The overuse or misuse of antibiotics has accelerated antibiotic resistance, creating a major challenge for the public health in the world. highlighted the prevalence of ARGs and MGEs in microbial community of STPs. Introduction The wide use of antibiotics results in environmental releases, accelerating antibiotic resistance development in hospital wastewater, domestic sewage and livestock manure. Sewage receives the gut bacteria previously exposed to antibiotics, so that sewage treatment plants (STPs) are considered SNS-032 as important reservoirs for antibiotic resistance genes (ARGs) [1]. Mobile genetic elements (MGEs), e.g. plasmids [2], [3], transposons [3] and integrons [4], [5], are often involved in horizontal transfer of ARGs among environmental SNS-032 bacteria. With the help of insertion SNS-032 sequences (ISs), transposons often jump randomly and occasionally on genome or plasmid, resulting in gene transfer and multiple resistances [6]. Integrons may capture, integrate and express resistance gene cassettes in their variable region and facilitate the transmission of resistance genes via transposons or conjugative plasmids [7], [8]. Integrons carrying multiple ARGs are frequently detected in the environments of STPs [4], [9], [10], [11]. Activated sludge and biofilm in STPs are characterized with high microbial density and diversity which may facilitate ARG horizontal transfer [12]. Previous studies have shown that plasmid diversity is extremely high in the microbial community of STPs [3], [9], [12], [13], [14], [15], [16]. However, these investigations were carried out using culture-dependent methods, thus genomic analysis is only possible for a limited selected subset of plasmids. Furthermore, investigation of antibiotic resistance in microbial communities based solely on cultivable bacteria makes the assessment results unrepresentative and biased [17]. Therefore, a metagenomic approach is needed for a more comprehensive overview of plasmids residing in STP microorganisms. Recent studies SNS-032 have shown SNS-032 that the high-throughput sequencing is a promising tool for the analysis of complex microbial communities [17], [18]. In this study, we employed the transposon aided capture (TRACA) system [19] to isolate novel plasmids from microbial communities in activated sludge of a STP of Hong Kong, China. High-throughput sequencing and metagenomic analyses were also employed to investigate the diversity and relative abundances of ARGs, virulent factors (VFs), as well as MGEs including plasmids, integrons and transposons. Results and Discussion Analysis of the plasmid metagenome of activated sludge using high-throughput sequencing In order to deeply explore the plasmid metagenome, we employed a plasmid purification kit and a Plasmid Safe DNase to concentrate the plasmids in the DNA samples. Quantitative real time PCR (qPCR) indicated that the level of tetracycline resistance gene type increased from 1.1105 copies/ng of total DNA to 4.1106 copies/ng of plasmid DNA after being purified by QIAGEN Plasmid Purification Kit and treated by Plasmid Safe DNase (Figure S1), indicating that the plasmids DNA were concentrated by about 37 fold after the treatments since the gene is found to be only located on MGEs, such as plasmids (see Belgian Biodafty Server – Antibioresistance Archive, http://www.antibioresistance.be). However, PCRs demonstrated that 16S rRNA gene still occurred in the plasmid DNA samples, showing that chromosomal DNA contamination was not completely removed. The plasmid metagenome of activated sludge was analyzed by Illumina Hiseq 2000 high-throughput sequencing, generating 11,550,210 clean reads comprising 1.2 Gb in total (Table S1). Annotation of all the reads showed that the majority was of bacterial origin while very small fractions came from fungi (548 reads, <0.005%) and protozoa (499 reads <0.005%). Nearly half (44.8%) of the Illumina reads was well assembled into a total of 4,641 contigs of over 500 bp, with N50 length of 3.0 kb and the total contig length of 7.1 Mb (Table S1). Qin et al. [20] established a human gut microbial gene catalogue by high-throughput sequencing and 42.7% of the Illumina reads was assembled into contigs of over 500 bp with an N50 length of only 2.2 kb. Comparatively, in this study more reads were well assembled and longer contigs were generated, indicating the validity of the high-throughput sequencing reads. In this study, Trp53inp1 MetaGene analyses revealed a total of 9,315 predicted open reading frames (ORFs) in the contigs longer than 100 bp (Table S2). They occupied 92.0% of the contigs, which is comparable to that in the human.