What Is Genomics?Genomics is the study of how the entire genome of a species functions as a unit and evolves over time.

It is the study of life’s blueprint, life’s diversity, and life’s history.Bioinformatics: Analyses the information content of genomes.

Comparative Genomics: Compares genome sequences with each other to infer evolutionary relationships and mechanisms of evolution.

Functional Genomics: Probes how genomes function, as a whole, to give rise to organisms.

Ecological Genomics: Understands how genomes, and the organisms they encode, fill specific environmental niches.

>500 complete microbial genomes 730 in progress

Why Sequence Whole Genomes?

• To speed characterization of genes mapped by linkage

• To obtain a "parts list" for what makes up an organism.

• To discover what sets of genes make organisms (and each of us) similar to and different from one another.

• To understand our evolutionary heritage. Our genomes are a reflection of our recent and ancient origins.

Dideoxy “chain termination” DNA sequencing-Sanger v1, continued

Dideoxy “chain termination” DNA sequencing-

Sanger v2 !!

Fred Sanger, SECOND Nobel Prize in 1980 (Chemistry; his first was in1958 for methods for determining amino acid sequences in proteins).

Made easily robot friendly.

High throughput DNA sequencing hasallowed the sequencing of whole Genomes.

This has driven the Genomics revolution.

Sheared and ssDNA Oil emulsion: 1 template/bead

Polony: clonal amplification

Polony + enzyme beads

Picoliter plate

The Next Gen Technologies: Pyrosequencing (454, Roche)

First of the ‘parallel’ sequencing platforms.

Sequencing-by-synthesisPyrosequencing

3’ATCGTTGCACGTCGACGTA5’TAGCAACG

dGTP PPi

ATP

ATP sulfurylase

Luciferase

400K reads X 400 bases = 16-20 Mb in 4 hrs!

So, 125 Mb is only 6 runs for 1X coverage!

Sequence by synthesis – one base at a time

Each base has a different color

Each base has a reversible terminator

The Next Gen Technologies: Illumina (Solexa)

DNA Single molecule array

Sample preparation Cluster growth

5’

5’3’

G

T

C

A

G

T

C

A

G

T

C

A

C

A

G

TC

A

T

C

A

C

C

TAG

CG

TA

GT

1 2 3 7 8 94 5 6

Image acquisition Base calling

T G C T A C G A T …

Sequencing

5 million clusters / channel; 8 channels / flow cell = 40 million reads times ~35 cycles (bases) = 120 billion bases in 3 days (1.2Gb)

The Next Gen Technologies: Illumina (Solexa)

Sanger Shotgun

454 Solexa

Cloning Yes No No

Chemistry Sanger pyrosequencing reversible terminators

DNA bases per $

Human (1X!!) $

1000

3,000,000

50,000

60,000

500,000

6,000

Time A really long time

4 hours 4 days

Accuracy Consensus 99.99%

Worst of the 3; not good with homopolymer repeats

1-3% errors

Assembly

Computational

tools

Best OK Horrible

Gap Closure and Finishing

Pain Pain in the rear ROYAL Pain in the rear

Technology Development drives Biology

First, we've learned that we have a lot to learn

Over 35% of genes in ANY organism (including Human) have no deducible function!

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