Following the identification of the "Golden Path" in Mapping Core, selected clones which comprise a complete tile path across the genome are sent to the Library Core laboratory so that we can construct individual subclone libraries needed for the sequencing process.

In the mapping group, the bacteria containing the clone to be sequenced are placed into a small agar plate containing the appropriate antibiotic and media. The plate of media provides a platform on which the bacteria can grow. The antibiotic ensures only the bacteria containing the necessary DNA will grow. These plates are placed in a 37 C overnight and during this time an individual bacteria will give rise to a whole colony of bacteria. The next day, Library Core removes the agar plates and picks up a single colony from each agar plate and adds it into liquid media. This liquid media is gently shaken at 37 C overnight and during this time. Since each bacterial cell contains one copy of the clone, large volume liquid cultures are required to obtain the millions of copies of the clone needed to make the library.

The clone DNA must be extracted and purified from the bacterial culture. In the process the cells are broke open or lysed, the proteins removed, and clone DNA captured by precipitation with ethanol. To be sure the correct clone is being processed, a fingerprint is generated from the clone DNA and compared to the fingerprint originally generated in the mapping group. If the fingerprint matches the clone DNA is sent for library construction, however, if the fingerprint does not match the clone DNA is abandoned.

The confirmed DNA is sheared and randomly broken into fragments of a selected size, usually 4.0 to 4.3 kb. Shearing accomplished by forcing the DNA through a very small orifice under high pressure. The shearing process causes damage to the ends of the clone DNA so they are treated with enzymes that repair the damage and make the ends of the fragments blunt. The repaired DNA fragments are separated by size on an agarose gel and specific size fragments are excised from the gel. The clone DNA is isolated from the gel matrix and ligated, or attached to a sequencing vector. The ligated subclones are transformed or placed into bacterial cells. Each bacterial cell contains one subclone and due to an antibiotic resistance gene located on the sequencing vector only bacterial cells containing a subclone are able to grow on a plate containing antibiotic. The transformations are plated and each bacterial cell containing a subclone gives rise to one colony. The resulting colonies are then sent into the Genome Sequencing Center's Production Pipeline for sequencing and assembly.

Whole Genome Library Construction

In a process similar to that of BAC Subclone Library Construction, Whole Genome Library Construction shears DNA, sizes the fragments using a gel, ligates the fragments into a vector and puts the fragments into a competent cell using electroporation.

The primary difference between the two, however, is the source DNA used to make the library. BAC DNA is usually about 150,000 base pairs in length, while whole genome DNA is just that; the DNA from the entire genome of an organism. A large genome, like the Human genome or Chimpanzee genome, is about three billion base pairs, while a small genome, like the bacteria Salmonella's genome, is about 5 million base pairs.

The DNA, once sized, is ligated primarily into one of two different types of vector to form either a plasmid (which holds a DNA fragment about 4,000 base pairs) or a fosmid (which holds a DNA fragment about 30,000 base pairs).

Once created, the library is plated onto large agar plates, and the resulting colonies are sent into the Genome Sequencing Center's Production Pipeline for sequencing and assembly.