Next Generation Sequencing Facilitates Quantitative Analysis of Klebsiella pneumoniae strain HKE9, HKE9-M-rpoS,HKE9-C-M-ropS
Compared with HKE9, HKE9-M-rpoS (rpoS knockout in hypervirulent Klebsiella pneumoniae strain HKE9) had 624 genes expressed significantly different, among which 148 genes were significantly up-regulated and 476 genes were significantly down-regulated, 586 genes on chromosomes, 10 genes on circular plasmid and 28 genes on linear plasmid; HKE9-C-M-rpoS had 746 genes expressed significantly different, among which 286 genes were significantly up-regulated and 460 genes were significantly down-regulated, 727 genes on chromosomes, 11 genes on circular plasmid and 8 genes on linear plasmid. Compared with HKE9-M-rpoS, HKE9-C-M-rpoS (rpoS complement in strain HKE9-M-rpoS) had 509 genes expressed significantly different, among which 324 genes were significantly up-regulated and 185 genes were significantly down-regulated, 458 genes on chromosomes, 3 genes on circular plasmid and 48 genes on linear plasmid. Overall design: A fresh culture of hypervirulent Klebsiella pneumoniae strain HKE9, HKE9-M-rpoS (rpoS knockout in strain HKE9), and HKE9-C-M- rpoS (rpoS complement in strain HKE9-M-rpoS) strains were grown to OD600 of 0.6. Total RNA was extracted using RNAprotect Bacteria Reagent and RNeasy minikit (Qiagen, DE). RNA quality was monitored on 1% agarose gels. RNA purity (OD260/OD280, OD260/OD230) was checked using the NanoPhotometer® spectrophotometer (Implen, USA). RNA integrity was measured using Bioanalyzer 2100 (Agilent, CA). A total amount of 3 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations, and then transcriptome sequencing was performed at Novaseq 6000 in Gene Denovo Co. Ltd(Guangzhou, China). Quality trimmed reads were mapped to the HKE9 using Bowtie2 (version 2.2.8), reads mapped to ribosome RNA were removed. Retained reads were aligned with reference genome to identify known genes and calculated gene expression by RSEM. To evaluate reproducibility between samples, the correlation coefficient among replicas was calculated. Values closer to one indicated better reproducibility. Principle component analysis (PCA) was performed with the R package gmodels (http://www.r-project.org) to reveal the relationship between samples. The gene expression level was further normalized by using the fragments per kilobase of transcript per million (FPKM) mapped reads method to eliminate the influence of different gene lengths and amount of sequencing data on the calculation of gene expression. The edgeR package (http://www.r-project.org/) was used to identify differentially expressed genes (DEGs) across samples with fold changes = 2 and a false discovery rate-adjusted P (q value) < 0.05. DEGs were then subjected to an enrichment analysis of GO functions (http://www.geneontology.org) and KEGG pathways (http://www.genome.jp/kegg), and q values < 0.05 were using as threshold.