Limitations of Sanger sequencing include: 

• Inability to detect large deletions or duplications of sequences
• Can only sequence short DNA fragments of about 300 to 1000 base pairs
• Sequence quality is not very good in the first 15 to 40 bases as that is where the primer binds
• Sequence quality degrades after 700 to 900 bases
• Not suitable for parallel testing
• Not suitable for sequencing large genomes
• High infrastructure costs and high cost per test

Despite its limitations, Sanger sequencing is still a good choice when sequencing single genes or amplicon targets up to 100 base pairs, analyzing fragments or short tandem repeats, or identifying microbes.