RNA sequencing (RNA-SEQ) of RRD1 knockout yeast strains with Rapamycin treatment

Purpose: We develop a multivariate polynomial temporal genetic association (MPTGA) approach for detecting temporal genetic loci of quantitative traits monitored over time in a population. To dissect causal regulators under each temporal genetic locus, we develop a temporal genetic causality test (TGCT) for inferring causal relationships between traits linked to the locus. We applied MPTGA and TGCT to simulated data sets and a yeast population that was molecularly profiled over six time points in response to treatment with the drug rapamycin. We demonstrate significantly increased power to detect genetic loci modulating gene expression traits over time in this population and to resolve the causal regulators of dynamic expression quantitative loci hot spots, thereby increasing the power to elucidate regulatory networks. We experimentally validated a teQTL (temporal expression Quantitative Trait Locus) hot spot locus interacting with rapamycin treatment over time, which could only be identified using our temporal genetic association procedure. We experimentally validated RRD1, a cis regulated gene in this locus, as the causal regulator for this teQTL hot spot. Methods: The wild type strain BY4730 and RRD1 knockout strain YSC6273-201925697 were obtained from Thermo Scientific Open Biosystems. Yeast was grown in YPD medium to log-phase in shaken flasks at 30°C. Total RNA was extracted as described previously. For rapamycin treatment, 100nM rapamycin (Cayman Chemical, Ann Arbor, MI) was added to the medium after yeast grew to log-phase. After culture for 50 minutes, total RNA was extracted the same as above. All experiments were repeated 3 times on three different days. Approximately 250 ng of total RNA per sample were used for library construction by the TruSeq RNA Sample Prep Kit (Illumina) and sequenced using the Illumina HiSeq 2500 instrument with 100nt single read setting according to the manufacturer's instructions. Sequence reads were aligned to yeast genome assembly using Tophat . Total 6,932 yeast transcripts were quantified using Cufflinks, and 5,542 of them overlap with transcripts on Yeast Genome 2.0 Arrays from Affymatrix, which was used for generating the yeast F2 time course data. The 5,542 transcripts were used in further analysis. Differentially expressed genes were defined by CuffDiff. At q-value <0.01, 64 and 581 were in RRD1 ko signature without rapamycin (RRD1 ko no treatment vs. wild type no treatment) and RRD1 ko signature with rapamycin (RRD1 ko with rapamycin vs. wild type with rapamycin), respectively. Conclusions: Integration of temporal and genetic data has the potential to enhance the power to resolve causal relationships and to provide a more accurate view of regulatory networks in complex biological systems.研究目的：本研究开发了多变量多项式时序遗传关联方法（Multivariate Polynomial Temporal Genetic Association, MPTGA），用于检测研究群体中随时间监测的数量性状的时序遗传位点。为解析每个时序遗传位点下的因果调控因子，我们开发了时序遗传因果检验（Temporal Genetic Causality Test, TGCT），以推断该位点关联性状间的因果关系。我们将MPTGA与TGCT应用于模拟数据集，以及一个经雷帕霉素药物处理、在六个时间点完成分子谱分析的酵母群体。本研究证实，该方法可显著提升在该群体中检测随时间调控基因表达性状的遗传位点的效能，并解析动态表达数量性状位点热点的因果调控因子，从而增强阐明调控网络的能力。本研究通过实验验证了一个随时间与雷帕霉素处理产生相互作用的时序表达数量性状位点热点（temporal expression Quantitative Trait Locus, teQTL），该位点仅能通过本研究提出的时序遗传关联流程被识别，并验证了该位点中的顺式调控基因（cis-regulated gene）RRD1为该teQTL热点的因果调控因子。 研究方法：本研究从赛默飞世尔Open Biosystems（Thermo Scientific Open Biosystems）获取野生型菌株BY4730与RRD1敲除菌株YSC6273-201925697。将酵母置于YPD培养基中，于30℃摇瓶培养至对数生长期。总RNA提取参照既往实验方案进行。雷帕霉素处理组：待酵母生长至对数生长期后，向培养基中加入终浓度100nM的雷帕霉素（Cayman Chemical，密歇根州安娜堡市），培养50分钟后，按前述方法提取总RNA。所有实验均于3个不同日期重复3次。每份样本取约250ng总RNA，使用TruSeq RNA样本制备试剂盒（Illumina）构建文库，并依照制造商说明书采用Illumina HiSeq 2500测序仪以100nt单端读长模式进行测序。使用Tophat将测序reads比对至酵母基因组组装版本。采用Cufflinks对共计6932条酵母转录本进行定量，其中5542条与Affymatrix公司Yeast Genome 2.0芯片的转录本存在重叠，该芯片曾用于生成酵母F2时序数据集。后续分析均基于这5542条转录本展开。差异表达基因通过CuffDiff进行鉴定。当q值<0.01时，无雷帕霉素处理的RRD1敲除特征基因集（RRD1敲除未处理组 vs 野生型未处理组）与有雷帕霉素处理的RRD1敲除特征基因集（RRD1敲除处理组 vs 野生型处理组）分别包含64个与581个差异表达基因。 研究结论：整合时序数据与遗传数据，有望提升解析因果关系的效能，并为复杂生物系统中的调控网络提供更为精准的认知。