Construction of a shotgun library
The construction of the shotgun library was one of the critical steps of the sequencing project. Our goal was to reach about 90% of the genome and a coverage of about 4X with as few as 8,000 clones. This meant that we needed to construct a shotgun library as less biased as possible.
DNA from low-GC Gram-positive bacteria are known to give instability in E. coli. Therefore we choosed a very narrow range of insert size (from 1kb to 2kb) to avoid strong cloning bias. Additional tricks were used to decrease the probability of creating a biased shotgun library :
- DNA was isolated from cells in stationary phase to avoid bias due to replication copies
- DNA from several sonicating conditions were pooled before size selection
- The clones library was checked using physical mapping and hybridisation and by PCR
- Only one ligation mixture of 10 microliter was used to obtain the 8,500 clones
Most of the clones had an insert of 1.3 kb and could be easily sequenced with only universal primers.