To test if 89K can excise from the S. suis 05ZYH33 chromosome, a PCR strategy was designed to detect the unoccupied rplL locus and an extrachromosomal circular form of 89K using specific primers that face outward from the integrated island or anneal to chromosomal DNA flanking the insertion site ( Fig. 2A) . Primer combinations P1/P2 and P3/P4 were used to amplify the left and right junction regions respectively. Primer pair P2/P3 was designed to detect the presence of a circular 89K, and primer pair P1/P4 was used to amplify the repaired chromosomal junction formed upon excision. The primers used are listed in Table S2 , and PCRs were performed with a standard protocol consisting of 30 cycles. The resulting amplicons were analysed by electrophoresis and were cloned into the pMD18-T vector (TaKaRa) for sequencing.