RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary...

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I presented these slides at the Plant Metabolic Network workshop held at the Plant Animal Genome Conference (PAG) XXII, January, 2014. The main goals of the talk were to describe RNA-Seq based annotation of a blueberry genome assembly and explain how we used PlantCyc enzyme data to associate blueberry genes with metabolic pathways.

Transcript of RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary...

  • RNA-Seq Analysis of Blueberry Fruit Identies Candidate Genes Involved in Ripening and Secondary Metabolism Ann Loraine presenting work of: Vikas Gupta, University of Aarhus April Roberts, Ivory Clabaugh, Ketan Patel, Nate Watson, University of North Carolina, Charlotte and many more....
  • Archana Raja Ra'ad Gharabaih Nate Watson Rob Reid April Estrada Stig Andersen Stig Andersen Plants for Health Institute Ann Loraine Ketan Patel Mary Ann Lila Mark Burke Vikas Gupta Ivory Clabaugh Funded by University of North Carolina Mike Wang General Administration Allan Brown Cory Brouwer
  • Transcriptome project goals Identify genes expressed in blueberry during fruit development and ripening. Identify genes encoding enzymes responsible for synthesis of bioactive compounds. Resources for breeding, genomics.
  • Why blueberry? Popular food with health benets - functional food. Antioxidant, thanks to anthocyanin (other compounds) Other pathways? diabetes learning & memory heart disease
  • 454 sequencing 2009 Harvest green ripe 2010 Harvest green ripe 2.5 million 454 ESTs, average size around 300 bases ... not enough to form complete transcript models Ketan Patel
  • Illumina RNA-Seq - 5 stages, 3 bioreps pad cup green pink April 19 April 28 May 20 ripe 3 plants 2-41 2-42 3-33 [row][plant] April 4 June 2 ~ 2 months Stages similar to Zifkin, 2012 (Constable lab) April Estrada
  • Data Processing, Data Analysis Align to genome, and then annotate. May 2013 Assembly Work of Rob Reid, Allan Brown, et al Predicted: 600 Mb (ow cyto) Assembly: ~400 Mb (aDer removing N's) ~13K Scaolds N50 145 Kb Mark Burke Mike Wang Rob Reid Allan Brown Cory Brouwer
  • Annotate: GABox pipeline RNASeq Illumina TopHat, CuLinks RNASeq 454 Align w/ GMAP 60K protein- coding genes 70K transcripts Ab iniTo predicTon Genewise Augustus Glimmer Filter based on expression, structure ,etc. nr gene set MUCH alternaTve splicing, some of it developmental regulated Developed by: Vikas Gupta Used it to annotate 3 genomes:
  • Blueberry Resources Integrated Genome Browser with Illumina RNA-Seq alignments, coverage graphs, splice juncTons features (alternaTve splicing) 454, Sanger EST alignments (GMAP) Gene models (GABox pipeline) Func7onal annota7ons for data analysis Gene Ontology annotaTons Pathway enzyme annotaTons PlantCyc
  • IGB and RNA-Seq Free from BioViz.org
  • Click to open blueberry
  • Gene models load automaTcally open data sets keyword search 12
  • ripe pink mature green cups pads involved in splicing gene models
  • DierenTal gene expression 11 days 9 days 22 days 460 DE genes 1,049 DE genes 5,117 DE genes early high MG high 13 days 22 DE genes ripe high
  • FuncTonal AnnotaTon Gene Ontology annota
  • FuncTonal AnnotaTon - PlantCyc Searched berry sequences against PlantCyc Filtered hits % 65 subj. coverage, % 60 idenTty Iden