Integrated Analysis of Population Genomics, Transcriptomics and Virulence Provides Novel Insights into Streptococcus pyogenes Pathogenesis [442 strains]

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: We chose a subset of isolates from the sequenced population of 2,101 emm28 strains that would approximately span the genetic variation present in the entire population. 442 emm28 strains grown as singleton cultures and harvested at one time point (early-stationary phase), and libraries were prepared using RNAtag-seq. 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. Overall design: We made 57 pools containing total RNA from 8 strains each, and 1 additional pool with total RNA from 5 strains. Thus, single-stranded cDNAs were organized into 58 pools. Subsequently, each set of four pools was combined into one superpool rendering 14 superpools, and one additional superpool containing only 2 pools. 15 superpools were sequenced on Illumina NextSeq 500 instrument. Demultiplexing of reads from superpools into separate pools was performed with bcl2fastq. Read quality assessment and read quality trimming were done with FASTQC and Trimmomatic, respectively. Reads from each pool were demultiplexed into separate fastq files corresponding to individual samples based on the inline barcodes using FASTX-toolkit.