How Sanger Sequencing Was Done

Whole-genome shotgun sequencing is a technique used for determining the DNA sequence of a genome by randomly shearing the DNA, sequencing multiple fragments whose sequences overlap, and inferring the original sequence by reassembling the fragments. Three sizes of fragments are sequenced, 2-4 kb (kilobase, or 1000 bases), 8-10 kb, and 40 kb. This explanation follows the procedure for 2-4-kb fragments. Procedures for the other sizes are similar.

oneLibrary Creation

1. Shearing of DNA

2. Insertion of Fragments into a Plasmid

3. Transformation

4. Subcloning the Sheared Fragment

5. Colony Picking

twoProduction Sequencing

6. Lysing the Cell

7. Rolling-Circle Amplification

8. Sequencing Chemistry

9. Post-Sequencing-Reaction Cleanup

10. Capillary Sequencing

threeAssembly and QA

11. Assembly

12. Quality Assessment