TM3’seq: A Tagmentation-Mediated 3’ Sequencing Approach for Improving Scalability of RNAseq Experiments

Abstract

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 we 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. We expect that the cost- and time-efficient features of TM3′seq make large-scale RNA-seq experiments more permissive for the entire scientific community.

Transposase-assisted tagmentation of RNA/DNA hybrid duplexes

“…For instance, the libraries generated by TRACE-seq in its current form are not strand-specific, which is a significant drawback for RNA-Seq experiments. Yet, TRACE-seq should be able to be converted to 5’ RNA-seq or 3’ RNA-seq, which can directionally preserve the 5’ or 3’ end information of transcripts ( Cole et al, 2018 ; Pallares et al, 2020 ). In addition, TRACE-seq could be used in multiplex profiling when utilizing Tn5 transposase containing barcoded adaptors ( Cusanovich et al, 2015 ; Zhu et al, 2019 ).…”

Wild flies hedge their thermal preference bets in response to seasonal fluctuations

“…DNA was made into libraries using a liquid handling robot (Analytic Jena CyBio-Felix Model 30-5015-100-24). Library preparation was done using a tagmentation protocol with Tn5 transposase 47,48 . Genomic DNA from 273 individual flies was made into per-individual libraries.…”

The Detection and Bioinformatic Analysis of Alternative 3′ UTR Isoforms as Potential Cancer Biomarkers

“…The single cell methods include early cell-barcoding of samples which allows individual samples to be pooled and processed as a single sample. Early pooling (or early multiplexing) of samples significantly reduces the costs and increases sequencing-throughput [ 69 ]. Another interesting feature of single-cell RNA-seq methods is the use of UMIs, which allows detection of PCR duplicates while reporting the unique transcript counts and thus, removes PCR amplification bias [ 79 , 80 ].…”