High efficiency application of a mate-paired library from next-generation sequencing to postlight sequencing: Corynebacterium pseudotuberculosis as a case study for microbial de novo genome assembly

• A new methodology for the Ion Torrent PGM with mate-paired library is presented.
• De novo assembly of a prokaryotic genome from a single sequencing run.
• The Ion Torrent with mate-paired library is more efficient than the SOLiD platform.
• The equilibrium between read length and throughput improve the assembly results.
• The Ion Torrent PGM data reduced the effort to finish the genome assembly.

With the advent of high-throughput DNA sequencing platforms, there has been a reduction in the cost and time of sequencing. With these advantages, new challenges have emerged, such as the handling of large amounts of data, quality assessment, and the assembly of short reads. Currently, benchtop high-throughput sequencers enable the genomes of prokaryotic organisms to be sequenced within two hours with a reduction in coverage compared with the SOLiD, Illumina and 454 FLX Titanium platforms, making it necessary to evaluate the efficiency of less expensive benchtop instruments for prokaryotic genomics. In the present work, we evaluate and propose a methodology for the use of the Ion Torrent PGM platform for decoding the gram-positive bacterium Corynebacterium pseudotuberculosis, for which 15 complete genome sequences have already been deposited based on fragment and mate-paired libraries with a 3-kb insert size. Despite the low coverage, a single sequencing run using a mate-paired library generated 39 scaffolds after de novo assembly without data curation. This result is superior to that obtained by sequencing using libraries generated from fragments marketed by the equipment's manufacturer, as well as that observed for mate-pairs sequenced by SOLiD. The generated sequence added an extra 91 kb to the genome available at NCBI.