Computational protocol: A novel ammonia-oxidizing archaeon from wastewater treatment plant: Its enrichment, physiological and genomic characteristics

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Protocol publication

[…] Cells were harvested from the enrichment culture by passing through a 0.2 μm GTTP membrane (Millipore, USA), immediately frozen and stored at −80 °C until further analysis. DNA was extracted using a FastDNA SPIN Kit for soil (MP Biomedicals, USA) following the manufacturer’s protocol. The concentration of DNA was determined using Nanodrop spectrophotometer ND-1000 (ThermoFisher Scientific, USA). The bacteria and archaeal 16S rRNA and amoA genes were amplified by PCR using the primers listed in in the . All the PCR conditions were as follows except bacterial amoA gene: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 60 s; and 72 °C for 10 min. For bacterial amoA gene, a condition of 94 °C for 2 min; 35 cycles of 94 °C for 40 s, 51 °C for 60 s, and 72 °C for 60 s; and 72 °C for 5 min was used. Cloning into pGEM-Teasy vector (Promega, USA) and sequencing with T7 and SP6 vector primers was performed using standard procedures. Phylogenetic analysis of archaeal 16S rRNA and amoA gene was performed by using MEGA version 5.2, and the neighbor-joining tree was constructed using Maximum Composite Likelihood method. Bootstrap value of 1,000 replicates was set to estimate the reliability of phylogenetic reconstruction. [...] DNA for genome sequencing was extracted using PowerSoil DNA Isolation Kit (MO BiO, USA) from 500 ml culture of strain SAT1. Two DNA libraries were constructed: a paired-end library with an insert size of 500 bp and a mate-pair library with an insert size of 5 kb. Both libraries were sequenced using an Illumina HiSeq2500 by PE125 strategy. Library construction and Sequencing was performed at the Beijing Novogene Bioinformatics Technology Co., Ltd. Illumina PCR adapter reads and low quality reads were filtered using in-house program. The archaeal and bacterial communities of Metagenomic DNA were analyzed by using MG-RAST online tools. The filtered reads were assembled by SOAPdenovo, which yielded 537 contigs in total. The contaminated bacterial contigs were removed through analyzing the correlation between sequencing depth and GC content (), and aligning with known contaminated bacterial genomes (e.g. Ralstonia pickettii 12D). The remaining gaps were closed using PCR and Sanger sequencing, which finally resulted in one completed scaffold of 1.6 Mb. Gene prediction was performed on the SAT1 genome assembly by GeneMarkS with integrated model which combine the GeneMarkS generated (native) and Heuristic model parameters. Transfer RNA (tRNA) genes were predicted with tRNAscan-SE, Ribosome RNA (rRNA) genes were predicted with rRNAmmer. A whole genome Blast search (E-value less than 1e-5, minimal alignment length percentage larger than 40%) was performed against 3 databases. They are KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Clusters of Orthologous Groups), and NR (Non-Redundant Protein Database). Comparison of the SAT1 and other AOA genome was performed using MUMmer 3.0. The average nucleotide identity (ANI) and average amino acid identity (AAI) was calculated online (http://enve-omics.ce.gatech.edu/ani/). […]

Pipeline specifications

Software tools MEGA, SOAPdenovo, GeneMarkS, tRNAscan-SE, MUMmer
Databases KEGG MG-RAST
Applications Genome annotation, Phylogenetics, Metagenomic sequencing analysis, Nucleotide sequence alignment
Diseases Sprains and Strains
Chemicals Ammonia, Urea