The steps in genome sequencing

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Wendy Crawford
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1 Genome Annotation

2 The steps in genome sequencing Generate genome sequence Assemby ORF caing trna identification rrna identification Functiona annotation

3 Annotating Genomes Identifying which protein performs which function

5 Why annotate a genome? Cataog what's there Identify what's missing but shoud be there! Things you don't know In vitro growth Mycopasma pneumoniae Comparative genomics Hypothesis generation

6 The goas of annotation Exchange information with others Compare annotations between organisms

7 How to annotate a genome? Sequence Assembe Identify open reading frames Putative proteins

8 Putative protein Open Reading Frame (ORF) A stretch of amino acids with no stop codon Coding Sequence (CDS) An ORF that coud encode a protein Protein encoding gene (PEG) An ORF that coud encode a protein Hypothetica protein = putative protein Something that has not been experimentay shown Poypeptide

9 PEGS E. coi 4,391 genes 4,288 genes that make proteins (pegs)

10 How to annotate a genome? Sequence Assembe Identify open reading frames Putative proteins Assign functions to proteins BLAST HMMR Psi-BLAST

11 Traditiona genome annotation

12 BLAST Simiarities Traditiona genome annotation

13 BLAST Simiarities Traditiona genome annotation

14 BLAST Simiarities Traditiona genome annotation

15 BLAST Simiarities Traditiona genome annotation

16 BLAST Simiarities Traditiona genome annotation

17 BLAST Simiarities Traditiona genome annotation

18 BLAST Simiarities Traditiona genome annotation

19 BLAST Simiarities Traditiona genome annotation

20 BLAST Simiarities Traditiona genome annotation

21 BLAST Simiarities Traditiona genome annotation

22 BLAST Simiarities Traditiona genome annotation

23 BLAST Simiarities Traditiona genome annotation

24 BLAST Simiarities Traditiona genome annotation

25 Protein Famiies

26 Protein Famiies

27 Protein Famiies

28 Protein Famiies

29 Gene Ontoogy Ontoogy A hierarchy of functions Does not need to be inear - Directed Acycic Graph Controed Vocabuary Decides which words or phrases to use

30 GO Gene ontoogy A eukaryotic focus - Drosophia - Mus - Saccharomyces - Homo

31 GO Ceuar component The parts of a ce Moecuar function e.g. igand binding Bioogica processes What things do

32 GO Terms [GO ID, function] e.g: GO: Ontoogy: moecuar function Name: pyruvate kinase activity

33 GO Terms [GO ID, function] e.g: GO: Ontoogy: moecuar function Name: pyruvate kinase activity Mainy assigned by BLAST/HMMER/... etc

34 Directed Acycic Graph Moecuar function Cataytic activity Transferase activity Transferase activity, transferring phosphorous Kinase activity phosphotransferase activity, acoho group as acceptor Pyruvate kinase activity

35 Probems Annotation by committee Eukaryotic focus Some efforts to counter that - Owen White - Arriane Toussaint Not very deep Strict controed vocabuary

36 Aternatives

37 Basic bioogy aci acz acy aca Jacob & Monod, 1961

38 Basic bioogy aci acz acy aca

39 Different types of custering < 80 % < 80 % < 80%

40 Different types of custering < 80 % < 80 % < 80%

42 Different types of custering < 80 % < 80 % < 80%

43 Heme / chorophy metaboism is conserved They are both porphyrins

44 Occurrence of custering in different genomes 1 Custers of genes w/ maximum 80% identity Genes in subsystems in custers Tota number of genomes in group 120 Fraction of genes in custers Number of genomes

45 The Subsystems Approach to Annotation Subsystem is a generaization of pathway coection of functiona roes jointy invoved in a bioogica process or compex Functiona Roe is the abstract bioogica function of a gene product atomic, or user-defined, exampes: - 6-phosphofructokinase (EC ) - LSU ribosoma protein L31p - Streptococca viruence factors - Shoud not contain putative, thermostabe, etc Popuated subsystem is compete spreadsheet of functions and roes

46 Histidine Degradation Conversion of histidine to gutamate Functiona roes defined in tabe Incusion in subsystem is ony by functiona roe Controed vocabuary

