Data from: Evaluation of TagSeq, a reliable low-cost alternative for RNAseq

RNAseq is a relatively new tool for ecological genetics that offers researchers insight into changes in gene expression in response to a myriad of natural or experimental conditions. However, standard RNAseq methods (e.g., Illumina TruSeq® or NEBNext®) can be cost prohibitive, especially when study designs require large sample sizes. Consequently, RNAseq is often underused as a method, or is applied to small sample sizes that confer poor statistical power. Low cost RNAseq methods could therefore enable far greater and more powerful applications of transcriptomics in ecological genetics and beyond. Standard mRNAseq is costly partly because one sequences portions of the full length of all transcripts. Such whole-mRNA data is redundant for estimates of relative gene expression. TagSeq is an alternative method that focuses sequencing effort on mRNAs’ 3’ end, reducing the necessary sequencing depth per sample, and thus cost. We present a revised TagSeq library construction procedure, and compare its performance against NEBNext®, the “gold-standard” whole mRNAseq method. We built both TagSeq and NEBNext® libraries from the same biological samples, each spiked with control RNAs. We found that TagSeq measured the control RNA distribution more accurately than NEBNext®, for a fraction of the cost per sample (~10%). The higher accuracy of TagSeq was particularly apparent for transcripts of moderate to low abundance. Technical replicates of TagSeq libraries are highly correlated, and were correlated with NEBNext® results. Overall, we show that our modified TagSeq protocol is an efficient alternative to traditional whole mRNAseq, offering researchers comparable data at greatly reduced cost.RNA测序（RNAseq）是生态学遗传学领域一款相对新兴的研究工具，可帮助研究人员洞悉生物体在各类自然或实验条件下的基因表达变化。然而，标准RNA测序方法（如Illumina TruSeq®或NEBNext®）往往成本高昂，尤其当研究设计需要大样本量时。因此，RNA测序常未能被充分应用，或仅应用于样本量过小的研究，导致统计效能低下。低成本的RNA测序方案，有望极大拓展转录组学在生态学遗传学及其他相关领域的应用广度与效能。标准mRNA测序（mRNAseq）成本较高的部分原因在于，其需对所有转录本的全长片段进行测序，而这类全mRNA测序数据对于相对基因表达量的估算而言存在冗余。TagSeq作为一种替代方法，将测序重心聚焦于mRNA的3'端，可降低单样本所需的测序深度，从而压缩实验成本。本研究提出了一种改良后的TagSeq文库构建流程，并将其性能与被视为“金标准”的全mRNA测序方法NEBNext®进行对比。我们从同一批掺入了对照RNA的生物样本中，分别构建了TagSeq与NEBNext®文库。实验结果显示，在单样本成本仅约为NEBNext®的10%的情况下，TagSeq对对照RNA分布的定量准确性更优。这种准确性优势在中低丰度转录本中尤为显著。TagSeq文库的技术重复样本间相关性极强，且其测序结果与NEBNext®的结果也具有良好相关性。综上，本研究证实，经改良的TagSeq实验方案可作为传统全mRNA测序的高效替代方案，能够在大幅降低成本的同时，为研究人员提供可媲美的测序数据。