RNASeq Analysis of Blueberry Fruit Identifies 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....
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RNA-Seq analysis of blueberry fruit identifies candidate genes involved in ripening and secondary metabolism
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.
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RNA-‐Seq Analysis of
Blueberry Fruit Identifies 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....
Mike Wang
Allan Brown
Mark Burke
Cory Brouwer
Rob ReidApril
Estrada
Ketan Patel
Vikas GuptaIvory
Clabaugh
Stig AndersenStig AndersenPlants for Health Institute
Ann Loraine
Ra'adGharabaih
Nate Watson
Mary Ann Lila
Archana Raja
Funded by University of North Carolina GeneralAdministration
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 benefits - functional food.
• Antioxidant, thanks to anthocyanin (other compounds)
• Other pathways?
diabetes
heart disease
learning & memory
454 sequencing
green ripe
Ketan Patel
2009 Harvestgreen ripe
2010 Harvest
2.5 million 454 ESTs, average size around 300 bases... not enough to form
complete transcript models
Illumina RNA-Seq - 5 stages, 3 bioreps3
plan
ts
2-41
2-42
3-33
pad cup green pink ripe
April Estrada
[row]-[plant] April 4 April 19 April 28 May 20 June 2
~ 2 months
Stages similar to Zifkin, 2012 (Constable lab)
Data Processing, Data Analysis
• Align to genome, and then annotate.• May 2013 Assembly –Work of Rob Reid, Allan Brown, et al– Predicted: 600 Mb (flow cyto)– Assembly: ~400 Mb (aDer removing N's)• ~13K Scaffolds• N50 145 Kb
Rob Reid
Allan Brown
Cory Brouwer
Mike Wang
Mark Burke
RNASeqIllumina
60K protein-‐coding genes 70K transcripts
RNASeq454
Ab iniTopredicTon
GenewiseAugustusGlimmer
Annotate: GABox pipeline
Align w/ GMAP
TopHat, CuffLinks
Filter based on expression, structure ,etc.
nr gene set
Developed by: Vikas GuptaUsed it to annotate 3 genomes:
MUCH alternaTve splicing,some of it developmental regulated