Artifactual Palindrome and Mutations Resulting from the Stable Secondary Structure of Templates in Sanger Sequencing

摘要

Due to its advantages of high precision and low cost, automated Sanger sequencing technology remains useful for many applications despite the emergence of next-generation sequencing (NGS) platforms. Few studies revealed the sequence errors in sanger sequencing because the instrumentation and chemistry currently used for Sanger sequencing is highly reliable and reproducible. Unexpectedly, in our study, the sequencing artifacts were identified in sequencing the PCR product of rAAV-ITR (recombination Adeno-Associated Virus-inverted terminal repeat) which formed highly stable T-shaped hairpin and played critical role in virus production. The artifactual sequences were characterized by the 3' end palindromic sequence and nucleotide substitutions. We confirmed that the presence of stable hairpin in template results in sequencing artifacts by employing disruptor to eliminate the structure, sequencing the PCR product and the synthezized strands containing stable or weak hairpin respectively for comparation. Furthermore, we proposed that endonuclease activity of Taq polymerase cleaves the templates containing stable structure to generate annealed 3'end which acts as primer to synthesize the artifactual palindrome.