Evaluation of TagSeq, a reliable low‐cost alternative for RNA seq

Abstract RNA seq 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 RNA seq methods (e.g., Illumina TruSeq ® or NEBN ext ® ) can be cost prohibitive, especially when study designs require large sample sizes. Consequently, RNA seq is often underused as a method, or is applied to small sample sizes that confer poor statistical power. Low cost RNA seq methods could therefore enable far greater and more powerful applications of transcriptomics in ecological genetics and beyond. Standard mRNA seq is costly partly because one sequences portions of the full length of all transcripts. Such whole‐ mRNA data are redundant for estimates of relative gene expression. TagSeq is an alternative method that focuses sequencing effort on mRNA s’ 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 NEBN ext ® , the ‘gold‐standard’ whole mRNA seq method. We built both TagSeq and NEBN ext ® libraries from the same biological samples, each spiked with control RNA s. We found that TagSeq measured the control RNA distribution more accurately than NEBN ext ® , 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 NEBN ext ® results. Overall, we show that our modified TagSeq protocol is an efficient alternative to traditional whole mRNA seq, offering researchers comparable data at greatly reduced cost.