Transcriptomics-Tobacco

Tobacco Pipeline Target

• Get the differentially expressed Genes in tobacco leaf
• Author: Guru and Rajkumar and et al
• Timeline
  • Sequencing of ESTs: SEP 2009
  • Assemble / pre annotation: NOV 2009
  • Printing Array  : JAN 2010
  • Combining the protein data: MAR 2010
  • Re annotation of hybridised genes  : APR 2010
  • Consenses of the 2 data for the selected genes (MAY 2010)
  • Validate selected genes (100) by RT: JUN 2010
  • Manuscript (after approval from ITC): JUL 2010
  • Lab validation of selected genes: DEC 2010
  • Get the final list of genes characterised : JAN 2011
  • Manuscript MAR 2011

Strategy

• Gene Prediction
  • Flavour +/- tobacco samples
  • Extract mRNA and made ESTs
  • Sequencing and EST short reads
  • Assemble the EST reads (contigs and singlets)
  • Annotated the contigs
  • Meaningful EST pieces
  • Implanted them on micro array
  • Further annotate them to spot reliable genes to the existing models
  • Use the contrasting samples to hybridise them to assign gene status and differentially expressed
• Validate the identified genes

Data

• TGI downloaded Sequences
  • tgi_contig : 81,959
  • tgi_singlet: 5,58,987
• EST Unigene assembled
  • est_contig: 38,261
  • est_singlet: ~2 lakh (damaged)
• TOBEA
  • EST contigs: 40642

Implementation

• Mapping ESTs on tgi
  • Genome threading or Blat
    • Impractical because tgi_singlet vs est_contig -> >8 days of continuous run in quadcore machine
    • Impractical because tgi_singlet vs est_singlet ->> month of continuous run in quadcore machine
• Gene prediction if TGI contig
  • Genome threading
    • TGI_CONTIG vs EST_CONTIG
    • Multithreaded

• Some Tools
  • gth_run
  • trim_seq | Filter "N", size
  • multifasta-fastas