Sequencing and assembly of Bacillus thuringiensis INTA Fr7-4 genome.

LAURA NAVAS; ELIO ORTIZ; DIEGO SAUKA; GRACIELA BENINTENDE; MARCELO BERRETTA; RUBEN ZANDOMENI; ARIEL AMADIO

Bariloche

Conferencia; 5th Argentinian conference on bioinformatics and computational biology; 2014

Asociación Argentina de Bioinformática y Biología Computacional

In recent years, there has been growing interest in sequencing isolates of the Gram positive bacterium Bacillus thuringiensis (B. thuringiensis) to discover new insecticidal proteins useful for biocontrol of agricultural pests and mosquitoes. B. thuringiensis INTA Fr7-4 is a native strain isolated from a soil sample in the province of Misiones. We have previously reported the complete sequence of three plasmids of this strain and characterized three insecticidal genes of the crystal (cry) family of proteins. This work reports on the sequencing of the genomic DNA from B. thuringiensis INTA Fr7-4 and the assembly of the readings to obtain the sequence of the chromosome. Genomic DNA from B. thuringiensis INTA Fr7-4 was sequenced using a 8 Kb long jumping distance library and 2x150 bp run on a MiSeq Illumina apparatus. After applying the appropriate quality clipping, 2,442,414 paired end readings (total of 4,884,828) were obtained, with an average length of 129 bp, and 4,962,965 singleton reads averaging 124 nt length. A de novo assembly was done using Velvet. The longest scaffold was 3.9 Mb long, and a total of 10 scaffolds longer than 10 kb were obtained. Scaffolds were compared with the GenBank database using blastn showing high identity with the chromosome of Bacillus bombysepticus str. Wang, a closely related species. For this reason, using it as a reference genome, we were able to build a map of B. thuringiensis INTA Fr7-4 chromosome consisting of 5 scaffolds, giving a total size of 5.2 Mb. Annotation of chromosome scaffolds using the RAST server was performed. We identified 5,300 CDS and 105 tRNA and rRNA gene-encoding regions. However, 187 CDS related to sporulation process in bacteria and 103 with chromosomal DNA replication attracted our attention. We did not find any insecticidal gene in the chromosome of B. thuringiensis INTA Fr7-4. The scaffolds not located within the chromosome belong to plasmid DNA. All plasmids previously sequenced were reconstructed, and a new plasmid of 259 Kb long was identified. This plasmid contains the previous detected insecticidal genes.