Project description:In this study, we provided the first genome-wide, base pair-resolution map of 6mA in Tetrahymena by applying single-molecule real-time (SMRT) sequencing.
Project description:Single Molecule Real Time (SMRT) sequencing was utilized to map the genome-wide m6A methylation pattern in B. mayonii. Consensus modified motifs were identified in wildtype B. mayonii as well as clonal isolates lacking plasmids that encode predicted methyltransferases. Two conserved m6A modification motifs were identified, and were fully attributable to the presence of specific methyltransferases.
Project description:Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and Campylobacter jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N6-methyladenine (m6A) and N4-methylcytosine (m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTases genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. No attempt was made to detect 5-methylcytosine (m5C) recognition motifs from the SMRT sequencing data because this modification produces weaker signals using current methods. However, all predicted m6A and m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active, but also revealing their recognition sequences. Examination of the methylomes of six different strains of bacteria using kinetic data from single-molecule, real-time (SMRT) sequencing on the PacBio RS.
Project description:Bifidobacterium breve represents one of the most abundant (bifido)bacterial species in the gastro-intestinal tract of (breast-fed) infants, where their presence is believed to be beneficial. In the present study whole genome sequencing, employing PacBio’s Single Molecule, Real-Time (SMRT) sequencing platform, combined with comparative genome analysis allowed the most extensive genetic investigation of this taxon. Our findings demonstrate that Restriction/Modification (R/M) systems constitute a substantial part of the B. breve variome. Availing of the methylome data generated by SMRT sequencing, combined with targeted Illumina bisulfite sequencing (BiSeq) and comparative genome analysis, we were able to detect methylation recognition motifs and assign these to identified B. breve R/M systems, where in several cases such assignments were confirmed by restriction analysis. Furthermore, cloning of a selected methyltransferase-encoding gene validated the activity of the corresponding R/M system, and was shown to overcome the barrier they impose to genetic accessibility, thus allowing future genetic manipulation of members of this species.
