Insertion site analysis is very important in functional genomics research of Flammulina filiformis. The analysis methods commonly used are reverse PCR, thermal asymmetric interlaced PCR, Tail-PCR, chromosome walking, etc., and these methods are hard to operate, time-consuming, poorly specific and inefficient. In recent years, the next generation sequencing has been applied in identification insertion site that transformants were sequenced one by one, but it has a large workload and a high cost. In this study, a matrix design was used to mix the DNA of multiple transformants to form a sample pool. After resequencing, the insertion sites were analyzed. The sequencing data of M sample pools can analyze the insertion sites of M×(M+1)/2 transformants. The matrix design was used to detect 21 transformants, and 21 insertion sites were identified. This method indicates that matrix design is practicable for insertion site identification and suitable for large sample analysis, such as mutant library.
next generation sequencing