediction algorithm is calibrated to return QVs that conform to the industry-standard relationship established by the Phred software. If your pipeline involves data analysis with Phred software to assign QVs after the data is basecalled, you can simplify your workflow and use the KB basecaller instead. The KB basecaller can perform basecalling and assign QVs. Then, you can generate *phd.1 or *.scf files using the KB basecaller to integrate with your downstream pipeline.
Sanger Sequencing Secondary Analysis
These tools allow you to further refine your sequencing results. Algorithms in our secondary data analysis software products perform a number of functions supporting applications such as mutation detection and genotyping, and produce graphical outputs.
Product Selection Guide: Sanger Sequencing Data Analysis Software
|Sequencing Analysis Software v5.3 with KB™ Basecaller v1.4||Primary Analysis Tool||
|Sequence Scanner Software v2.0||Software Viewing Tool||
|SeqScape® Software for Mutation Profiling v2.6||Secondary Analysis Tool||
|Variant Reporter™ Software v1.0||Secondary Analysis Tool||