47 Subsystem Spreadsheet Subsystem Spreadsheet Organism Variant HutH HutU HutI GuF HutG NfoD ForI Bacteroides thetaiotaomicron 1 Desufotea psychrophia 1 Haobacterium sp. 2 Deinococcus radiodurans 2 Bacius subtiis 2 Cauobacter crescentus 3 Pseudomonas putida 3 Xanthomonas campestris 3 Q8A4B3 Q8A4A9 Q8A4B1 Q8A4B0 gi gi gi gi Q9HQD5 Q9HQD8 Q9HQD6 Q9HQD7 Q9RZ06 Q9RZ02 Q9RZ05 Q9RZ04 P10944 P25503 P42084 P42068 P58082 Q9A9MI P58079 Q9A9M0 Q9A9L9 Q88CZ7 Q88CZ6 Q88CZ9 Q88D00 Q88CZ3 Q8PAA7 P58988 Q8PAA6 Q8PAA8 Q8PAA5 Listeria monocytogenes -1 Coumn headers taken from tabe of functiona roes Rows are seected genomes or organisms Ces are popuated with specific, annotated genes Functiona variants defined by the annotated roes Variant code -1 indicates subsystem is not functiona Custering shown by coor

48 The Popuated Subsystem Subsystem Spreadsheet Organism Variant HutH HutU HutI GuF HutG NfoD ForI Bacteroides thetaiotaomicron 1 Desufotea psychrophia 1 Haobacterium sp. 2 Deinococcus radiodurans 2 Bacius subtiis 2 Cauobacter crescentus 3 Pseudomonas putida 3 Xanthomonas campestris 3 Q8A4B3 Q8A4A9 Q8A4B1 Q8A4B0 gi gi gi gi Q9HQD5 Q9HQD8 Q9HQD6 Q9HQD7 Q9RZ06 Q9RZ02 Q9RZ05 Q9RZ04 P10944 P25503 P42084 P42068 P58082 Q9A9MI P58079 Q9A9M0 Q9A9L9 Q88CZ7 Q88CZ6 Q88CZ9 Q88D00 Q88CZ3 Q8PAA7 P58988 Q8PAA6 Q8PAA8 Q8PAA5 Listeria monocytogenes -1

49 Nan-operon, a key pathway within the Siaic Acid Metaboism subsystem Microbia siaic acid metaboism has now been firmy estabished as a viruence determinant in a range of infectious diseases

50 The nan-operon

51 Comparison of annotations within the conserved custer (nan-operon) Coor coding for annotations: - green, consistent - yeow; genera cass; - gray, inconsistent or not informative

52 Methionine Biosynthesis From here You need to get to here

55 ?

56 ? Missing genes?

57 Hypothesis testing that eads to the wet ab...

58 Subsystems deveoped based on Wet ab Chromosoma context Metaboic context Phyogenetic context Microarray data Proteomics data

59 How can we compare annotations There are severa groups doing annotations of microbia genomes How do we compare them?

60 Caveat emptor!

61 Natura Metrics Number of subsystems defined Number of functiona roes defined Number of genes connected to functiona roes

62 Annotations for some genomes

63 Appied Metrics Number of soid connections of gene to functiona roe where soid is 1. supported by experimenta data 2. connected to functiona roe and in chromosoma custer with genes impementing functiona roes from the same subsystem 3. ony gene in genome connected to a functiona roe in an active variant of a subsystem Reactions, GO terms, Artices, Other databases cross references (number and diversity)

64 Appied Metrics

65 Comparison of annotations within the conserved custer (nan-operon) Coor coding for annotations: - green, consistent - yeow; genera cass; - gray, inconsistent or not informative

66 Tamudic question * If I find the identica protein sequence in two different organisms, is it doing the same function in both organisms? Per: Eio Schaecter, Sma Things Considered. A tamudic question is unanswerabe

