Quality Values in Mutation Surveyor Software

Summary

This webinar focuses on the importance of quality values in sequencing and mutation calling. It explores various quality scores such as Fred score, signal-to-noise ratio, and mutation score, explaining their roles in assessing trace quality and minimizing false positives. The webinar also discusses the software's tools for visualizing these scores in chromatograms, reports, and graphical displays. These quality metrics, including base score, lane quality, and mutation scores, are crucial for ensuring the accuracy of sequencing results and mutation detection. The session highlights how these values can be utilized in reports for better decision-making and analysis.

Takeaways

• 😀 Quality values like Fred score, signal-to-noise ratio, and mutation scores are critical in assessing sequencing confidence and accuracy.
• 😀 Fred score is a fundamental metric for nucleotide base quality, with higher scores indicating more accurate base calls and lower error probability.
• 😀 Mutation Surveyor offers settings for handling Fred scores, allowing users to display original scores or those calculated after mobility correction.
• 😀 Signal-to-noise ratio is a key indicator for mutation peak detection, used to calculate mutation scores and identify regions of trace files with potential quality issues.
• 😀 Lane quality scores assess the overall trace quality, calculated using signal-to-noise ratio for the entire trace and providing insight into reference trace selection.
• 😀 Mutation scores, including mutation score for point mutations and indels (insertions and deletions), are calculated based on error probabilities and signal quality factors.
• 😀 The mutation score display in graphical reports allows users to evaluate the quality of mutation calls, with options to view both individual and summed mutation scores.
• 😀 Custom reports allow users to filter and report quality values like Fred score, signal-to-noise ratio, and mutation score based on user-defined thresholds.
• 😀 The NM score helps assess the contribution of normal alleles versus mutant alleles at specific positions, offering insights into trace quality and mutation confidence.
• 😀 Reports such as the Project Reviewer, GAD (Graphical Analysis Display), and Custom Reports can include various quality values, offering flexibility in reporting trace and nucleotide quality.
• 😀 Mutation Surveyor supports visual and tabular reporting of quality metrics such as Fred score, lane quality, and mutation score, helping users analyze sequence quality and refine mutation calls.

Q & A

What is the primary purpose of quality values in sequencing?

Quality values are used to assess the confidence and accuracy of sequencing, as well as mutation calling, helping minimize false positive calls by identifying regions of low quality in the trace files.

How does the Fred score function in sequencing?

The Fred score measures the quality of nucleotide identification in DNA sequencing. It is logarithmically linked to the error probability of the base call, with a score assigned to each base call. The higher the Fred score, the higher the accuracy of the base call.

What are the different ways to display Fred scores in Mutation Surveyor?

Fred scores can be displayed in chromatograms, including the sample file view, as well as in graphical analysis displays. They can also be included in various reports like the Project Reviewer Report, the GAD Report, and the Custom Report.

What does the Signal-to-Noise ratio indicate in sequencing?

The Signal-to-Noise ratio compares the intensity of the mutation peak (signal) to the median noise intensity of the minor peaks, determining how large the signal must be for a mutation to be recognized as valid.

How does the Signal-to-Noise ratio affect mutation calls?

A higher Signal-to-Noise ratio indicates a clearer and more accurate mutation peak. It is used to assess trace quality and is factored into the calculation of several scores, such as the mutation score and indel quality scores.

What is the Base Score and how is it calculated?

The Base Score measures the quality of a comparison region for a sample. It is calculated as the average of the Fred scores for the bases within the comparison region and can be displayed in standard reports when selected.

What role does Lane Quality play in Mutation Surveyor?

Lane Quality is used to assess the overall trace quality before alignment. It helps select between multiple reference trace files and can be displayed in reports like the Project Reviewer Report and the GAD Report. Low Lane Quality may indicate poor trace quality or heterozygous indel events.

What does the Mutation Score represent?

The Mutation Score evaluates the confidence in mutation calls. It is calculated based on factors such as the Fred score, signal-to-noise ratio, and overlapping factors. A higher mutation score indicates higher confidence in the mutation call.

How are mutation scores reported in Mutation Surveyor?

Mutation scores are reported in various graphical and tabular reports. In the Project Reviewer Report, both individual and total mutation scores are displayed, while other reports may show an average mutation score for specific positions.

What is the NM score and how is it used?

The NM score indicates the contribution of the normal allele at a mutation position relative to the mutant alleles. It is based on the height ratio of the fluorescence peaks and can help determine if a sample contains a normal allele or a mutation.