De novo Assembly of a Medium Sized Eukaryotic Genome Using Pacific Biosciences Single Molecule Real Time Sequencing Technology

Lates calcarifer (Asian seabass/barramundi) is a carnivorous, marine teleost belonging to the order Perciformes. It is an important fish species from an aquaculture perspective, its farming initiated in the 1970s in Thailand followed by a rapid spread to a large number of countries extending beyond its native range. The Asian seabass is euryhaline and catandromous with a wide geographic distribution stretching from Northern Australia to at least the Western part of India.

The Asian seabass has an estimated genome size of ~700Mb and a poorly represented transcriptome in GenBank. We embarked on this project with the main aim of employing next generation sequencing (NGS) platforms to produce an “improved high-quality draft of the genome”. A backcrossed individual with decreased genomic polymorphism was chosen to serve as a resource for the Asian seabass genome project. Sequences were generated on multiple platforms: Illumina (80X coverage), Sanger sequencing (1X coverage of BAC end sequences) and Pacific Biosciences, single-molecule, real-time sequencing system (>80X coverage).

The initial plan was to use a hybrid strategy where the first draft of the genome would be generated from the short reads generated on the Illumina platform (80X coverage) and long reads from the PacBio platform (20-30X coverage). However, we were unable to use this approach due to the requirement of high computational capacity for the error correction of PacBio sequences. As an alternative approach, we generated a much higher coverage of PacBio data (totally >80X) in order to be able to self-correct the reads using Hierarchical Genome Assembly Process (HGAP). In addition, we also made use of the assembly generated from high quality Illumina reads and BAC end sequences to error correct the PacBio reads. The outcome of the different approaches used to assemble the Asian seabass genome is presented.

We have also sequenced and assembled the transcriptome and characterized repetitive elements to aid genome assembly as well as annotation. We aim to use the genome sequence information for genetic/genomic selection assisted by ‘state of the art’ large-scale platform technologies to enable the development of elite Asian seabass varieties.