Genomic tips 100 g columns

To con rm the absence of unexpected mutations around the deleted gene, Illumina sequencing was performed for all three single-gene knockout mutants (BID1, BID2, and BID3) and the parent strain BHY606. Qiagen Genomic-tips 100/G columns and Genomic DNA Buffer Set were used to extract the genomic DNA for NGS. The library was prepared using a TruSeq DNA PCR-free kit (Illumina, Inc., San Diego, CA, USA) and was sequenced on the NovaSeq 6000 platform at NovogeneAIT Genomics (Singapore). The raw reads were trimmed using fastp [66] and assembled using the Unicycler [67] . To investigate the sequence heterogeneity in the joint region (attS) of C and C′ on the circular SEs, PCR products of attS were obtained by PCR ampli cation of total DNA with KOD plus neo polymerase using the primers listed in Additional le 10. Amplicons were then puri ed, indexed for multiplex sequencing, and sequenced on the MiSeq platform (Illumina, Inc.) at Fasmac Co., Ltd. (Atsugi, Kanagawa, Japan) to give 236270 to 488664 paired reads per amplicon. The raw reads were trimmed, merged using fastp [66] , and then ltered to select the reads containing the correct primer sequence using the seqkit [68] . The number of unique merged reads was counted using the fastp-uniq function of the fastq-tools [69] . The commands used in NGS data analysis are described in the README le available in Figshare [70] (link).