Computational protocol: High Levels of Antibiotic Resistance Genes and Their Correlations with Bacterial Community and Mobile Genetic Elements in Pharmaceutical Wastewater Treatment Bioreactors

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Protocol publication

[…] The DNA samples extracted from activated sludge were sent to Jiangsu Zhongyijinda Analytical & Testing Co., Ltd for library construction and high-throughput sequencing on a Hiseq2500 platform (Illumina, San Diego, CA, USA). The sequencing strategy of Index 101 PE (Paired-End sequencing, 101-bp reads and 8-bp index sequence) was applied to generate raw sequencing reads. For quality control, the raw sequences contaminated by adapter or containing three or more unknown nucleotides (‘N’) were firstly removed using the quality control (QC) pipeline recommended by sequencing institutions. A strict filtration strategy was then conducted by using Galaxy (https://usegalaxy.org/). ‘FASTQ Groomer’ tool was used to convert quality formats, and ‘Filter by quality’ tool was then used to remove low quality sequences to ensure that more than 75% bases of each filtered read with quality greater than 30. The quality-filtered reads were used for subsequent analysis. The metagenomic sequencing data have been deposited into sequence read archive (SRA) database under accession number PRJNA 316198. [...] In order to evaluate the distribution of ARGs and MGEs in PWWTPs and STP sludge, the quality-filtered reads were separately aligned to databases including Antibiotic Resistance Genes Database (ARDB, http://ardb.cbcb.umd.edu/index.html) [], The Integron Database (INTEGRALL, http://integrall.bio.ua.pt/) [], Insertion Sequences Database (IS Finder, https://www-is.biotoul.fr//) [] and NCBI Reference Sequence Database (NCBI RefSeq database, http://www.ncbi.nlm.nih.gov/refseq) []. A read was annotated as ARG sequences according to its best BLAST hit in ARDB with a threshold of amino acid sequence identity≥90% and sequence alignment length≥25 amino acids []. A read was identified as integron or insertion sequence if the sequence had an identity ≥90% with its best BLAST (BLASTn with the E-value cut-off at 10−5) hit over an alignment of at least 50 bp []. The plasmids identification was determined by using BLAST (BLASTn with the E-value cut-off at 10−5) with a threshold of nucleotide sequence identity ≥95% over an alignment of at least 90 bp [].The quality-filtered Illumina reads of the eight sludge samples were submitted to Metagenomics RAST server (MG-RAST) (http://metagenomics.anl.gov/) []. The MG-RAST server was used to compare the reads of the PWWTP sludge samples using ‘best hit classification’ function with Ribosomal Database Project (RDP) database as the annotation source with a maximum Evalue cutoff of 10−5, a minimum identity of 97%, and a minimum alignment length of 50 bp [, ]. […]

Pipeline specifications

Software tools Galaxy, BLASTN, RAST
Databases ARDB INTEGRALL MG-RAST ARDB
Application Metagenomic sequencing analysis
Organisms Bacteria
Chemicals Aminoglycosides, Oxygen, Tetracycline, Streptogramins