1. What do you with a whole genome sequence?

2. Translate it into all 6 reading frames……

3. Identify all of the stop codons..…

4. And the start codons……
Can then identify all Open Reading Frames (ORFs)
But are all real genes?

5. Three major prokayotic gene modelers:
Generation uses predominantly 6-mer statistics to recognize coding regions; it uses a proximity rule-based start call with ATG and GTG as potential starts.
Glimmer uses interpolated Markov models (IMMs) to identify the coding regions; it uses ATG, GTG, and TTG as potential starts.
Critica uses blastn to produce alignments from the entire dataset and derives dicodon statistics to recognize coding sequences. It uses an SD sensor with ATG, GTG, and TTG as potential starts.

6. Now what?
BLAST genes:
To assign functions based on similarity with known genes

7. Basic Local Alignment Search Tool
BLAST
Basic Local Alignment Search Tool
finds regions of local similarity between sequences
>my favorite gene
Atgtcgctagctagctsctagctag
Database of many gene sequences
GenBank is one example
Answers the questions—
Is there a match?
And how good is it?

8. But--Gene D has only 20% identity to gene A!
What are the genes doing?
Function is assigned based on degree of similarity of an already characterized gene in the database
2 potential problems with this approach
Transitive catastrophe
Gene A
Assigned function based on mutant phenotype or biochemical characterization of protein product
Gene B
From genome sequence:
70% identity to gene A
Gene C
From genome sequence:
60% identity to gene B
Gene D
From genome sequence:
70% identity to gene C
But--Gene D has only 20% identity to gene A!

9. Would like to propagate function only to orthologous genes
Homolog– genes sharing a common origin
note: two genes are homologs or they or not
no such thing as %homology or “more homologous”
Two main kinds of homologs
Orthologs-genes orginating from a single ancestral gene in the last common ancestor of the compared genomes
Paralogs-genes related via duplication

10. X,Y,Z are genes in the same family
A, B, C are three species

11. Two more complicated cases:
Xenologs-genes orginating from a HGT of an ortholog in a distant lineage
Pseudoparalogs- homologous genes that appear to paralogs in a single genome analysis but have arisen due to a combination of vertical and lateral descent

12. How to identify orthologs:
One way: Reciprocal BLAST analysis
>Genome A gene1
AGTGCATGTCCC
>Genome A gene 2
TGTGCGTAGTCCAAA
Database:
Genome B
AND
>Genome B gene1
GGTTTTTACA
>Genome B gene 2
AAACCTCTCTGA
Database:
Genome A
ASK: are two genes each other’s Best BLAST hit?

13. Can be confounded by lineage specific gene loss

14. What if there is nothing at all similar in the database?4% 4% 2%
20%
Call it a “hypothetical” gene
If it has a match but that is to another hypothetical gene?
“conserved hypothetical” 1% 4% 1% 2%
32% 1%
Conserved Hypothetical
25%
Hypothetical 1% 4%
DNA Replication & Repair
Energy Metabolism
Nucleotide Metabolism
Lipid Metabolism
Transcription
Amino Acid Metabolism
Translation
Carbohydrate Metabolism
Transport
Cofactor Metabolism
Unassigned