1. Annotation of eukaryotic genomes
Genomic DNA
ab initio gene prediction
transcription
Unprocessed RNA
RNA processing
Mature mRNA
Gm3
AAAAAAA
Comparative gene prediction
translation
Nascent polypeptide
folding
Active enzyme
Functional identification
Function
Reactant A
Product B

2. Genome analysis overview: C.elegans

3. Gene finding: ab initio
What features of a ORF can we use?
Size - large open reading frames
DNA composition - codon usage / 3rd position codon bias
Other features:
Kozak sequence CCGCCAUGG
Ribosome binding sites
Termination signal (stops)
Splice junction boundaries

4. Gene finding: comparative
Use knowledge of known coding sequences to identify region of genomic DNA by similarity
transcribed DNA sequence
peptide sequence
related genomic sequence

5. Annotation of eukaryotic genomes
Genomic DNA
ab initio gene prediction
transcription
Unprocessed RNA
RNA processing
Mature mRNA
Gm3
AAAAAAA
Comparative gene prediction
translation
Nascent polypeptide
folding
Active enzyme
Functional identification
Function
Reactant A
Product B

7. Artemis display for S.pombe cosmid

10. Methods for searching Needleman & Wunsch - global alignment
Pairwise alignments: matching a query sequence against a database of subject sequences
Needleman & Wunsch - global alignment
Smith-Waterman - local alignment
FastA
BLAST
Others: SSAHA, WABA
see Chapter 7 Developing Bionformatics Computer Skills

11. BLAST - local similarity searches
BLAST (Basic Local Alignment Search Tool) is the workhorse of genome annotation due to it’s early optimisation for the UNIX platform
Underlies most of the web-based servers world-wide
Comes in many flavours:
BLASTN - DNA against DNA
BLASTX - DNA against Protein
BLASTP - Protein against Protein
TBLASTN - Protein against DNA
TBLASTX - DNA against DNA at the peptide level

12. BLAST - results
BLAST returns high-scoring pairs (HSPs) with a score and p-value. Blast output files can be large and difficult to interpret.
Hence we need tools to make sense of the data - both to filter/process the file and to visualise the resulting multiple sequence alignments.
MSPcrunch - a post-processor for BLAST with a number of different output types.
BioPerl - modules for handling sequences and BLAST output

13. Standard similarity searches for first-pass annotation
genomic DNA v transcript data
BLASTN / EST_GENOME
TBLASTX
genomic DNA v genomic DNA
BLASTN
genomic DNA v non-redundant protein data
BLASTX

14. Data for gene prediction
EST/mRNA - intra-species matches
TBLASTX - inter-species matches
BLASTX - intra-species matches
BLASTX - inter-species matches
Coding measures - genefinder, hexamer
Splice sites - consensus sequences

16. Multiple Sequence alignments in ACEDB

17. Manual review of gene predictions
Check concordance with transcript data
Check concordance with peptide similarity data
Check splice site usage (intron / exon boundaries)
Set of human appraised gene predictions. The translations of the CDS sequences are used for protein feature analysis and initial assignment (ID, function)