What are the limitations of the Sanger Sequencing method?

There are several limitations of the Sanger Sequencing method. One main limitation is in terms of the length and quality of DNA sequences it can effectively handle. It is typically suitable for sequencing short DNA fragments, ranging from approximately 300-1000 base pairs. Another limitation is that as the sequencing process progresses, the quality of the sequence tends to degrade especially after reaching 700-900 bases. A third limitation is that the initial portion (15-40 bases) of the sequence may exhibit lower quality as this is where the primer binds. Additionally, when sequencing cloned DNA fragments, there is a risk of including sequences from the cloning vector into the final sequence. This method also exhibits difficulty in distinguishing single base pair differences in longer segments. Sanger Sequencing may also not be effective in detecting mosaicism and has limitations in throughput. Moreover, it may use a larger quantity of input DNA compared to NGS and is also not cost-efficient for sequencing genes in the same genomic region or genes in parallel.