# Evaluation of TagSeq, a reliable low-cost alternative for RNAseq

## TL;DR

TagSeq is a low-cost RNA sequencing method that provides accurate gene expression data at about 10% of the cost of standard methods.

## Contribution

A revised TagSeq protocol is presented as a cost-effective and accurate alternative to traditional RNAseq methods.

## Key findings

- TagSeq accurately measured control RNA distribution more effectively than NEBNext®.
- TagSeq is approximately 10% of the cost of standard RNAseq methods.
- TagSeq technical replicates are highly correlated with each other and with NEBNext® results.

## Abstract

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-prime end, thereby reducing the necessary sequencing depth per sample, and thus cost. Here we present a revised TagSeq protocol, 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.