Combinatory use of distinct single-cell RNA-seq analytical platforms reveals the heterogeneous transcriptome response

Yukie Kashima,Masataka Shirai,Masahito Hosokawa,Hiroko Matsunaga,Ying Liu,Katsuya Tsuchihara,Takashi Kohno,Ayako Suzuki,Kohji Arikawa,Yutaka Suzuki,Haruko Takeyama,Sumio Sugano

doi:10.1038/s41598-018-21161-y

Yukie Kashima, Masataka Shirai + Show 10 more

Open Access

https://doi.org/10.1038/s41598-018-21161-y

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Abstract

Single-cell RNA-seq is a powerful tool for revealing heterogeneity in cancer cells. However, each of the current single-cell RNA-seq platforms has inherent advantages and disadvantages. Here, we show that combining the different single-cell RNA-seq platforms can be an effective approach to obtaining complete information about expression differences and a sufficient cellular population to understand transcriptional heterogeneity in cancers. We demonstrate that it is possible to estimate missing expression information. We further demonstrate that even in the cases where precise information for an individual gene cannot be inferred, the activity of given transcriptional modules can be analyzed. Interestingly, we found that two distinct transcriptional modules, one associated with the Aurora kinase gene and the other with the DUSP gene, are aberrantly regulated in a minor population of cells and may thus contribute to the possible emergence of dormancy or eventual drug resistance within the population.

Highlights

Recent developments in single-cell sequencing technologies have opened the possibility of analyzing individual single cells
After removing PCR duplicates and separating the scRNA-seq tags for individual cells based on unique molecular identifiers (UMIs), 83,649,012 reads remained on average
With the micro-droplet platform, we separated the cells by confining the cells into micro-droplets, where the mRNAs of each cell were labeled with distinct barcodes in individual droplets

Summary

Introduction

Recent developments in single-cell sequencing technologies have opened the possibility of analyzing individual single cells. It is important to focus on the molecular diversity of cancer cells to understand the mechanisms underlying the emergence of drug-resistant and metastasizing cells Detailed knowledge of such intra-tumor heterogeneity would provide crucial information for understanding the eventual development of drug-resistant cells or metastatic dissemination in cancer and generate potential opportunities for novel pharmaceutical interventions[6,7]. PC9 and II-18 cells harbor driver mutations in the EGFR gene and a 15-base-pair-deletion in exon 19 and L858R, respectively These cell lines are sensitive to gefitinib. H2228 cells have an ALK-fusion as a driver mutation, so gefitinib should be ineffective[38,39] Using these cell lines, we collected scRNA-seq data from a total of 536 and 54,631 single cells using a micro-chamber-based platform, bead-seq, and a micro-droplet-based platform, Chromium of 10× Genomics, respectively. We describe our attempts to elucidate the heterogeneous transcriptomic responses of cancer cells in response to an anti-cancer drug

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