Next Generation Sequencing Facilitates Quantitative Analysis of S. aureus subsp. aureus ST398 and ST239 transcriptomes. Next Generation Sequencing Facilitates Quantitative Analysis of S. aureus subsp. aureus ST398 and ST239 transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goal of this study is to investigate the significantly different pathways and genes between ST398 and ST239. Methods: mRNA profiles of ST398 and ST239 at mid-logarithmic growth phase (4h) were generated by deep sequencing, respectively in quadruplicate and duplicate samples, using the Hiseq2000 (Illumina, CA) sequencer. The four samples of ST398 are J-92 (Sample1), W-604 (Sample2), R-1025 (Sample3) and R-1089 (Sample4) and grouped to G1, while the two samples of ST239 are J-95 (Sample5) and J-99 (Sample6) and grouped to G2. The sequence reads of ST398 and ST239 that passed quality filters were respectively aligned to S. aureus subsp. aureus ST398 (RefSeq accession number AM990992) and S. aureus subsp. aureus TW20 (RefSeq accession number NC _017331) using the Burrows-Wheeler Alignment tool (BWA) followed by ANOVA (ANOVA). Only the consistent data between the four ST398 samples and two ST239 samples were reserved for further analysis. qRT–PCR validation was performed using SYBR Green assays. Results: Using an optimized data analysis workflow, RNA-seq analyses revealed four types of significantly differentially expressed genes between ST398 and ST239 (G1 only, G2 only, G1/G2>2, G2/G1>2), and only the type of G1/G2>2 was included in this study. The type of G1/G2>2 included 164 genes in total, in which there are 14 top genes showing G1/G2>5 including essB gene. Conclusions: Our data provide new information to the signicantly different genes between ST239 and ST398, especially the highly expressed genes in ST398 compared to ST239 which might be closely related to the high virulence of ST398. Overall design: mRNA profiles of ST398 and ST239 were generated by deep sequencing, in duplicate, using the Hiseq2000 (Illumina, CA) sequencer.

研究目的：下一代测序（Next-generation sequencing, NGS）已彻底革新了基于系统视角的细胞通路分析范式。本研究旨在探究ST398与ST239之间存在显著差异的细胞通路与基因。

研究方法：本研究采用Illumina Hiseq2000测序仪（Illumina, 加利福尼亚州），分别对处于对数中期生长阶段（培养4小时）的ST398与ST239的信使RNA（messenger RNA, mRNA）表达谱进行深度测序：ST398设置4个生物学重复样本，ST239设置2个生物学重复样本。ST398的4个样本分别为J-92（样本1）、W-604（样本2）、R-1025（样本3）及R-1089（样本4），并归为G1组；ST239的2个样本分别为J-95（样本5）与J-99（样本6），归为G2组。将通过质量过滤的ST398与ST239测序读段，分别比对至金黄色葡萄球菌ST398亚种（RefSeq登录号：AM990992）与金黄色葡萄球菌TW20亚种（RefSeq登录号：NC_017331），比对工具采用Burrows-Wheeler比对工具（Burrows-Wheeler Alignment tool, BWA），后续进行方差分析（Analysis of Variance, ANOVA）。仅保留ST398的4个样本与ST239的2个样本的一致数据用于后续分析。采用SYBR Green法进行实时定量逆转录PCR（qRT–PCR）验证实验。

研究结果：通过优化的数据分析流程，RNA测序分析揭示了ST398与ST239之间存在四类显著差异表达基因（仅G1组表达、仅G2组表达、G1/G2>2、G2/G1>2），本研究仅纳入G1/G2>2类型的基因。该类型共包含164个基因，其中14个核心基因的G1/G2表达比值大于5，涵盖essB基因。

研究结论：本研究数据为ST239与ST398之间的显著差异基因提供了新的研究依据，相较于ST239，ST398中高表达的基因可能与ST398的高毒力特性密切相关。

整体实验设计：采用Illumina Hiseq2000测序仪（Illumina, 加利福尼亚州），对ST398与ST239的信使RNA（messenger RNA, mRNA）表达谱进行深度测序，设置2个生物学重复。