When a fragment of gene was cloned into a vector for sequencing, two scenarios may lead to change of the sequence.
1) Errors in the PCR amplification, and wrong fragments can be cloned into vectors. ABI promises that the sequencing accuracy of its instrument can reach over 98.5% within a certain range.
2) Because of the limitation of the accuracy of the instrument, it is difficult to avoid a base sequence error in a longer sequence. After confirming the accuracy of cloning, bidirectional sequencing can minimize the sequencing errors. If you want to achieve the highest accuracy of sequence, it is necessary to conduct bidirectional sequencing. With simple unidirectional sequencing, we can not guarantee the complete accuracy of the measured sequence, which is determined by the accuracy of the instrument.