Whole-Genome Sequencing of Cyanobacterial Strains

Total DNA of PCC6803 and tolerant strains were extracted using the Genomic DNA Buffer Set and QIAGEN Genomic-tips 100/G (Qiagen, CA, USA). A DNA library was prepared using Truseq DNA PCR-Free LT sample prep kit (Illumina, CA, USA) and KAPA DNA library quantification kit (KK4824, KAPA Biosystems, MA, USA), and the sample was sequenced using MiSeq sequencer with MiSeq Reagent kit v2 (Illumina) generating 150 bp paired-end reads. The sequence data were mapped to the genome sequence of PCC6803 GT-I strain (NCBI Reference Sequence No.: NC_017038.1)^{27 (link)} using Bowtie 2 software ver. 2.2.3 with default parameters^{41 (link)}. SNPs were identified using SAMtools ver 1.0 and BCFtools ver. 1.0^{42 (link)} with a threshold of a quality score of >150. The sequence data obtained from MiSeq was deposited in DDBJ Sequence Read Archive under accession number DRA010198. The mutations identified by whole-genome sequencing were confirmed by Sanger sequencing using DNA fragments, including the mutation site amplified by PCR using the primer pair listed in Supplementary Table 4.