DNA Sequencing – “Plus and Minus” Plus –Incubate with T4 DNA Polymerase and single dNTP –T4 Polymerase degrades 3’ ends in absence of dNTP –Fractionated.

DNA Sequencing – “Plus and Minus”

• Plus– Incubate with T4 DNA Polymerase and single dNTP– T4 Polymerase degrades 3’ ends in absence of dNTP– Fractionated on PAGE w/ladder

• Minus– Incubate with DNA Polymerase I and 3 dNTPs– Denatured from template– PAGE electrophoresis

• Disadvantage– Neither method completely accurate– Both must be used together

DNA Sequencing – Chemical Cleavage

Maxam and Gilbert

DNA Sequencing – Dideoxy Sequencing

DNA sequencing with chain-terminating inhibitorsF. Sanger, S. Nickelen, and A.R. Coulson (1977)

Sanger et al

• Bacteriophage X174• Restriction enzyme

frag.– A12d and A14

• ddGTP, ddATP, ddTTP and araCTP– Arabinonucleoside

• dATP chase

Sanger et al

• R4 fragment as primer• Digest with restriction

enzyme• ddNTPs used

• Problem– What if second restriction site

close to first one– New bands arise

Sanger et al

• A8 fragment as primer

• Single ribonucleotide incorporated at end of primer

• ddTTP, araCTP, ddCTP, ddGTP, and ddATP

Shotgun Sequencing

Genomic DNA sheared

Cloned into vectors

Sequence

Fragment Contigs

Determining the sequence of both ends of the shotgun library

End-sequencing large-insert clones to find one that spans the region of interest

Shotgun Sequencing (cont.)

• Pilot project for Human Genome

• 2 kbp and 10 kbp mate pairs• BACs were end-sequenced to generate set of mate

pairs• Problem with assembly

– Confusion with repetitive DNA stretches

• Solution – Scaffolds of contigs

Myers et al

• High quality input data needed• Accuracy of pair mate distances• Reliablity of true pairing

The Drosophila Data Sets• WGS – 2 kbp, 10 kbp and BAC• Joint Data Set

– WGS– 2nd and 3rd chromosome– 340 BAC and P1 inserts– X chromosome– 4th chromosome

Myers et al• Screen for rep.

elements– Ribosomal DNA– Histones– Heterochromatin– Retrotransposons

• Overlap: true/repeat induced

Myers et al• Unitigger

– Unique or repetitive seq– Correct subassembly or fragments– Detect repeat boundaries so can remove repetitive overlap

between the unitigs

Myers et al• Scaffolder• Repeat Resolutions

– Rocks - two mate pairs – Stones - single mate pair– Pebbles – find best tiling across gap

• Consensus

Myers et al

Myers et al

Myers et al