Transcriptomics-Tobacco: Difference between revisions

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* Get the differentially expressed Genes in tobacco leaf
* Get the differentially expressed Genes in tobacco leaf
* Author: Guru and Rajkumar and et al  
* Author: Guru and Rajkumar and et al  
* Timeline
* Time line
** Sequencing of ESTs: SEP 2009
** Sequencing of ESTs: SEP 2009
** Assemble / pre annotation: NOV 2009
** Assemble / pre annotation: NOV 2009
** Print Array  : JAN 2010
** Printing Array  : JAN 2010
** Combining the protein data: MAR 2010
** Combining the protein data: MAR 2010
** Re annotation of hybridised '''genes'''  : APR 2010
** Re annotation of hybridized '''genes'''  : APR 2010
** Consenses of the 2 data for the selected genes (MAY 2010)
** Con senses of the 2 data for the selected genes (MAY 2010)
** Validate selected genes (100) by RT: JUN 2010
** Validate selected genes (100) by RT: JUN 2010
** '''Manuscript''' (after approval):  JUL 2010
** '''Manuscript''' (after approval from ITC):  JUL 2010
** Lab validation of selected genes: DEC 2010
** Lab validation of selected genes: DEC 2010
** Get the final list of genes characterised : JAN 2011
** Get the final list of genes characterized : JAN 2011
** '''Manuscript''' MAR 2011
** '''Manuscript''' MAR 2011
=Scientific Assumptions=
=Scientific Assumptions=
=Strategy=
=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=
=Implementation=
# Some Tools
=== Mapping ESTs on Genomics DNA ===
## [[gth_run]]
:Impractical because tgi_singlet vs est_contig -> >8 days of continuous run in quad core machine
## [[trim_seq]] | Filter "N", size
* Strategy
## [[multifasta-fastas]]
** EST
## [[Mapping ESTs on tgi]]
*** 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]]




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[[Category:Bioinformatics]]
[[:Category:Bioinformatics]]

Latest revision as of 10:24, 5 July 2013

Tobacco Pipeline Target

  • Get the differentially expressed Genes in tobacco leaf
  • Author: Guru and Rajkumar and et al
  • Time line
    • Sequencing of ESTs: SEP 2009
    • Assemble / pre annotation: NOV 2009
    • Printing Array  : JAN 2010
    • Combining the protein data: MAR 2010
    • Re annotation of hybridized genes  : APR 2010
    • Con senses 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 characterized : JAN 2011
    • Manuscript MAR 2011

Scientific Assumptions

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
  • 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
  • Some Tools






Category:Bioinformatics

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