Sequencing of eight evolved Klebsiella pneumoniae ATCC25955 strains with lactic acid tolerance

We employed microbial adaptive laboratory evolution to bolster lactic acid tolerance in Klebsiella pneumoniae ATCC25955 and acquired eight evovled strains A1, A2, A3, A4, A8, B1, C8, and D4. The genetic alterations in the genomes of A1, A2, A3, A4, A8, B1, C8, and D4 were analyzed using a DNBSEQ platform. Comparative analysis of genome sequences of A1, A2, A3, A4, A8, B1, C8, and D4 with that of the wild type identified several single nucleotide polymorphism (SNP) mutations. With alignment software MUMmer, each query sequence is aligned with reference sequence. The variation sites between the query sequence and reference sequence are found out and filtered preliminarily to detect potential SNP sites. The sequences with the length of 100 bp at both sides of SNP in the reference sequence are extracted and aligned with assembly results to verify SNP sites by using BLAT. If the length of aligned sequence is shorter than 101 bp, this SNP is considered as incredible and it will be removed; if the extracted sequence can be aligned with the assembly results several times, this SNP is considered locate in repeat region and it will also be removed. Blast, TRF, Repeatmask software are used to predict SNP in repeat regions. The credible SNP can be obtained through filtering SNP located in repeat regions.