Usually, template DNA or sequencing primers can result in the formation of overlapping peaks. Here are the summary for these reasons:
1) Sequencing primer has alternative matching site on the template DNA
2) Presence of impurities in template DNA. If the template DNA is a plasmid or isolated from E.coli culture, the clone can be contaminated. If the template DNA is a PCR product, it might contain some unrelated DNA but with similar size in the sample.
3) Presence of unique structures on the template DNA, such as poly A or poly G, hairpin structure, etc.
4) Primer degradation, impure primer, or primer having poor binding specificity.
