Data Analysis

After electrophoresis, data collection software creates a sample file of the raw data. Using downstream software applications, further data analysis is required to translate the collected color-data images into the corresponding nucleotide bases.

Primary Analysis

These tools convert the images gathered during Data Collection into all four colors, representing the four corresponding nucleotide bases (Figure 1). For example, our Sequence Analysis Software is a primary analysis tool that must be used after collection is completed. The Sequence Analysis software application allows users to basecall and re-basecall, trim data ends, display, edit and print sample files. Primary analysis software processes the your raw data in an *.ab1 file using algorithms and applies the following analysis settings to the results:

• Basecalling
  
  
  

  Figure 1: Primary Analysis Software results display each of the 4 bases as a different color.

  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 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 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.

Secondary Analysis

These tools allow you to further refine your results. Algorithms in our secondary analysis software products perform a number of functions supporting applications such as mutation detection and genotyping, and produce graphical outputs.

Product Selection Guide: Data Analysis Software

Software	Type	Features	Applications
Sequencing Analysis Software v5.3 with KB™ Basecaller v1.4	Primary Analysis Tool	Longer read lengths, including accuracy at 5' end Low signal to noise Basecalling quality values Accurate mixed basecall	De novo Sequencing
Sequence Scanner Software v1.0	Software Viewing Tool	Review traces in thumbnail format and sort by trace quality Effective quality metrics Simultaneously view raw and analyzed data Results at a glance with Plate Report view	De novo Sequencing Quality Control
SeqScape® Software for Mutation Profiling v2.6	Secondary Analysis Tool	Obtain quality values for each base pair, consensus sequence and mutation Automatically process raw data and generate reports with mutations linked to source electropherograms Pre-configure templates Custom data views	Resequencing Medical sequencing SNP/Mutation detection SNP/Mutation/Pathogen subtyping
Variant Reporter™ Software v1.0	Secondary Analysis Tool	Handle > 10 times more sample files than competing software products Accelerate data review Simple to learn Robust data filtering w/ effective quality metrics	Resequencing Comparative sequencing and direct sequencing Medical sequencing SNP/Mutation Detection

Next Generation Technology

A History of Innovation in Genetic Analysis (PDF)