Flx titanium sequencing chemistry

To confirm the purity of genomic DNA, the 16S rDNA specific region of K. oxytoca HKOPL1 was amplified and cloned. Then, 20 positive clones were submitted for Sanger sequencing. BLASTN analysis revealed that the K. oxytoca HKOPL1 16S rDNA sequences highly correlate with the current Klebsiella genus database. Evaluation of the quality of genomic DNA was done by using the Quant-iT™ PicoGreen dsDNA kit (Invitrogen).
A whole genome shotgun library of K. oxytoca HKOPL1 was generated with 0.5 μg of the genomic DNA; the shotgun sequencing was done with 454 GS Junior General Library Preparation Kit (Roche). In addition, an 8kb insert paired-end library was generated with 15 μg of the genomic DNA; the paired-end sequencing was done with 454 GS Junior Paired-end Library Preparation Kit (Roche). Paired-end reads were used to orientate the contigs into scaffolds.
The DNA libraries were amplified by emPCR and sequenced with FLX Titanium Sequencing Chemistry (Roche). Two shotgun runs and two paired-end runs were performed for each library. After sequencing, the raw data was assembled by Newbler 2.7 (Roche) with default parameters. Primer pairs were designed to amplify the gaps between contigs, and the resulting PCR products were directly sequenced by Sanger sequencer ABI 3130.

A whole genome shotgun library was produced with 5 µg of the genomic DNA of IMT5155. The shotgun sequencing procedure followed the instruction of 454 GS Junior General Library Preparation Kit (Roche). In addition, an 8 kb insert paired end library was produced with 15 µg of the genomic DNA of IMT5155. The paired end sequencing procedure followed the instruction of 454 GS Junior Paired-end Library Preparation Kit (Roche). Paired-end reads were used to orientate the contigs into scaffolds. The DNA libraries were amplified by emPCR and sequenced by FLX Titanium sequencing chemistry (Roche). Two shotgun runs and one paired-end runs were performed based on their individual library. After sequencing, the raw data were assembled by Newbler 2.7 (Roche) with default parameters. Primer pairs were designed along the sequences flanking the gap regions for PCR gap filling. The complete sequences of IMT5155 chromosome and two plasmids have been deposited in GenBank (Accession numbers: CP005930, CP005931, and CP005932, respectively).