Sequencing Strategies In Conifer

The large size and complexity of conifer genomes present formidable challenges to available genome sequencing strategies. This talk describes the PineRefSeq project's sequencing strategies based on different approaches to complexity reduction. These complementary approaches enable high quality reference genome assemblies for loblolly pine while serving as a model for sequencing other large, complex genomes such as Douglas-fir and sugar pine. 

Whole-genome shotgun sequencing of a single haploid megagametophyte offers a major reduction in complexity while severely constraining the quantity of available DNA. The resulting haploid data is also well suited for additional computational complexity reduction. Short-insert megagametophyte coverage is augmented with deep sequencing of multiple long-insert libraries (fosmid DiTags and Illumina jumping libraries) from the parental diploid DNA.

The second and complementary approach to the sequencing of the loblolly genome is based on the high-throughput processing and sequencing of transient fosmid pools from the genome of the diploid parent. This approach allows us to tune the complexity of the effectively haploid assembly so as to apply the best-in-class short read assemblers. While this genome sequencing method presents particular challenges, such as cost and potential representation bias, it does robustly yield large contigs and scaffolds by a wholly separate procedure. On a smaller scale, fosmid pools have many applications such as the characterizing novel repetitive elements. Full genomic coverage with fosmid pools would greatly augment the loblolly reference genome assembly.