We provide data for 9 bacteria from the Nextera Mate pair protocol. Sequencing was performed using MiSeq v2 reagents and 2 × 150 bp reads.

Genomic DNA samples from nine bacterial specimens were obtained from ATCC3 (Table 1). Using the gel-free workflow of the Nextera Mate Pair Sample Preparation Kit, DNA libraries were constructed from 1 µg genomic DNA in less than 2 days. The protocol used an initial “tagmentation” reaction to simultaneously fragment the DNA to > 1 kb and add biotinylated adapters to the ends of the molecules. Next, the tagmented DNA molecules were circularized and the ends of each fragment were joined. Circularized molecules were fragmented again and enriched by streptavidin beads, yielding smaller fragments (< 1 kb) containing the mate pair ends. Adapters were then added to these fragments for cluster generation and sequencing (Figure 2). Sequencing these smaller mate pair fragments provided information about genomic regions separated by large distances, enabling complete genome assembly. All bacterial libraries were pooled together for cluster generation and sequencing. The pool contained 2 replicates of each of the 9 libraries, yielding a total of 18 genomes. Libraries were loaded onto a MiSeq reagent cartridge and clustered on the MiSeq System. Paired-end sequencing was performed using MiSeq v2 reagents and 2 × 150 bp reads. Although longer read lengths are possible with the MiSeq System, 2 × 150 bp was determined to be the optimal read length for these samples. Sequencing generated 13 million reads passing filter, with a total run time of 32 hours.