Method of amplifying single cell transcriptome

Abstract

The present disclosure provides methods for amplifying RNA by using a combination of reverse transcription and amplification cycles based on multiple annealing and cyclization. Primers are used such that the resulting amplicons comprise a first cell-specific barcode sequence, a second cell-specific barcode sequence, and a unique molecular identifier barcode sequence.

Description

[0001] Related application information [0002] This application claims priority to U.S. Provisional Application No. 62 / 512,144, filed May 29, 2017, which is hereby incorporated by reference in its entirety for all purposes. [0003] Statement of Government Interests [0004] This invention was made with government support under CA174560 and CA186693 from the National Institutes of Health. The government has certain rights in this invention. [0005] background [0006] field of invention [0007] Embodiments of the invention generally relate to methods and compositions for amplification of single cell messenger RNA, such as messenger RNA from a single cell. Background technique [0008] Single cell RNA sequencing techniques are known. See Wen et al., Genome Biology (2016) 17:17, DOI10.1186 / s13059-016-0941-0; Mortazavi et al., Nature Methods DOI:10.1038 / nmeth.1226; Chapman et al., PLoS ONE 10(3):e0120889, doi: 10.1371 / journal.pone.0120889 (2015); and Sheng et al., Nature...

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Problems solved by technology

[0009] Despite these advances, a common limitation of these methods is low RNA detection efficiency, which is typically 20% or less (see Ziegenhain, C., Vieth, B., Parekh, S., Reinius, B., Guillaumet- Adkins, A., Smets, M., Leonhardt, H., Heyn, H., Hellmann, I. and Enard, W. (2017) Comparative Analysis of Single-Cell RNA Sequencing Methods. ) MolCell, 65, 631-643e634; Liu, S. and Trapnell, C. (2016) Single-cell transcriptome sequencing: recent advances and remaining challenges. F1000Res, 5)

This increases uncertainty in RNA quantification due to sampling noise and leads to loss of lowly expressed transcripts

Another limitation is that despite the addition of UMIs, RNA quantification is still inaccurate due to UMI error counts

The reason for this phenomenon is that the UMI-containing reverse transcription primer may not be completely removed before cDNA amplification, and the removal efficiency cannot be measured by existing methods

Finally, for methods that use PCR to amplify cDNA, the exponential amplification process can lead to amplification bias

Taken together, these issues limit the completeness, accuracy, and cost-effectiveness of existing scRNA-seq methods

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