Systematic assessment of next generation sequencing for quantitative small RNA profiling: a multiple protocol study across multiple laboratories
Abstract
Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols.
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Another recent paper found relative quantification of microRNA RNA-seq across multiple laboratories to be remarkably accurate and reproducible, consistent with this work.
“As well, another recent paper investigating microRNA RNA-seq across multiple laboratories found relative quantification to be “remarkably accurate and reproducible” (Giraldez et al. 2017), consistent with our work.”
From Toward the human cellular microRNAome (2017) · cites this paper as Giraldez et al. 2017
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1,500 scientists lift the lid on reproducibility
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What does research reproducibility mean?
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Reproducibility of high-throughput mRNA and small RNA sequencing across laboratories
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Self-citationDiverse human extracellular RNAs are widely detected in human plasma
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Cited by
Sorted by citations
Toward the human cellular microRNAome
McCall, M. N., Kim, M.-S., Adil, M., Patil, A. H., Lu, Y., Mitchell, C. J., Leal-Rojas, P., Xu, J., Kumar, M., Dawson, V. L., et al. (2017) · Genome Research · doi:10.1101/gr.222067.117 · Cited by 151 · see citing works
- Authors
- Giraldez, M., Spengler, R., Etheridge, A., Godoy, P., Barczak, A., Srinivasan, S., De Hoff, P., Tanriverdi, K., Courtright, A., Lu, S., Khoory, J., Rubio, R., Baxter, D., Driedonks, T., Buermans, H., Nolte-‘t Hoen, E., Jiang, H., Wang, K., Ghiran, I., Wang, Y., Van Keuren-Jensen, K., Freedman, J., Woodruff, P., Laurent, L., Erle, D., Galas, D., & Tewari, M.
- Publisher
- openRxiv
- Published
- 2017-05-17
- Cited by
- 6
- References
- 5
- Type
- Preprint
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