Data Analysis — Quiz Part 2

Sample Answer: Possible errors include: (1) No overlapping regions between forward and reverse primers — caused by missing primers, homopolymers, poor quality sequences, multicopy genes, or low primer efficiency; (2) Mixed peaks/high background noise — indicating uncertain bases or possible insertions/deletions; (3) IUPAC ambiguity codes appearing instead of standard A/T/G/C bases. Troubleshooting involves: verifying sequences against both forward and reverse chromatograms, re-sequencing problematic regions, trying alternative primers, and checking for contamination.

Sample Answer: Key points: (1) Check the Trace QC / Q-score box — values should be 20+ for good quality; (2) Examine peak intensity — peaks should be clean, single, and well-separated; (3) Trim poor-quality bases from both ends (typically ±50 bp); (4) Look for ambiguous bases or IUPAC codes; (5) Reverse complement the reverse primer sequence before alignment; (6) Verify both forward and reverse sequences against each other; (7) Discard and re-sequence if quality is consistently poor.