Annotation of eukaryotic genomes

transcription

RNA processing

translation

AAAAAAA

Genomic DNA

Unprocessed RNA

Mature mRNA

Nascent polypeptide

folding

Reactant A Product BFunction

Active enzyme

ab initio gene prediction

Comparative gene prediction

Functional identification

Gm3

Genome analysis overview: C.elegans

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

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

Annotation of eukaryotic genomes

transcription

RNA processing

translation

AAAAAAA

Genomic DNA

Unprocessed RNA

Mature mRNA

Nascent polypeptide

folding

Reactant A Product BFunction

Active enzyme

ab initio gene prediction

Comparative gene prediction

Functional identification

Gm3

Artemis display for S.pombe cosmid

Methods for searching• 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

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

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

Standard similarity searches for first-pass annotation

• genomic DNA v transcript data

• BLASTN / EST_GENOME

• TBLASTX

• genomic DNA v genomic DNA

• BLASTN

• TBLASTX

• genomic DNA v non-redundant protein data

• BLASTX

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

Multiple Sequence alignments in ACEDB

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)