Lecture on bacterial genome analysis and annotation for Bio305 course at the University of Birmingham

1. Bio305 Bacterial Genome Annotation and Analysis Professor Mark Pallen

6. Whole-Genome Shotgun Sanger Sequencing Random shearing Size selection Cloning Sequence each insert with two primers Pick colonies to create shotgun library bacterial chromosome plasmid vector Plasmid preps

8. High-Throughput Shotgun Sequencing Random shearing Size selection bacterial chromosome Add adapters Amplify Sequence

9. Illumina Sequencing

13. Genome Assembly Scaffold Contig 3 Contig 2 Contig 1 Physical Gap Sequence Gap

19. Or this?

23. Base composition aids genome analysis GC skew (G-C)/G+C) Identifies origin of replication and leading lagging strands Genes coded by location & function %G+C Genes shared with E. coli Genes unique to S. typhi

27. The problem of conflicting ORFs Non-coding ORFs CDSs (note ORF can extend upstream of start codon)

28. The Problem of Frameshift Errors Actual sequence 10 20 30 40 50 60 70 | | | | | | | ATGAGTACCGCTAAATTAGTTAAATCAAAAGCGACCAATCTGCTTTATACCCGCAACGATGTCTCCGACAGCGAGAAA M S T A K L V K S K A T N L L Y T R N D V S D S E K • V P L N • L N Q K R P I C F I P A T M S P T A R K E Y R • I S • I K S D Q S A L Y P Q R C L R Q R E K 10 20 30 40 50 60 70 | | | | | | | ATGAGTACCGCTAAATTAGTTAAATCAAAAAGCGACCAATCTGCTTTATACCCGCAACGATGTCTCCGACAGCGAGAA M S T A K L V K S K S D Q S A L Y P Q R C L R Q R E • V P L N • L N Q K A T N L L Y T R N D V S D S E K E Y R • I S • I K K R P I C F I P A T M S P T A R K Frameshifted sequence after single base error

29. CDS Prediction: Graphical Plots GC content by reading frame Amino-acid composition by reading frame, compared to average for globular proteins

41. Bit Scores high is good E-values low is good http://www.ncbi.nlm.nih.gov/BLAST/tutorial/

42. Typical Blast Output Sum Reading High Probability Sequences producing High-scoring Segment Pairs: Frame Score P(N) N emb|X69337|ECDPS E.coli dps gene for binding protein +2 834 6.4e-109 1 gb|U04242|ECU04242 Escherichia coli core starvation p... +3 828 2.7e-106 1 emb|X14180|ECGLNHPQ Escherichia coli glutamine permeas... +3 443 2.8e-53 1 gb|U18769|HDU18769 Haemophilus ducreyi fine tangled p... +1 150 4.0e-18 2 dbj|D01016|ANALTI46 Anabaena variabilis lti46 gene. >e... +2 129 4.8e-12 2 gb|M84990|P26BPO Plasmid pOP2621 ORF1 gene, 5' end;... -2 131 6.7e-09 1 gb|U16121|HPU16121 Helicobacter pylori neutrophil act... +1 112 1.8e-06 1 gb|M32401|TRPTYF1 T.pallidum pallidum antigen TyF1 g... +3 101 5.6e-06 2 emb|X71436|RPNTRB R.phaseoli ntrB gene +1 67 0.76 2 gb|L35598|DRODGC1A Drosophila melanogaster receptor g... +1 48 0.97 3

43. Typical Blast Output gb|U18769|HDU18769 Haemophilus ducreyi fine tangled pili major pilin subunit gene Length = 780 Plus Strand HSPs: Score = 150 (68.0 bits), Expect = 4.0e-18, Sum P(2) = 4.0e-18 Identities = 36/89 (40%), Positives = 46/89 (51%), Frame = +1 Query: 30 ELLNRQVIQFIDLSLITKQAHWNMRGANFIAVHEMLDGFRTALIDHLDTMAERAVQLGGV 89 E L ++ +L+LI K AHWN+ G FIAVHEMLD + D +D +AER LG Sbjct: 253 EALQMRLQGLNELALILKHAHWNVVGPQFIAVHEMLDSQVDEVRDFIDEIAERMATLGVA 432 Query: 90 ALGTTQVINSKTPLKSYPLDIHNVQDHLK 118 G + + YPL QDHLK Sbjct: 433 PNGLSGNLVETRQSPEYPLGRATAQDHLK 519

45. Pfam domains

46. Pfam search results

47. The Annotation Catastrophe Signal Peptide A protease B Coiled coil domain C Homology lies in one domain Signal Peptide Protein A “ a protease” Protein B Protein C But functional assignment for whole of protein A comes from another domain, carried across in error, so proteins B and C get misannotated as proteases