The base-calling program becomes unsure of a sequence in the following conditions:
- Extremely broad peak: Sometimes the peak given in automated sequencing program is too broad. There is always confusion whether a broad peak signifies one base or two bases. The base-calling program becomes unsure of the base in such a case.
- Extremely small peak: Small peaks, generally arise due to human error. So, it becomes confusing for the software to consider a small peak to be a nucleotide or not.
- Heterozygosity: When a diploid genome is sequenced, due to heterozygosity, two peaks of different colors appear at the same position.
In all the above three cases, the software becomes confused regarding the exact base at particular positions in the genome. For such positions, base calling program marks the position as ‘N’.