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Why bother with a new technology?

Why bother with a new technology?. Current (2005) $1.00 per kilobase 20 bp / second 100 Gb / year. NHGRI Goals $ ↓ 100x, 5yrs $ ↓ 10,000x, 15yrs. Collins, Morgan, Patrinos (2003). Growth of GenBank. 60. 50. 40. 30. Base pairs of DNA (billions). Sequences (millions). 20. 10. 0.

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Why bother with a new technology?

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  1. Why bother with a new technology? Current (2005) $1.00 per kilobase 20 bp / second 100 Gb / year NHGRI Goals $ ↓ 100x, 5yrs $ ↓ 10,000x, 15yrs Collins, Morgan, Patrinos (2003)

  2. Growth of GenBank 60 50 40 30 Base pairs of DNA (billions) Sequences (millions) 20 10 0 1985 1990 1995 2000 December 1982 September 2005

  3. Completed Genome Sequences (roughly) And growing…

  4. >100,000 species are represented in GenBank all species 128,941 viruses 6,137 bacteria 31,262 archaea 2,100 eukaryota 87,147 Table 2-1 Page 17

  5. Absolutely requires a correctly paired nucleotide so as not to allow errors This requirement means can’t elongate if there is NO nucleotide either - thus a primer is necessary

  6. Genome Sequencing Visit: http://jgi.doe.gov/education/how/index.html for more explanation Hydroshearing

  7. Insertion of fragments into bacterial plasmids

  8. Transformation of plasmids into bacteria via electroporation

  9. Growth of colonies on an agar plate

  10. Robotic colony picking and distribution to 384 well plate (one colony per well)

  11. Lysis of bacterial cultures at 95 C

  12. Rolling circle amplification of plasmid DNA Use phi29 polymerase

  13. Transfer amplified DNA to new plate for cyclesequencing Add cycle sequencing reagents

  14. Purify DNA from sequencing reaction with magnetic beads

  15. Bar coding and capillary electrophoresis and laser scanning

  16. Computational assembly of fragments into contigs

  17. Quality control

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