Annotating the Genome (H)

Transcription

1 Annotating the Genome (H)

2 Annotation principles (H1) What is annotation? In general: annotation = explanatory note* What could be useful as an annotation of a DNA sequence? an amino acid sequence? What else? Any other sequences that we might want to annotate? *A note is a message. It may be written in any language, on paper, clay tablet, computer file, etc. It may contain various kinds of information.

3 Annotation principles (H1) Annotation For DNA: annotations could describe beginnings and ends of coding regions, gene names, gene functions, etc. For proteins: annotations could describe functions locations of genes, etc.

4 Types of Annotation We can distinguish Structural annotation E.g. locations of genes/coding regions Functional annotation What the sequence does

5 Types of Annotation II Structural annotation E.g. locations of genes/coding regions What else? Functional annotation What the sequence does What else?

6 Types of Annotation II (cont.) Structural annotation E.g. locations of genes/coding regions What else? ORFs, folding, motif locations: any properties of the molecule itself Functional annotation What the sequence does What else? Biological, biochemical functions, regulation, interactions, expression parameters: any biological information

7 How to Annotate the Genome Can start by looking for telltale features Signals: localized motifs of various kinds Contents: longer ( non-localized ) sequences Homologies: molecules with the same ancestor as x Let s look at these in more detail

8 Telltale Features Signals: localized motifs of various kinds Contents: longer ( non-localized ) sequences Homologies: molecules with the same ancestor as x Signals include: promoters splice sites start codons stop codons polyadenylation sites

9 Telltale Features Signals I: Promoters Signals include: promoters binds transcription factor protein thereby enables the gene downstream to be expressed not all promoters are identical, but they are similar differences affect their binding effectivenesses sometimes less effective binding is better (why?) a theory about promoter mutations They explain many differences between closely related species (e.g. humans, chimps) See e.g. pp of The Agile Gene splice sites, start codons, stop codons, polyadenylation sites

10 Telltale Features Signals I: Promoters (cont.) Promoters On anti-sense strand (why?) Near gene they control (why?) Upstream of gene they control (why?) bp Various programs exist that try to find them They have characteristics supporting this Let's search for tools and see what they are splice sites, start codons, stop codons, polyadenylation sites

11 Telltale Features Signals II: Splice Sites Signals include: promoters splice sites are at the boundaries between introns and exons (which are?) most introns start with GT and end with AG D. Gusfield, Algorithms on Strings, Trees, and Sequences, 1997 but not all! And GT & AG also appear for other reasons Like what? Yet, those short sequences often suggest a boundary start codons, stop codons, polyadenylation sites

12 Telltale Features Signals II: Splice Sites (cont.) Signals include: promoters splice sites So what is a splice site? There is a donor splice site (at 5' end) Begins with GU (mrna) or (antisense DNA) There is an acceptor splice site (at 3' end) Ends with AG (mrna) or (antisense DNA) There is about pyrimidines (C, U) near the AG Near that is a branch point with an A

13 Telltale Features Signals II: Splice Sites (cont. ii) Signals include: promoters splice sites -

14 Telltale Features Signals II: Splice Sites (cont. ii) Signals include: promoters splice sites Here it is in IUPAC notation: - M-A-G-[cut]-G-U-R-A-G-U (donor site)... intron sequence... C-U-R-[A]-Y (branch sequence nucleotides upstream of acceptor site)... Y-rich-N-C-A-G-[cut]-G (acceptor site) This has some items besides ACGU

15 Telltale Features Signals II: Splice Sites (cont. iii) splice sites Here it is in IUPAC notation: - M-A-G-[cut]-G-U-R-A-G-U (donor site)... intron sequence... C-U-R-[A]-Y (branch sequence nucleotides upstream of acceptor site)... Y-rich-N-C-A-G-[cut]-G (acceptor site) This has some items besides ACGU See table at Combinatorial question: does this table have any missing entries?

