DNA sequence-ULTIMATE Map DNA sequencing-methods Assembly/sequencing. Genome Characterization. Assigned reading: Service 2006 review paper Assigned listening: Ecic Lander genomics lecture. BIO520 Bioinformatics Jim Lund. DNA Sequence Project Size/Type. 500 bases 2500 bases 10 kbp
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BAC, big virus
Bacterial genome, YAC-size
Nematode (Caenorhabditis elegans): 100 Mb
Thale cress (Arabidopsis thaliana): 160 Mb
Fruit fly (Drosophila melanogaster): 180 Mb
Puffer fish (Takifugu rubripes): 400 Mb
Rice (Oryza sativa): 490 Mb
Human (Homo sapiens): 3.5 Gb
Leopard frog (Rana pipiens): 6.5 Gb
Onion (Allium cepa): 16.4 Gb
Mountain grasshopper(Podisma pedestris): 16.5 Gb
Tiger salamander (Ambystoma tigrinum): 31 Gb
Easter lily (Lilium longiflorum): 34 Gb
Marbled lungfish (Protopterus aethiopicus): 130 Gb
Fluorescence paradigm, ABI
Next generation sequencing
Polymerase addition sequencing
454 Sequencing, Illumina
dN : ddN
100 : 1
Polyacrylamide Gel Electrophoresis
suited to automation
rapid (2 hrs vs 12 hrs)
simple temperature control
96 well format
migration ~1/log N
ABI3730XL (2002, 96 samples, 1000 base reads, ~$350,000, higher sensitivity, lower reagent cost, ~$1/reaction)
700 Kbp / 24 hours.
384 capillary sequencers
5700 sequences / 24 hr day
2.8 Mbp / 24 hours.
Results are shown as an electropherogram showing a peak for each base. From the peak
heights and widths, a Phred score is assigned to each individual base. A high Phred
score indicates a high certainty as to the identity of that particular base.
ABI: 1000 bp reads
Illumina: 50-100 bp reads
454 Sequencing: 300-400 bp reads
How do we cover a genome?
DIVIDE AND CONQUER: assemble these short sequence fragments.
EditView (ABI PRISM)
Chromas (free/pay versions)
Divide and Conquer
The random approach now predominates for big projects
Shear DNA (nebulize)
finish ends, ligate into vector
Sequence to 8X – 10X coverage
Sequence both ends of templates.
Read length (1,000bp typical)
Accuracy (99% good)
Only 1 strand
UK’s sequencing center has one:
Pyrosequencing (sequencing by detection of nucleotides added during DNA synthesis.
350-400 million bases per run (10 hrs.).
400 bp sequence reads.
1,000,000 reads per run.
$6,600 per run, 60kb/$1, or $0.00165/bp.