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Transcriptome Analysis : Case studies from Chlamydomonas

Transcriptome Analysis : Case studies from Chlamydomonas

JGI User Meeting 8 @ 2013: Genome Tech Workshop, RNA-Seq talk

Abhishek Pratap

March 26, 2013
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  1. Cu Mn N P Zn Fe S Cu Mn P

    Zn Fe S Nutrient depletion Experiment: Chlamydomonas as a biofuel model Lack of Nutrients induces lipid accumulation.
  2. 6h 24h 48h 8h 2h 1h 30’ 10’ 0 ~1

    cell cycle Control Visible lipid accumulation by microscopy Log phase 3days N Nitrogen depletion Sulfur depletion S Design
  3. Examples of candidate TF genes Transcriptional co-activator Up-regulated Down-regulated Expression

    (FPKM) Expression (FPKM) Time Time Transcription factor regulating nitrogen metabolism
  4. Approach • Build a catalogue of transcripts for an organism

    – Decipher transcriptional structure of genes – New discoveries w.r.t to reference • Novel genes/isoforoms • Gene fusions • Gene boundary extensions • Study abundance of genes/transcripts between different conditions • Find Key Regulator/s responsible for change in transcriptome dynamics RNA-Seq
  5. Strand Specificity: power to resolve strand specific expression Forward strand

    cov Reverse strand cov Non stranded cDNA stranded cDNA
  6. Data Summary & Quality Replicate Correlation Background noise in strand

    specific data 10 • Sequenced 9 time points • 2 biological replicates / cond • ~100 million raw reads / cond • Strand Specific library • with ERCC Spike-in : quality control
  7. Assembled Transcripts Inside ref transcript model same- strand opp- strand

    Outside ref model Ref transcripts not found in assembly Ref gene model Inside – Ref: same – strand Inside – Ref : Opposite – strand Outside Ref: New genes Analysis
  8. Reference RNA-Seq based #genes 18,773 16,467 # transcripts 19,529 32,395

    Uniq Transcriptome size 94.6 Mb 96.1 Mb Mean transcript length 5411 +/- 4283 bp 6203 +/- 4925 bp Bioinformatics Challenges • gene fusions • Antisense transcripts • Low abundant real signal v/s noise
  9. Analysis and design of RNA sequencing experiments for identifying isoform

    regulation. Nature Methods(2010). Visualizing Alternative Splicing
  10. Chlamy’s Transcriptome Reference RNA-Seq #genes 18,773 16,467 # transcripts 19,529

    32,395 Uniq Transcriptome size 94.6 Mb 96.1 Mb Mean transcript length 5411 +/- 4283 6203 +/- 4925 Discoveries made #novel genes 184 #novel exons inside ref genes 544 #novel Antisense genes 1727 #mergers (ref genes) ~685 #extensions (ref genes) 6100
  11. Comparison with known targets Reported down-regulated genes fatty acid synthesis

    Carboxytransferase : syn of fatty acid de-novo fatty acid synthesis
  12. Summary • data quality control for RNA-Seq • Building a

    transcriptome to capture – new genes/transcripts – gene fusions – Improve annotation (if available) • response of the genetic machinery to a stimulus: differential expression • Picking the possible key regulators
  13. The work conducted by the U.S. Department of Energy Joint

    Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231 Chew Yee Ngan, Chee Wong, James Han, Chia-Lin Wei, Simon Prochnik