TM3’Seq: a tagmentation-mediated 3′ sequencing approach for improving scalability of RNA-Seq experiments

RNA-seq has become the standard tool for collecting genome-wide expression data in diverse fields, from quantitative genetics and medical genomics to ecology and developmental biology. However, RNA-seq library preparation is still prohibitive for many laboratories. Recently, the field of single-cell transcriptomics has reduced costs and increased throughput by adopting early barcoding and pooling of individual samples-producing a single final library containing all samples. In contrast, RNA-seq protocols where each sample is processed individually are significantly more expensive and lower throughput than single-cell approaches. Yet, many projects depend on individual library generation to preserve important samples or for follow-up re-sequencing experiments.

Improving on currently available RNA-seq methods Princeton University researchers have developed TM3’seq, a 3′-enriched library preparation protocol that uses Tn5 transposase and preserves sample identity at each step. TM3’seq is designed for high-throughput processing of individual samples (96 samples in 6h, with only 3h hands-on time) at a fraction of the cost of commercial kits ($1.5 per sample). The protocol was tested in a range of human and Drosophila melanogaster RNA samples, recovering transcriptomes of the same quality and reliability than the commercial NEBNext® kit. The researchers expect that the cost- and time-efficient features of TM3’seq make large-scale RNA-seq experiments more permissive for the entire scientific community.

Tagmentation-mediated 3’seq protocol –TM3’seq


The steps of the library preparation procedure are shown. Pooling of individual libraries is done in step 6, after final PCR amplification; this is known as late multiplexing, and it allows the preservation of individual sample libraries for follow up experiments like re-sequencing of specific samples.

Pallares LF, Picard S, Ayroles JF. (2019) TM3’seq: A Tagmentation-Mediated 3′ Sequencing Approach for Improving Scalability of RNAseq Experiments. G3 (Bethesda) [Epub ahead of print]. [article]

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