Computational protocol: Streptomyces colonosanans sp. nov., A Novel Actinobacterium Isolated from Malaysia Mangrove Soil Exhibiting Antioxidative Activity and Cytotoxic Potential against Human Colon Cancer Cell Lines

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Protocol publication

[…] Genomic DNA extraction for PCR was performed as described by Hong et al. (). PCR amplification of the 16S rRNA gene was conducted according to the protocol described by Lee et al. (). The 16S rRNA gene sequence of strain MUSC 93JT was aligned with representative sequences of related type strains in the genus Streptomyces retrieved from the GenBank/EMBL/DDBJ databases using CLUSTAL-X software (Thompson et al., ). The alignment was first verified manually and adjusted, followed by construction of phylogenetic trees with neighbor-joining (Saitou and Nei, ; Figure ) and maximum-likelihood algorithms (Felsenstein, ; Figure ), utilizing the MEGA version 6.0 (Tamura et al., ). For neighbor-joining algorithm, the evolutionary distances were computed using the Kimura's two-parameter model (Kimura, ). The calculations of level of sequence similarity were performed by EzTaxon-e server (http://eztaxon-e.ezbiocloud.net/; Kim et al., ). Bootstrap based on 1,000 resampling method of Felsenstein () was used to analyze the stability of the resultant tree topologies.For DNA-DNA hybridization, the extraction of genomic DNA of strain MUSC 93JT, Streptomyces malachitofuscus JCM 4493T, Streptomyces misionensis NBRC 13063T and Streptomyces phaeoluteichromatogenes DSM 41898T were conducted according to the protocol described by Cashion et al. (). DNA-DNA hybridization was performed by the Identification Service of the DSMZ, Braunschweig, Germany based on the procedure as described by De Ley et al. () with slight modifications according to Huss et al. (). The G + C content of strain MUSC 93JT was determined by HPLC (Mesbah et al., ).BOX-PCR fingerprint analysis was performed for the characterization of strain MUSC 93JT and the closely related strains with the use of primer BOX-A1R (5′-CTACGGCAAGGCGACGCTGACG-3′; Versalovic et al., ; Lee et al., ). The BOX-PCR cycling parameters were performed as described by Lee et al. () and the PCR products were visualized using 2% agarose gel electrophoresis. [...] Genomic DNA of MUSC 93JT was extracted using Masterpure™ DNA purification kit (Epicentre, Illumina Inc., Madison, WI, USA) followed by RNase (Qiagen, USA) treatment (Ser et al., , ) Subsequently, the DNA quality was examined using NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and a Qubit version 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA). DNA library construction was performed using Nextera™ DNA Sample Preparation kit (Nextera, USA) and the library quality was validated by Bioanalyzer 2100 high sensitivity DNA kit (Agilent Technologies, Palo Alto, CA). Paired-end sequencing was carried out on MiSeq platform with MiSeq Reagent Kit 2 (2 × 250 bp; Illumina Inc., Madison, WI, USA). The paired-end reads were then trimmed and de novo assembled with CLC Genomics Workbench version 7 (CLC bio, Denmark). Gene prediction was carried out using Prodigal version 2.6, whereas rRNA and tRNA were predicted using RNAmmer and tRNAscan SE version 1.21 (Lowe and Eddy, ; Lagesen et al., ; Hyatt et al., ). The assembly was annotated using Rapid Annotation using Subsystem Technology (RAST) and by the NCBI Prokaryotic Genomes Annotation Pipeline (Angiuoli et al., ; Aziz et al., ). Results of genome sequencing and bioinformatics analysis were presented in the description of Streptomyces colonosanans sp. nov. […]

Pipeline specifications

Software tools Clustal W, MEGA, CLC Genomics Workbench, Prodigal, RNAmmer, tRNAscan-SE, RAST
Databases DDBJ EzBioCloud
Applications Genome annotation, Phylogenetics
Organisms Homo sapiens