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/workshop2010/– 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.