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The genome of Mycoplasma pulmonis (strain UAB CTIP) has a size of 963 879 pb and a G+C% of 26.6%. It has been sequenced at a depth of 9.8X. The 12,352 assembled reads were obtained from four different libraries. Libraries A (first created for the pilot study) and C were constructed with Sau3AI fragments (partial digestion of the genome) : fragments with a size of 1 to 2 kb (library A) and 2 to 3 kb (library C) were cloned into the pUC18 vector. Partial digestion of genomic DNA with a second enzyme (Tsp509I) yielded fragments of 5-6 kb which were cloned in the pBAM3 vector to give library R. The fourth library (library B, mini-BACs) was constructed with Tsp509I fragments longer than 15 kb which were cloned in the pBACe3.6 vector.
Sequencing was performed at both ends of inserts on 500 clones from library A, 2,000 clones from library C, 1,500 clones from library B and 4,000 clones from library R. For the first three libraries, ABI 377 sequencers were used (mean length of reads : 600 nucleotides) and 4 genome equivalents were retained for assembly ; for library R, Li-Cor 4200L sequencers were used (mean length of reads : 930 nucleotides) and 5.8 genome equivalents were retained for assembly. Finishing work included the sequencing of several PCR product in order to fill a cloning gap and to resolve regions with uncertainties. To achieve a quality up to Bermuda standards, 481 primers were synthesised and 1,144 supplementary sequencing reactions were performed. Assembly was validated by a comparison between the theoretical restriction profile and the experimental profile obtained with a) total genomic DNA, and b) a selection of overlapping clones.