Komparativní genomová sekvenace (CGS): nový typ sekvenování bakteriálních genomů.
| Title in English | Comparative genome sequencing (CGS): New sequencing strategy for bacterial genomes. |
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| Authors | |
| Year of publication | 2006 |
| Type | Article in Proceedings |
| Conference | X. Pracovní setkání biochemiků a molekulárních biologů. Sborník příspěvků. |
| MU Faculty or unit | |
| Citation | |
| Field | Genetics and molecular biology |
| Keywords | Comparative genome sequencing; bacterial genomes; T. pallidum |
| Description | Oligonucleotide hybridization chip was constructed based on the genome sequence of T. pallidum ssp. pallidum Nichols. This array contains a set of 29-mers covering both strands and spaced 7 nucleotides apart (i.e. 325,138 hybridization probes). After hybridization of fluorescently labeled DNA of the target strain to the chip, genome regions of different sequence were identified. Results of the mapping phase were used to design resequencing chip. Resequencing chip was used to identify individual single nucleotide polymorphisms (SNPs). Regions where SNP identification was not successful contained short indels and/or SNP clusters and these regions were sequenced using dideoxyterminator method (DDT). To obtain complete genome sequence of examined strains CGS found changes were combined with WGF results. WGF represents complementary method to hybridization comparison, because it enables detection of inserted sequences which is a limitation of hybridization methods in general. We performed comparison of SS14, Samoan D and Cuniculi A strains to Nichols strain. Mapping phase of T. pallidum ssp. pallidum SS14 genome showed 20 hypervariable regions not suitable for further hybridization analysis. Resequencing array revealed 231 SNPs. We found altogether 320 SNPs, 11 deletions and 15 insertions (1-1255 bp in lenght). In T. pallidum ssp. pertenue Samoan D genome, 904 SNPs were identified and 79 regions need to be DDT sequenced. In T. paraluiscuniculi Cuniculi A genome 5933 SNPs were discovered and more than 500 regions are recommended for DDT sequencing. Sequence differences found by CGS and WGF were used to define intra- and inter-subspecies variability of T. pallidum and to map candidate loci for screening of clinical isolates of individual subspecies. Detection of sequence changes in hypervariable regions was already used for identification of novel strains in clinical material. |
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