Assembly and Validation of an Evolutionary Reference Genome for Flowering Plants (Amborella trichopoda)

Amborella trichopoda (Amborellaceae), as the sister to all other extant angiosperms, occupies a crucial phylogenetic position, and its genome sequence is therefore an important reference for comparative genomic studies across the angiosperms. A complete genome sequence of Amborella will help in understanding the evolution of key angiosperm traits and provide a baseline to examine genome organization throughout the angiosperms. We are using a whole-genome shotgun strategy to sequence the ~731-Mb Amborella genome. We assembled over 48 Gb of DNA sequence, comprising single-end (SE) 454-FLX, SE 454-FLX+, 11-kb paired-end (PE) 454-FLX, and 3-kb PE Illumina HiSeq, and 69,466 BAC-end reads. These sequences were extensively screened and filtered, for organellar contaminants, short read lengths, poor quality, artificial duplicates, and chimeras. De novo sequence assembly was performed on these filtered sequence sets (~23 Gb) using Newbler.  This resulted in 5,745 scaffolds totaling 706 Mb with a mean scaffold size of 123 kb and N50 size of 4.9 Mb covering over 96% of the Amborella genome. Significantly, about 90% of our assembled sequence resides within 155 scaffolds, each greater than 1.1 Mb in length. In the absence of genetic and physical maps for Amborella, we employed a combination of chromosomal FISH (fluorescent in situ hybridization) analysis and optical mapping (OpGen) to evaluate the fidelity and the chromosomal positioning of our current scaffolds.  Using this approach, our assembly continuity increased by 2X (N50 from 4.9 Mb to 9.3 Mb and N90 from 1.2 Mb to 2.9 Mb). We are further evaluating the potential of this approach to serve as a surrogate to using long-range PE libraries (like BAC ends) to increase the contiguity of a genome assembly.