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Pipeline publication

[…] th 30 ml of peptone-yeast extract glucose broth. Pr. naegleriophila were purified from amoebae by a first centrifugation step at 120 × g for 10 min. Then, remnants from amoebae were removed from the resuspended bacterial pellet by centrifugation at 6500 × g for 30 min onto 25% sucrose (Sigma Aldrich, St Louis, USA) and finally at 32000 × g for 70 min onto a discontinuous Gastrographin (Bayer Schering Pharma, Zurich, Switzerland) gradient (48%/36%/28%)., Pr. naegleriophila genomic DNA was isolated with the Wizard Genomic DNA purification kit (Promega Corporation, Madison, USA). Reads obtained with Genome Sequencer 20TM by Roche Applied Science (Penzberg, Germany) were assembled de-novo using Newbler V1.1.02.15 yielding 93 large contigs with a mean 16× coverage. Scaffolding on Pr. amoebophila strain UWE25 and PCR-based techniques were used to close the gaps between those contigs. Solexa 35 bp reads obtained from sequencing with Genome Analyzer GaIIx (Illumina, San Diego, USA) by Fasteris (Plan les Ouates, Switzerland) were then mapped to the final assembly with BWA () and visualized with Consed (). Homopolymer errors were corrected in the plasmid and the chromosome sequence after manual inspection of discrepancies covered by >2 reads with a Phred base quality score of >10. Sequence start was placed in an intergenic region closest to the minimum of the GC skew, as determined with a sliding window of 100 nt., GenDB 2.4 pipeline () was used for a first automatic annotation of the genome that was followed by manual curation of annotation. Coding sequence (CDS) prediction was performed using Prodigal (). All predicted CDS were submitted to similarity searches against nr, Swissprot, InterPro, Pfam, TIGRfam, and KEGG databases. Putative signal peptides, transmembrane helices, and nucleic acid binding domains were predicted using SignalP (), TMHMM (), and Helix-Turn-Helix (), respectively. Protein domain identification was used to manually curate genome annotations with a scheme as proposed in . The complete and annotated genome sequences have been deposited in the European Nucleic Archive under the project PRJEB7990 with accession numbers LN879502 and LN879503., To identify CRISPR repeats, the genome sequences were submitted to CRISPRFinder (). The spacers within the CRISPR locus […]

Pipeline specifications

Software tools Newbler, BWA, Consed, GenDB, Prodigal