Genomics An Introduction to Genomics Lab

BIO 481/Genomics :: Goals Determine all or part of the genome sequence of a novel organism Create a shotgun sequencing library Assemble sequence reads into contigs Analyse and annotate the sequence Genes (protein coding, pseudogenes, tRNAs, etc)‏ Sequence motifs (direct repeats, inverted repeats, palindromes, etc)‏

What to sequence? Why a phage? Biological importance Cost Technical simplicity Safety Why a phage of mycobacteria? Useful to other scientists Comparative genomics

There are an estimated bacteriophages in the world! 10,000,000,000,000, 000,000,000,000, 000,000 Phage Facts

As many seconds as if the entire world population counted 1 number per second for 50 trillion years. As many words spoken if every person in the world spoke their daily quota for trillion years. As many as the miles of the distance of 5.8 hundred trillion times around the planet Jupiter. As many as the dollars it would take for the entire world population to pay back the national debt 250 billion times. As many as the number of metric tons of 1.8 billion earths. Phage Facts

As many as the picometers to measure the distance of 21 ½ trips from Jacksonville, Florida to Juneau, Alaska. As many as the miles of 425 billion round trips to the nearest galaxy, the Andromeda Galaxy. As many as milliliters of water as the daily amount of water to pass through a human intestine of the entire world population at the rate of 9 liters/day for 50 trillion years. As many as the number of words if you typed 1 quadrillion words/minute for 3.29 quadrillion years. As many as the number of base pairs as found in all human cells in the total world population. Phage Facts

What to sequence? Why a phage? Biological importance Cost Technical simplicity Safety Why a phage of mycobacteria? Useful to other scientists Comparative genomics

Of interest as tools to study their important bacterial hosts (M. tuberculosis, M. leprae, M. avium, etc)‏ Not anticipated to be systematically different from phage of other hosts Isolated as plaque formers on lawns of M. smegmatis Isolated by direct plating: No amplification Have complete sequences for 50 genomes; 30 studied in further detail Mycobacteriophages

Image courtesy Jonathan King and Sherwood Casjens Salmonella Infected with Bacteriophage P22

From Dirt to Data and Beyond

Restriction Digests and Gel Electrophoresis

Mycobacteriophage Electron Micrographs

Mycobacteriophage BPs

Genome Analysis Tools BLAST ClustalW Glimmer GeneMark HMMer Artemis tRNAscan Dotter and others...

Mycobacteriophage Genomes

Bioinformatics visualizing and comparing genome sequences

Mycobacteriophage Dot Plot

Searching for Sequence Repeats

Horizontal Gene Transfer Mycobacteriophages as a case study

Cluster of Related Mycobacteriophages

A BC E BF E BC Homologous recombination A BC K LM K BC Targeted homologous recombination A BC Illegitimate recombination AB/Y Z X YZ Models for Recombination

Rv0002 (DnaN) ‏ Rv0024 Rv0054 (SSB) ‏ Rv0058 (DnaB) ‏ Rv0220 (LipC) ‏ Rv0320 Rv0344c (lpqJ) ‏ Rv0357c (PurA) ‏ Rv0399c (lpqK) ‏ Rv0474 Rv0430 Rv0570 (NrdZ) ‏ Rv0861c Rv0867c Rv0907 Rv0937 Rv1271c Rv1291c Rv1299 (PrfA) ‏ Rv1367c Rv1547 (DnaE1) ‏ Rv1615 Rv1629 (PolI) ‏ Rv1701 (XerD) ‏ Rv1728c Rv1730 Rv1922 Rv2017 Rv2101 (HelZ) ‏ Rv2119 Rv2176 (PknL) ‏ Rv2179c Rv2307 Rv2343c (DnaG) ‏ Rv2461c (ClpP) ‏ Rv2469c Rv2715 Rv2721c Rv2734 Rv2737c (RecA) ‏ Rv2748c (FtsK) ‏ Rv2754c Rv3053c (NrdH) ‏ Rv3090 Rv3125c (PPE protein) ‏ Rv3201c Rv3202c Rv3219 (WhiB1) ‏ Rv3333c Rv3427c Rv3437 Rv3447c Rv3482 Rv3514 Rv3532 (PPE protein) ‏ Rv3585 (RadA) ‏ Rv3594 Rv3597c (Lsr2) ‏ Rv3609c (FolE) ‏ Rv3610c (FtsH) ‏ Rv3766 Rv3811 (Csp) ‏ Rv3894c Rv3915 (CwlM) ‏ MT2779 >12 M. avium. M. tuberculosis genes in Mycobacteriophages