|Lindsey, Rebecca - Centers For Disease Control And Prevention (CDCP) - United States|
|Meinersmann, Richard - Rick|
|Loparev, Vladimir - Centers For Disease Control And Prevention (CDCP) - United States|
Submitted to: United States-Japan Cooperative Program in Natural Resources
Publication Type: Abstract Only
Publication Acceptance Date: 11/15/2013
Publication Date: 12/8/2013
Citation: Oakley, B., Lindsey, R.L., Meinersmann, R.J., Loparev, V. 2013. Microbial genome sequencing using optical mapping and Illumina sequencing. United States-Japan Cooperative Program in Natural Resources. December 7-12, 2013. Tsukuba, Japan. P.43.
Technical Abstract: Introduction Optical mapping is a technique in which strands of genomic DNA are digested with one or more restriction enzymes, and a physical map of the genome constructed from the resulting image. In outline, genomic DNA is extracted from a pure culture, linearly arrayed on a specialized glass slide, cut with a restriction enzyme, and the resulting set of fragments imaged at high resolution. Gaps at the restriction sites provide reference marks to determine the size of fragments in comparison to standards. Finally, fragments from multiple strands are assembled by aligning restriction sites to build a contiguous map of the genome. In essence, an optical map is truly a barcode – a pattern comprised of particular fragment sizes in a particular order. Optical maps are based on whole genomes similar to other so-called barcoding techniques such as PFGE, but contain much more information as the order of fragments is preserved in correspondence to the physical location of restriction sites on the genome. When used in combination with high-throughput sequencing techniques, optical mapping can provide a powerful tool for assembly of sequence fragments, identification of missing fragments, and validation of the final sequence. An example of this approach from a recent genome sequencing project is provided here. Materials and Methods Whole genome maps were generated according to the OpGen protocol using Argus consumables (www.opgen.com). Briefly, high molecular mass genomic DNA was isolated from axenic cultures using a gentle cell lysis procedure and diluted with minimal pipetting. DNA molecules were spread and immobilized along microfluidic channels on QCard glass slide surfaces and digested with the restriction enzyme NcoI. DNA fragments were stained with fluorescent dye and scanned using a fluorescent microscope. Automated image analysis software was used to create Single Molecule Restriction Maps (SMRMs) which were assembled by overlapping restriction fragment patterns to produce a whole genome map. For genome sequencing, DNA was sequenced on an Illumina MiSeq instrument in a paired-end run pooled with approximately 130 16S rRNA amplicon samples. Raw sequence data were pre-processed with the software tool trimmomatic and the fastx toolkit, merged with flash, and assembled with Velvet and Geneious. Assembled contigs were mapped to the optical map with MapSolver. Results and Discussion A physical map of the approximately 2M bp genome of a novel isolate belonging to the Epsilonproteobacteria genus Arcobacter used here as a case study was assembled to completion using optical mapping. Sequencing of genomic DNA on an Illumina MiSeq instrument produced >6,000,000 paired-end reads in a sequencing run combined with >100 samples of 16S rRNA amplicons. de novo assemblies of the genomic sequences produced N50 values up to 150 kb with the largest contig >500 kb. Preliminary assembly of these reads to the optical map showed areas of missing coverage for subsequent re-sequencing. Conclusions Optical mapping is a powerful whole-genome barcoding method that can be a useful addition to de novo genome sequencing projects. Availability of an optical map greatly facilitates genome assembly and re-sequencing for gap closure, particularly for isolates without a closely-related genome sequence available. The rapid decreases in cost and increases in throughput of modern sequencing instruments allow bacterial genomes to be sequenced relatively trivially as part of larger sequencing projects.