Flossie Roberts You're likely viewing Oxford Nanopore sequencing data in a chromatogram-like visualization, which is more common with Sanger sequencing. In Nanopore data, a raised black line that isn't labeled as a base (A/T/G/C) often represents a low-confidence or ambiguous signal. This occurs when the basecaller detects some signal at that position but cannot confidently assign a nucleotide, possibly due to noise, a modified base, or a borderline quality score.

If the raised signal consistently appears across runs and resembles a G in height or shape, it may indicate a real variant (e.g., SNP), but the software isn't confident enough to call it outright. Nanopore data is inherently noisier than other sequencing technologies, and some visualization tools show intermediate or uncalled signals to reflect uncertainty.

This isn't necessarily an error. It could represent a true base that didn't meet the basecaller's threshold or a modified base that disrupts the usual signal pattern. To investigate further, you can check the raw FASTQ file for quality scores at that position, inspect read alignments in IGV or similar tools, and use variant callers like Medaka or Nanopolish to assess whether it's a SNP or sequencing artifact.