Expression profiling by high throughput sequencing
Summary
The goals of this study are to use Next-generation sequencing (NGS) to detect bacterial mRNA profiles of wild-type E. coli K-12 MG1655 and its mutants, and their mRNA response under the exposure of five antidepressants, including sertraline, duloxetine, bupropion, escitalopram and agomelatine. The concentrations were 50 mg/L for the treatment. For sertraline, in addition to 50 mg/L, 1 mg/L was also applied. The group without dosing antidepressants was the control group. Each concentration was conducted in triplicate. By comparing the mRNA profiles of experimental groups and control group, the effects of these five antidepressants on transcriptional levels can be revealed. Illumina HiSeq 2500 was applied. 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 MG1655 reference genome (NC_000913.3) 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.
Overall design
Bacterial mRNA profiles of wild-type E. coli K-12 MG1655 in response to 50 mg/L of antidepressants for 2 hours. Bacteria without dosing drugs are the control group, in triplicate, using Illumina HiSeq 2500
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