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Hands on exercise day1

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Sucheta Tripathy

This document provides instructions for hands-on genetics exercises to identify coding sequences unique to Phytophthora. Students will analyze RNA-Seq reads assembled into the P. sojae reference sequence to discover transcripts. They will then annotate the sequences by blasting against gene models and databases to determine if they represent new genes or correct errors in other organism's gene models. The goal is to find a coding sequence that is unique in Phytophthora.

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Hands on Exercises – Day1 Sucheta Tripathy, VBI
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
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.
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.
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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Hands on exercise day1

  • 1. Hands on Exercises – Day1 Sucheta Tripathy, VBI
  • 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. 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. 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. 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.