Hands on exercise day1

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Hands on exercise

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Hands on exercise day1

  1. 1. Hands on Exercises – Day1 Sucheta Tripathy, VBI
  2. 2. Genetics of being Phytophthora? • Objective: Find a coding sequence that is unique in Phytophthora. • What is starting material? – 16 million RNASeq reads are assembled into P.sojae reference sequence to generate junctions. These junctions are judged using some of the best available algorithms. • http://vmd.vbi.vt.edu/download/data/workshop 2010/ – Coverage.wig – ps1V1.fasta
  3. 3. Transcript discovery • Sort the coverage file on the basis of the number of hits to the reads on column 4. • Find the upper 25% percentile. • Remove sequences larger than 1000 or less than 10 bases long. • Fetch data from ps1V1 file. • Split fasta file into N equal parts.
  4. 4. Annotation Steps • Blast against P.sojae gene models(vmd.vbi.vt.edu/toolkit). • Check coding potential with P.sojae codon usage tables. - If found hit, then get the gene model and compare the splice sites and correct it. - If not found, then blast against P.ramorum/H.arabidopsidis/P.infestans coding sequences. - See if matches with the splice junctions correctly – if not, the gene models in those organisms are INCORRECT.
  5. 5. Annotation • Blast against nr database. If blast hit is not found with any coding sequences in nr database, then most probably you found a new gene.. • Check if the sequence is a signal peptide/target peptide to determine if it is secretory in nature. • Run MEME motif analysis search on the sequence.

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