Project description:Purpose: We used GAS as a model bacterial pathogen to investigate the complex relationship between genome, transcriptome, and virulence in a large population of type emm28 strains recovered from humans over 26 years in geographically diverse locations. Methods: An isogenic mutant strain (MGAS27961-10T) with a 10-T homopolymeric tract was generated using allelic exchange. Primers were used to amplify a 2,690-bp fragment using genomic DNA of MGAS28085 an emm28 clinical isolate with the naturally occurring 10 “T” variant region. The resulting PCR product was cloned into suicide plasmid pBBL740 and transformed into the parental strain MGAS27961. RNA-seq was performed on the isogenic mutant strain MGAS27961-10T and parental strain (MGAS27961-9T). Quality of the total RNA, rRNA-depleted RNA, and cDNA libraries was evaluated with RNA Nano, RNA Pico, and DNA high-sensitivity kits (Agilent Technologies), respectively, using an Agilent 2100 Bioanalyzer. For each sample, the cDNA library concentration was measured fluorometrically with Qubit™ dsDNA HS assay kits (Invitrogen). The cDNA libraries were diluted, pooled, and sequenced with an Illumina NextSeq 500 instrument.
Project description:PFGRC has developed a cost effective alternative to complete genome sequencing in order to study the genetic differences between closely related species and/or strains. The comparative genomics approach combines Gene Discovery (GD) and Comparative Genomic Hybridization (CGH) techniques, resulting in the design and production of species microarrays that represent the diversity of a species beyond just the sequenced reference strain(s) used in the initial microarray design. These species arrays may then be used to interrogate hundreds of closely related strains in order to further unravel their evolutionary relationships. The Pneumococcus are among most deadly pathogens world-wide. The infections and outbreaks caused by this pathogens is quite frequent despite existing diagnostic network and therapeutic means. Therefore, developing reliable diagnostic tools and efficient (broad-spectrum) therapeutics for Streptococcus pneumoniae remain a public health priority for every country in world today. The comparative genomics study will provide the largest hitherto genomic data sets regarding this pathogen.These large data sets will enable us as well as other members of scientific community to conduct comprehensive data mining in the form of gene association studies with statistical power and significance.