Utilizing the Illumina deep sequencing technique to define

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Utilizing the Illumina deep sequencing technique to define the plant transcriptome Pinghua Li Institute of Tropical Biosciences & Biotechnology (ITBB) Chinese Academy of Tropical Agriculture Sciences (CATAS) GCP21-II, Kampala, Uganda June 19, 2012

Acknowledgments Brutnell Lab (Danforth) Xiang Lab (Weill Cornell Medical College) Jenny Xiang Diana Shao Wang Lab (ITBB, CATAS) Wenquan Wang Zhiqiang Xia Xin Chen Kun Pan Yang Zhang Giovannoni Lab (Cornell) Jim Giovannoni Silin Zhong Wang Lab (CAS) Ying Wang Gong Xiao Brutnell Lab (Danforth) Tom Brutnell Lin Wang Tesfamichael Kebrom Nelson Lab (Yale University) Tim Nelson (Yale) Neeru Gandotra Lori Tausta Turgeon Lab (Cornell University) Bob Turgeon Edwin Reidel Sun Lab (Cornell University) Qi Sun Lalit Ponnala Liu Lab (Iowa State University) Peng Liu Yaqing Si

High throughput sequencing – a new paradigm Illumina (Solexa) SOLiD 454/Roche Genome Sequencer Single Molecule Sequencing/Pacific Biosciences Applications Genome sequencing microRNA screening Gene expression (RNA-seq) ChIP-seq RNA-seq profiles vs. microarray • Less dependent on gene annotation • Resolves closely related members of a gene family • Identifies alternative splicing patterns • Capture low abundant expression

Outline RNA-seq library construction What to expect from RNA-seq (an example from Maize leaf transcriptome) Application in wild type vs. cultivar comparison of Cassava

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RNA-seq library construction Wang et al. PLoS One. 2011

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PCR amplification with Bar-coded primers RNA-seq library construction PCR amplification with Bar-coded primers Pool libraries Illumina Hiseq 2000 Sequence how deep? (Tomato) ZhangJun Fei, Cornell

Aconitase RNA-seq application From maize leaf transcriptome Genome annotation correction Differential expressed genes discovery (Biological stories) Aconitase Exon skipping

RNA-seq application-- Gene annotation correction Wang et al. PLoS One. 2011

RNA-seq application-Differential expressed (DE)gene-biological stories RNA-seq raw read Align to the reference genome Quantitative gene expression Determine the DE gene Clustering and pathway analysis Biological stories-paper writing Base -1cm +4cm Tip Li et al, Nature Genetics, 2010

RNA-seq application-Cassava transcriptome Lines: Arg 7 vs. W14 Tissue: Roots, leaves and stems Platform: Illumina Hiseq 2000 KU50 & Arg7 (cultivar) vs. W14 (wild type) Starch biosynthesis Root 1480 390 345 378 Leaf 2234 Stem 2381 1545 DE genes in root, leaf and stem Leaf Root

Thank you for your attention!

Maize leaf developmental gradient for 9 day Leaf 3 RNA-seq application Leaf 1 Maize leaf developmental gradient for 9 day Leaf 3 Base -1 cm +4 cm Tip Source Sink Transition Base -1 cm +4 cm Tip Li et al, Nature Genetics, 2010