Computational protocol: Genome-Wide Study of the Defective Sucrose Fermenter Strain of Vibrio cholerae from the Latin American Cholera Epidemic

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Protocol publication

[…] Two Next Generation Sequencing platforms were used to generate the whole genome sequence of the V. cholerae IEC224 strain. A hybrid strategy was used to assemble the sequencing reads: first, a de novo method was used to assemble long reads from the GS FLX 454 system (Roche, Applied Science); then a resequencing methodology was used to assemble short reads from the SOLiD 3 Plus platform (Life Technologies). In order to close the genome, deep sequencing strategies were used, including reads on a mated-paired library.The data generated by the GS FLX 454 was assembled de novo with the Newbler software (version 2.6, Roche). Ninety-two contigs were generated from 319,825 reads with an average size of 400 nucleotides. The sequencing depth was of 35.5×, which covered approximately 95% of the genome in comparison to the V. cholerae strain N16961 (chromosome I: NC_002505.1 and chromosome II: NC_002506.1 ).Furthermore, in order to close the genome, a mated-paired library was sequenced in 23 million short reads by the SOLiD 3 Plus platform, yielding a genome coverage of 268× in comparison to the reference. We used the SOLiD™ Bioscope software (Applied Biosystems) for resequencing and the N16961 strain as a reference. A final consensus sequence was generated with the scaffolds of both sequencers. The hybrid sequencing strategy, using short reads of SOLiD for re-sequencing, allowed us to close the gaps of the de novo sequence generated from the assembly of the long pyrosequencing reads. Through this pipeline we were able to close difficult sequencing regions such as the class 4 integron cassette array called V. cholerae superintegron (SI). This was also achieved because a phylogenetically close reference was available to support the re-sequencing assembly.The final sequence was submitted to automatic annotation by the Rapid Annotation using Subsystem Technology (RAST) pipeline . The resulting annotation was manually curated based in the V. cholerae Genome Blast (NCBI). Both chromosome sequences were deposited in the GenBank database under the accession numbers CP003330 and CP003331, respectively for chromosome I and II. […]

Pipeline specifications

Software tools Newbler, SOLiD BioScope Software, RAST
Application WGS analysis
Organisms Vibrio cholerae, Filamentous phage
Diseases Cholera
Chemicals Alanine, Nucleotides, Sucrose