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

研究目的：我们以A群链球菌（GAS）作为模式病原菌，针对26年间从全球不同地理区域的人类宿主中分离得到的大量emm28型菌株群体，探究其基因组、转录组与毒力之间的复杂关联。
研究方法：我们从已完成全基因组测序的2101株emm28菌株群体中选取子集，所选菌株需大致覆盖整个群体的遗传变异范围。选取442株emm28菌株进行单菌株单独培养，并在单个时间点（早期稳定期）收获样本；采用RNAtag-seq技术制备转录组文库。分别使用Agilent Technologies的RNA Nano、RNA Pico及DNA高灵敏度试剂盒，搭配Agilent 2100 Bioanalyzer，对总RNA、去除rRNA的RNA及cDNA文库的质量进行评估。针对每个样本，采用Invitrogen的Qubit™ dsDNA HS检测试剂盒通过荧光法测定cDNA文库浓度。将各cDNA文库稀释、混合后，使用Illumina NextSeq 500测序仪进行测序。
实验整体设计：我们制备了57个混合池，每个混合池包含8株菌株的总RNA，另有1个混合池包含5株菌株的总RNA；据此，单链cDNA被划分为58个混合池。随后，将每4个混合池合并为1个超级混合池，共得到14个超级混合池；另有1个仅包含2个混合池的超级混合池，总计15个超级混合池。使用Illumina NextSeq 500测序仪对这15个超级混合池进行测序。通过bcl2fastq软件将超级混合池的测序reads拆分至各个独立混合池。分别使用FASTQC和Trimmomatic完成测序reads的质量评估与质量修剪。利用FASTX-toolkit，根据内嵌条形码将每个混合池的reads拆分至对应单个样本的独立fastq文件中。