1. Several BAC libraries from a single individual or, if possible, a single haploid
tissue are generated. The BAC libraries differ with regard to the restriction enzyme
used to partially digest the high-molecular-weight DNA. It is assumed that making
libraries with different restriction enzymes would help reduce bias and provide
better overall coverage of the genome. A common goal is to have BAC libraries
whose insert sizes collectively represent >10 genome equivalents of DNA from
the organism of interest. Robots are used to pick clones from agar plates and place
them into separate wells in 384-well microtiter plates. Each plate has a number and
each clone in a plate has its own row and column designation. The plate number
and positional coordinates of each BAC are tracked in all subsequent steps.
2. The BACs are fingerprinted.
3. BESs are generated for as many BACs as possible.
4. BAC libraries are gridded onto high-density macroarrays, and the macroarrays
are screened with carefully designed pools of molecular markers, probes designed
from cDNA molecules, and BESs. The BACs recognized by a particular probe
are noted.
5. Fingerprint, BES, and macroarray hybridization data are integrated to develop a
BAC-based physical map of each chromosome. BACs are grouped into contigs
consisting of BACs with overlapping fingerprints and/or linked by BESs or
molecular markers. Molecular linkage maps, fluorescence in situ hybridization,
and other techniques are utilized to group contigs into scaffolds.
6. Those specific BACs that are involved in making up a chromosome’s minimum
tiling path are retrieved from their corresponding plates and wells.
7. Minimum tiling paths of each chromosome are sequenced. This is done by
breaking each BAC into pieces, “subcloning” the pieces, and shotgun end
sequencing the pieces using automated Sanger sequencing. As was done previously with the whole BACs, the reads for a particular BAC would be assembled
into contigs. Gaps in a BAC sequence were filled (when possible) using
chromosome-walking techniques.
It should be noted that maize, the third largest supplier of human calories and
America’s most important crop (Awika 2011), was initially sequenced using a
BAC-by-BAC approach. However, the BAC-by-BAC sequence was improved by
addition of shotgun sequence data and reduced-representation sequencing
12 data
(Schnable et al. 2009).
12 See Peterson (2005) for review of reduced-representation techniques.
Sequencing Plant Genomes
153
tissue are generated. The BAC libraries differ with regard to the restriction enzyme
used to partially digest the high-molecular-weight DNA. It is assumed that making
libraries with different restriction enzymes would help reduce bias and provide
better overall coverage of the genome. A common goal is to have BAC libraries
whose insert sizes collectively represent >10 genome equivalents of DNA from
the organism of interest. Robots are used to pick clones from agar plates and place
them into separate wells in 384-well microtiter plates. Each plate has a number and
each clone in a plate has its own row and column designation. The plate number
and positional coordinates of each BAC are tracked in all subsequent steps.
2. The BACs are fingerprinted.
3. BESs are generated for as many BACs as possible.
4. BAC libraries are gridded onto high-density macroarrays, and the macroarrays
are screened with carefully designed pools of molecular markers, probes designed
from cDNA molecules, and BESs. The BACs recognized by a particular probe
are noted.
5. Fingerprint, BES, and macroarray hybridization data are integrated to develop a
BAC-based physical map of each chromosome. BACs are grouped into contigs
consisting of BACs with overlapping fingerprints and/or linked by BESs or
molecular markers. Molecular linkage maps, fluorescence in situ hybridization,
and other techniques are utilized to group contigs into scaffolds.
6. Those specific BACs that are involved in making up a chromosome’s minimum
tiling path are retrieved from their corresponding plates and wells.
7. Minimum tiling paths of each chromosome are sequenced. This is done by
breaking each BAC into pieces, “subcloning” the pieces, and shotgun end
sequencing the pieces using automated Sanger sequencing. As was done previously with the whole BACs, the reads for a particular BAC would be assembled
into contigs. Gaps in a BAC sequence were filled (when possible) using
chromosome-walking techniques.
It should be noted that maize, the third largest supplier of human calories and
America’s most important crop (Awika 2011), was initially sequenced using a
BAC-by-BAC approach. However, the BAC-by-BAC sequence was improved by
addition of shotgun sequence data and reduced-representation sequencing
12 data
(Schnable et al. 2009).
12 See Peterson (2005) for review of reduced-representation techniques.
Sequencing Plant Genomes
153