The invention provides a multi-primer designed AAV vector ITR sequence integrity determination method, and belongs to the technical field of next-generation
sequencing data analysis and application, and the method comprises the following steps: designing an AAV vector infection host
cell with n primers in an ITR region, extracting
intracellular fluid, and carrying out next-generation sequencing to obtain a primary Fastq file; the method comprises the following steps: carrying out coarse classification on sequences according to primer information, screening by using a biological information method to obtain a chimeric sequence, aligning one part of the chimeric sequence to a
host genome, aligning the other part of the chimeric sequence to an AAV vector
genome, and determining the junction of two sequences with different sources in the chimeric sequence as an ITR
breakpoint; according to the samples, the primers and the sequence information corresponding to the ITR breakpoints, the distribution condition of the ITR breakpoints in the ITR area is counted, and a statistical table is obtained; and a qualitative and
quantitative result of the ITR fracture position is obtained. According to the method, the integrity of the ITR region of the AAV vector sequence can be accurately qualitative and quantitative.