67 hisa FIG function: Phosphoribosyformimino-5-aminoimidazoe carboxamide ribotide isomerase (EC ) Other functions in RefSeq: phosphoribosyformimino-5-aminoimidazoe carboxamide phosphoribosyformimino-5-aminoimidazoe carboxamide ribotide isomerase phosphoribosyformimino-5-aminoimidazoe carboxamide ribotide... 1-(5-phosphoribosy)-5-[(5- phosphoribosyamino)methyideneamino] imidazoe-4-carboxamide isomerase N-(5-phospho-L-ribosy-formimino)-5-amino-1-(5- phosphoribosy)-4-imidazoecarboxamide isomerase N-(5'-phospho-L-ribosy-formimino)-5-amino-1-(5'-phosphoribosy)-4-imidazoecarboxamide isomerase N-(5'-phospho-L-ribosy-formimino)-5-amino-1- (5'- phosphoribosy)-4-imidazoecarboxamide isomerase N-(5'-phospho-L-ribosy-formimino)-5-amino-1- (5'-phosphoribosy)-4-imidazoecarboxamide isomerase N-(5'-phospho-L-ribosy-formimino)-5-amino-1- (5'-phosphoribosy)-4- imidazoecarboxamide isomerase Phosphoribosy isomerase A [1-[5-phosphoribosy]-5-[[5-phosphoribosyamino]methyideneamino] imidazoe-4-carboxamide isomerase]

68 Measuring Consistency Define a set of protein famiies such that each famiy contains genes paying the same function Attach functiona roes to protein famiies Measure the consistency of the annotations made to genes within each famiy 1. "consistency" is the odds that two proteins from the same famiy have the same function 2. Evauate both famiies and functions.

69 Consistency among databases (2008)

70 Number of RefSeq proteins in famiies

71 How to measure accuracy If everything was caed hypothetica protein the database woud be 100% consistent Need to measure accuracy (specificity) as we as consistency Sampe 100 proteins at random from curated set (i.e. that are beieved to be correct) Manuay inspect annotations to score correctness

72 Probems Subsytems are biased! Subsystems are inaccurate! Merging annotations between different groups is poitica/psychoogica not technica!

73 Probems E. coi 4,391 genes 4,288 genes that make proteins (pegs) 676 genes that make enzymes 15% of genes encode enzymes!

74 The SEED Famiy

75 Three eve hierarchy Over 1,000 Subsystems Amino Acids and Derivatives Aanine, serine, and gycine Serine Biosynthesis Amino Acids and Derivatives Lysine, threonine, methionine, and cysteine Methionine Biosynthesis Make your own subsystems!

76 Cass # SS Cass # SS Amino Acids and Deriva/ves 56 Nuceosides and Nuceo/des 14 Carbohydrates 97 Phosphorus Metaboism 6 Ce Division / Cyce 10 Photosynthesis 9 Ce Wa and Capsue 50 Potassium metaboism 3 Custering-based ss 193 Protein Metaboism 52 Cofactors, Vitamins, Pigments 43 RNA Metaboism 39 DNA Metaboism 30 Regua/on/signaing 23 FaMy Acids, Lipids, and Isoprenoids 22 Respira/on 44 Membrane Transport 41 Secondary Metaboism 24 Metaboism of Aroma/c Compounds 30 Stress Response 37 Mo/ity and Chemotaxis 8 Sufur Metaboism 12 Nitrogen Metaboism 11 Viruence 116

Annotation of Compete Genomes Automated user originated processing Takes 1-7 hours depending on size and compexity of the genome ~2,000 externa submissions,

77 Annotation of Compete Genomes Automated user originated processing Takes 1-7 hours depending on size and compexity of the genome ~2,000 externa submissions, incuding hundreds of genomes not yet pubicy reeased. Reannotation of >500 genomes compete 1,000 users, 200 organizations, 25 countries. hmp://rast.nmpdr.org/

78 The annotation process (compete genomes) Find the phyogenetic neighborhood of your genome Look for proteins that reated organisms have Core proteins Subset of a subsystems Use those cas as a training set for critica/ gimmer Intrinsic training set!

79 This one s for Gary

80 Automatic metaboic reconstruction Subsystem, GO, and KEGG connections KEGG EC numbers KEGG reaction numbers SEED reaction numbers (Chris Henry) Metaboic fux modes Automaticay generate FBA matrices (Aaron Best/ Matt DeJongh; Hope Coege)

82 The Popuated Subsystem

83 Automaticay compare metaboic reconstructions

84 Find and suggest candidate functions Rapidy correct missing annotations Add more members to subsystems Improves future genome annotations! (especiay with new subsystems)

85 RAST usage grows...

86 RAST coverage...

The SEED Family

The SEED Family www.nmpdr.org www.theseed.org Number of known sequences How much has been sequenced? 100 Environmentalbacteria sequencing l genome s First bacterial genome Year 1,000 bacteria l genome