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 | ||
* | * Time line | ||
** Sequencing of ESTs: SEP 2009 | ** Sequencing of ESTs: SEP 2009 | ||
** Assemble / pre annotation: NOV 2009 | ** Assemble / pre annotation: NOV 2009 | ||
** Printing Array : JAN 2010 | ** Printing Array : JAN 2010 | ||
** Combining the protein data: MAR 2010 | ** Combining the protein data: MAR 2010 | ||
** Re annotation of | ** 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 | ** Validate selected genes (100) by RT: JUN 2010 | ||
** '''Manuscript''' (after approval from ITC): 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 | ** Get the final list of genes characterized : JAN 2011 | ||
** '''Manuscript''' MAR 2011 | ** '''Manuscript''' MAR 2011 | ||
Revision as of 07:34, 4 June 2010
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
- EST
- 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
- Genome threading
- 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
- gth_run
- trim_seq | Filter "N", size
- multifasta-fastas