16 Telltale Features Signals II: Splice Sites (cont. iv) splice sites Here it is in IUPAC notation: - M-A-G-[cut]-G-U-R-A-G-U (donor site)... intron sequence... C-U-R-[A]-Y (branch sequence nucleotides upstream of acceptor site)... Y-rich-N-C-A-G-[cut]-G (acceptor site) You can see that there is hope for: Automatic detection of splice sites therefore... Automatic detection of introns

17 Telltale Features Signals III: Start Codons Signals include: Promoters, splice sites, start codons TAC (AUG) in most eukaryotes This also codes for amino acid M (which is?) Would you guess that many proteins start with a methionine? How good a signal is AUG? All start codons are methionine codons, but not all methionine codons are start codons - So AUG suggests but does not require something stop codons, polyadenylation sites

18 Telltale Features Signals IV: Stop Codons Signals include: Promoters, splice sites, start codons, Stop codons They are (RNA version) UAA, UAG and UGA They do not code for an amino acid They tell a ribosome to stop making a protein Therefore, they indicate the end of a gene All start codons are methionine codons, but not all methionine codons are start codons - Is it analogous for stop codons? Which are more reliable signals, start or stop codons? Does the genome have about 3x more STOP occurrences than STARTs?

19 Telltale Features Signals IV: Polyadenylation sites Signals include: Promoters, splice sites, start codons, stop codons, and polyadenylation sites Adenine is a nucleobase (or just base) Adenine becomes part of adenosine (a nucleoside=nucleobase+ribose) Adenosine becomes part of AMP, damp (add in a phosphate backbone molecule) ADP, ATP are also made from adenosine termination of mature eukaryotic mrnas occurs at polyadenylation sites downstream from stop codons delineation of these sites is essential for the study of gene regulation and the design of probes for transcriptome analysis. typical poly(a) sites are between 0 and 2 kb from the stop codon /23/06 Are polyadenylation sites before or after the stop codon?

20 Source:

21 Polyadenylation The tail of an mrna is typically a long string of A s (in eukaryotes, sometimes in bacteria) Poly- = many -aden- = adenine/adenosine/amp/adp/atp -ylation = process of converting Polyadenylation helps determine the lifetime of an mrna hence how many protein molecules it makes hence polyadenylation regulates gene activity Promoters also do this in another way

22 How Polyadenylation Works How does a car engine work? There is a simpler essence, and many complex details How does polyadenylation work? The essence: The stop codon is not important It is for synthesizing protein, not mrna! Enzymes recognize short mrna sequences (What is an enzyme?) They cleave the mrna, then add A s to the new end

23 How Polyadenylation Works (cont.) How does polyadenylation work? The essence: The stop codon is not important It is for synthesizing protein, not mrna! Enzymes recognize short mrna sequences (What is an enzyme?) They cleave the mrna, then add A s to the new end Sequences they look for: AAUAAA (typical for animals) Repetitions of UGUAA (mammals) Such short sequences are called signals

24 Polyadenylation: an interesting fact Some genes have multiple polyadenylation sites So the gene can produce different length mrnas Same gene, different transcripts Different transcripts, different translations Some genes have alternative exons Same gene, different transcripts This is one reason why we have more proteins than genes DNA provides options but doesn t say which will happen! So DNA does not determine everything

25 Finding Signals Signals can be found using consensus sequences which describe which codons are conserved and which are variable We've seen ones like: C[AT]D{P} Is this a protein or DNA consensus sequence? For DNA signals, use DNA consensus sequences

26 DNA Signals from DNA Consensus Sequences Signals can be found using DNA consensus sequences Can careful examination of an individual s sequences reveal what is conserved? For DNA signals, use DNA (not protein) consensus sequences Example: C[AG]RY{T}N R for purine (A or G) Y for pyrimidine (C or T or U) N for any nucleobase (recall IUPAC notation)

27 Recap: How to Annotate the Genome Can start by looking for telltale features Signals: localized motifs of various kinds Contents: longer ( non-localized ) sequences Homologies: molecules with the same ancestor as x Let s look at contents in more detail next

28 Telltale Features Contents The most important contents sequence: a coding region

29 Telltale Features Contents II A coding region is a key contents sequence Recall that signals can be found using consensus sequences Do you expect homologous introns to have consensus sequences? Let s understand the vocabulary, then answer the question

30 Telltale Features Contents II Another contents sequence type: CpG islands: regions of DNA near and in approximately 40% of promoters of mammalian genes. They are regions where a large concentration of phosphodiester-linked cytosine and guanine pairs exist. - Hence, CpG from cytosine phosphodiester guanine CpG means C next to G in the same strand CG pair refers to a base pair across 2 strands The usual formal definition of a CpG island is a region with at least 200 bp and with a GC percentage that is greater than 50% [humans overall have 42%] and with an observed/expected CpG ratio that is greater than 60%. CpG=CG. What is the expected % of CGs in a sequence? Oddly, observed % is only about 1%! Biomolecular reasons.

