454 sequencing system software v 2 7 and v 3

WGS with 454 Roche technology was performed for Russian epidemic strains A. ruhlandii SCCH3:Ach33–1365 ST36 (AruhST36) and B. cenocepacia GIMC4560:Bcn122 ST709 (BcenST709). Shotgun and paired-end libraries were prepared. Paired-end libraries were built according to the 3 kb protocol. The sequencing procedure was performed with a GS Junior Titanium Sequencing Kit, and a GS Junior+ Series XL+ Kit, according to the manufacturer’s guidelines. Genome assembly was performed with 454 Sequencing System Software v.2.7 and v.3.0 (Roche).

Two types of libraries were used for BCG Russia 368 genome sequencing by 454 Roche platform: shortgun and paired-end. The latter was built according to the 3 kb protocol. Sequencing procedure was performed using the GS Junior Titanium Sequencing Kit, i.e. GS Junior + Series XL+ Kit according to the manufacturer’s guidelines.
Assembly was performed with 454 Sequencing System Software v.2.7 and v.3.0 (Roche), yielding five scaffolds. PCR and Sanger sequencing were used for gap closure with primers presented in Additional file 1: Table S1. Most gaps were found in the sequences of PPE/PE-PGRS genes. Prediction of the secondary structures of this DNA sequences and calculation of the minimum free energy (MFE) structure were performed using RNAfold web server [11 ]. GC-reach DNA fragments amplification was optimized by both the involvement of 10% DMSO (Sigma) and 5% D-(+)-Trehalose dehydrate (Sigma). Sanger sequencing of amplicons was successful only in 5% DMSO presence.

DNA sequence assembly into scaffolds was performed with 454 Sequencing System Software v.2.7 and v.3.0 (Roche). To aid in assembling individual chromosome we used data from reference strains: B. lata strain Burkholderia sp. 383; B. contaminans strain MS14; B. ubonensis strain MSMB22; B. cenocepacia strains: J2315, DDS 22E-1; B. cepacia strains: DDS 7H-2, ATCC 25416. The software Rapid Annotations Subsystems Technology and SEED [10 (link), 11 (link)] were used for annotating the genome of B. contaminans strains. BioProjects PRJNA349796 and PRJNA349797 were registered in GenBank with BioSample Accessions SAMN05933033 for GIMC4509:Bct370 and SAMN05933042 for GIMC4587:Bct370-19. Now the genomes are in the process of the chromosomes assembling.