Next Generation Sequencing Facilitates Quantitative Analysis of Pseudomonas aeruginosa PAO1 Transcriptomes under AgNP and AgNR exposure

The goals of this study are to use Next-generation sequencing (NGS)to detect bacterial mRNA profiles of Pseudomonas aeruginosa PAO1 in response to 0, 1, 20 and 25 mg/L AgNPs or 0, 1,30 and 300 mg/L AgNRs for 2 h, using Illumina HiSeq 2500.The NGS QC toolkit (version 2.3.3) was used to treat the raw sequence reads to trim the 3’-end residual adaptors and primers, and the ambiguous characters in the reads were removed. Then, the sequence reads consisting of at least 85% bases were progressively trimmed at the 3’-ends until a quality value ≥ 20 were kept. Downstream analyses were performed using the generated clean reads of no shorter than 75 bp. The clean reads of each sample were aligned to the E. coli reference genome (NC_000913) using SeqAlto (version 0.5). Cufflinks (version 2.2.1) was used to calculate the strand-specific coverage for each gene, and to analyze the differential expression in triplicate bacterial cell cultures. The statistical analyses and visualization were conducted using CummeRbund package in R (http://compbio.mit.edu/cummeRbund/). Gene expression was calculated as fragments per kilobase of a gene per million mapped reads (FPKM, a normalized value generated from the frequency of detection and the length of a given gene. Bacterial mRNA profiles of Pseudomonas aeruginosa PAO1 in response to 0, 1, 20 and 25 mg/L AgNPs or 0, 1,30 and 300 mg/L AgNRs for 2 h, using Illumina HiSeq 2500

本研究旨在采用下一代测序（Next-generation sequencing, NGS）技术，依托Illumina HiSeq 2500平台，检测铜绿假单胞菌PAO1（Pseudomonas aeruginosa PAO1）分别暴露于0、1、20、25 mg/L银纳米颗粒（AgNPs）以及0、1、30、300 mg/L银纳米棒（AgNRs）环境中2小时后的细菌mRNA表达谱。研究使用NGS QC Toolkit（版本2.3.3）对原始序列读段进行预处理：修剪3'端残留的接头与引物，并去除读段中的模糊碱基。随后对序列读段的3'端进行逐步修剪，直至保留的碱基质量值≥20，且保留至少85%碱基比例的序列。后续分析采用长度不短于75 bp的clean reads开展。使用SeqAlto（版本0.5）将每个样本的clean reads比对至大肠杆菌参考基因组（NC_000913）。采用Cufflinks（版本2.2.1）计算每个基因的链特异性覆盖度，并对三组生物学重复的细菌细胞培养物进行差异表达分析。使用R语言中的CummeRbund包（http://compbio.mit.edu/cummeRbund/）完成统计分析与可视化工作。基因表达量以每百万比对reads中每个基因每千碱基的片段数（FPKM，即由检测频率与目标基因长度生成的标准化数值）进行量化。本研究采用Illumina HiSeq 2500平台，检测铜绿假单胞菌PAO1（Pseudomonas aeruginosa PAO1）分别暴露于0、1、20、25 mg/L银纳米颗粒（AgNPs）以及0、1、30、300 mg/L银纳米棒（AgNRs）环境中2小时后的细菌mRNA表达谱。