31 Telltale Features Contents III If we do not have consensus sequences associated with a contents sequence type, how to determine them? Recall our objective: distinguish genes from their surrounding nucleotides Nucleotide frequencies are different for coding sequences Nucleotide dependencies are different for coding sequ's Why? Consider frequencies of stop codons in gene vs. non-gene sequences

32 Recap: How to Annotate the Genome Can start by looking for telltale features Signals: localized motifs of various kinds Contents: longer ( non-localized ) sequences Homology: Molecules with the same ancestor Let s look at homologies more next

33 Telltale Features Homologies If a sequence is homologous with a known gene, is the sequence is a gene? exon, is the sequence an exon? intron, is the sequence an intron? intergenic sequence, is the sequence an intergenic sequence? Can proteins be homologous too?

34 Combining Evidence for Genome Annotation Consider the three telltale features Signals: localized motifs of various kinds Contents: longer ( non-localized ) sequences Homologies: molecules with the same ancestor as x Ideally, software would use all of these to determine what s a gene and what s not

35 ORFs Another Way to Detect Genes ORF Open Reading Frame Nucleotide sequence beginning with a START triplet and ending with the next STOP triplet Can code for a protein, therefore Compare the lengths of ORFs that are genes with ORFs that are not! How can that be used to decide which of the 6 reading frames is the right one? Could this method miss short genes? long genes?

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37 ORFs for Detecting Genes - Warnings When might looking for long ORFs miss a gene?

38 ORFs for Detecting Genes - Warnings When might looking for long ORFs miss a gene? Very short genes They might make peptides for example Some bacteria use non-standard start codons Sometimes TGA/UGA is not a stop codon Instead it codes for selenocysteine Sometimes it looks like a gene used to be a gene is no longer expressed (so it isn t a gene)

39 What About Distinguishing Between Exons and Introns? Recall: what are introns & exons? Introns can split two expressed codons One codon is at the end of one exon The other codon begins the next Introns can even split a single codon! Distinguishing exons and introns is tricky Use content detection Look for splice sites

40 What About RNA Genes? These genes are not expressed I.e. they don t produce proteins rrna trna To identify them, search possible sequences for homologies RNA genes tend to change very slowly due to the extra complications in RNA genes Physical structure, molecular variations

41 What is the Result of Finding all these Genes in Humans? Humans have about thousand genes Human DNA is 98.5% the same as bonobo DNA, 99% the same as chimp DNA Chimps and Bonobos diverged after the split with proto-humans Why the difference then?

42 What is the Result of Finding all these Genes in Humans? Humans: about 20,000 to 25,000 genes Does this seem low? Consider: we re coded with 20 amino acids That s even less than 20-25k! We re coded with 4 nucleotides That s even less than 20, much less 20-25k! So what is the explanation?!?

43 Annotation: Software and Databases (H2) These are for annotating the genome They do it by finding genes why is finding genes, annotation? because this information may be associated with the corresponding pieces of the genome Given a giant string, we label parts of it ORF Finder is one tool available on-line at NCBI works best for genes without introns (e.g. bacterial genomes) How would you guess ORF Finder works?

44 How Well Does a Gene Finder Work? Have it classify each nucleotide in a test sequence PP=predicted positive (i.e. is in a gene) PN=predicted negative (i.e. not in a gene) AP=actual positive (is really in a gene) AN=actual negative (isn t really in one) TP=true positive (finder got it right) TN=true negative (finder got it right) Now calculate TP,TN, FP, & FN (how?) Then get sensitivity=tp/ap=tp/(tp+fn) and specificity=tp/pp=tp/(tp+fp) You can combine sensitivity and specificity (see footnote 1 p. 120, Westhead et al.)