Exercise 4 : Sequence alignment for gene prediction

Objective: identify genes in genomic sequences

We want to identify genes within a chicken genomic sequence and to determine the precise positions of their exons, introns, translation initiation sites and translation termination sites. We will use two different approaches, based on sequence comparison, to identify genes . You will have to compare des results of the both methods, to try to understand the discrepancies between the different predictions.

1- First of all, it is necessary to identify and mask repeated sequences

• RepeatMasker (return format: html) ( ***    *** )
Mirror sites: Germany, USA, UK (Zebrafish specific)

2- Search for transcribed regions (EST, cDNA) in genomic DNA

• Search for ESTs matching the genomic sequence (masked): MEGABLAST (NCBI)  (use database est_others) ( *** )
• Select all matching ESTs (at least 98% identity, > 70 bp) from chicken (Gallus gallus), and retrieve sequences from NCBI BLAST output  ( *** )
• Assemble ESTs with CAP3  ( ***    *** ) (save Contigs in a text file)
• Align each cDNA to the genomic DNA with  SIM4 ( ***    ***  *** )

 

3- Comparative approach: search protein coding regions by similarity

• Search for proteins matching the genomic sequence (masked) with BLASTX (translated BLAST searches): BLAST (NCBI)( *** )
• Retrieve the closest homologue ( *** )
• Alignment this protein to the genomic DNA with  GENEWISE (Sanger), or GENEWISE (Pasteur) (parameter: genewise protein to genomic DNA)( *** )

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Now repeat the same exercise with two other chicken genomic sequences: genomic1   genomic2  (see results )