DNA sequencing: Basic idea

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DNA sequencing Basic idea

• Background test tube DNA synthesis
• DNA polymerase (a natural enzyme) extends
  2-stranded DNA over a 1-stranded template
• primer extension polymerase
• 5 TTACAGGTCCATACTA ?
• 3 AATGTCCAGGTATGATACATAGG 5
• Template
• Can buy DNA polymerase and do this in a tube.

Quicktime animation
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DNA sequencing, cont
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DNA sequencing, cont
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DNA sequencing, cont
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Quicktime animation
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Modern Sanger sequencing

• Dye terminator sequencing
• Flourescent label on terminator, not primer
• Different colors for ddA, ddC, ddG, ddT
• Run all 4 reactions in a single lane
• Image under 4 colors of laser
• Capillary electrophoresis
• Each sequence is sized thru a separate, thin tube
  (capillary)
• Avoids lane tracking errors
• Automated readout -- Phred

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Limitations of technology

• Error prone, especially at beginning end
• But Phred estimates error probability
• Not useful beyond 500-800 bp

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Whole chromatogram (trace)
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Start of trace
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End of trace
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Base calling, assembly, editing

• Software tools
• PHRED calls bases from traces. Reads.
• Estimates error probability for each base
  (quality values)
• PHRAP assembles reads a longer sequence
• Uses quality values
• Not intended for whole-genome assembly
• Research on assembly algorithms is ongoing

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Sequencing Genomes
Michael Brent Dept. of Computer
Science Washington University
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Why sequence a genome?

• Cool technology
• Infrastructure for molecular science
• E.g. Cloning studying a gene of interest
• Parts list for the human body
• Genome science
• Evolution and dynamics of genomes
• Medicine
• Genomic causes of disease and health

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Which genomes?
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How can I sequence a genome?

• Shotgun sequencing simple version
• Cut your DNA at random locations
• Get 700-800 bp of sequence from the end of each
  fragment AAGTCGTGGG.
• Use overlapping sequences to reassemble

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Step 1 cutting cloning

• Cut/break the DNA
• Physical shear put it in a blender, or
• Restriction digest
• B. Separate fragments by size select

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1C. Clone select fragments
Quicktime animation
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2. Sequence random clones

• Pick a clone containing copies of 1 insert from
  the plate
• Separate the plasmids from the cells
• Sequence the inserts using primers complementary
  to the vector

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3. Assemble fragments

• Idea
• Common end sequences may indicate overlap in
  original sequence
• overlapping shotgun sequences
• CTGACTAAGTCAUGTTACAG
• TTACAGCAGGTATGATA
• assembled sequence
• CTGACTAAGTCAUGTTACAGCAGGTATGATA

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3. Assemble fragments

• Problems
• Sequencing error may obscure true overlap
• Common end sequences can occur by chance
• Repeats DNA of higher eukaryotes contains many
  copies of nearly identical sequences
• This means overlaps are often from different
  copies of the same repeat element
• Repeats are the major issue in sequencing
• Polymorphism

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Genome assembly

• Challenge
• Cant assemble sequencing reads based on
  overlapping ends in long repeats
• CTGACTAAGTCAUGTTACAG
• TTACAGCAGGTATGATA
• Overlaps may be from different repeat copies
• Leading to large-scale misassembly
• Polymorphic mismatches may prevent good joins

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Single-molecule sequencing

• Since 2007, we can sequence individual molecules
  without cloning
• Many molecules are attached to a surface and
  copied, forming a cluster of identical templates
• Reversible dye terminators are incor-porated
  according to templates (1 bp)
• Slide is imaged sequentially under 4 color
  lasers, showing which dye was incorporated at
  each cluster

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Single-molecule sequencing

• Terminator is cleaved off and 2nd-strand
  synthesis continues for next cycle
• Each cycle is one position in the sequence
• 108 50 nt reads / 2-day run (Solexa)
• 106 400 nt reads / 5-day run (454)
• For Sanger, 103 700 nt reads / day
• Read-length vs. throughput tradeoff