Genome sequencing methods
Genome sequencing. The source of genomic DNA was the type strain
Thermoplasma acidophilum DSM 1728 (Deutsche Stammsammlung, BraunschweigSöckheim, Germany). Cells of Thermoplasma were cultivated as described by Freundt 1.
Isolation of genomic DNA (Stratagene) was performed using the Marmur procedure 2.
Genomic libraries. Four phagemid libraries were obtained by complete cleavage
of genomic DNA with the restriction enzymes BamH I, Bgl II, Sac I and EcoR I,
respectively. DNA fragments in the range from 3 kbp to 8 kbp were cloned into the
Lambda ZAP express vector (Stratagene). Construction of the phagemid libraries were
performed according to the instructions of the manufacturer. Another phagemid library
was constructed with genomic DNA that was partially cleaved with Sau3A I. The further
steps were performed as described above. The cosmid library using the SuperCos I
cosmid vector was constructed by Stratagene.
Long range PCR. Four areas of the genome were not covered by clones of the
DNA libraries. The corresponding DNA fragments, ranging between 4.5 and 15.9 kbp,
were amplified using long range PCR (Expand Long Template PCR System, Roche),
performed according to the instructions of the manufacturer.
DNA sequencing. All DNA sequencing was performed using dye terminator
chemistry (Big Dye RR Terminator Cycle Sequencing kit, Perkin-Elmer). Analysis of the
DNA sequencing reactions were performed with an ABI 373 and an ABI 377 sequencer,
respectively. 1100 DNA sequencing reactions were performed by a company
(Medigenomix, Munich, Germany).
Assembly und Editing. Editing and assembly of the DNA sequencing reactions
was performed using the Sequencher 3.0 software (Gene Codes Corporation, Ann Arbor,
MI) on a MacIntosh G3 computer.
References
1. Christiansen, C., Freundt, E.A. & Black, F.T. Genome Size and DeoxyribonucleicAcid Base Composition of Thermoplasma acidophilum. Int. J. Syst. Bacteriol. 25, 99-101
(1975).
2. Marmur, J. A Procedure for the Isolation of Desoxyribonucleic Acid from MicroOrganisms. J. Mol. Biol. 3, 208-218 (1961).