QualTrace II

QualTrace II is a DNA sequencing quality control analysis program developed by Nucleics, designed for production QC monitoring in high-throughput genomics centers and core DNA sequencing facilities. The software examines raw dideoxy sequencing data within ABI trace files to automatically identify problems with DNA sequencing reaction preparation or with the sequencer itself, enabling rapid detection of issues that limit sequence read length.

QualTrace II is compatible with trace files produced by ABI base callers on ABI and MegaBACE DNA sequencers, and can process either single .ab1 trace files or entire folders of trace files. It is designed to integrate into existing sequencing data pipelines and bioinformatics infrastructure, and can be used for both troubleshooting sequencing problems and optimizing production protocols.

Sequencing Problem Detection

• No signal data due to failed reactions or capillary blockage
• Mixed trace signal resulting from multiple DNA templates in the sequencing reaction
• Noisy or very low signal data traces producing only short reads
• Weak signal strength at the end of the trace resulting in short reads
• Early mixed trace signal due to template contamination by PCR products
• Delayed starts of trace signal indicating overloading of the sequencer capillary with template DNA or other contaminants
• Problems with the sequencer's spectral calibration resulting in major channel cross-talk signal
• Presence of significant leftover BigDye sequencing mix in loaded reactions (dye blobs)
• Traces showing a rapid decline in peak signal

Additional Trace Metrics Reported

• Indels (insertions and deletions)
• Total number of high quality bases
• Peak signal start position
• The point in the trace where signal reaches the noise threshold
• Level of peak blur (poor peak resolution)
• Signal-to-noise ratio for early, mid, and late regions of each trace, enabling tracking of trace signal decay
• Instrument diagnostics including machine name, run module, capillary length, run date, tray name, tray well, polymer lot, sample, sample ID, and user

Output and Reporting

• Generates a log file report containing the QualTrace category type and code for each trace
• Records signal-to-noise ratios across early, mid, and late trace regions to support optimization of template concentration and BigDye mix
• Supports batch processing of folders of trace files as well as individual .ab1 files

Performance in DNA Sequencing Facilities

• In a validated analysis of 11,745 ABI 3730xl traces, QualTrace II correctly categorized traces into nine quality categories
• The analysis identified that approximately 17% of traces showed mixed signal from multiple DNA templates, with those traces averaging only 322 phred Q20+ aligned bases compared to 647 for excellent traces
• Approximately 7% of traces were identified as failed; continuous monitoring by capillary number enabled identification of blocked capillaries requiring replacement
• Traces were base called using PHRED and aligned to known consensus sequences using CROSS_MATCH to validate QualTrace category predictions

Auto QualTrace Variant

• A graphical version of QualTrace II, called Auto QualTrace, is available for small to medium sized DNA sequencing facilities operating one to ten ABI DNA sequencers
• Auto QualTrace provides an easier-to-use interface and additional features aimed at lower-volume facilities

QualTrace II has been superseded by QualTrace III and is no longer sold or supported. Documentation is available through the QualTrace DNA sequencing user manual, and technical support inquiries can be directed to Nucleics at [email protected].