High-frequency, low-coverage "false positives" mutations may be true in GS Junior sequencing studies.

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• The GS Junior sequencer offers streamlined library preparation and data processing.
• Pyrosequencing errors can introduce biases during library construction and emulsion PCR.
• Distinguishing true positives from false positives among low-frequency or low-coverage mutations is challenging.

Purpose of the Study:

• To enhance the accuracy of identifying true positive mutations among those with borderline false-positive characteristics detected by GS Junior sequencing.
• To establish reliable criteria for validating low-frequency and low-coverage variants.

Main Methods:

• Sanger sequencing was employed to validate mutations identified by GS Junior sequencing.
• Mutations were categorized into two groups based on coverage (<20-fold) and frequency (>30%) or vice versa.
• Statistical analysis was performed to compare false-positive rates between groups.

Main Results:

• Mutations with borderline characteristics were evaluated using Sanger sequencing.
• Group A (coverage <20-fold, frequency >30%): 2 of 10 mutations were unconfirmed; 2 heterozygous alleles were confirmed.
• Group B (coverage >20-fold, frequency <30%): None of the 16 mutations were confirmed.
• Significantly different false-positive prevalences were observed between the two groups (p=0.001).

Conclusions:

• Mutations detected at frequencies below 30% can be confidently classified as false positives.
• Mutations with frequencies over 30%, even with coverage below 20-fold, require verification via Sanger sequencing for accurate identification.