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 Genomics DNA

Impractical because tgi_singlet vs est_contig -> >8 days of continuous run in quad core machine

• Strategy
  • EST
    • Assembling TOBEA EST and ITC-EST (~80,000 sequences) | Merge singlet and contigs and name it.
  • Genomic DNA
    • Merge TGI contig and TGI singlet (6.5 lakhs sequences) | name individual fasta
    • Merge All Bacs and name individual fasta
    • Merge all genomic DNA
  • Mapping (Genome Threader) EST on gDNA
• Pipes
  • uncompress BACs | few sec
  • rename BACs | few sec
  • rename TGI | 12 min
  • rename ESTs | 5 min
  • assemble ITC_EST-TOBEA_EST | 26min | 16core implementation, but after 3 min only one core was used
  • parse assemblage ITC_EST-TOBEA_EST | 3 sec
  • GTH_EST-gDNA | 3 days
• Results
  • count_fasta.pl | Counting the frequency
  • Total number of ITC-ESTs:
  • Total number of TOBEA-ESTs:
  • Total number of Unigenes:

Gene prediction if TGI contig

• • Genome threading
    • TGI_CONTIG vs EST_CONTIG
    • Multithreaded
• Comparing Unique TOBEA EST contigs
  • Tobea ESTs are from all the tissues, while our Tobacco ESTs are from Leaf. Getting the unique TOBEA EST will help to add to the present array
  • Process
  • Script
    • blast_tobea
    • parse_tobea
• Some Tools
  • gth_run
  • trim_seq | Filter "N", size
  • multifasta-fastas