Annotating the cytochrome P450 CYP720B gene family in white spruce

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1. 2012-11-23 Annotating the cytochrome P450 CYP720B gene family in white spruce Shaun Jackman http://en.wikipedia.org/wiki/Picea_glauca 1
2. White spruce Picea glauca http://en.wikipedia.org/wiki/Picea_glauca 2
3. Cytochrome P450 ✤ Enzymes involved in biosynthesis and metabolism ✤ Catalyze the oxidation of organic compounds ✤ Contain a heme cofactor ✤ Primarily membrane bound 3
4. CYP720B gene family ✤ Conifer-specific gene family ✤ Involved in the biosynthesis of diterpene resin acids, important in conifer defence against insects and fungus http://en.wikipedia.org/wiki/Mountain_pine_beetle 4 http://en.wikipedia.org/wiki/Resin
5. CYP720B in sitka spruce Picea sitchensis ✤ Twelve CYP720B genes identified in sitka spruce ✤ Full-length transcript sequences available ✤ Use these sequences to identify orthologs in white spruce (Picea glauca) http://en.wikipedia.org/wiki/Picea_sitchensis Katrin Geisler 5
6. White spruce sequencing data Picea glauca ✤ RNA-seq of eight tissues: bark, embryo, flush bud, mature needle, megagametophyte, seedling, xylem and young bud ✤ Whole genome sequencing 24 lanes of Illumina HiSeq for 64-fold coverage 6 Hamberger, Ohnishi et al. (2011) Plant Phys. 157: 1677-1695
7. Identify white spruce transcripts ✤ Assemble the white spruce RNA-seq data usingTrinity (Grabherr et al. 2011) (assembled by Mack Yuen) ✤ Identify the white spruce CYP720B transcripts by aligning the sitka spruce transcripts to the white spruce RNA-seq contigs using BWA-SW (Li and Durbin 2010) ✤ Cluster the sitka spruce and white spruce transcripts using CLC Genomics Workbench 7
8. 8 White spruce CYP720B transcripts Sitka spruce White spruce
9. Assemble the white spruce genome ✤ Assemble the white spruce genome sequence using ABySS (Simpson et al. 2009) ✤ Assembled 8.4 billion reads using 996 processors on 83 machines with an aggregate 4 TB of RAM ✤ Assembled 18 Gbp in 5 million scaffolds larger than 500 bp with a scaffold N50 of 6 kbp 9
10. Identify white spruce genes ✤ Identify CYP720B genomic contigs by aligning the sitka spruce and white spruce transcripts to the white spruce genome assembly using BWA-SW ✤ Align the sitka spruce and white spruce transcripts to the CYP720B genome contigs using gmap (Wu and Watanabe 2005) 10
11. CYP720B4 11 ✤ Expressed in both sitka spruce and white spruce Sitka spruce White spruce
12. CYP720B7 12 ✤ Expressed only in sitka spruce Sitka spruce White spruce
13. CYP720B15/16/17 related 13 ✤ White spruce transcripts align, but no sitka spruce transcripts Sitka spruce White spruce
14. Cytochrome P450 CYP720B Genes Sitka spruce EST White spruce RNA-seq White spruce genome assembly CYP720B2 yes yes CYP720B4 yes yes CYP720B5 yes yes CYP720B7 no yes CYP720B8 no partial CYP720B9 no yes CYP720B10 no partial CYP720B12 yes yes CYP720B15 yes partial CYP720B16 no partial CYP720B17 no partial 14