Computational protocol: The presence of nitrate dramatically changed the predominant microbial community in perchlorate degrading cultures under saline conditions

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[…] 16S rRNA 16S rRNAThe closest relatives of the partial 16S rRNA gene sequences were identified in the NCBI nt database (ftp://ftp.ncbi.nih.gov/blast/db) using BLAST 2.2.27 + []. Taxonomic affiliations were derived from the best hits excluding matches to uncultured or unidentified organisms.(2)Metagenome AnalysisMetagenome AnalysisThe overall strategy of metagenomic datasets analysis is presented on Figure . The quality of the collected sequencing reads was assessed with the fastQC 0.10.1 tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc). The reads were processed with the FASTX toolkit (http://hannonlab.cshl.edu/fastx_toolkit) to remove terminal low-quality nucleotides, and then assembled using Metavelvet 1.2.02 which is an extension of the Velvet assembler designed to do de novo metagenome assembly from short sequence reads []. It is bundled with Velvet 1.2.08 []. The k-mer length and the minimum contig size were set to 19 and 100, respectively. The assembled contigs were submitted to the MG-RAST 3.3 server [] for taxonomic assignment and functional annotation. Alternatively, the contigs were evaluated batchwise using BLASTN searches against the Silva rRNA database (version 111) [] and/or the NCBI nt and 16SMicrobial databases (ftp://ftp.ncbi.nih.gov/blast/db). BLASTX searches against the NCBI nr database (ftp://ftp.ncbi.nih.gov/blast/db) were also conducted. Primary sorting of BLAST search results was performed using BlastParser 1.2 (http://geneproject.altervista.org). Contigs that matched database entries with E-value < 10−10 (NCBI nt and nr databases) or < 10−5 (NCBI 16SMicrobial and Silva rRNA databases) were selected for further analysis. Taxonomic classification of BLASTX hits was performed with MEGAN 4.70.4 [] using the lowest common ancestor (LCA) algorithm. The same approach was used to evaluate BLASTN matches with the Silva rRNA and NCBI 16SMicrobial databases. Annotation of BLASTN hits in NCBI nt database was performed using proprietary Perl scripts (ContigEval pipeline) for sorting the BLAST output, retrieval of taxonomic information from NCBI nodes.dmp and names.dmp files (ftp://ftp.ncbi.nih.gov/pub/taxonomy), and identification of the hit genes on subject nucleotide sequences by parsing NCBI gene2accession and gene_info files (ftp://ftp.ncbi.nlm.nih.gov/gene/DATA). Contigs mapped to sequences of particular interest were further extended using targeted assembly software, Mapsembler 1.3.16 []. In addition, some contig lengthening was achieved by running a transcriptome assembler, Oases 0.2.08 [] in metagenomic context with k-mers ranging from 19 to 31 and coverage cut-off set to 3. Oases was used only in the reconstruction of rRNA and perchlorate reductase gene sequences. Due to the significant probability of chimeric assembly, the Oasis-produced contigs were extensively filtered using the Uchime tool from the Usearch 6.0 software package [] and ChimeraSlayer release 20110519 [].Figure 3(3)GenBank Accession NumbersGenBank Accession NumbersPartial 16S rRNA gene sequences of isolated strains NWO, NY, NYO, PW, PWO, PY and PYO were deposited in GenBank under accession numbers KF135667-KF135673, respectively. Nucleotide sequences encompassing pcrABCD gene clusters recovered from NP30 and P30 mixed cultures are assigned accession numbers KF135674 and KF135675, respectively. Chlorite dismutase (cld) gene sequences from NP30 and P30 mixed cultures are placed under accession numbers KF135676 and KF135677, […]

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