Benchmarking full-length transcript single cell mRNA sequencing protocols

Abstract

Background Single cell mRNA sequencing technologies have transformed our understanding of cellular heterogeneity and identity. For sensitive discovery or clinical marker estimation where high transcript capture per cell is needed only plate-based techniques currently offer sufficient resolution. Results Here, we present a performance evaluation of four different plate-based scRNA-seq protocols. Our evaluation is aimed towards applications taxing high gene detection sensitivity, reproducibility between samples, and minimum hands-on time, as is required, for example, in clinical use. We included two commercial kits, NEBNext® Single Cell/ Low Input RNA Library Prep Kit (NEB®), SMART-seq® HT kit (Takara®), and the non-commercial protocols Genome & Transcriptome sequencing (G&T) and SMART-seq3 (SS3). G&T delivered the highest detection of genes per single cell. SS3 presented the highest gene detection per single cell at the lowest price. Takara® kit presented similar high gene detection per single cell, and high reproducibility between samples, but at the absolute highest price. NEB® delivered a lower detection of genes but remains an alternative to more expensive commercial kits. Conclusion For the tested kits we found that ease-of-use came at higher prices. Takara can be selected for its ease-of-use to analyse a few samples, but we recommend the cheaper G&T-seq or SS3 for laboratories where a substantial sample flow can be expected.

CapTrap-seq: a platform-agnostic and quantitative approach for high-fidelity full-length RNA sequencing

“…However, it should be noted that these approaches are often designed for specific platforms and may require additional sample preparation steps, such as efficient ribodepletion. Consequently, their effectiveness in targeting all transcript types may vary 21 , 22 .…”

A Protocol for Low-Input RNA-Sequencing of Patients with Febrile Neutropenia Captures Relevant Immunological Information

“…Here, we hypothesise that full-transcript plate-based low-input protocol can be both sensitive enough in terms of gene capture and practical in terms of handling in the laboratory to be used for patients with low WBC counts. We previously found that the SMART-Seq ® HT kit protocol performed well for single-cell sequencing, with high robustness and ease of implementation [ 18 ].…”